UNITED STATES ENVIRONMENTAL PROTECTION AGENCY 

WASHINGTON, D.C. 20460 

OFFICE OF CHEMICAL SAFETY AND 

POLLUTION PREVENTION 

MEMORANDUM 

Date: 6/29/2015 

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

PC Code: 056801 

Decision No.: 4600006, 468314, 468316 

Petition No.: NA 

DP Barcode: D397990, D404352, D404353 

Registration No.: NA 

Risk Assessment Type: NA 

TXR No.: 0056268 

MRID No.: See Table 

Regulatory Action: NA 

Case No.: NA 

CAS No.: 63-25-2 

40 CFR: NA 

FROM: 

Immediate 

Greg Akerman, 

Office 

Ph.D. ~ ~ 

Health Effects Division (7509P) 

THROUGH: Jess 

Deputy 

Rowland 

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 carbaryl: Test Order# EDSP-056801-57. 

Page I of2 

Page 1 of 149
II. RESPONSE

Attached are the EDSP Tier 1 assay DERs for carbaryl.

III. MRID Table

Chemical: Carbaryl PC Code: 056801

Guideline Assay MRID 

890.1100 Amphibian Metamorphosis Assay (Frog) NA

890.1150 Androgen Receptor Binding (Rat Prostate) 48615201

890.1200 Aromatase Assay (Human Recombinant) 48615202

890.1250 Estrogen Receptor Binding 48615203

890.1300 Estrogen Receptor Transcriptional Activation 

(Human Cell Line HeLa-9903) 48615204 

890.1350 Fish Short-Term Reproduction 48669601

890.1400 Hershberger (Rat) 47987701

890.1450 Female Pubertal (Rat) NA

890.1500 Male Pubertal (Rat) NA

890.1550 Steroidogenesis (Human Cell Line – H295R) 48615205

890.1600 Uterotrophic (Rat) 47987702

NA= Not Applicable (requirement satisfied by other scientific relevant information (OSRI))

Page 2 of 2 

Page 2 of 149
DATA EVALUATION RECORD

CARBARYL

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

EPA Contract No. EP10H001452 

Task Assignment No. 2-20-2012 (MRID 48615201) 

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: 2/16/2012

Secondary Reviewer: Signature:

David A. McEwen, B.S. Date: 2/29/2012

Program Manager: Signature:

Jack D. Early, M.S. Date: 3/06/2012

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 3/06/2012

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

signing by CSS-Dynamac Corporation personnel. 

Page 3 of 149
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 4 of 149
CARBARYL/ 056801 

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

OCSPP 890.1150/ OECD None 

Primary Reviewer: Khin Oo M.D. 

Health Effects Division, Office of Pesticide Programs 

Secondary Reviewer: Greg Akerman, Ph.D. 

Health Effects Division, Office of Pesticide Programs 

~ •• 11;I:.•• Signature:-~~=::::::::::::--:-----

Date: c I .t i I 2. c 1£ 

Signature: -~~· .... ~___ ;a_ ___ _ Date: _<fl_~/.;..="'"'"~h~'---- Template version 08/2011 

DAT A EVALUATION RECORD 

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

PC CODE: 056801 DP BARCODE: D397990 

TXR#: 0056268 CAS No.: 63-25-2 

TEST MATERIAL (PURITY): Carbary! (100% a.i.) 

SYNONYMS: 1-naphthyl methylcarbamate 

CITATION: Rouquie, D. (2011 ). Evaluation of Carbary I in the Androgen Receptor Binding 

Assay. Bayer SAS, Sophia Antipolis Cedex, France. Laboratory Study No. : SA 

11189, October 26, 2011. MRID 48615201. Unpublished. 

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

TEST ORDER#: EDSP-056801-57 

EXECUTIVE SUMMARY: In an androgen receptor (AR) binding assay (MRID 48615201 ), 

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

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

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

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

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

competitive binding assay was conducted to measure the binding of a single concentration of 

[3H]-R 1881 (1 nM) in the presence of increasing concentrations of carbaryl (logarithmic increase 

from 10- 10 to 10-3 M). DMSO was used as the vehicle at a final assay concentration of .S3 .3%. 

The assay included dexamethasone as a weak positive control, and Rl 881 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 (Kd) was 1.0 I nM. The Kd obtained 

from Saturation Assay #2 was slightly 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 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%). The saturation binding experiment 

demonstrated that the AR was present in a reasonable concentration and functioning with 

appropriate affinity for the Rl 881 ligand. 

Page 5 of 149
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 2 of 14

CARBARYL/ 056801 OCSPP 890.1150/ OECD None

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

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

reduction in [3

H]-R1881binding occurred at the highest concentration tested (10−3 M), with 

specific binding being reduced to 28-34%. Specific binding was also reduced at 10−4 M (60-

85%), but not at concentrations ≤10−5 M (87-111% binding). Carbaryl was classified as a binder 

in all three competitive binding runs.

The estimated mean log IC50 was −3.5 M for carbaryl, −4.4 M for the weak positive control and 

−9.1 M for R1881. Compared to the R1881 ligand, the mean relative binding affinity (RBA) 

was 0.00029% for carbaryl and 0.0020% for the positive control. The solvent control responses 

indicated no drift in the study assay. All performance criteria were met, with the exception that 

ligand depletion and non-specific binding (NSB) were not reported, but the IC50s for R1881 and 

the weak positive control were within the expected ranges. These deviations are considered 

minor; the performance criteria were met.

Based on the results from the three runs, carbaryl 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 6 of 149
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 3 of 14

CARBARYL/ 056801 OCSPP 890.1150/ OECD None

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Bayer S.A.S., Bayer CropScience

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); M. Vagner (Report 

Unit Assistant)

Study Period: June 29, 2011 to July 22, 2011

2. Test substance: Carbaryl

Description: Beige to pink powder

Source: Bayer CropScience

Batch #: NNCAAX0025 (expiry date: February 16, 2013)

Purity: 100% a.i.

Solubility: Not reported

Volatility: Not reported

Stability: Not reported

Storage conditions: 2 to 30°C

CAS #: 63-25-2

Molecular weight: 201.22 g/mol

Structure:

3. Non-labeled ligand: R1881

Supplier: Fountain Limited (Malta)

Catalog #:

Batch #:

E3164-000

B1023

Purity: 98.8%

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)

July 4, 2011 to July 19, 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

6. Solvent/vehicle control: Dimethyl sulfoxide (DMSO), batch no. 73296KMV; Sigma.

Justification for choice of 

solvent:

DMSO is one of the solvents recommended in the guideline (OPPTS 890.1150) and 

carbaryl is reasonably soluble in this solvent.

Final Concentration: ≤3.3%

Page 7 of 149
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 4 of 14

CARBARYL/ 056801 OCSPP 890.1150/ OECD None

B. METHODS

1. Preparation of Rat Ventral Prostate Cytosol: The preparation of rat prostate cytosol for 

androgen receptor binding assays was detailed under Study SA 11021 which included the 

saturation binding experiment (Attachment 4 of the subject study). 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 ± 3oC (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 71, 73, and 115-122 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 

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 

Page 8 of 149
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 5 of 14

CARBARYL/ 056801 OCSPP 890.1150/ OECD None

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 [3

H]-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 115-122 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, [3H]-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. 

Page 9 of 149
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 6 of 14

CARBARYL/ 056801 OCSPP 890.1150/ OECD None

TABLE 3. Summary of Conditions for Competitive Binding Experiment a

Source of receptor Rat ventral prostate cytosol

Concentration of radioligand 1 nM

Optimization of receptor concentration Not reported b 

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

Incubation Temperature 5 ± 3oC (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 pages 12, 14 and 71 of the study report.

b 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. 

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

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

the binding of a single concentration of [3

H]-R1881 (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 vehicle, and no precipitation was visibly observed at any 

concentration (≤10−3

 M). 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 (carbaryl), 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 10 of 149
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 7 of 14

CARBARYL/ 056801 OCSPP 890.1150/ OECD None

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 Carbaryl (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 [3

H]-R1881 

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

performed as described. 

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

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

Page 11 of 149
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 8 of 14

CARBARYL/ 056801 OCSPP 890.1150/ OECD None

b. Descriptors for receptor binding

Bmax: maximal binding capacity

Kd: dissociation constants

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

by the competitor

Relative Binding Affinity (RBA): IC50 of R1881 × 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 used for the assay. The goodness of fit (r2

) was not reported and the Kd

obtained from Saturation Assay #2 was slightly 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 R-1881 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 75 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 [3

H]-

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

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

each run that illustrate the binding of [3H]-R1881 to the androgen receptor. The data fit 

results in a linear plot.

Page 12 of 149
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 9 of 14

CARBARYL/ 056801 OCSPP 890.1150/ OECD None

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

Binding Experiment.

Saturation Assay #1 – Non-Specific and

Total Binding

Saturation Assay #1 – Specific Binding

Saturation Assay #2 – Non-Specific and

Total Binding

Saturation Assay #2 – Specific Binding

Saturation Assay #3 – Non-Specific and

Total Binding

Saturation Assay #3 – Specific Binding

Page 13 of 149
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 10 of 14

CARBARYL/ 056801 OCSPP 890.1150/ OECD None

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

 

B. COMPETITIVE BINDING EXPERIMENT: Competitive binding experiment

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

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

binding inhibition occurred at the highest concentration tested (10−3 M), with specific 

binding being reduced to 28-34%. Specific binding was also reduced at 10−4 M (60-85%), 

but not at concentrations ≤10−5 M (87-111% binding). Because the test chemical reduced 

[

3

H]-R1881 binding to <50% in all three runs, carbaryl classified as an AR binder in this 

assay. 

The estimated mean log IC50 was −3.5 M for carbaryl, −9.1 M for R1881, and −4.4 M for 

the weak positive control. When compared to R1881, the mean RBA was 0.00029% for 

carbaryl and 0.0020% 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 #1 was discarded (and repeated) due to the use by error of an invalid control 

substance.

Page 14 of 149
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 11 of 14

CARBARYL/ 056801 OCSPP 890.1150/ OECD None

TABLE 6. Competitive Binding Assay of Carbaryl 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

Carbaryl NR NR NR NA

Log IC50 (M) R1881 −9.1 −9.1 −9.1 −9.1

Positive control −4.4 −4.4 −4.4 −4.4

Carbaryl ~−3.8 −3.3 −3.4 −3.5

IC50 (M) R1881 7.6 × 10−10 8.8 × 10−10 8.7 × 10−10 8.3 × 10−10

Positive control 4.0 × 10−5 4.3 × 10−5 4.0 × 10−5 4.1 × 10−5

Carbaryl ~1.5 × 10−4 5.6 × 10−4 4.3 × 10−4 3.8 × 10−4

Log RBA b Positive control −4.7 −4.7 −4.7 −4.7

Carbaryl −5.3 −5.8 −5.7 −5.6

RBA (%) Positive control 0.0019 0.0020 0.0022 0.0020

Carbaryl 0.00052 0.00016 0.00020 0.00029

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 carbaryl 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 concentration (10−3 M) of carbaryl. A graph of 

the mean values of carbaryl across the three runs is provided in Figure 4. 

Page 15 of 149
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 12 of 14

CARBARYL/ 056801 OCSPP 890.1150/ OECD None

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

Compounds.

Page 16 of 149
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 13 of 14

CARBARYL/ 056801 OCSPP 890.1150/ OECD None

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

 Carbaryl in Three Competitive Binding Assays.

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

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

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

Ligand depletion is minimal. The recommended ratio of 

total binding in the absence of competitor to total amount 

of [

3

H]-R1881 added per assay tube.

≤15% Not reported c X 

Carbaryl Top (% binding) 80 to 115 104 to 111 X

R1881 fitted curve parameters

Top (% binding) 82 to 114 93 to 103 X

Bottom (% binding) −2.0 to 2.0 −1.14 to 1.95 X

Hill Slope −1.2 to −0.8 −1.11 to −0.89 X

Weak positive control (dexamethasone) fitted curve parameters

Top (% binding) 87 to 106 97 to 104 X

Bottom (% binding) −12 to 12 −1.27 to 6.30 X

Hill Slope −1.4 to −0.6 −1.07 to −0.93 X

Saturation Binding Experiment Kd (nM) 0.685 to 1.57 0.59 to 1.25 d X

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

a Data were obtained from pages 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 Kd was slightly low (0.59 nM) in Run 2, but within the acceptable range in the other two runs (Saturation 

Binding Experiment; SA 11021).

e The non-specific binding was not reported and could not be calculated from the raw data provided.

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

unlabeled R1881 displaced [3

H]-R1881 in a manner consistent with one-site binding, as 

indicated by a Hill slope of −1.1 to −0.89. Examination across the runs indicated 

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

Page 17 of 149
Androgen Receptor Binding (Rat Prostate Cytosol) (2011) / Page 14 of 14

CARBARYL/ 056801 OCSPP 890.1150/ OECD None

III. DISCUSSION AND CONCLUSIONS

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

competitive assays, carbaryl was considered to be a very 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 highest concentration of carbaryl. The IC50 values for 

carbaryl in the three evaluations were determined to be ~1.5×10−4

, 5.6×10−4 and 4.3×10−4

M. The RBA values for carbaryl in comparison to R1881 were 0.00051%, 0.00016% and 

0.00020%.

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

androgen receptor was present in a reasonable concentration and functioning with 

appropriate affinity for the R1881 ligand. The 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 slightly 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 plots for 

the three saturation assays were linear. The specific binding reached a plateau, and nonspecific binding was less than 20% of total binding (4.6-12.6%). 

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

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

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

specific binding being reduced to 28-34%. Specific binding was also reduced at 10−4 M 

(60-85%), but not at concentrations ≤10−5 M (87-111% binding). Carbaryl is classified as a 

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

tested in all three runs. 

The mean log IC50 was −3.5 M for carbaryl compared to −9.1 M for R1881 and −4.4 M for 

the weak positive control. Compared to R1881, the mean RBA was 0.00029% for carbaryl 

and 0.0020% for the weak 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 tested. The solvent control responses indicated no drift in the study assay. 

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 are considered minor; the performance criteria were considered 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 18 of 149
DATA EVALUATION RECORD

CARBARYL 

Study Type: OCSPP 890.1200, Aromatase Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-20-2012 (MRID 48615202) 

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: 3/02/2012

Secondary Reviewer Signature:

Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 3/05/2012

Program Manager: Signature:

Jack D. Early, M.S. Date: 3/07/2012

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 3/07/2012

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

signing by CSS-Dynamac Corporation personnel. 

Page 19 of 149
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 20 of 149
Aromatase (Human Recombinant) Assay (2011) I Page I of 12 

CARBARYL I 056801 OCSPP 890.1200 I OECD None 

Primary Reviewer: Vincent Chen M.S. Signature: ~-- Health Effects Division (7509P) Date: z.y -svtJ z_O.J..S 

Secondary Reviewer: Greg Akerman, Ph.D. Signature: L9 ~l'-4..,..__ 

Risk Assessment Branch Ill, Health Effects Division (7509P) Date: ' (; 'h.rhJ- Template version 08/2011 

DAT A EVALUATION RECORD 

STUDY TYPE: Aromatase (Human Recombinant); OCSPP 890.1200 

PC CODE: 056801 DP BARCODE: D397990 

TXR: 0056268 CAS No.: 63-25-2 

TEST MATERIAL (PURITY): Carbaryl Technical (100%) 

SYNONYMS: 

IUPAC: 1-naphthyl methylcarbamate 

CAS: 1-naphthalenyl N-methylcarbamate 

CITATION: Tinwell, H. (2011). Evaluation ofCarbaryl in the Aromatase Assay. Bayer S.A.S. 

(Bayer CropScience [355, Rue Dosto'ievski, BP 153, 06903 Sophia Antipolis 

Cedex, France]). Laboratory Report No.: SA 11060, October 20, 2011. MRID 

48615202. Unpublished. 

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

TEST ORDER: EDSP-056801-57 

EXECUTIVE SUMMARY: In an in vitro aromatase (CYP 19) assay (MRID 48615202), 

Carbary I (100% a.i., Batch# NNCAAX0025) was incubated with human recombinant aromatase 

and tritiated androstenedione ( 1-~ [3H(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 carbaryl 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 carbaryl. 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 c10- 10 to 10-5

) using a 

known inhibitor (4-hydroxyandrostenedione; 4-0H ASDN; formestane), and the test carbaryl 

series c10- 10 to 10-3) with three repetitions per concentration. 

Aromatase activity in the full activity controls ranged from 0.078 to 0.106 nmol/mg-protein/min 

for the 3 test runs, with a mean(± SD) of 0.091±0.009 nmol/mg-protein/min. Activity in the 

background controls ranged from 6.8 to 8.2% and averaged 7.4% of the full control activity. The 

response of each full activity control within a run was 89 to 122% of the average full activity. 

The response of the full activity controls and background controls was acceptable, although 

activity in the full controls was marginally acceptable. 

Page 21 of 149
Aromatase (Human Recombinant) Assay (2011) / Page 2 of 12

CARBARYL / 056801 OCSPP 890.1200 / OECD None

For the positive control (4-OH ASDN), aromatase activity averaged 0.0849 ± 0.0216 nmol/mgprotein/min at the lowest tested concentration (10−10 M) and 0.0027 ± 0.0008 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. The 

overall %CV exceeded the recommended 15% at the 10−10 to 10−6 M test concentrations (18-

27%), primarily due to the large difference in activity between the third run and the first two 

runs. The top curve (% activity) was 104.3% (Run 1), 100.7% (Run 2), and 65.5% (Run 3) 

compared to the specified guideline range of 90-110%. For 4-OH ASDN, the estimated log IC50

averaged −6.83 M (outside of recommended −7.3 to −7.0 M range). 

For carbaryl, aromatase activity averaged 0.0785 ± 0.0190 nmol/mg-protein/min at the lowest 

tested concentration (10−10 M) and 0.0382 ± 0.0024 nmol/mg-protein/min at the highest tested 

concentration (10−3 M). Carbaryl reduced aromatase activity compared to controls, on average, 

to approximately 65% at 10−4 M and 42% at 10−3 M. 

Although carbaryl exhibited a concentration-dependent inhibition of aromatase activity and 

decreased activity by more than 50% (log IC50 = −3.83 M), the average slope of the inhibition 

curve was steeper than –2 (-2.92). Therefore, carbaryl is classified as Equivocal for aromatase

inhibition in this assay. 

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

890.1200). 

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

Assurance statements were provided. 

Page 22 of 149
Aromatase (Human Recombinant) Assay (2011) / Page 3 of 12

CARBARYL / 056801 OCSPP 890.1200 / OECD None

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Substance: Carbaryl Technical

Description: Beige to pink powder

Source: Bayer CropScience, Germany

Lot/Batch #: NNCAAX0025 (expires February 16, 2013) 

Purity: 100%

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: 201.22 g/mol

CAS #: 63-25-2

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%

3. Radiolabeled Substrate: 1-β [

3

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

3

H]ASDN)

Source: Perkin Elmer (Catalog # NET-926)

Lot/Batch #: 3615304 

Radiochemical Purity (Supplier): 97%

Specific Activity: 20.7 Ci/mmol (0.766 TBq/mmol)

Radiochemical Purity (In-lab Determination): Not provided

4. Positive Control:

4-hydroxyandrostenedione (4-OH ASDN; 

Formestane) 

CAS #: 566-48-3

Source: Sigma-Aldrich (Catalog # F2552)

Lot/Batch #: 081K2133 

Purity: 99.6 %

5. Solvent (Vehicle Control): DMSO

Source: Sigma-Aldrich (Ref. # 154938)

Lot/Batch #: 07796KK and 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: 0.113 mmol/mg protein/min

Page 23 of 149
Aromatase (Human Recombinant) Assay (2011) / Page 4 of 12

CARBARYL / 056801 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.

Carbaryl 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, carbaryl 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 ca. 

−74 oC 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 °C for no more than six months. 

Other assay components 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) 0.1 M

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 0.113 nmol/mg protein/min. The minimum aromatase 

activity in the full activity control samples of the proficiency test was determined to be 

0.0982 nmol/mg protein/min, which was slightly less than the minimum acceptable 

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 24 of 149
Aromatase (Human Recombinant) Assay (2011) / Page 5 of 12

CARBARYL / 056801 OCSPP 890.1200 / OECD None

control (duplicate) and carbaryl (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

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.

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 

for the log IC50 of −6.7 M was slightly greater than the guideline range (−7.3 to −7.0 M).

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: Data were not provided. 

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 25 of 149
Aromatase (Human Recombinant) Assay (2011) / Page 6 of 12

CARBARYL / 056801 OCSPP 890.1200 / OECD None

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

activity to the presence of eight concentrations of carbaryl 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. The full enzymatic activity (≥95% for the 

means of each run) was obtained at ≤10−5

 M carbaryl in Runs 1 and 2, defining the top of 

the concentration-response curve. In Run 3, the top of the curve only reached 65% full 

activity, similar to the positive control. 

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

Carbaryl Conc 1b 3 All test components plus carbaryl 1×10−3

Carbaryl Conc 2b 3 All test components plus carbaryl 1×10−4

Carbaryl Conc 3b 3 All test components plus carbaryl 1×10−5

Carbaryl Conc 4b 3 All test components plus carbaryl 1×10−6

Carbaryl Conc 5b 3 All test components plus carbaryl 1×10−7

Carbaryl Conc 6b 3 All test components plus carbaryl 1×10−8

Carbaryl Conc 7b 3 All test components plus carbaryl 1×10−9

Carbaryl Conc 8b 3 All test components plus carbaryl 1×10−10

a The complete assay contained buffer, propylene glycol, microsomal protein, [3

H]ASDN, and NADPH. b Proficiency chemicals or tests chemical(s). 

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

Page 26 of 149
Aromatase (Human Recombinant) Assay (2011) / Page 7 of 12

CARBARYL / 056801 OCSPP 890.1200 / OECD None

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 

(SD), standard error of the mean (SEM), and coefficient of variation (CV). 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 were 

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 

0.078 to 0.106 nmol/mg-protein/min for the three test runs, with a mean and standard 

deviation of 0.091±0.009 nmol/mg-protein/min. Activity in the background controls ranged 

from 6.8 to 8.2% and averaged 7.4% of the full control activity. 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. The response of 

each full activity control within a run was 89 to 122% of the average full activity. The 

response of the full activity controls and background controls was acceptable for each run, 

although activity in the full controls was just marginally acceptable. 

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

activity averaged 0.0849 ± 0.0216 nmol/mg-protein/min at the lowest tested concentration 

Page 27 of 149
Aromatase (Human Recombinant) Assay (2011) / Page 8 of 12

CARBARYL / 056801 OCSPP 890.1200 / OECD None

(10−10 M) and 0.0027 ± 0.0008 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 SD, SEM, and %CV. Inhibition response 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 10−10 to 10−6 M test concentrations (18-27%); 

however, the higher CV values were due solely to the usually low activity observed during 

Run 3. 

TABLE 7. Effect of Carbaryl 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 2.9 0.38 0.22 13

−6 3 18.7 3.90 2.25 21

−6.5 3 39.8 7.16 4.13 18

−7 3 61.3 16.02 9.25 26

−7.5 3 77.8 19.46 11.24 25

−8 3 84.5 20.53 11.85 24

−9 3 87.1 23.29 13.45 27

−10 3 92.2 20.04 11.57 22

Carbaryl −3 3 42.2 5.55 3.2 13

−4 3 64.8 12.80 7.4 20

−5 3 85.2 22.65 13.0 27

−6 3 87.7 21.58 12.5 25

−7 3 85.3 21.27 12.3 25

−8 3 84.6 21.28 12.3 25

−9 3 83.3 18.06 10.4 22

−10 3 85.2 17.77 10.3 21 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

Page 28 of 149
Aromatase (Human Recombinant) Assay (2011) / Page 9 of 12

CARBARYL / 056801 OCSPP 890.1200 / OECD None

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

 

C. TEST SUBSTANCE: For carbaryl, aromatase activity averaged 0.0785 ± 0.0190 

nmol/mg-protein/min at the lowest tested concentration (10−10 M) and 0.0382 ± 0.0024 

nmol/mg-protein/min at the highest tested concentration (10−3 M). The mean aromatase 

activity of carbaryl (expressed as % full control activity) for each concentration tested across 

all 3 runs is presented in Table 7, along with the overall SD, SEM, and %CV. Inhibition 

response curves for carbaryl from each run are shown in Figure 2, and the average inhibition 

response curve across all runs is shown in Figure 3. Carbaryl reduced aromatase activity

compared to control, on average, to ca. 65% at 10−4 M and 42% at 10−3 M. At lower 

concentrations of carbaryl, aromatase activity was 83.3-87.7% of control. As with the 

positive control, the high degree of variation in the carbaryl data was the result of the low 

levels of aromatase activity observed across all concentrations. Carbaryl exhibited a 

concentration dependent inhibition of aromatase activity and decreased activity by more 

than 50% at 10–3 M (log IC50 = −3.83 M); however, the average slope of the inhibition 

curves was steeper than −2 (−2.92); indicating an equivocal for aromatase inhibition in this 

assay. 

Page 29 of 149
Aromatase (Human Recombinant) Assay (2011) / Page 10 of 12

CARBARYL / 056801 OCSPP 890.1200 / OECD None

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

FIGURE 3. Mean Inhibition Response Curve for Carbaryl. 

Page 30 of 149
Aromatase (Human Recombinant) Assay (2011) / Page 11 of 12

CARBARYL / 056801 OCSPP 890.1200 / OECD None

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

(as %CV) was >15%, except for 10−3 M carbaryl. The estimated log IC50 ranged from 

−3.95 to −3.70 M for the test material and averaged −3.83 M compared to the positive 

control 4-OH ASDN, which averaged −6.83 M. The log IC50 values for the positive control 

4-OH ASDN (−7.25 to −6.60 M) were just outside the guideline range of −7.3 to −7.0 M. 

The average slope of the concentration response curve was −2.92 for carbaryl and −0.88 for 

4-OH ASDN; however, confidence in the mean slope is low due to the large variation (70% 

CV for carbaryl and 15% CV for 4-OH ASDN).

TABLE 8. Effect of Carbaryl 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)

Carbaryl −3.84 −3.70 −3.95 −3.83 0.12 3.2

4-OH ASDN −6.63 −6.60 −7.25 −6.83 0.37 5.4

Slope

Carbaryl −5.22 −2.17 −1.36 −2.92 2.04 69.8

4-OH ASDN −0.83 −1.032 −0.78 −0.88 0.13 15.2 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

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATORS CONCLUSIONS: For each run the data for the full activity and 

background activity controls were within the acceptable ranges proposed in the guideline. 

Furthermore there was no assay drift recorded in any of the three runs. The data generated 

for the positive control evaluations run concurrently with the carbaryl evaluations confirmed 

the responsiveness of the aromatase to a reference inhibitor. Specifically, the log IC50 values 

for the positive control, formestane were −6.63, −6.60, −7.25 for each of the three 

independent evaluations with an overall log IC50 of −6.73. Although the log IC50 values 

were slightly outside the range (−7.3 to −7.0) proposed in the guideline all other criteria 

were met. The log IC50 for carbaryl was between −3.7 and −3.947 with an overall log IC50 

of approximately −3.84, which equates to an IC50 of ~1.45 × 10−4 M. However, the overall

slope of the concentration curve (Figure 3) was >−2 (~−4.12). Based on the data and 

consideration of the criteria for data interpretation, the activity of carbaryl in the aromatase 

assay is considered as being equivocal.

B. AGENCY COMMENTS: Aromatase activity in the full activity controls ranged from 

0.078 to 0.106 nmol/mg-protein/min for the 3 test runs, with a mean (± SD) of 0.091 ± 

0.009 nmol/mg-protein/min. Activity in the background controls ranged from 6.8 to 8.2% 

and averaged 7.4% of the full control activity. The response of the full activity controls and 

background controls was acceptable, although activity in the full controls was marginally

acceptable. 

For the positive control substance (4-OH ASDN), aromatase activity averaged 0.0849 ± 

0.0216 nmol/mg-protein/min at the lowest tested concentration (10−10 M) and 0.0027 ± 

0.0008 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 10−10 to 10−6

Page 31 of 149
Aromatase (Human Recombinant) Assay (2011) / Page 12 of 12

CARBARYL / 056801 OCSPP 890.1200 / OECD None

M test concentrations (18-27%), due to the large difference between Run 3 and the first two 

runs. The top curve (% activity) was 104.3% (Run 1), 100.7% (Run 2), and 65.5% (Run 3) 

compared to the specified guideline range of 90-110%. 

For carbaryl, aromatase activity averaged 0.0785 ± 0.0190 nmol∙mg-protein−1∙min−1 at the 

lowest tested concentration (10−10 M) and 0.0382 ± 0.0024 nmol∙mg-protein−1∙min−1 at the 

highest tested concentration (10−3 M). Carbaryl reduced aromatase activity compared to 

controls, on average, to approximately 65% at 10−4 M and 42% at 10−3 M. The estimated 

log IC50 ranged from −3.95 to −3.70 M for carbaryl and averaged −3.83 M compared to the 

positive control 4-OH ASDN, which averaged −6.83 M. The log IC50 values for the 

positive control 4-OH ASDN (−7.25 to −6.60 M) were outside the guideline range of −7.3 to 

−7.0 M. The average of the slopes for the concentration response curve was −2.92 for 

carbaryl and −0.88 for 4-OH ASDN; however, confidence in the mean slope is low due to 

the large variation (70% CV for carbaryl and 15% CV for 4-OH ASDN).

Although carbaryl exhibited a concentration dependent inhibition of aromatase activity and 

decreased activity by more than 50% at 10–3 M (log IC50 = −3.83 M), the average slope of 

the inhibition curves was steeper than −2 (−2.92); therefore, carbaryl is classified as 

equivocal for aromatase inhibition in this assay. 

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

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

• The required proficiency data were not provided. 

• The SD and %CV were not provided for the positive control for the Hill slope and log

IC50. The reviewers were able to calculate these values from the individual data.

• For the positive control, the log IC50 values (−7.25 to −6.60 M) were outside the 

guideline range of −7.3 to −7.0 M, and the overall %CV exceeded the recommended 

15% at the 10–10 to 10–6 test concentrations (18-27%).

• Given the low level of activity observed for both the test chemical and positive control 

in Run 3, data from this run should not have been used for analysis and a fourth run 

should have been conducted. 

Page 32 of 149
DATA EVALUATION RECORD

CARBARYL

Study Type: OCSPP 890.1250, Estrogen Receptor Binding Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-20-2012 (MRID 48615203) 

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: 2/17/2012

Secondary Reviewer: Signature:

David A. McEwen, B.S. Date: 2/28/2012

Program Manager: Signature:

Jack D. Early, M.S. Date: 3/06/2012

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 3/06/2012

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

signing by CSS-Dynamac Corporation personnel. 

Page 33 of 149
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 34 of 149
CARBARY LI 056801 

Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) I Pagel of 18 

OCSPP 890.1250/ OECD None 

Primary Reviewer: Patience Browne, Ph.D. 

Office of Science Coordination and Policy 

Secondary Reviewer: Greg Akerman, Ph.D. 

Health Effects Division, Office of Pesticide Programs 

Signature:~ Date: 2j.2kj 1$ 

Signature: 07 ~ _.,J.,._ _ _ 

Date: c;fjw .c 

Template version ()8/2011 

DATA EVALUATION RECORD 

STUDY TYPE: Estrogen Receptor Binding Assay Using Rat Uterine Cytosol (ER-RUC); 

OCSPP 890.1250 

PC CODE: 056801 DP BARCODE: D397990 

TXR#: 0056268 CAS No.: 63-25-2 

TEST MATERIAL (PURITY): Carbaryl (100% a.i.) 

SYNONYMS: 1-naphthyl methylcarbamate 

CITATION: Rouquie, D. (2011 ). Evaluation of Carbary I in the Estrogen Receptor Binding 

Assay. Bayer SAS, Sophia Antipolis Cedex, France. Laboratory Study No.: SA 

11209, October 26, 2011. MRID 48615203 . Unpublished 

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

TEST ORDER#: EDSP-056801-57 

EXECUTIVE SUMMARY: In an estrogen receptor (ER) binding assay (MRID 48615203), 

uterine cytosol from Sprague-Dawley rats was used as the source of ER to conduct saturation 

and competitive binding experiments. A saturation binding experiment was conducted to 

demonstrate that the ER isolated from rat uterine cytosol was present in reasonable numbers and 

was functioning with appropriate affinity for the radiolabeled reference estrogen prior to 

conducting ER competitive binding experiments. The competitive binding experiment was 

conducted to measure the binding of a single concentration of [3H]- I 7P-estradiol (I nM) in the 

presence of increasing concentrations of carbaryl (logarithmic increase from 10- 10 to 10- 3 M). 

DMSO was used as the vehicle at a final concentration of S2%. The assay included 19-

norethindrone as a weak positive control, octyltriethoxysilane as a negative control, and I 7Pestradiol as the natural ligand reference material. 

In the saturation binding experiment, the appropriate protein concentration was determined to be 

0.5 mg/mL as it bound 25-35% of the [3H]-l 7P-estradiol. The Kd for [3H]-I 7P-estradiol was 

0.070 nM, and the estimated Bmax was 34.3 fmol/l 00 μg protein for the prepared uterine cytosol. 

The Kd for each run was within the expected range of 0.03 to 1.5 nM. 

In the competitive binding experiment, the reference compounds generally performed as 

expected. The negative control, octyltriethoxysilane, had no effect on binding, and there were 

concentration dependent decreases in binding for the natural ligand and the weak positive 

control. The mean IC so was 1.8 x I 0- 9 M for the natural ligand and 9.0 x l o-6 M for the weak 

--

Page 35 of 149
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 2 of 18

CARBARYL/ 056801 OCSPP 890.1250/ OECD None

positive control. The relative binding affinity (RBA) for the weak positive control was 0.019%, 

compared to the natural ligand. The Hill Slope for the natural ligand was outside the acceptable 

range, at −2.0 to −1.7, indicating that the receptors were behaving as though there was more than 

one binding site for 17β-estradiol. Carbaryl was tested over a concentration range that fully 

defined the top of the curve. The maximum percent binding 109.0-117.9% was within 

approximately 25 percentage points of the lowest concentration of the estradiol standard 83.5-

116.2%. 

An estimated mean IC50 was not calculated for carbaryl as it did not achieve 50% binding 

inhibition. In two assay runs, carbaryl reduced the specific binding of the radiolabeled estradiol 

at the highest concentration tested (10−3 M) to 62-71%; in Run 3 percent binding was ≥75% at 

10−3 M. 

Based on the results from the three runs, carbaryl is classified as Equivocal in the Estrogen 

Receptor Binding Assay. 

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

assay (OCSPP 890.1250).

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

Page 36 of 149
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 3 of 18

CARBARYL/ 056801 OCSPP 890.1250/ OECD None

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Bayer S.A.S., Bayer CropScience

Location: 355 Rue Dostoievski, BP 153, 06903 Sophia Antipolis Cedex, France

Study Director: D. Rouquie

Other Personnel: H. Tinwell (replacement Study Director); M.P. Come (Technician); M. Vagner 

(Report Unit Assistant)

Study Period: July 5, 2011 to July 20, 2011

2. Test substance: Carbaryl

Description: Beige to pink powder

Source: Bayer CropScience

Lot/Batch #: NNCAAX0025 (expiry date: February 16, 2013)

Purity: 100 % a.i.

Solubility: Not reported

Volatility: Not reported

Stability: Not reported

Storage conditions: 2 to 30°C

CAS #: 63-25-2

Molecular weight: 201.22 g/mol

Structure:

3. Non-labeled ligand: 17β-estradiol

Supplier: Sigma-Aldrich, St. Louis, MO

Catalog #: E8875

Batch #: 010M0142

Purity: ≥98% 

CAS #: 50-28-2

4. Radioactive ligand: [

3

H]-17β-estradiol

Supplier: Perkin Elmer, Boston, MA

Catalog #: NET517

Batch#: 650702

Radiochemical purity: >97%

Specific activity: 130.2 Ci/mmol

Concentration of stock: 1 nM 

5. Positive control: 19-norethindrone

Supplier: Sigma-Aldrich, St. Louis, MO

Catalog #: N4128

Batch #: 019K1735

Purity: ≥98% 

CAS # : 66-22-4

Page 37 of 149
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 4 of 18

CARBARYL/ 056801 OCSPP 890.1250/ OECD None

6. Negative control: Octyltriethoxysilane

Supplier: Alfa Aesar

Catalog #: L04407

Batch #: 10153935

Purity: 97.3%

CAS # : 2943-75-1

7. 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 

carbaryl is reasonably soluble in this solvent.

Final Concentration: ≤2%

B. METHODS 

1. Preparation of Rat Uterine Cytosol (RUC): The preparation of rat uterine cytosol for the 

ER binding assays was detailed under Study SA 11020 which included three independent 

saturation binding experiments (Attachment 4 of the subject study). A total of 90 

ovariectomized Sprague Dawley (Crl:CD(SD)) rats were obtained from Charles River 

Laboratories (St. Germain-sur-l’Abresle, France). Animals were 13 weeks old at the time of 

euthanasia, and rats had been ovariectomized 7 days prior to tissue harvest. The uteri were 

weighed, homogenized in TEDG (Tris, EDTA, DTT, glycerol) + PMSF 

(phenylmethylsulfonyl fluoride) buffer, and then centrifuged for 10 min at 2500×g at 4 oC. 

The supernatant was transferred and centrifuged for 60 minutes at 105,000×g, discarding the 

resulting pellet. Protein concentration of the cytosol was determined to be 3.35 mg/mL 

using a DC protein assay kit (BioRad). The cytosol was divided into aliquots (volume not 

reported) for storage at −74 ± 10 oC until use.

2. Saturation (radioligand) Binding Experiment: Three independent saturation binding 

experiments measuring total and non-specific binding of [3H]-17β-estradiol were performed 

in a previous study (SA 11020) for the cytosol used in the subject competitive binding 

experiment to demonstrate that the ER was present in reasonable concentrations and had the 

appropriate affinity for the native 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 uterine cytosol

Concentration of radioligand (as serial dilutions) 0.03-3.0 nM 

Concentration of non-labeled ligand (100X [radioligand]) 3-300 nM

Concentration of receptor Sufficient to bind 25-35% of radioligand 

Temperature Refrigerated (4oC)

Incubation time 16-20 hours

Composition of assay buffer Tris 10 mM (Tris/HCl)

EDTA 1.5 mM 

Glycerol 10% (v/v)

Phenylmethylsulfonyl fluoride 1 mM 

DTT 1 mM

a Data were obtained from pages 77-80 of the study report.

Page 38 of 149
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 5 of 18

CARBARYL/ 056801 OCSPP 890.1250/ OECD None

On the day of the assay, the specific activity of the stock solution [3H]-17β-estradiol was 

adjusted for decay over time, and serial dilutions in TEDG + PMSF buffer were prepared to 

achieve the final concentrations of 0.03, 0.06, 0.08, 0.1, 0.3, 0.6, 1, and 3 nM. Solutions of 

non-labeled 17β-estradiol were prepared in a similar manner to achieve concentrations that 

were 100-fold greater than each respective radiolabeled concentration to result in final 

concentrations of 3, 6, 8, 10, 30, 60, 100, and 300 nM. The optimal protein concentration 

for the cytosol batch was determined by testing serial amounts of protein per tube, using 

0.03 nM radiolabeled estradiol, until a concentration was reached that bound 25-35% of the 

total radioactivity added. Each assay consisted of three non-concurrent runs, and each run 

contained three concurrent replicates at each concentration (Table 2).

TABLE 2. Saturation Binding Experiment Run a

Total binding b Non-specific 

binding c Radioligand alone d Assay Components

350 μL 300 μL --- TEDG + PMSF buffer

50 μL 50 μL 50 μL [

3

H]-17β-estradiol (8 serial dilutions) e

--- 50 μL --- Non-labeled 17β-estradiol (8 serial dilutions, 

100x each respective labeled concentration) f

100 μL 100 μL --- Uterine cytosol (diluted to appropriate conc.)

500 μL 500 μL 50 μL Total volume in each assay tube

a Data were obtained from pages 123-130 of the study report. Tube numbers corresponding to assay 

conditions were not provided.

b Total binding = [

3

H]-17β-estradiol bound to ER

c Non-specific binding = [3

H]-17β-estradiol and 100-fold greater non-labeled bound to ER

d Total [

3

H]-17β-estradiol alone for dpm determination at each concentration

e Final concentrations of [3

H]-17β-estradiol = 0.03, 0.06, 0.08, 0.1, 0.3, 0.6, 1, and 3 nM.

f Final concentrations of non-labeled 17β-estradiol = 3, 6, 8, 10, 30, 60, 100, and 300 nM.

Tubes were incubated with gentle vortexing for 16-20 hours at 4°C. To separate bound 

from free estradiol, hydroxyapatite (HAP) slurry was added to each tube and incubated in a 

water bath for 15 minutes, then centrifuged for 10 min at 1000×g. The remaining pellet was 

washed three times with TEDG + PMSF buffer. Ethanol was added to the HAP pellet 

remaining in each tube to extract the [

3

H]-17β-estradiol. An aliquot of supernatant was 

radioassayed by scintillation counting. The temperature was maintained at 4 oC throughout 

the assay prior to extraction with ethanol. 

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

competitive binding experiment is included in Table 3. 

Page 39 of 149
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 6 of 18

CARBARYL/ 056801 OCSPP 890.1250/ OECD None

TABLE 3. Summary of Conditions for Competitive Binding Experiment a

Source of receptor Rat Uterine Cytosol 

Concentration of radioligand 1 nM 

Concentration of receptor Sufficient to bind ≤10-15% of radioligand

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

Temperature 4±3 °C 

Incubation time 16-20 hours 

Composition of assay buffer Tris 10 mM 

EDTA 1.5 mM 

Glycerol 10% (v/v) 

Phenylmethylsulfonyl fluoride 1 mM

DTT 1 mM 

a Data were obtained from pages 12, 14 and 17 of the study report.

A solubility test was performed to determine the appropriate solvent (DMSO) using visual 

observation to examine for precipitation (use of magnification as not reported). On the day 

of the assay, the specific activity of the stock solution [3

H]-17β-estradiol was adjusted for 

decay over time, and diluted in TEDG+PMSF buffer to achieve the final appropriate 

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

by testing serial amounts of protein per tube, using 1.0 nM radiolabeled estradiol, until a 

concentration was reached that bound 10-15% of the total radioactivity added (125 μg 

protein per tube). Serial dilutions of carbaryl, the positive control (19-norethindrone is 

substituted for the weak positive norethynodrel because it is not commercially available), the 

negative control (octyltriethoxysilane), and the reference material (non-labeled 17β-estradiol) 

were prepared to achieve the concentrations shown in Table 4. Each assay consisted of three 

runs, and each run contained three replicates at each concentration, six replicates measuring 

total activity (50 μL master mix) plus six measuring total binding (solvent control) resulting 

in 105 samples/assay for a total of 315 samples. 

Page 40 of 149
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 7 of 18

CARBARYL/ 056801 OCSPP 890.1250/ OECD None

TABLE 4. Molar (M) concentrations in Competitive Binding Assay Run a b

Carbaryl

Weak Positive control Negative control Reference Chemical

19-Norethindrone Octyltriethoxysilane Non-labeled

17β-estradiol

Tubes 76-99 c Tubes 28-51 c Tubes 52-75 c Tubes 1-27 and 100-105 c

10−10 10−8.5 10−10 Solvent control d

10−9 10−7.5 10−9 10−11

10−8 10−7 10−8 10−10

10−7 10−6.5 10−7 10−9.5

10−6 10−6 10−6 10−9

10−5 10−5.5 10−5 10−8.5

10−4 10−4.5 10−4 10−8

10−3 10−4 10−3 10−7

a Data were obtained from pages 12-13 and 36-46 of the study report. 

b Each tube contains: 10μL of the test substance, positive control, negative control, solvent control, or nonlabeled 17β-estradiol; 390 μL of TEDG + PMSF buffer with [3

H]-17β-estradiol; and 100 μL of uterine 

cytosol (with ER), for a total of 500 μL.

c Each concentration of each chemical was run in triplicate, for a total of 105 tubes per run. Tube numbers 

were not provided, but were assigned by the reviewer based on data spreadsheets on pages 36-46. 

d Solvent was DMSO (2%) in Tubes 1-3 and 100-102.

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

buffer and extraction with ethanol), followed by scintillation counting of bound [3H]-17βestradiol were similar to those used in the saturation binding experiment. 

C. DATA ANALYSIS: For both the competitive binding and saturation binding assays, 

GraphPad Prism was used to generate nonlinear regression used to fit a curve (for 17βestradiol, the positive control, and the test substance) to the Hill equation formula which 

incorporated IC50 as a parameter to be estimated. For parameters reported from the 

saturation binding experiment (Kd and Bmax) and competitive binding experiment (log IC50

and RBA), the means were calculated for each run. 

1. Definitions

a. Classification of test material: Classification of the test material is based on the average of 

three runs. Each run was first individually classified as follows:

Interactive = lowest point on the fitted curve within the range of the data is less than 50% 

(i.e., >50% of the radiolabeled estradiol has been displaced from the ER). 

Not interactive = there are usable data points at or above 10−6 M and either the lowest point 

on the fitted response curve within the range of the data is above 75% (i.e., 

<25% of the radiolabeled estradiol has been displaced from the ER) or a 

binding curve cannot be fitted and the lowest average percent binding among 

concentration groups in the data is above 75%. 

Equivocal up to the limit of concentrations tested = If there are no data points at or above 

a test chemical concentration of 10−6

M and either a binding curve can be fit 

Page 41 of 149
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 8 of 18

CARBARYL/ 056801 OCSPP 890.1250/ OECD None

but ≤50% of the radiolabeled estradiol has been displaced from the ER or a 

binding curve cannot be fit and the lowest average percent binding among 

concentration groups in the data is >50%. 

Equivocal = A run is classified as equivocal if it does not fall into any of the categories 

above. 

The categorical classification of each run was assigned a numerical value as follows:

Run Classification Numerical Value

Interactive 2

Equivocal 1

Not interactive 0

Equivocal up to the limit of concentrations tested “missing”

The values for each run were then averaged across runs and the chemical classified using the 

following ranges: 

Test Material Classification Numerical Range

Interactive average ≥1.5

Equivocal 0.5≥ average <1.5

Not interactive average <0.5

Equivocal up to the limit of concentrations tested “missing”

b. Descriptors for receptor binding: 

Bmax: maximum specific binding number (fmol ER/100 μg cytosol protein) measures the 

concentration of active receptor sites

Kd: dissociation constant (nM), measures the affinity of the receptor for its natural ligand

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

the receptor

Relative Binding Affinity (RBA %): IC50 of 17β-estradiol ÷ IC50 of test substance × 100 

II. RESULTS

A. SATURATION BINDING EXPERIMENT: The appropriate protein concentration for 

the saturation binding assay was determined to be 0.5 mg/mL as it bound 25-35% of the 

[

3

H]-17β-estradiol (within the Guideline range of 25-35% binding). The results from the 

three saturation binding assays are summarized in Table 5 and shown graphically in Figures 

1-9. The mean Kd for [

3

H]-17β-estradiol was 0.070 nM, and the mean estimated Bmax was 

34.3 fmol/100 μg protein for the prepared rat uterine cytosol. The Kd and Bmaxfor each run 

fell within the expected range (0.03 to 1.5 nM and 10 to 150 fmol/100 μg uterine cytosolic 

protein, respectively). In addition, non-specific binding was less than 50% total binding and 

variability between non-specific and total binding were relatively low. The data produced 

consistent and linear Scatchard plots. The goodness of fit (r2

) was not reported. 

Page 42 of 149
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 9 of 18

CARBARYL/ 056801 OCSPP 890.1250/ OECD None

TABLE 5. Saturation Binding Experiment of 17β-estradiol with Estrogen Receptor from Rat Uterine 

Cytosol a

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

r

2 (unweighted) NR NR NR NA

Bmax (nM) 0.027 0.037 0.039 0.034 ± 0.011

Bmax (fmol/100 μg protein) 27 37 39 34.3 ± 11.1

Kd (nM) 0.089 0.073 0.049 0.070 ± 0.035

a Data were obtained from page 81 of the study report. 

b Calculated by reviewers (Mean ± SE)

r

2 Goodness of fit 

NR Not reported

NA Not applicable

FIGURE 1. Total and Non-specific binding Run 1.

FIGURE 2. Specific binding Run 1.

Page 43 of 149
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 10 of 18

CARBARYL/ 056801 OCSPP 890.1250/ OECD None

FIGURE 3. Scatchard plot for Specific binding Run 1.

FIGURE 4. Total and Non-specific binding Run 2.

Page 44 of 149
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 11 of 18

CARBARYL/ 056801 OCSPP 890.1250/ OECD None

FIGURE 5. Specific binding Run 2.

FIGURE 6. Scatchard plot for Specific binding Run 2.

Page 45 of 149
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 12 of 18

CARBARYL/ 056801 OCSPP 890.1250/ OECD None

FIGURE 7. Total and Non-specific binding Run 3.

FIGURE 8. Specific binding Run 3.

Page 46 of 149
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 13 of 18

CARBARYL/ 056801 OCSPP 890.1250/ OECD None

FIGURE 9. Scatchard plot for Specific binding Run 3.

B. COMPETITIVE BINDING EXPERIMENT: The results from the three competitive 

binding experiments are summarized in Table 6 and presented graphically in Figures 10-13. 

Precipitation was not observed at any concentration. In two assay runs, carbaryl reduced the

specific binding of the radiolabeled estradiol at the highest concentration tested (10−3 M) to 

62-71%; in Run 3 percent binding was ≥75% at 10−3 M. Therefore carbaryl was classified 

as equivocal (value= 1) in runs 1 and 2 and non-interaction (value= 0) in run 3. Overall 

carbaryl is classified as “equivocal” (overall value: 0.67) in this assay (Table 7).

 The estimated mean log IC50 was not calculated for carbaryl as the percent binding 

inhibition did not reach 50% for any run. The mean IC50 for the weak positive control was 

0.9 × 10−5

 M, and the RBA was 0.019% for the positive control, compared to R1881. RBA 

could not be calculated for carbaryl. 

Page 47 of 149
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 14 of 18

CARBARYL/ 056801 OCSPP 890.1250/ OECD None

TABLE 6. Competitive Binding Assay of Carbaryl with Estrogen Receptor from Rat Uterine Cytosol a

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

r

2 (unweighted) 17β-estradiol NR NR NR NA

19-norethindrone NR NR NR NA

Carbaryl NR NR NR NA

Log IC50 (M) 17β-estradiol −8.75 −8.74 −8.76 −8.75

19-norethindrone −5.05 −5.00 −5.04 −5.03

Carbaryl NA NA NA NA

IC50 (M) 17β-estradiol 1.8 × 10−9 1.8 × 10−9 1.7 × 10−9 1.8 × 10−9

19-norethindrone 0.9 × 10−5 1.0 × 10−5 0.9 × 10−5 0.9 × 10−5

Carbaryl NA NA NA NA

Log (RBA) c 19-norethindrone −3.70 −3.74 −3.72 −3.72

Carbaryl NA NA NA NA

RBA (%) 19-norethindrone 0.020 0.018 0.019 0.019

Carbaryl NA NA NA NA

a Data were obtained from page 17 of the study report.

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

c Calculated by reviewers

r

2 Goodness of fit

RBA Relative binding affinity

NR Not reported

NA Not applicable

TABLE 7. Binding Classification of Carbaryl with Estrogen Receptor a

Run 1 2 3 Mean Binding Classification c

Classification category value b 1 1 0 0.67 Equivocal

a Data were obtained from page 18 of the study report.

b Classification category value: Interactive = 2; Equivocal = 1; Not interactive = 0; Equivocal up to the limit of 

concentrations tested (“missing”, i.e., not included in calculation of mean).

c Interactive = mean ≥1.5; Equivocal = 0.5≤ mean <1.5; Not interactive = mean <0.5

Page 48 of 149
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 15 of 18

CARBARYL/ 056801 OCSPP 890.1250/ OECD None

FIGURE 10. Percentage [3H]-E2 Bound to the Estrogen Receptor in the Presence of 

Carbaryl, Unlabeled E2, 19-Norethindrone or Octyltriethoxysilane. Run 1 

FIGURE 11. Percentage [3H]-E2 Bound to the Estrogen Receptor in the Presence of 

Carbaryl, Unlabeled E2, 19-Norethindrone or Octyltriethoxysilane. Run 2 

Page 49 of 149
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 16 of 18

CARBARYL/ 056801 OCSPP 890.1250/ OECD None

FIGURE 12. Percentage [3H]-E2 Bound to the Estrogen Receptor in the Presence of 

Carbaryl, Unlabeled E2, 19-Norethindrone or Octyltriethoxysilane. Run 3

FIGURE 13. Mean Percentage [3H]-E2 Bound to the Estrogen Receptor in the Presence of 

Carbaryl in Three Competitive Assays.

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

properly, each run was evaluated using the following criteria (Table 8): 

Page 50 of 149
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 17 of 18

CARBARYL/ 056801 OCSPP 890.1250/ OECD None

TABLE 8. Criterion a Tolerance Limit(s) Value Yes No

17β-estradiol fitted curve parameters

Loge residual SD ≤2.35 NR

Top (% binding) b 94 to 111 97 to 111 X

Bottom (% binding) −4 to 1 0.9 to 2.7 X

Hill Slope (log10(M)-1) −1.1 to −0.7 −2.0 to −1.7 X

Weak Positive control (19-norethindrone) fitted curve parameters b

Loge residual SD NA NR

Top (% binding) NA 103 to 110 X

Bottom (% binding) NA −4.4 to 7.5 X

Hill Slope (log10(M)-1) NA −1.0 to −0.7 X

Solvent concentration

DMSO ≤10% ≤2% X

Negative control (octyltriethoxysilane) does not displace 

more than 25% of [

3

H]-17β-estradiol from the ER on average 

across all concentrations

≤25% 13.1-15.7% X

a Data were obtained from pages 17, 25, 27 and 29 of the study report. 

b The EPA Guideline does not define a set of tolerance limits for 19-norethindrone. Acceptance criteria were only 

defined for norethynodrel, which cannot be obtained commercially. The values reported were considered 

acceptable as they show 19-norethindrone to be an acceptable weak positive control.

NR Not reported

NA Not applicable

The Hill slope was outside the acceptable range, at −2.0 to −1.7, indicating that the receptors 

were behaving as though there was more than one binding site for 17β-estradiol. The 

bottom % binding of 0.9 to 2.7% was also outside the acceptable range (−4 to 1). 

Carbaryl was tested over a concentration range that fully defined the top of the curve. The 

maximum percent binding 109.0-117.9% was within approximately 25% of the lowest 

concentration of the estradiol standard 83.5-116.2%. Placements along the X-axis, and top 

and bottom plateaus were consistent across runs. 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: A total of three independent competitive binding 

experiments were conducted. The amount of cytosolic protein used in each competitive 

binding experiment was 125 μg/assay tube. The IC50 values for 17β-estradiol were 

determined to be 1.8×10−9, 1.8×10−9 and 1.7×10−9 M. The IC50 values for 19-norethindrone 

were determined to be 0.9×10−5, 1.0×10−5 and 0.9×10−5 M. The RBA values for 19-

norethindrone, in comparison to 17β-estradiol, were 0.020%, 0.018% and 0.019%. Based 

on the results of the three competitive experiments, carbaryl was considered equivocal for 

ER binding based on the average value of 0.67. 

B. AGENCY COMMENTS: For the saturation binding experiment, the appropriate protein 

concentration was determined to be 0.5 mg/mL as it bound 25-35% of the [3H]-17βestradiol. The mean Kd for [

3

H]-17β-estradiol was 0.070 nM, and the mean estimated Bmax

was 34.3 fmol/100 μg protein for the prepared uterine cytosol. The Kd for each run was 

Page 51 of 149
Estrogen Receptor Binding (Rat Uterine Cytosol) (2011) / Page 18 of 18

CARBARYL/ 056801 OCSPP 890.1250/ OECD None

within the expected range of 0.03 to 1.5 nM. The data produced consistent and linear 

Scatchard plots. The goodness of fit (r2

) was not reported. 

For the competitive binding experiment, the estimated mean IC50 was not calculated for 

carbaryl as it did not achieve 50% binding. The mean IC50 for the weak positive control 

was 9.0×10−6 M, with a RBA of 0.019%. The Hill slope for the natural ligand was outside 

the acceptable range, at −2.0 to −1.7. Carbaryl was tested over a concentration range that 

fully defined the top of the curve. The maximum percent binding 109.0-117.9% was within 

approximately 25 percentage points of the lowest concentration of the estradiol standard 

83.5-116.2%. 

In two assay runs, carbaryl reduced the specific binding of the radiolabeled estradiol at the 

highest concentration tested (10−3 M) to 62-71%; in Run 3 percent binding was ≥75% at 

10−3 M. Carbaryl is classified as equivocal in this assay as the mean specific ER binding 

was >50% and <75% at carbaryl concentrations up to 10–3 M in two of the three independent 

runs. 

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: 

• Several performance criteria were not met for 17β-estradiol.

• The criteria of loge residual SD for the competitive binding assay was not reported. 

• Animal number 2829 had a uterine weight that was two times higher than the upper 

limit of the remaining animals. It was likely that the ovariectomy of this animal was 

not complete and there was still some endogenous estrogen present (minor concern as it 

was a single animal).

Page 52 of 149
DATA EVALUATION RECORD

CARBARYL

Study Type: OCSPP 890.1300, Estrogen Receptor Transcriptional Activation 

EPA Contract No. EP10H001452 

Task Assignment No. 2-20-2012 (MRID 48615204) 

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: 2/20/2012

Secondary Reviewer Signature:

Scott D. Studenberg, Ph.D., 

D.A.B.T. Date: 3/6/2012 

Program Manager: Signature:

Jack D. Early, M.S. Date: 3/7/2012

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 3/7/2012

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

signing by CSS-Dynamac Corporation personnel. 

Page 53 of 149
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 54 of 149
CARBARYL /056801 

Estrogen Receptor Transcriptional Activation (2011) I Page 1 of 10 

OCSPP 890.1300/ OECD 455 

Primary Reviewer: Patience Browne, Ph.D. Signature~ Health Effects Division 

Secondary Reviewer: Minerva Mercado Ph.D., DABT Signatur 

Date:~ 

Dat~e. .~

Template version 08/2011 

Health Effects Division 

DATA EVALUATION RECORD 

STUDY TYPE: Estrogen Receptor Transcriptional Activation (Human cell Line, HeLa-9903); 

OCSPP 890.1300; OECD 455. 

PC CODE: 056801 DP BARCODE: D397990 

TXR#: 0056268 CAS No.: 63-25-2 

TEST MATERIAL (PURITY): Carbaryl (100% a.i.) 

SYNONYMS: 1-naphthalenyl N-methylcarbamate 

CITATION: Blanck, 0. (2011). Carbary[: Evaluation in the in vitro (HeLa-9903) estrogen 

receptor transcriptional activation assay. Bayer SAS, Sophia Antipolis Cedex, 

France. Laboratory Report No.: SA 11155, October 17, 2011. MRID 

48615204. Unpublished. 

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

TEST ORDER#: EDSP-053801-57 

EXECUTIVE SUMMARY: In an estrogen receptor transcriptional activation assay (MRID 

48615204), hERa-HeLa-9903 cells cultured in vitro were exposed to carbaryl (100% a.i., 

Batch# NNCAAX0025) at serial log concentrations of 10- 10 M to 10-

4 Min DMSO (0.1 % v/v) 

for approximately 24 hours. The experiments were performed using 96-well plates and each 

carbaryl concentration was tested in triplicate (3 wells/plate). Cells were exposed to the test 

agent for 23 ± l hr to induce reporter (luciferase) gene products. Luciferase expression in 

response to activation of the estrogen receptor by carbaryl was measured upon addition of a 

luciferase substrate and detection with a luminometer with acceptable sensitivity. Carbary! was 

tested up to the limit of solubility, 10-4 M. The laboratory proficiency assays using the required 

reference compounds adequately demonstrated that the assay was functioning properly. The 

mean RPCMax for carbaryl was 2.7% for the first run and 0.5% for the second run, and the 

associated PCMax was 10-

4

M for both runs. Because the RPCMax < PC10 in both assay runs, 

carbaryl was considered negative for estrogen receptor transcriptional activation in this test 

system. 

This assay satisfies the EDSP Tier 1 Test Order requirement for an Estrogen Receptor 

Transcriptional Activation assay (OCSPP 890.1300). 

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

statements were provided. 

.. 

Page 55 of 149
Estrogen Receptor Transcriptional Activation (2011) / Page 2 of 10

CARBARYL /056801 OCSPP 890.1300/ OECD 455

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Substance: Carbaryl

Description: Beige to pink powder

Source: Bayer Crop science

Lot/Batch #: NNCAAX0025

Purity: 100%

Solubility: Not reported (NR)

Volatility: NR

Stability: NR

Storage conditions: NR

Vapor pressure: NR

CAS #: 63-25-2

Structure:

O

O

N

H

3CH

2. Reference substances

17β-estradiol (strong estrogen; positive control)

Supplier: Sigma-Aldrich St. Louis, MO

Catalogue and Batch #: Batch # 101M0142

Purity: 100%

CAS # : 50-28-2

17α-estradiol (weak estrogen)

Supplier: Sigma-Aldrich St. Louis, MO

Catalogue and Batch #: 029K4124

Purity: 99.9%

CAS # : 57-91-0

Corticosterone (negative compound)

Supplier: Sigma-Aldrich St. Louis, MO

Catalogue and Batch #: 039K0268

Purity: 100%

CAS # : 50-22-6

17α-methyltestosterone (very weak agonist)

Supplier: Sigma-Aldrich St. Louis, MO

Catalogue and Batch #: BCBB5955

Purity: 100%

CAS # : 58-18-4

3. Vehicle(s)

Solvent: DMSO

Solvent control 

(final concentration):

0.1%

Page 56 of 149
Estrogen Receptor Transcriptional Activation (2011) / Page 3 of 10

CARBARYL /056801 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 (JCRB) Cell Bank and were verified to be free of 

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

Minimum Essential Medium (EMEM) without phenol red, supplemented with kanamycin 

(60 mg/L) and 10% dextran-coated charcoal-treated fetal bovine serum (DCC-FBS; Thermo 

Scientific, Batch Number ATF32351), in an incubator under 5% CO2 at 37 oC. Upon 

reaching 75-90% confluency, 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. The Growth 

media was replaced with media containing serial log dilutions of carbaryl in DMSO. Cells 

were incubated for 22-24 hours at 37±1°C. The total final concentration of DMSO was 

0.1%. Cytotoxicity was determined using the ATPlite 1 step kit from Perkin Elmer. The 

laboratory report states that each concentration of carbaryl or reference chemical was tested 

in triplicate wells, except the positive control (1 nM 17β-estradiol) and vehicle control were 

tested in six wells each. Raw data for vehicle control wells was not provided. While it was 

stated that the PC was tested in six wells, raw data for only 3 wells was provided. The lab 

report states that two plates per run were used, however raw data provided seems to belong 

to only one plate per run (i.e. 3 wells per concentration). It was not specified if the VC and 

PC test wells were in the same plate as the carbaryl and/or reference chemical wells.

Transcriptional activation of the estrogen receptor was determined using the NeoLite kit 

from Perkin Elmer. Luminescence was measured using a Wallac 1450 MicroBeta Windows 

Workstation, V 4.01.014, with a detection limit of 32 fg luciferase/well. 

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

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

concentrations resulting in insolubility and/or cytotoxicity. 

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

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

of proficiency chemicals. The details of the proficiency test were not provided, only the 

results. 

Page 57 of 149
Estrogen Receptor Transcriptional Activation (2011) / Page 4 of 10

CARBARYL /056801 OCSPP 890.1300/ OECD 455

Compound CAS No. Concentration

Range (M)

Expected

Response a Notes 

Diethylstilbestrol (DES) 56-53-1 10−14 to 10−8 Positive ---

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

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

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

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

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

1,3,5-Tris(4-hydroxyphenyl)benzene c 15797-52-1 10−12 to 10−5 Positive Cytotoxic at 100μM. PCmax

approx. 50% of PC. Binds to 

hERα and has ER antagonistic 

activity

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

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

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

that should be the highest tested 

concentration

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

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

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

c Compound selected to challenge solubility and cytotoxicity

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

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

therefore unacceptable.

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

concentrations of each of the following reference chemicals were tested although it is not 

clear if they 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 carbaryl when applicable. 

The test material was defined as negative for inducing estrogen receptor transcriptional 

activation if the RPCMax < PC10 in at least 2 of 2 (or 2 of 3) runs. Log EC50 and Hill slope 

values are calculated only if a positive response is observed. Coefficients of variation (CV) 

were calculated for the luminescence data triplicates. Concentrations showing >20% 

cytotoxicity or evidence of insolubility were excluded from analyses. 

4. Definitions

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

(bottom) and maximum (top) response 

Page 58 of 149
Estrogen Receptor Transcriptional Activation (2011) / Page 5 of 10

CARBARYL /056801 OCSPP 890.1300/ OECD 455

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 carbaryl solutions at 1 M in DMSO, therefore the 

definite 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 

82%. Based on these results, logarithmically increasing concentrations from 10−11 M to 

10−5 M were selected for the assay.

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

Concentration (M) % Viability b Commentsc

10−4 82.1

10−5 108.3

10−6 109.7

SDS 1% 0.0 Positive control for cytotoxicity

VC b 100.0

a Data were obtained from page 17 of the study report.

b VC = Vehicle control

B. POSITIVE AND NEGATIVE REFERENCE CHEMICALS

1. Proficiency Chemicals: The responsiveness of cells to the required proficiency chemicals 

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

different days. The data are reported in Table 2. 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. 

Page 59 of 149
Estrogen Receptor Transcriptional Activation (2011) / Page 6 of 10

CARBARYL /056801 OCSPP 890.1300/ OECD 455

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 36 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 17β-estradiol and corticosterone. 

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

the Hill Slope. The log PC50 and log PC10 for 17α-methyltestosterone were lower than the 

expected range for both assays; such deviation in the results shows an increased sensitivity 

to very weak agonists. In run 1, the log PC10 for 17α-estradiol was lower than log PC10 for

17β-estradiol in the same run, which is an unexplained anomaly. 

TABLE 3. Performance Criteria for Reference Chemicals

Reference Chemical Parameter Acceptable 

Range

Values Acceptable

Run 1 Run 2 Yes No

17β-estradiola

LogPC50 −11.4 to −10.1 −10.4 −10.7 X

LogPC10 <−11 −11.8 −11.4 X

LogEC50 −11.3 to −10.1 −10.4 −10.7 X

Hill Slope 0.7 to 1.5 0.7 1.4 X

Test range 10−14 to 10−8 M 10−14 to 10−8 M 10−14 to 10−8 M X

17α-estradiola

LogPC50 −9.6 to −8.1 −8.9 −8.9 X

LogPC10 −10.7 to −9.3 −12.3 −10.1 X

LogEC50 −9.6 to −8.4 −8.9 −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

Corticosteronea

Test range 10−10 to 10−4 M 10−10 to 10−4 M 10−10 to 10−4 M X

17α-methyltestosteroneb

LogPC50 −6.0 to −5.1 −7.02 −5.99 X

LogPC10 −8.0 to −6.2 −9.60 −8.48 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 15 and 19 of the study report. 

b Data for 17α-methyltestosterone were calculated by the reviewers, based on normalized individual well data from pages 

42 and 55 of the study report, as there were apparent errors in the reported calculated data in the study report. 

Page 60 of 149
Estrogen Receptor Transcriptional Activation (2011) / Page 7 of 10

CARBARYL /056801 OCSPP 890.1300/ OECD 455

C. DEFINITIVE ASSAY

1. Vehicle and Positive Controls: Data for the vehicle and positive controls as reported on 

page 38 of the study report are included in Table 4. The mean TA values, expressed as 

arbitrary light units for the vehicle control were 461.1 and 573.0 for the first run, and 468.2 

and 582.5 for the second run. The mean normalized values for the positive control were

2053.3 and 2207.2 for the first run, and 2153.3 and 2080.8 for the second run. The PC50 for 

E2 in this assay was 1591.7 and 1634.2 for the first run, and 1685.7 and 1498.3 for the 

second run. The PC10 was 205.3 and 220.7 for the first run, and 215.4 and 208.1 for the 

second run. While mean values were provided for each of two plates on page 38 of the 

study report, raw data for only 3 replicates per tested chemical was reported in the 

appendices, suggesting that raw data for only one plate was reported. Mean PC values 

calculated by the reviewer using raw data from the appendices are close to but do not match 

those provided by the investigator for one plate. Since VC raw data is not provided, the 

reviewer inferred average VC for one plate base on normalized test and reference chemical 

values; the calculated values match summary data provided in the study report.

TABLE 4. Transcriptional Activation (TA) Response of Vehicle and Positive Control

Sample Vehicle Control Positive Control b

Runs Mean SD Mean SD Fold Induction c

1 – Plate 1 a

– Plate 2 a

per appendix

Ref chems raw-norm

Carbaryl raw-norm

461.6

573.0

461.6

573.0

44.7

76.9

NC

NC

2053.3

2207.2

2070.2

NR

NR

87.2

4.4

3.9

4.5

2 – Plate 1 a

– Plate 2 a

per appendix

468.2

582.5

468.2

39.5

68.3

NC

2153.9

2080.8

1908.0

NR

NR

173.7

4.6

3.6

4.1

a Data were obtained from page 38 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)

NR Not reported

NC Could not be calculated

Per appendix Calculated by reviewer based on data from pages 42 and 55 of the study report

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

carbaryl in the two assay runs is presented in Table 5. The concentration-response curves 

depicting fold induction of relative transcriptional activation are presented in Figures 1 and 

2 below. The mean RPCMax was 2.7% for the first run and 0.5% for the second run, and the 

associated PCMax was 10−4 M for both runs. Because the RPCMax < PC10 in both runs, 

carbaryl is considered negative for estrogen receptor transcriptional activation in this test 

system. 

Page 61 of 149
Estrogen Receptor Transcriptional Activation (2011) / Page 8 of 10

CARBARYL /056801 OCSPP 890.1300/ OECD 455

TABLE 5. Relative Transcriptional Activation (RTA) of Carbaryla

Parameter RTA (mean ± SD); % of Positive Control (PC)

Run 1 Run 2

Conc. (M) Mean SD Mean SD

10−4 2.7 2.7 0.5 7.5

10−5 −5.5 6.6 −5.4 2.5

10−6 −3.7 7.6 −1.9 3.6

10−7 −7.0 3.8 −6.2 3.3

10−8 −7.3 4.4 −4.4 2.1

10−9 −7.0 3.8 −3.0 4.6

10−10 −5.9 4.1 −5.1 3.1

LogEC50b 1.987 −3.841

Hill Slopeb 0.7105 7.528

RPCMax 2.7 0.5

PCMax 10−4 10−4

PC50 1.987 −3.841

PC10 0.7024 −4.187

a Data were calculated by the reviewer, using data obtained from pages 52 and 65 of the study report.

b Data were obtained from pages 28 and 33.

NA Not Applicable

FIGURE 1. Relative Transcription Activation (RTA) of Carbaryl: Run 1.

Page 62 of 149
Estrogen Receptor Transcriptional Activation (2011) / Page 9 of 10

CARBARYL /056801 OCSPP 890.1300/ OECD 455

FIGURE 2. Relative Transcription Activation (RTA) of Carbaryl: Run 2. 

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

though corticosterone did induce weak transcriptional activation at a 10-9 M dose in run 2. 

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

the Hill Slope. The log PC50 and log PC10 for 17α-methyltestosterone were lower than the 

acceptable range for both assays, although these results indicate that the assay had an 

increased sensitivity to weak agonists. In run 1, the log PC10 for 17α-estradiol was less than

log PC10 for17β-estradiol in the same run indicating potential spurious results. Mean 

luciferase activity was 4.2-fold for the first run and 4.1-fold for the second run. The foldinduction 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. For 17α-methyltestosterone, the mean RPCMax was 83.5% for the 

first run and 69.5% for the second run. For corticosterone, the apparent RPCMax was 15.5% 

for the first run and 16.8% for the second run, however these elevated values were due to 

single well readings that were very high and outside the variability of the rest of the 

corticosteroid data in the same assays, and they occurred at 10-9 in Run 1 and at 10-8 M in 

Run 2, therefore did not show a dose-response trend. When the apparent outliers are taken 

out of the analysis, RPCMax for corticosteroid are 1.1% in Run 1 and 0.2% in Run 2. 

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 

carbaryl was less than 10% of the positive control 17β-estradiol in both runs. Therefore, 

carbaryl was negative in this human estrogen receptor transcriptional activation assay. 

B. AGENCY COMMENTS: Carbaryl was tested up to the limit of solubility, 10−4 M. The 

summary data for laboratory proficiency assays indicated correct classification of required 

proficiency compounds. Deviations from performance criteria for reference compounds 

Page 63 of 149
Estrogen Receptor Transcriptional Activation (2011) / Page 10 of 10

CARBARYL /056801 OCSPP 890.1300/ OECD 455

included lower Hill slope and log PC10 values for 17α-estradiol in one run, and log PC10 for 

17α-estradiol was less than Log PC10 for17β-estradiol in run 1. In addition, the log PC50

and log PC10 for 17α-methyltestosterone were lower than the acceptable range for both 

assays, indicating potential increased sensitivity to weak agonists. The mean RPCMax for 

carbaryl was 2.7% for the first run and 0.5% for the second run, and the associated PCMax

was 10−4 M for both runs. Because the RPCMax < PC10 in both runs, carbaryl was 

considered negative for estrogen receptor transcriptional activation in this test system. 

C. STUDY DEFICIENCIES: No deficiencies were noted.

Page 64 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀ

ԡԹՌԹԯԽՉՍՁՊԽՅԽՆՌ Ԣ

ԬԢԠԡԡԹՌԹԭՇՁՆՌ

ԢԭԞԪԯԦԡ

ԢԭԞԤՍՁԼԽՄՁՆԽ

ԱԽՋՌԪԹՌԽՊՁԹՄ ԠԹՊԺԹՊՑՄ

ԠՇՅՅՇՆԫԹՅԽ ԠԹՊԺԹՊՑՄ

ԠՀԽՅՁԻԹՄԫԹՅԽ ԦԲԭԞԠ

 ԠԞ԰ԫԹՅԽ

 ԠԞ԰ԫՇ

 ԰ՑՆՇՆՑՅՋ

ԭՊՁՅԹՊՑԯԽՎՁԽՏԽՊ ԧՇՀՆԪԹՊՌՇՆ

ԢՆՎՁՊՇՆՅԽՆՌԹՄ԰ԻՁԽՆՌՁՋՌԠԡԪ԰ՅՁՌՀ

԰ԽԻՇՆԼԹՊՑԯԽՎՁԽՏԽՊ
ՋԱԽՊՁ԰ԪՑԽ

ԢՆՎՁՊՇՆՅԽՆՌԹՄ԰ԻՁԽՆՌՁՋՌԠԡԪ԰ՅՁՌՀ

ԭՊՁՅԹՊՑԯԽՎՁԽՏԽՊԞՆԼՊԽՏ԰ԹՑԽՊԭ

ԟՁՇՄՇԿՁՋՌԢԭԞԬԭԭԢԣԢԡԢԯԟ

԰ԽԻՇՆԼԹՊՑԯԽՎՁԽՏԽՊԢԼՏԹՊԼ԰ԻՇՄՄՇ

ԱՇՐՁԻՇՄՇԿՁՋՌԢԭԞԬԭԭԢԣԢԡԢԯԟ

ԞԼԼՁՌՁՇՆԹՄԯԽՎՁԽՏԽՊԠԹՌՀԽՊՁՆԽԞՍԺԽ

԰ԽՆՁՇՊ԰ԻՁԽՆՌՁՋՌԢԭԞԬԭԭԢԣԢԡԢ

ԢԭԞԭԠԠՇԼԽ 

ԡԹՌԽԢՎԹՄՍԹՌՁՇՆԠՇՅՈՄԽՌԽԼ#ԼԼՅՅ

ԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀ

ԢԭԞԪԯԦԡ

ԭԹԿԽՇԾ

ԢԭԞԡԭԟԹՊԻՇԼԽ 

ԬԢԠԡԤՍՁԼԽՄՁՆԽ





ԭՍՊՁՌՑ


ՆԹՈՀՌՀԹՄԽՆՑՄԫՅԽՌՀՑՄԻԹՊԺԹՅԹՌԽ

ՆԹՈՀՌՀԹՄԽՆՑՄՅԽՌՀՑՄԻԹՊԺԹՅԹՌԽ



ԠԹՊԺԹՊՑՄ

ԭՀԡ ԡԹՌԽ

Հ

ԽՊՋԭՀԡ ԡԹՌԽ

Հ

ԭՀԡ ԰ՁԿՆԹՌՍՊԽ

ԡԹՌԽ

ՇՆԭՀԡ ԰ՁԿՆԹՌՍՊԽՆՇՄՇՆԿԽՊԹՌԢ

 ԡԹՌԽ

ԽԽ ԰ՁԿՆԹՌՍՊԽ

Ԣԯԟ ԡԹՌԽ

ՑՑՑՑ%

ՀԠԹՊԺԹՊՑՄ

ԫՍՅԺԽՊ

ԢԭԞ

6/8/2015

Digitally signed by 

ANDREW SAYER 

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

Government, 

ou=USEPA, ou=Staff, 

cn=ANDREW SAYER, 

dnQualifier=000005140

9 

Date: 2015.06.09 

15:09:23 -04'00'

Page 65 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԠԦԱԞԱԦԬԫԶՇՊՃԡԬԠԹՊԺԹՊՑՄ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԣԹՌՀԽԹԼԪՁՆՆՇՏ
ԭՁՅԽՈՀԹՄԽՋ

ՈՊՇՅԽՄԹՋԣՇՄՄՇՏՁՆԿԬԭԭԱ԰ԹՆԼԬԢԠԡԤՍՁԼԽՄՁՆԽՋԲՆՈՍԺՄՁՋՀԽԼՋՌՍԼՑՈԽՊԾՇՊՅԽԼԺՑ

԰ՅՁՌՀԽՊՋ ԳՁՋԻՁԽՆՌԴԹՊԽՀԹՅ ԪԹՋՋԹԻՀՍՋԽՌՌՋ  ԩԹԺՇՊԹՌՇՊՑ ՊԽՈՇՊՌ ՆՍՅԺԽՊ  ԰ՌՍԼՑ

ՋՈՇՆՋՇՊԽԼԺՑԟԹՑԽՊԠՊՇՈ԰ԻՁԽՆԻԽԯԽՋԽԹՊԻՀԱՊՁԹՆԿՄԽԭԹՊՃԫՇՊՌՀԠԹՊՇՄՁՆԹ԰ՌՍԼՑԻՇՅՈՄԽՌԽԼ

ԧՍՆԽ

ԫՇՌԽԱՀԽԲ԰ԢԭԞԢՆԼՇԻՊՁՆԽԡՁՋՊՍՈՌՇՊ԰ԻՊԽԽՆՁՆԿԭՊՇԿՊԹՅ
Ԣԡ԰ԭԱՁԽՊՋԻՊԽԽՆՁՆԿԺԹՌՌԽՊՑՁՋԻՇՅՈՊՁՋԽԼՇԾ

ԽՄԽՎԽՆՋԻՊԽԽՆՁՆԿԹՋՋԹՑՋՁՆՌԽՆԼԽԼՌՇՁԼԽՆՌՁԾՑԹԻՀԽՅՁԻԹՄ0ՋՄՁՃԽՄՑԽՆԼՇԻՊՁՆԽԺՁՇԹԻՌՁՎՁՌՑՁԽՁՌՋՈՇՌԽՆՌՁԹՄՌՇ

ՁՆՌԽՊԹԻՌՏՁՌՀՌՀԽԽՋՌՊՇԿԽՆԹՆԼՊՇԿԽՆՇՊՌՀՑՊՇՁԼ
ԢԞՇՊԱՈԹՌՀՏԹՑՋԱՀԽՊՇԺՍՋՌՆԽՋՋՇԾՌՀԽԱՁԽՊ

ԺԹՌՌԽՊՑՁՋԺԹՋԽԼՇՆՌՀԽՋՌՊԽՆԿՌՀՋՇԾԽԹԻՀՁՆԼՁՎՁԼՍԹՄԹՋՋԹՑՌՇՁԼԽՆՌՁԾՑՈՇՌԽՆՌՁԹՄԽՆԼՇԻՊՁՆԽԺՁՇԹԻՌՁՎՁՌՑՏՁՌՀ

ԻՇՅՈՄԽՅԽՆՌԹՊՑԽՆԼՈՇՁՆՌՋՏՁՌՀՁՆՌՀԽԹՋՋԹՑՏՀԽՊԽԹՎԹՁՄԹԺՄԽԹՆԼՊԽԼՍՆԼԹՆԻՑԹԻՊՇՋՋՌՀԽԺԹՌՌԽՊՑԱՀՍՋ

ՌՀԽՊԽՋՍՄՌՋՇԾԽԹԻՀՁՆԼՁՎՁԼՍԹՄԹՋՋԹՑՋՀՇՍՄԼՆՇՌԺԽԻՇՆՋՁԼԽՊԽԼՁՆՁՋՇՄԹՌՁՇՆԺՍՌՊԹՌՀԽՊՋՀՇՍՄԼԺԽԻՇՆՋՁԼԽՊԽԼ

ՁՆՌՀԽԻՇՆՌԽՐՌՇԾՇՌՀԽՊԹՋՋԹՑՋՁՆՌՀԽԺԹՌՌԽՊՑԹՋՏԽՄՄԹՋԬՌՀԽՊ԰ԻՁԽՆՌՁԾՁԻԹՄՄՑԯԽՄԽՎԹՆՌԦՆԾՇՊՅԹՌՁՇՆ
Ԭ԰ԯԦ

ԦՆՇՊԼԽՊՌՇԼԽՌԽՊՅՁՆԽՁԾԹԻՀԽՅՁԻԹՄՀԹՋՌՀԽՈՇՌԽՆՌՁԹՄՌՇՁՆՌԽՊԹԻՌՏՁՌՀՌՀԽԢԞՇՊԱՈԹՌՀՏԹՑՋԹԴԽՁԿՀՌՇԾ

ԢՎՁԼԽՆԻԽ
ԴՇԢԽՎԹՄՍԹՌՁՇՆՇԾԱՁԽՊԹՋՋԹՑՊԽՋՍՄՌՋՁՆԻՇՅԺՁՆԹՌՁՇՆՏՁՌՀՌՀԽԾՁՆԼՁՆԿՋՁՆՌՀԽԬ԰ԯԦՋՀՇՍՄԼ

ԺԽՍՆԼԽՊՌԹՃԽՆ
ՊԽԾԽՊՌՇՌՀԽԴՇԢԡՇԻՍՅԽՆՌ

ԢԵԢԠԲԱԦԳԢ԰ԲԪԪԞԯԶ

ԱՀԽ ԼԹՑ ՋՀՇՊՌՌԽՊՅ ՊԽՈՊՇԼՍԻՌՁՇՆ ԹՋՋԹՑ ՇԾ ԻԹՊԺԹՊՑՄ ՏՁՌՀ ԾԹՌՀԽԹԼ ՅՁՆՆՇՏ 
ԭՁՅԽՈՀԹՄԽՋ ՈՊՇՅԽՄԹՋ ՏԹՋ

ԻՇՆԼՍԻՌԽԼՍՆԼԽՊԾՄՇՏՌՀՊՇՍԿՀԻՇՆԼՁՌՁՇՆՋԞԼՍՄՌԾՁՋՀ
ՏԽԽՃՋՇՄԼՅԹՄԽՋԹՆԼԾԽՅԹՄԽՋՁՆԽԹԻՀԿՊՇՍՈ

ԿՊՇՍՈՋՈԽՊՌՊԽԹՌՅԽՆՌՏԽՊԽԽՐՈՇՋԽԼՌՇԻԹՊԺԹՊՑՄ
ՈՍՊՁՌՑԹՌՆՇՅՁՆԹՄԻՇՆԻԽՆՌՊԹՌՁՇՆՋՇԾ
ՆԽԿԹՌՁՎԽ

ԻՇՆՌՊՇՄ   ԹՆԼ  ՅԿ ԹՁԩ ՅԽԹՆՅԽԹՋՍՊԽԼ ԻՇՆԻԽՆՌՊԹՌՁՇՆՋ ՏԽՊԽ  
ԩԬԮ

ԻՇՆՌՊՇՄԹՆԼՅԿԹՁԩԱՀԽՌԽՋՌՋՑՋՌԽՅՏԹՋՅԹՁՆՌԹՁՆԽԼԹՌՌՇոԠԹՆԼԹՈԥՇԾ

ՌՇ

ԞՄՄԽԾԾԽԻՌՋԹՊԽՊԽՈՇՊՌԽԼԺԹՋԽԼՇՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԪԽԹՆՋՍՊՎՁՎԹՄՁՆՅԹՄԽՋՏԹՋՁՆ

ՌՀԽ ԻՇՆՌՊՇՄ  ԹՆԼ  ՅԿ ԹՁԩ ԿՊՇՍՈՋ ՋՍՊՎՁՎԹՄ ՁՆ ՌՀԽ  ՅԿ ԹՁԩ ԿՊՇՍՈ ՏԹՋ  


ԣԽՅԹՄԽՋՍՊՎՁՎԹՄՏԹՋ


ԹՆԼ
ՁՆՌՀԽԻՇՆՌՊՇՄ

 ԹՆԼՅԿ ԹՁԩԿՊՇՍՈՋ ՊԽՋՈԽԻՌՁՎԽՄՑԠՇՅԺՁՆԽԼՋԽՐՋՍՊՎՁՎԹՄՏԹՋ ԹՆԼ ՁՆՌՀԽ

  ԹՆԼ  ՅԿ ԹՁԩ ԿՊՇՍՈՋ ՊԽՋՈԽԻՌՁՎԽՄՑ ԾՇՊ ՏՀՁԻՀ ՌՀԽՊԽ ՏԽՊԽ ՆՇ ՇԺՋԽՊՎԽԼ ՋՁԿՆՁԾՁԻԹՆՌ

ԼՁԾԾԽՊԽՆԻԽՋ ԱՀԽՊԽ ՏԽՊԽ ՆՇ ՆՇՌԹԺՄԽ ՇԺՋԽՊՎԹՌՁՇՆՋ ՏՁՌՀ ՊԽԿԹՊԼՋ ՌՇ ԺԽՀԹՎՁՇՊ ՇՊ ԻՀԹՆԿԽՋ ՁՆ ԹՈՈԽԹՊԹՆԻԽ

Page 66 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ


ՋՈԽԻՁԾՁԻԹՄՄՑ ԻՇՄՇՊԺԹՆԼՁՆԿ ՇՎՁՈՇՋՁՌՇՊ ԹՈՈԽԹՊԹՆԻԽ ՇՊ ՋՁՒԽ ՇԾ ԼՇՊՋԹՄ ՆԹՈԽ ՈԹԼ ՁՆ ՌՀԽ ԻՇՆՌՊՇՄ ՇՊ ՌՊԽԹՌԽԼ

ԿՊՇՍՈՋԹՆԼՆՇ ՊԽՈՇՊՌԽԼԻՄՁՆՁԻԹՄՋՁԿՆՋՇԾՌՇՐՁԻՁՌՑԪԹՄԽ ԺՇԼՑՄԽՆԿՌՀՏԹՋԿՊԽԹՌԽՊ
ՈՁՆՌՀԽ

ՅԿ ԹՁԩ ՌՊԽԹՌՅԽՆՌ ԿՊՇՍՈ ԫԽՁՌՀԽՊ ԾԽՅԹՄԽ ԺՇԼՑ ՄԽՆԿՌՀ ՆՇՊ ԺՇԼՑ ՏԽՁԿՀՌ ԾՇՊ ԽՁՌՀԽՊ ՋԽՐ ՏԹՋ ԹԾԾԽԻՌԽԼ ԺՑ

ԻԹՊԺԹՊՑՄԽՐՈՇՋՍՊԽ

ԠԹՊԺԹՊՑՄ ՀԹԼ ՆՇ ԽԾԾԽԻՌ ՇՆ ԾԽԻՍՆԼՁՌՑ ՇՊ ԾԽՊՌՁՄՁՌՑ ՁՆ ԹՆՑ ՌՊԽԹՌՅԽՆՌ ԿՊՇՍՈ ԞԼԼՁՌՁՇՆԹՄՄՑ ՈՄԹՋՅԹ ՎՁՌԽՄՄՇԿԽՆՁՆ


ԳԱԤԻՇՆԻԽՆՌՊԹՌՁՇՆՋՏԽՊԽՍՆԹԾԾԽԻՌԽԼՁՆՅԹՄԽՋԹՆԼԾԽՅԹՄԽՋԹՆԼՌՀԽՊԽՏԽՊԽՆՇՌՊԽԹՌՅԽՆՌՊԽՄԹՌԽԼԽԾԾԽԻՌՋՇՆ

Ԥ԰ԦՁՆԽՁՌՀԽՊՋԽՐԥՇՏԽՎԽՊՅԽԼՁԹՆՅԹՄԽՆՍՈՌՁԹՄՌՍԺԽՊԻՄԽՋԻՇՊԽՏԹՋՄՇՏԽՊՁՆՌՀԽՅԹՁԩՌՊԽԹՌՅԽՆՌ

ԿՊՇՍՈ
ՌՀԹՆՁՆԻՇՆՌՊՇՄԾՁՋՀ

ՈԥՁՋՌՇՈԹՌՀՇՄՇԿՁԻԹՄՇԺՋԽՊՎԹՌՁՇՆՋՁՆՅԹՄԽՋՁՆԻՄՍԼԽԼՁՆԻՊԽԹՋԽՋ

ՁՆ ՅՁՆՁՅԹՄ ՌԽՋՌՁԻՍՄԹՊ ԼԽԿԽՆԽՊԹՌՁՇՆ ԹՆԼ ՌՀԽ ՈԽՊԻԽՆՌԹԿԽ ՇԾ ՋՈԽՊՅԹՌՇԿՇՆՁԹ ԹՌ ՌՀԽ ՅԽԹՆՅԽԹՋՍՊԽԼ  ՅԿ

ԹՁԩՌՊԽԹՌՅԽՆՌՄԽՎԽՄԫՇՊԽՅԹՊՃԹԺՄԽՀՁՋՌՇՈԹՌՀՇՄՇԿՁԻԹՄՇԺՋԽՊՎԹՌՁՇՆՋՏԽՊԽՆՇՌԽԼՁՆՅԹՄԽՋԹՌՇՌՀԽՊՌՊԽԹՌՅԽՆՌ

ՄԽՎԽՄՋՇՊՁՆԾԽՅԹՄԽՋԭՄԹՋՅԹՋԽՐՋՌԽՊՇՁԼՋՏԽՊԽՆՇՌՅԽԹՋՍՊԽԼ

ԞՄՄՈԽՊԾՇՊՅԹՆԻԽԻՊՁՌԽՊՁԹՏԽՊԽՅԽՌՁՆՌՀՁՋՋՌՍԼՑԽՐԻԽՈՌՌՀԹՌԾՇՊԻՇՆՌՊՇՄՊԽՈՄՁԻԹՌԽԡՋՈԹՏՆՁՆԿԼՁԼՆՇՌՇԻԻՍՊԹՌ

ՄԽԹՋՌԽՎԽՊՑԾՇՍՊԼԹՑՋՆՇՊՏԹՋՌՀԽԹՎԽՊԹԿԽԾԽԻՍՆԼՁՌՑԽԿԿՋԾԽՅԹՄԽԼԹՑՇՊԿՊԽԹՌԽՊԥՇՏԽՎԽՊՌՀԽԹՎԽՊԹԿԽ

ԾԽԻՍՆԼՁՌՑԾՇՊՌՀԹՌ ՊԽՈՄՁԻԹՌԽՏԹՋԽԿԿՋԾԽՅԹՄԽԼԹՑԼՍՊՁՆԿՌՀԽԾՁՊՋՌՏԽԽՃՇԾԽՐՈՇՋՍՊԽԹՆԼԾԽԻՍՆԼՁՌՑՏԹՋ

ՁՆԻՊԽԹՋԽԼ ՇՎԽՊՌՀԽ ԼՍՊԹՌՁՇՆ ՇԾՌԽՋՌՁՆԿԞԼԼՁՌՁՇՆԹՄՄՑՌՀԽԾԽԻՍՆԼՁՌՑ ՊԹՌԽՋ ՁՆՌՀԽ ՇՌՀԽՊ ՊԽՈՄՁԻԹՌԽՋՏԽՊԽ 

ԽԿԿՋԾԽՅԹՄԽԼԹՑՁՆԼՁԻԹՌՁՆԿՌՀԹՌԾԽԻՍՆԼՁՌՑՏԹՋԹԼԽՉՍԹՌԽՁՆՌՀԽԻՇՆՌՊՇՄԿՊՇՍՈ

ԱՀՁՋԹՋՋԹՑՋԹՌՁՋԾՁԽՋՌՀԽԢԡ԰ԭԱՁԽՊԱԽՋՌԬՊԼԽՊ ՊԽՉՍՁՊԽՅԽՆՌՋԾՇՊԹԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑ


ԬԠ԰ԭԭԤՍՁԼԽՄՁՆԽ

ԯԽՋՍՄՌՋ԰ՑՆՇՈՋՁՋ

ԱԽՋՌՇՊԿԹՆՁՋՅԹԿԽԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆՏԽԽՃՋ

ԪԽԹՆԺՇԼՑՏԽՁԿՀՌԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ
ԿԾՇՊՅԹՄԽՋ
ԿԾՇՊԾԽՅԹՄԽՋ

ԪԽԹՆՄԽՆԿՌՀԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆԫՇՌԯԽՈՇՊՌԽԼ

ԱԽՋՌՌՑՈԽԾՄՇՏՌՀՊՇՍԿՀ

Page 67 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԱԹԺՄԽ԰ՍՅՅԹՊՑՇԾԯԽՈՊՇԼՍԻՌՁՎԽԹՆԼԥԭԤԢԾԾԽԻՌՋՁՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑ
ԣ԰ԱԯԞՏՁՌՀԠԹՊԺԹՊՑՄ

ԱՊԽԹՌՅԽՆՌ


ՅԿԹՁԩ

#ՅԽԹՆ

ՅԽԹՋՍՊԽԼ%

ԣԽԻՍՆԼՁՌՑ

ԣԽՊՌ

԰ՍԻԻԽՋՋ

ԱՍԺԽՊԻՄԽ԰ԻՇՊԽ Ԥ԰Ԧ ԤՇՆԹԼԹՄԥՁՋՌՇ ԭՄԹՋՅԹԳԱԤ ԭՄԹՋՅԹԱ ԭՄԹՋՅԹԢ

Ԫ ԣ Ԫ ԣ Ԫ ԣ Ԫ ԣ Ԫ ԣ Ԫ ԣ

 ԫՇ ԫՇ ԫՇ ԫՇ ԫՇ ԫՇ ԫՇ ԫՇ ԫՇ ԫՇ     ԫՇ ԫՇ ԫՇ ԫՇ ԫՇ ԫՇ ԫՇ ԫՇ ԫՇ ԫՇ     ԫՇ ԫՇ ԶԽՋ ԫՇ ԫՇ ԫՇ ԶԽՋ ԫՇ ԫՇ ԫՇ    

ԞԺԺՊԽՎՁԹՌՁՇՆՋԠՇՆԻԠՇՆԻԽՆՌՊԹՌՁՇՆԡՁԾԾԡՁԾԾԽՊԽՆԻԽԢ֦ԽՋՌՊԹԼՁՇՄԣԣԽՅԹՄԽԣԽՊՌԣԽՊՌՁՄՁՒԹՌՁՇՆԤ԰ԦԤՇՆԹԼՇ԰ՇՅԹՌՁԻԦՆԼԽՐԥՁՋՌՇԥՁՋՌՇՈԹՌՀՇՄՇԿՑ

ԪԪԹՄԽԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽԱԱԽՋՌՇՋՌԽՊՇՆԽԳԱԤԳՁՌԽՄՄՇԿԽՆՁՆ

 Ԟ՟ՑԽՋՠՁՆԼՁԻԹՌԽՋԹՋՁԿՆՁԾՁԻԹՆՌԼՁԾԾԽՊԽՆԻԽԺԹՋԽԼՇՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽՆԽԿԹՌՁՎԽ
ԻՄԽԹՆՏԹՌԽՊԻՇՆՌՊՇՄՍՆՄԽՋՋՇՌՀԽՊՏՁՋԽՋՈԽԻՁԾՁԽԼ

 ԱՀԽԻՊՁՌԽՊՁԹԾՇՊՋՁԿՆՁԾՁԻԹՆԻԽԹՊԽԼԽՋԻՊՁԺԽԼՁՆՌՀԽԯԽՎՁԽՏԽՊ0ՋԞՆԹՄՑՋՁՋԹՆԼ԰ՌԹՌՁՋՌՁԻԹՄԳԽՊՁԾՁԻԹՌՁՇՆՋԽԻՌՁՇՆՋՇԾՌՀԽԡԢԯԠՇՆԻՄՍՋՁՇՆՋՊԽԿԹՊԼՁՆԿ

ՀՁՋՌՇՈԹՌՀՇՄՇԿՑՅԹՑԺԽՀԽԹՎՁՄՑՏԽՁԿՀՌԽԼԺՑՌՀԽԽՐՈԽՊՌՇՈՁՆՁՇՆՇԾԹԺՇԹՊԼԻԽՊՌՁԾՁԽԼՈԹՌՀՇՄՇԿՁՋՌ

Page 68 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

Ԧ ԪԞԱԢԯԦԞԩ԰ԞԫԡԪԢԱԥԬԡ԰

ԤՍՁԼԽՄՁՆԽԣՇՄՄՇՏԽԼԱՀՁՋՋՌՍԼՑՏԹՋԻՇՆԼՍԻՌԽԼԾՇՄՄՇՏՁՆԿՌՀԽՈՊՇՌՇԻՇՄԼԽՎԽՄՇՈԽԼԺՑԞՆՃՄԽՑԽՌԹՄ

ԹՆԼՅԽԽՌՋՌՀԽՊԽՉՍՁՊԽՅԽՆՌՋՇԾԬԠ԰ԭԭԤՍՁԼԽՄՁՆԽԹՆԼԬԢԠԡ

ԤՍՁԼԽՄՁՆԽԾՇՊՌՀԽԾՁՋՀՋՀՇՊՌՌԽՊՅՊԽՈՊՇԼՍԻՌՁՇՆԹՋՋԹՑԱՀԽԾՇՄՄՇՏՁՆԿ

ԼԽՎՁԹՌՁՇՆՋՏԽՊԽՆՇՌԽԼ

 ԱՀՊԽԽՇԾՌՀԽՋՈԹՏՆՁՆԿԿՊՇՍՈՋՀԹԼՈՊԽԽՐՈՇՋՍՊԽԾԽԻՍՆԼՁՌՑՇԾՌՇ

ԽԿԿՋԾԽՅԹՄԽԼԹՑԿՍՁԼԹՆԻԽՊԽԻՇՅՅԽՆԼՋԹՌՄԽԹՋՌԽԿԿՋԾԽՅԹՄԽԼԹՑ

ԥՇՏԽՎԽՊԽԹԻՀՇԾՌՀԽՋԽԿՊՇՍՈՋՋՈԹՏՆԽԼԹՌՄԽԹՋՌԽՎԽՊՑԼԹՑՋ

 ԠՇՆԻԽՆՌՊԹՌՁՇՆՋՇԾՍՆՁՇՆՁՒԽԼԹՅՅՇՆՁԹԹՆԼՊԽՋՁԼՍԹՄԻՀՄՇՊՁՆԽՁՆՌՀԽԼՁՄՍՌՁՇՆ

ՏԹՌԽՊՏԽՊԽՆՇՌՊԽՈՇՊՌԽԼ

 ԦՆՊԽՈՄՁԻԹՌԽԡՇԾՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄՋՈԹՏՆՁՆԿԼՁԼՆՇՌՇԻԻՍՊԹՌՄԽԹՋՌԽՎԽՊՑ

ԾՇՍՊԼԹՑՋՆՇՊՏԹՋՌՀԽԹՎԽՊԹԿԽԾԽԻՍՆԼՁՌՑԽԿԿՋԾԽՅԹՄԽԼԹՑՇՊԿՊԽԹՌԽՊ

ԥՇՏԽՎԽՊՌՀԽԹՎԽՊԹԿԽԾԽԻՍՆԼՁՌՑԾՇՊՌՀԹՌՊԽՈՄՁԻԹՌԽՏԹՋԽԿԿՋԾԽՅԹՄԽԼԹՑ

ՏՀՁԻՀՏԹՋՋՄՁԿՀՌՄՑՄԽՋՋՌՀԹՆՌՀԽՊԽԻՇՅՅԽՆԼԽԼՊԹՌԽԹՆԼՌՀԽԾԽԻՍՆԼՁՌՑՊԹՌԽՋՁՆ

ՌՀԽՇՌՀԽՊՊԽՈՄՁԻԹՌԽՋՊԹՆԿԽԼԾՊՇՅՌՇԽԿԿՋԾԽՅԹՄԽԼԹՑՁՆԼՁԻԹՌՁՆԿՌՀԹՌ

ԾԽԻՍՆԼՁՌՑՏԹՋԹԼԽՉՍԹՌԽՁՆՌՀԽԻՇՆՌՊՇՄՌՊԽԹՌՅԽՆՌ

ԱՀԽՋԽԼԽՎՁԹՌՁՇՆՋԼՇՆՇՌՁՅՈԹԻՌՌՀԽԹԻԻԽՈՌԹԺՁՄՁՌՑՇՊՊԽՋՍՄՌՋՇԾՌՀԽՋՌՍԼՑ

ԠՇՅՈՄՁԹՆԻԽ ԰ՁԿՆԽԼԹՆԼԼԹՌԽԼԫՇԡԹՌԹԠՇՆԾՁԼԽՆՌՁԹՄՁՌՑԤԩԭԹՆԼԮՍԹՄՁՌՑԞՋՋՍՊԹՆԻԽ

ՋՌԹՌԽՅԽՆՌՋՏԽՊԽՈՊՇՎՁԼԽԼԱՀՁՋՋՌՍԼՑՏԹՋԻՇՆԼՍԻՌԽԼՁՆԻՇՅՈՄՁԹՆԻԽՏՁՌՀ

ԹՄՄՈԽՊՌՁՆԽՆՌԲ԰ԢԭԞ
ԠԣԯԭԹՊՌԤՇՇԼԩԹԺՇՊԹՌՇՊՑԭՊԹԻՌՁԻԽ

ՊԽԿՍՄԹՌՁՇՆՋՏՁՌՀՌՀԽԾՇՄՄՇՏՁՆԿԽՐԻԽՈՌՁՇՆՋՊՇՍՌՁՆԽԾՇՇԼԹՆԼՏԹՌԽՊՋԻՊԽԽՆՁՆԿ

ԹՆԹՄՑՋԽՋՏԽՊԽԻՇՆԼՍԻՌԽԼԹՌԤԽՇԩԹԺՋԦՆԻԟՊԹՁՆՌՊԽԽԪԹՋՋԹԻՀՍՋԽՌՌՋ

ՍՋՁՆԿՋՌԹՆԼԹՊԼԲ԰ԢԭԞՈՊՇԻԽԼՍՊԽՋԹՆԼԹՊԽԻՇՆՋՁԼԽՊԽԼԾԹԻՁՄՁՌՑՊԽԻՇՊԼՋ

ՍՆԼԽՊ԰ՅՁՌՀԽՊՋԳՁՋԻՁԽՆՌ0Ջ԰ՌԹՆԼԹՊԼԬՈԽՊԹՌՁՆԿԭՊՇԻԽԼՍՊԽՋ

ԞԱԽՋՌԪԹՌԽՊՁԹՄ ԠԹՊԺԹՊՑՄ

ԡԽՋԻՊՁՈՌՁՇՆ ԞՈՀՑՋՁԻԹՄԼԽՋԻՊՁՈՌՁՇՆՏԹՋՆՇՌՈՊՇՎՁԼԽԼ

Page 69 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԬԢԠԡՊԽԻՇՅՅԽՆԼՋԼԽՋԻՊՁԺՁՆԿՏԹՌԽՊՋՇՄՍԺՁՄՁՌՑՅԽՄՌՁՆԿԺՇՁՄՁՆԿՈՇՁՆՌՋՌԹԺՁՄՁՌՑՁՆՏԹՌԽՊԹՆԼՄՁԿՀՌ

ՈԨԹԭՇՏՇՊԨՇՏՎԹՈՇՊՈՊԽՋՋՍՊԽՇԾՌԽՋՌԻՇՅՈՇՍՆԼԽՐՈՁՊԹՌՁՇՆԼԹՌԽ

ԩՇՌԫՇԟԹՌԻՀԫՇ ԡԠ
ԩՇՌԫՇ԰ԪԳԫՇ

ԭՍՊՁՌՑ 

ԦՅՈՍՊՁՌՁԽՋ ԫՇՆԽԯԽՈՇՊՌԽԼ

԰ՌԹԺՁՄՁՌՑՇԾԠՇՅՈՇՍՆԼ ԰ՌԹԺՄԽԪԽԹՆՅԽԹՋՍՊԽԼԻՇՆԻԽՆՌՊԹՌՁՇՆՋՑՁԽՄԼԽԼՊԽԻՇՎԽՊՁԽՋՇԾՌՇ

ՇԾՆՇՅՁՆԹՄՏՁՌՀԻՇԽԾԾՁԻՁԽՆՌՋՇԾՎԹՊՁԹՌՁՇՆ
ԠԳՇԾՌՇ

԰ՌՇՊԹԿԽԠՇՆԼՁՌՁՇՆՋՇԾ

ԱԽՋՌԠՀԽՅՁԻԹՄՋ ԰ՌՇՊԽԼԹՌՊՇՇՅՌԽՅՈԽՊԹՌՍՊԽՁՆՌՀԽՇՊՁԿՁՆԹՄԻՇՆՌԹՁՆԽՊՁՆԹԼԹՊՃՎԽՆՌՁՄԹՌԽԼ

ԻԹԺՁՆԽՌ

Page 70 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԟԱԽՋՌԬՊԿԹՆՁՋՅԱԹԺՄԽԤԽՆԽՊԹՄԦՆԾՇՊՅԹՌՁՇՆԞԺՇՍՌՌՀԽԱԽՋՌ԰ՈԽԻՁԽՋԹՆԼԞԻԻՄՁՅԹՌՁՇՆ

ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ
Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ

԰ՈԽԻՁԽՋԻՇՅՅՇՆՆԹՅԽ ԣԹՌՀԽԹԼԪՁՆՆՇՏ ԢԭԞՊԽԻՇՅՅԽՆԼՋԾԹՌՀԽԹԼՅՁՆՆՇՏ
ԭՁՅԽՈՀԹՄԽՋ

ՈՊՇՅԽՄԹՋ ԰ՈԽԻՁԽՋՋԻՁԽՆՌՁԾՁԻՆԹՅԽ ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ

԰ՈԽԻՁԽՋՋՌՊԹՁՆ
ՁԾՋՌԹՌԽԼ ԫՇՌԯԽՈՇՊՌԽԼ ԰ԪԳԩՇՌԫՇԞԴԽՊԽԾՁՋՀՇԺՌԹՁՆԽԼԾՊՇՅԹՋՁՆԿՄԽՄԹԺՇՊԹՌՇՊՑՋՌՇԻՃ!

ԶԽՋ ԰ՌՇԻՃԾՁՋՀՏԽՊԽՊԽՈՊՇԼՍԻՌՁՎԽՄՑՅԹՌՍՊԽԹՆՁՅԹՄՋ

ԹՈՈՊՇՐՁՅԹՌԽՄՑՏԽԽՃՋՇՄԼԹՆԼՁՆՋՈԹՏՆՁՆԿԻՇՆԼՁՌՁՇՆ

ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌԾՁՋՀԺԽԾՊՇՅԹ

ՋՁՆԿՄԽՄԹԺՇՊԹՌՇՊՑՋՌՇԻՃ

ԴԽՊԽԹԻԻՄՁՅԹՌՁՇՆԻՇՆԼՁՌՁՇՆՋՋԹՅԽԹՋԼԽԾՁՆՁՌՁՎԽՌԽՋՌ!

ԶԽՋ  ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌԾՁՋՀԺԽԹԻԻՄՁՅԹՌԽԼՍՆԼԽՊ

ՏԹՌԽՊՉՍԹՄՁՌՑԹՆԼՁՄՄՍՅՁՆԹՌՁՇՆԻՇՆԼՁՌՁՇՆՋՌՀԹՌԹՊԽ

ՋՁՅՁՄԹՊՌՇՌՀԽԼԽԾՁՆՁՌՁՎԽՌԽՋՌ

ԞԻԻՄՁՅԹՌՁՇՆՈԽՊՁՇԼ ՏԽԽՃՋ ԢԭԞ ՊԽԻՇՅՅԽՆԼՋ Թ ՅՁՆՁՅՍՅ ՌՏՇՏԽԽՃ

ԹԻԻՄՁՅԹՌՁՇՆ ՈԽՊՁՇԼ ԫՇՌԽ ՌՀԹՌ ՌՀԽ ԹԻԻՄՁՅԹՌՁՇՆ

ՈԽՊՁՇԼ ՁՋ ԼՁԾԾԽՊԽՆՌ ԾՊՇՅ ՌՀԽ ՋՍԺՋԽՉՍԽՆՌ ՁՆ ՋՁՌՍ

ՈՊԽԽՐՈՇՋՍՊԽՈՀԹՋԽ

Page 71 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ
Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ

ԡԽՌԹՁՄՋՇՆՀԽԹՄՌՀ ԫՇՋՁԿՆՁԾՁԻԹՆՌՅՇՊՌԹՄՁՌՑՏԹՋ

ՇԺՋԽՊՎԽԼՁՆՌՀՁՋՈՇՈՍՄԹՌՁՇՆՁՆՌՀԽՋԽՎԽՆԼԹՑՋՈՊՁՇՊՌՇՌՀԽՈՊԽԽՐՈՇՋՍՊԽՈԽՊՁՇԼ

ԣՁՋՀԼՁԼՆՇՌՊԽԻԽՁՎԽՌՊԽԹՌՅԽՆՌԾՇՊԼՁՋԽԹՋԽՁՆՌՀԽՌՏՇՏԽԽՃՋՈՊՁՇՊՌՇՌՀԽՋՈԹՏՆՁՆԿՉՍԹՄՁԾՁԻԹՌՁՇՆՈԽՊՁՇԼՈՊԽԻԽԼՁՆԿՌՀԽՌԽՋՌՇՊԼՍՊՁՆԿՌՀԽԽՐՈՇՋՍՊԽՈԽՊՁՇԼ

ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՅՇՊՌԹՄՁՌՑԼՍՊՁՆԿՌՀԽԼԹՑՋ

ՈՊՁՇՊՌՇՌՀԽՈՊԽԽՐՈՇՋՍՊԽՈՀԹՋԽԺԽՄԽՋՋՌՀԹՆ

ՇԾՌՀԽԻՍՄՌՍՊԽՈՇՈՍՄԹՌՁՇՆԦԾՅՇՊՌԹՄՁՌՑԼՍՊՁՆԿՌՀԽՋԽ

ԼԹՑՋՁՋԿՊԽԹՌԽՊՌՀԹՆԢԭԞՊԽԻՇՅՅԽՆԼՋ

ՌՀԹՌՌՀԽԾՁՋՀԺԽՊԽՂԽԻՌԽԼԦԾՅՇՊՌԹՄՁՌՑՁՋԺԽՌՏԽԽՆ

ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌԾՁՋՀԺԽՀԽՄԼԹՆՇՌՀԽՊ

ԼԹՑՋԦԾՅՇՊՌԹՄՁՌՁԽՋԿՊԽԹՌԽՊՌՀԹՆՇԻԻՍՊ

ԼՍՊՁՆԿՌՀՁՋԽՐՌԽՆԼԽԼԹԻԻՄՁՅԹՌՁՇՆՈԽՊՁՇԼԢԭԞ

ՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՀԽԾՁՋՀՆՇՌԺԽՍՋԽԼ

ԱՑՈԽՇԾԾՇՇԼ ԩՁՎԽԺՊՁՆԽՋՀՊՁՅՈՆԹՍՈՄՁՁ  ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌԾՁՋՀԺԽԾԽԼԾՊՇՒԽՆԺՊՁՆԽ

ՋՀՊՁՅՈՌՏՁԻԽՈԽՊԼԹՑՌՇՈՊՇՅՇՌԽԹԻՌՁՎԽ

ՊԽՈՊՇԼՍԻՌՁՇՆԹՆԼՅԹՁՆՌԹՁՆԺՇԼՑԻՇՆԼՁՌՁՇՆ

԰ՇՍՊԻԽՇԾԾՇՇԼ ԫՇՌՋՈԽԻՁԾՁԽԼ ԩՇՌԫՇԣՊԽՉՍԽՆԻՑՇԾԾԽԽԼՁՆԿ ՌՇՌՁՅԽՋԼԹՑ ԡԽՌԹՁՄՋՇՆԾԽԽԼՁՆԿ ԣՁՋՀՏԽՊԽԾԽԼԹՅԽԹՋՍՊԽԼ

ԹՅՇՍՆՌՇԾՄՁՎԽԺՊՁՆԽՋՀՊՁՅՈՆԹՍՈՄՁՁԣՁՋՀՏԽՊԽԾԽԼՌՏՁԻԽԼԹՁՄՑԼՍՊՁՆԿՌՀԽՈՊԽԽՐՈՇՋՍՊԽՍՈՌՇԼԹՑՆՁՆԽԹԾՌԽՊՏՀՁԻՀԾԽԽԼՁՆԿՏԹՋՁՆԻՊԽԹՋԽԼՌՇՌՀՊԽԽՌՁՅԽՋԼԹՁՄՑ

Page 72 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԱԹԺՄԽԣՁՋՀ԰ԽՄԽԻՌՁՇՆԹՆԼԭՊԽԢՐՈՇՋՍՊԽԭԽՊԾՇՊՅԹՆԻԽ

ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ
Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ

ԞԿԽԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ ՏԽԽՃՋ ԢԭԞՊԽԻՇՅՅԽՆԼՋՊԽՈՊՇԼՍԻՌՁՎԽՄՑՅԹՌՍՊԽ
ՋԽՐՍԹՄՄՑ

ԼՁՅՇՊՈՀՁԻԾՁՋՀՅՇՆՌՀՋՇՄԼ

ԪԽԹՆՏԽՁԿՀՌՇԾՅԹՄԽՋԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ
ՁԾԼԽՌԽՊՅՁՆԽԼ

Կ ԯԽՈՊԽՋԽՆՌԹՌՁՎԽՋԹՅՈՄԽ
ԫԾՊՇՅՌՀԽՌԽՋՌ

ՈՇՈՍՄԹՌՁՇՆ

ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌԹՋՍԺՋԹՅՈՄԽՇԾԾՁՋՀԺԽ

ՏԽՁԿՀԽԼԺԽԾՇՊԽՌՀԽՌԽՋՌՌՇԽՋՌՁՅԹՌԽՌՀԽՅԽԹՆ

ՏԽՁԿՀՌԾՇՊԽԹԻՀՋԽՐԦՌՁՋՊԽԻՇՅՅԽՆԼԽԼՌՀԹՌ

ՌՀԽՁՆԼՁՎՁԼՍԹՄՏԽՁԿՀՌՇԾԽԹԻՀԾՁՋՀՋԽՄԽԻՌԽԼԾՇՊՌՀԽՌԽՋՌԺԽՏՁՌՀՁՆչՇԾՌՀԽԽՋՌՁՅԹՌԽԼՅԽԹՆԾՇՊ

ԽԹԻՀՋԽՐ

ԯԹՆԿԽՇԾՁՆԼՁՎՁԼՍԹՄՏԽՁԿՀՌՋ
ՅԹՄԽՋԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ
ՁԾԼԽՌԽՊՅՁՆԽԼ

ԿԿ
ՏՁՌՀՁՆՇԾՌՀԽՅԽԹՆ

ԪԽԹՆՏԽՁԿՀՌՇԾԾԽՅԹՄԽՋԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ
ՁԾԼԽՌԽՊՅՁՆԽԼ

Կ ԯԽՈՊԽՋԽՆՌԹՌՁՎԽՋԹՅՈՄԽ
ԫԾՊՇՅՌՀԽՌԽՋՌ

ՈՇՈՍՄԹՌՁՇՆ

ԯԹՆԿԽՇԾՁՆԼՁՎՁԼՍԹՄՏԽՁԿՀՌՋ
ԾԽՅԹՄԽՋԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ
ՁԾԼԽՌԽՊՅՁՆԽԼ

ԿԿ
ՏՁՌՀՁՆՇԾՌՀԽՅԽԹՆ

ԪԽԹՆՄԽՆԿՌՀՇԾՅԹՄԽՋԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ
ՁԾԼԽՌԽՊՅՁՆԽԼ

ԫՇՌՊԽՈՇՊՌԽԼ

ԪԽԹՆՄԽՆԿՌՀՇԾԾԽՅԹՄԽՋԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ
ՁԾԼԽՌԽՊՅՁՆԽԼ

ԫՇՌՊԽՈՇՊՌԽԼ  

ԡՍՊԹՌՁՇՆՇԾՈՊԽԽՐՈՇՋՍՊԽՈՀԹՋԽ ԼԹՑՋ ԢԭԞՊԽԻՇՅՅԽՆԼՋԹՅՁՆՁՅՍՅՇԾԼԹՑՋԴԽՊԽՈՊԽԽՐՈՇՋՍՊԽԻՇՆԼՁՌՁՇՆՋՁԼԽՆՌՁԻԹՄՌՇՌՀԽԼԽԾՁՆՁՌՁՎԽՌԽՋՌ!

ԶԽՋ  ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՈՊԽԽՐՈՇՋՍՊԽԻՇՆԼՁՌՁՇՆՋ

ՁՆԻՄՍԼՁՆԿՌԽՅՈԽՊԹՌՍՊԽՈՀՇՌՇՈԽՊՁՇԼԾԽԽԼՁՆԿԽՌԻ

ԺԽՁԼԽՆՌՁԻԹՄՌՇԼԽԾՁՆՁՌՁՎԽՌԽՋՌԻՇՆԼՁՌՁՇՆՋ

Page 73 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ
Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ

ԫՍՅԺԽՊՇԾՈՊԽԽՐՈՇՋՍՊԽՌԹՆՃՋ  ԞԼԼՁՌՁՇՆԹՄԽՐՈՇՋՍՊԽԻՀԹՅԺԽՊՋՏԽՊԽՋԽՌՍՈ

ԼՍՊՁՆԿՌՀԽՈՊԽԽՐՈՇՋՍՊԽՈՀԹՋԽՌՇԹԻԻՇՍՆՌԾՇՊԹՈՇՌԽՆՌՁԹՄՄԹԻՃՇԾՋՈԹՏՆՁՆԿՁՆՋՇՅԽԻՀԹՅԺԽՊՋԹՆԼՇՊՅՇՊՌԹՄՁՌՑԼՍՊՁՆԿՌՀԽՈՀԹՋԽ

ԢԭԞ ՊԽԻՇՅՅԽՆԼՋ ՌՀԹՌ ԹԼԼՁՌՁՇՆԹՄ ՌԹՆՃՋ ՋԽՌ ՍՈ ԹՌ

ՌՀԽ ԺԽԿՁՆՆՁՆԿ ՇԾ ՈՊԽԽՐՈՇՋՍՊԽ ՏՁՄՄ ԽՆՋՍՊԽ ՌՀԹՌ

ՋՍԾԾՁԻՁԽՆՌ ՊԽՈՄՁԻԹՌԽՋ ՏՁՌՀ ՌՀԽ ԻՇՊՊԽԻՌ ՋԽՐ ՊԹՌՁՇ ԹՊԽ

ԹՎԹՁՄԹԺՄԽ

ԾՇՊՌՀԽԼԽԾՁՆՁՌՁՎԽՌԽՋՌ

ԫՍՅԺԽՊՇԾՅԹՄԽՋՈԽՊՌԹՆՃ ԫՍՅԺԽՊՇԾԾԽՅԹՄԽՋՈԽՊՌԹՆՃ ԭՊԽԽՐՈՇՋՍՊԽԾԽԻՍՆԼՁՌՑ ՇԾՌՀԽՋՈԹՏՆՁՆԿ

ԿՊՇՍՈՋՍՋԽԼՁՆՌՀԽԼԽԾՁՆՁՌՁՎԽԽՐՈՇՋՍՊԽՀԹԼՈՊԽԽՐՈՇՋՍՊԽԾԽԻՍՆԼՁՌՑՇԾ 

ԽԿԿՋԾԽՅԹՄԽՊԽՈՊՇԼՍԻՌՁՎԽԼԹՑՊԽՈՄՁԻԹՌԽ

ԱՀԽՊԽՅԹՁՆՁՆԿՌՀՊԽԽԿՊՇՍՈՋՀԹԼԾԽԻՍՆԼՁՌՑՊԹՆԿՁՆԿԾՊՇՅՌՇԽԿԿՋԾԽՅԹՄԽԼԹՑԹՄՌՀՇՍԿՀՌՀԽՋԽԿՊՇՍՈՋՋՈԹՏՆԽԼԹՌՄԽԹՋՌՇՆԻԽԽՎԽՊՑԼԹՑՋ

ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՈՊԽԽՐՈՇՋՍՊԽԾԽԻՍՆԼՁՌՑՁՆ

ԽԹԻՀՊԽՈՄՁԻԹՌԽ
ՌԹՆՃՋԽՄԽԻՌԽԼԾՇՊՍՋԽՁՆՌՀԽ

ԼԽԾՁՆՁՌՁՎԽՌԽՋՌԺԽԹՌՄԽԹՋՌ

ԽԿԿՋԾԽՅԹՄԽՊԽՈՊՇԼՍԻՌՁՎԽԼԹՑՊԽՈՄՁԻԹՌԽԼՍՊՁՆԿ

ՌՀԽԼԹՑՋՈՊՁՇՊՌՇՌՀԽԼԽԾՁՆՁՌՁՎԽՌԽՋՌ

ԫՍՅԺԽՊՇԾՋՈԹՏՆՋԼՍՊՁՆԿՈՊԽԽՐՈՇՋՍՊԽ

 ՌՁՅԽՋՁՆԼԹՑՋ  ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՋՈԹՏՆՁՆԿՇԻԻՍՊԹՌՄԽԹՋՌ

ՌՏՁԻԽՁՆՌՀԽԼԹՑՋՈՊՁՇՊՌՇՌՀԽԼԽԾՁՆՁՌՁՎԽՌԽՋՌ

Page 74 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ
Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ

ԡԽՌԹՁՄՋՇՆՈՊԽԽՐՈՇՋՍՊԽ ԡՍՊՁՆԿՌՀԽՈՊԽԽՐՈՇՋՍՊԽ

ՈՀԹՋԽՋՍՁՌԹԺՁՄՁՌՑԾՇՊՌԽՋՌՁՆԿՏԹՋԽՋՌԹԺՄՁՋՀԽԼՏՀԽՆՊԽԿՍՄԹՊՋՈԹՏՆՁՆԿՇԻԻՍՊՊԽԼՁՆԽԹԻՀՊԽՈՄՁԻԹՌԽԻՀԹՅԺԽՊԹՌՄԽԹՋՌՌՏՁԻԽՁՆՌՀԽՁՅՅԽԼՁԹՌԽԼԹՑՈԽՊՁՇԼՈՊԽԻԽԼՁՆԿՌԽՋՌՁՆՁՌՁԹՌՁՇՆՇՊՏՀԽՆԹՆԽԿԿՈՊՇԼՍԻՌՁՇՆՊԹՌԽՇԾ

ԽԿԿՋԾԽՅԹՄԽԼԹՑՏԹՋԹԻՀՁԽՎԽԼ

Page 75 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԠԢՐՈՇՋՍՊԽ԰ՑՋՌԽՅԱԹԺՄԽ԰ՍՅՅԹՊՑՇԾԦՆԾՇՊՅԹՌՁՇՆՇՆՌՀԽԢՐՈՇՋՍՊԽ԰ՑՋՌԽՅԹՆԼԱԽՋՌԳԽՋՋԽՄԠՀԹՊԹԻՌԽՊՁՋՌՁԻՋ

ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ
Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ

ԱՑՈԽՇԾԽՐՈՇՋՍՊԽ ԣՄՇՏՌՀՊՇՍԿՀ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԽՍՋԽՇԾԹԾՄՇՏՌՀՊՇՍԿՀ

ՋՑՋՌԽՅԞՋՆՇՌԽԼՁՆՌՀԽԠՇՊՊԽԻՌՁՇՆՋԹՆԼ

ԠՄԹՊՁԾՁԻԹՌՁՇՆՋԼՇԻՍՅԽՆՌ ՌՀԽՍՋԽՇԾԹՋՌԹՌՁԻ

ՊԽՆԽՏԹՄՋՑՋՌԽՅՁՋՆՇՌՊԽԻՇՅՅԽՆԼԽԼԾՇՊՌՀՁՋ

ԹՋՋԹՑ

ԱՑՈԽՇԾԾՄՇՏՌՀՊՇՍԿՀԼՁՄՍՌՁՇՆՋՑՋՌԽՅ

ԦՆՌԽՊՅՁՌՌԽՆՌԾՄՇՏՈՊՇՈՇՊՌՁՇՆԹՄԼՁՄՍՌԽՊ

ԦՆՌԽՊՅՁՌՌԽՆՌԾՄՇՏՈՊՇՈՇՊՌՁՇՆԹՄԼՁՄՍՌԽՊՋՇՊ

ԻՇՆՌՁՆՍՇՍՋԾՄՇՏՋԽՊՁԹՄԼՁՄՍՌԽՊՋԹՊԽ

ՊԽԻՇՅՅԽՆԼԽԼ

ԣՄՇՏՌՀՊՇՍԿՀՊԹՌԽ +ՅԩՅՁՆ ԱՀԽԾՄՇՏՊԹՌԽՎՇՄՍՅԽՊԽՈՄԹԻԽՅԽՆՌՋԽՎԽՊՑ

ՀՇՍՊՋՏՁՌՀԹՊԽՈՄԹԻԽՅԽՆՌՌՁՅԽՇԾՀՇՍՊՋ

ԯԽԻՇՅՅԽՆԼԽԼԾՄՇՏՌՀՊՇՍԿՀՊԹՌԽՁՋՅԩՅՁՆ


 ԩՀՊ ՇՊ ԹՌ ՄԽԹՋՌ  ՌՇՌԹՄ ՎՇՄՍՅԽ

ԽՐԻՀԹՆԿԽՋՈԽՊԼԹՑ

Բ԰ԢՆՎՁՊՇՆՅԽՆՌԹՄԭՊՇՌԽԻՌՁՇՆԞԿԽՆԻՑ
ԢԭԞ
ԠՇՊՊԽԻՌՁՇՆՋԹՆԼԠՄԹՊՁԾՁԻԹՌՁՇՆՋՇՆԱԽԻՀՆՁԻԹՄԞՋՈԽԻՌՋՇԾՌՀԽԱԽՋՌԤՍՁԼԽՄՁՆԽՋԾՇՊՌՀԽԢՆԼՇԻՊՁՆԽԡՁՋՊՍՈՌՇՊ԰ԻՊԽԽՆՁՆԿ

ԭՊՇԿՊԹՅԱՁԽՊԞՋՋԹՑՋ
ԬԠ԰ԭԭԱԽՋՌԤՍՁԼԽՄՁՆԽ԰ԽՊՁԽՋԪԹՊԻՀԬԾԾՁԻԽՇԾԠՀԽՅՁԻԹՄ԰ԹԾԽՌՑԹՆԼԭՇՄՄՍՌՁՇՆԭՊԽՎԽՆՌՁՇՆ
ԬԠ԰ԭԭԴԹՋՀՁՆԿՌՇՆԡԠ


ՀՌՌՈՏՏՏԽՈԹԿՇՎԽՆԼՇՈՍԺՋԹՋՋԹՑՎԹՄՁԼԹՌՁՇՆԻՄԹՊՁԾՁԻԹՌՁՇՆԼՇԻՈԼԾ

ԞԼԼՁՌՁՇՆԹՄԿՍՁԼԹՆԻԽԾՇՊԹՉՍԹՌՁԻՌԽՋՌԼԽՋՁԿՆՁՋՄՇԻԹՌԽԼՁՆԬԠ԰ԭԭԤՍՁԼԽՄՁՆԽ԰ՈԽԻՁԹՄԠՇՆՋՁԼԽՊԹՌՁՇՆՋԾՇՊԠՇՆԼՍԻՌՁՆԿԞՉՍԹՌՁԻԩԹԺՇՊԹՌՇՊՑ԰ՌՍԼՁԽՋ

Page 76 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ
Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ

ԡԽՌԹՁՄՋՇՆՌՇՐՁԻԹՆՌՅՁՐՁՆԿԾՇՊԾՄՇՏՌՀՊՇՍԿՀՋՑՋՌԽՅՋ

ԞՆԣԪԦՈՍՅՈՏԹՋՍՋԽԼՌՇ

ԼԽՄՁՎԽՊՋՌՇԻՃՋՇՄՍՌՁՇՆՌՇՅՁՐՁՆԿԻՀԹՅԺԽՊՋՈՇՋՁՌՁՇՆԽԼՇՎԽՊԹՅԹԿՆԽՌՁԻՋՌՁՊՈՄԹՌԽԣՄՇՏՋՈՄՁՌՌՁՆԿԻՀԹՅԺԽՊՋՏԽՊԽՍՋԽԼՌՇՈՊՇՅՇՌԽԾՍՊՌՀԽՊՅՁՐՁՆԿԹՆԼՌՇԽՉՍԹՄՄՑԼՁՋՌՊՁԺՍՌԽՌՀԽՌԽՋՌՋՇՄՍՌՁՇՆՋՌՇԽՐՈՇՋՍՊԽԹՉՍԹՊՁԹ

ԠԹՄՁԺՊԹՌՁՇՆՏԹՋԻՇՆԾՁՊՅԽԼՈՊՁՇՊՌՇՁՆՁՌՁԹՌՁՇՆԹՆԼՌՀԽԾՍՆԻՌՁՇՆՇԾՌՀԽԼՁՄՍՌԽՊՏԹՋՅՇՆՁՌՇՊԽԼԼԹՁՄՑԹՆԼԹՎՁՋՍԹՄԻՀԽԻՃՏԹՋՈԽՊԾՇՊՅԽԼՌՏՁԻԽԼԹՁՄՑ

ԯԽԻՇՅՅԽՆԼԽԼՌՇՐՁԻԹՆՌՅՁՐՁՆԿԾՇՊԾՄՇՏՌՀՊՇՍԿՀ

ՋՑՋՌԽՅՋԪՁՐՁՆԿԻՀԹՅԺԽՊՁՋՊԽԻՇՅՅԽՆԼԽԼ

ԺՍՌՆՇՌՊԽՉՍՁՊԽԼԞԽՊԹՌՁՇՆՁՋՆՇՌ

ՊԽԻՇՅՅԽՆԼԽԼ

ԾՇՊՅՁՐՁՆԿԞԼԽՅՇՆՋՌՊԹՌՁՇՆՌՀԹՌՌՀԽՌԽՋՌ

ՋՇՄՍՌՁՇՆՁՋԻՇՅՈՄԽՌԽՄՑՅՁՐԽԼԺԽԾՇՊԽՁՆՌՊՇԼՍԻԽԼ

ՁՆՌՇՌՀԽՌԽՋՌՋՑՋՌԽՅՁՋՊԽԻՇՅՅԽՆԼԽԼԱՀԽ

ՊԽԻՇՅՅԽՆԼԽԼԾՄՇՏՋՈՄՁՌՌՁՆԿԹԻԻՍՊԹԻՑՁՋՏՁՌՀՁՆ



ԞԽՊԹՌՁՇՆ! ԫՇ ԢԭԞՊԽԻՇՅՅԽՆԼՋԹԽՊԹՌՁՇՆՁԾԼՁՋՋՇՄՎԽԼՇՐՑԿԽՆ

ՊԽԹԻՀԽՋՅԿԩ
ՋԹՌՍՊԹՌՁՇՆ

԰ՇՍՊԻԽՇԾԼՁՄՍՌՁՇՆՏԹՌԽՊ ԴԽՄՄՏԹՌԽՊ ԢԭԞՊԽԻՇՅՅԽՆԼՋՆԹՌՍՊԹՄՇՊՊԽԻՇՆՋՌՁՌՍՌԽԼ

ՏԹՌԽՊ

ՁՌՁՋՊԽԻՇՅՅԽՆԼԽԼՌՀԹՌՆԹՌՍՊԹՄՏԹՌԽՊԺԽ

ՋՌԽՊՁՄՁՒԽԼՏՁՌՀԲԳԹՆԼՌԽՋՌԽԼԾՇՊՈԽՋՌՁԻՁԼԽՋ

ՀԽԹՎՑՅԽՌԹՄՋԹՆԼՇՌՀԽՊՈՇՋՋՁԺՄԽԻՇՆՌԹՅՁՆԹՆՌՋ

ԬԢԠԡԹԻԻԽՈՌՋԹՆՑՏԹՌԽՊՁՆՏՀՁԻՀՌՀԽՌԽՋՌ

ՋՈԽԻՁԽՋՋՀՇՏԻՇՆՌՊՇՄՋՍՊՎՁՎԹՄԹՌՄԽԹՋՌԹՋԿՇՇԼ

ԹՋՁՆԼՁԻԹՌԽԼՁՆՌՀԽՌԽՋՌԿՍՁԼԽՄՁՆԽ

ԴԹՋԼՁՄՍՌՁՇՆՏԹՌԽՊԹՆԹՄՑՒԽԼԾՇՊՈԽՋՌՁԻՁԼԽՋՀԽԹՎՑՅԽՌԹՄՋԹՆԼՇՌՀԽՊԻՇՆՌԹՅՁՆԹՆՌՋ!

ԶԽՋԫՇՈԽՋՌՁԻՁԼԽՋԭԠԟՋՇՊՌՇՐՁԻՅԽՌԹՄՋՏԽՊԽԼԽՌԽԻՌԽԼԹՌԻՇՆԻԽՆՌՊԹՌՁՇՆՋԻՇՆՋՁԼԽՊԽԼՌՇՐՁԻ

Page 77 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ
Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ

ԱԽՋՌՎԽՋՋԽՄՌՑՈԽՅԹՌԽՊՁԹՄՋ ԿՄԹՋՋ ԢԭԞԹՆԼԬԢԠԡՊԽԻՇՅՅԽՆԼՌՀԹՌՏԹՌԽՊԻՇՆՌԹԻՌ

ՈՇՊՌՁՇՆՋՇԾՌՀԽՋՑՋՌԽՅՆՇՌԻՇՅՈՊՇՅՁՋԽՌՀԽ

ՋՌՍԼՑ
ԽԿԹՄՄԿՄԹՋՋՎԽՋՋԽՄՋՇՊԿՄԹՋՋՎԽՋՋԽՄՋ

ՏՁՌՀՋՌԹՁՆՄԽՋՋՋՌԽԽՄԾՊԹՅԽՋԹՊԽԹԻԻԽՈՌԹԺՄԽ

ԽՐԹՅՈՄԽՋ

ԱԽՋՌՎԽՋՋԽՄՋՁՒԽ ԩ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԽՍՋԽՇԾԩՌԽՋՌ

ԻՀԹՅԺԽՊՋ


ԽԿՐՐԻՅ

ԣՁՄՄՎՇՄՍՅԽ ԩ ԢԭԞՊԽԻՇՅՅԽՆԼՋԩՋՇՄՍՌՁՇՆՈԽՊՌԹՆՃ԰ՈԹՏՆՁՆԿՋՍԺՋՌՊԹՌԽՅԹՌԽՊՁԹՄ ԞԿԽԼԭԳԠ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌԽԹԻՀՌԹՆՃԻՇՆՌԹՁՆՌՀՊԽԽ

ՋԽՅՁԻՁՊԻՍՄԹՊՋՈԹՏՆՁՆԿՋՍԺՋՌՊԹՌԽՋԽԿԹԿԽԼ

ԭԳԠՈՁՈԽԻՅՁՆՄԽՆԿՌՀՋՈՄՁՌ

ՄԽՆԿՌՀՏՁՋԽ

԰ՈԹՏՆՁՆԿՋՍԺՋՌՊԹՌԽՋՁՒԽ ԻՅ

ԞԼԼՁՌՁՇՆԹՄԼԽՌԹՁՄՋՇՆԽՐՈՇՋՍՊԽ

ՋՑՋՌԽՅ

ԫԞ  

ԞԺԺՊԽՎՁԹՌՁՇՆՋԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽ

ԱԹԺՄԽ԰ՍՅՅԹՊՑՇԾԴԹՌԽՊԮՍԹՄՁՌՑԠՀԹՊԹԻՌԽՊՁՋՌՁԻՋՁՆՌՀԽԱԽՋՌ԰ՑՋՌԽՅ

ԭԹՊԹՅԽՌԽՊ ԪՁՆՁՅՍՅ ԪԹՐՁՅՍՅ ԪԽԹՆ ԪԽԹՋՍՊԽՅԽՆՌԦՆՌԽՊՎԹՄ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ

ԱԽՅՈԽՊԹՌՍՊԽ
ոԠ    ԠՇՆՌՁՆՍՇՍՋՄՑ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌԽՅՈԽՊԹՌՍՊԽչՇԠՁՆՌԽՊ

ՊԽՈՄՁԻԹՌԽԹՆԼՁՆՌԽՊՌՊԽԹՌՅԽՆՌԼՁԾԾԽՊԽՆՌՁԹՄՋՋՀՇՍՄԼ

ՆՇՌԽՐԻԽԽԼ°Ԡ

Page 78 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԭԹՊԹՅԽՌԽՊ ԪՁՆՁՅՍՅ ԪԹՐՁՅՍՅ ԪԽԹՆ ԪԽԹՋՍՊԽՅԽՆՌԦՆՌԽՊՎԹՄ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ

Ոԥ    ԪԽԹՋՍՊԽԼՁՆՌԽՋՌՎԽՋՋԽՄ

ԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆԹՆԼՁՆ

ԹՄՌԽՊՆԹՌՁՆԿՊԽՈՄՁԻԹՌԽՋ

ԼԹՁՄՑՌՀԽՊԽԹԾՌԽՊ

ԢԭԞՊԽԻՇՅՅԽՆԼՋՈԥՌՇ

ԡՁՋՋՇՄՎԽԼՇՐՑԿԽՆ
ՅԿԩ    ԪԽԹՋՍՊԽԼՁՆՌԽՋՌՎԽՋՋԽՄ

ԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆԹՆԼՁՆ

ԹՄՌԽՊՆԹՌՁՆԿՊԽՈՄՁԻԹՌԽՋ

ԼԹՁՄՑՌՀԽՊԽԹԾՌԽՊ

ԢԭԞՊԽԻՇՅՅԽՆԼՋԼՁՋՋՇՄՎԽԼՇՐՑԿԽՆ
ԡԬ 

ՅԿԩ
 ԹՁՊՋԹՌՍՊԹՌՁՇՆ

ԱՇՌԹՄԹՄՃԹՄՁՆՁՌՑ
ՅԿԩԹՋԠԹԠԬ    ԱԽՋՌՁՆՁՌՁԹՌՁՇՆԹՆԼ

ՏԽԽՃՄՑՌՀԽՊԽԹԾՌԽՊ

ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՇՌԹՄԹՄՃԹՄՁՆՁՌՑ ՅԿԩԹՋ

ԠԹԠԬ

ԥԹՊԼՆԽՋՋ
ՅԿԩԹՋԠԹԠԬ    ԱԽՋՌՁՆՁՌՁԹՌՁՇՆԹՆԼ

ՏԽԽՃՄՑՌՀԽՊԽԹԾՌԽՊ

ԱՇՌԹՄՇՊԿԹՆՁԻԻԹՊԺՇՆ
ՅԿԩ   ԫԞ ԣԽԺՊՍԹՊՑԹՆԼԪԹՊԻՀ



ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՇՌԹՄՇՊԿԹՆՁԻԻԹՊԺՇՆՁՆ

ԼՁՄՍՌՁՇՆՏԹՌԽՊԺԽՅԿԩ

ԲՆՁՇՆՁՒԽԼԹՅՅՇՆՁԹ
սԿԩ ԫՇՌԯԽՈՇՊՌԽԼ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՍՆՁՇՆՁՒԽԼԹՅՅՇՆՁԹՁՆ

ՌՀԽԼՁՄՍՌՁՇՆՏԹՌԽՊԺԽսԿԩ

ԯԽՋՁԼՍԹՄԻՀՄՇՊՁՆԽ
սԿԩ ԫՇՌԯԽՈՇՊՌԽԼ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՊԽՋՁԼՍԹՄԻՀՄՇՊՁՆԽՁՆ

ԼՁՄՍՌՁՇՆՏԹՌԽՊԺԽսԿԩ

Page 79 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԭԹՊԹՅԽՌԽՊ ԪՁՆՁՅՍՅ ԪԹՐՁՅՍՅ ԪԽԹՆ ԪԽԹՋՍՊԽՅԽՆՌԦՆՌԽՊՎԹՄ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ

ԠՇՆԼՍԻՌՁՎՁՌՑ
ս԰ԻՅ    ԱԽՋՌՁՆՁՌՁԹՌՁՇՆԹՆԼ

ՏԽԽՃՄՑՌՀԽՊԽԹԾՌԽՊ

ԤԽՆԽՊԹՄՊԽԻՇՅՅԽՆԼԹՌՁՇՆՋԾՇՊԾՊԽՉՍԽՆԻՑՇԾ

ՅԽԹՋՍՊԽՅԽՆՌՋԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌ

ՌԽՅՈԽՊԹՌՍՊԽՈԥԹՆԼԼՁՋՋՇՄՎԽԼՇՐՑԿԽՆԺԽ

ՅԽԹՋՍՊԽԼՁՆԹՄՄՌԽՋՌՌԹՆՃՋԹՌՄԽԹՋՌՏԽԽՃՄՑԹՆԼ

ՌՀԹՌՀԹՊԼՆԽՋՋԹՆԼԹՄՃԹՄՁՆՁՌՑԺԽՅԽԹՋՍՊԽԼՁՆ

ԻՇՆՌՊՇՄՋԹՆԼՁՆՇՆԽՌԹՆՃԹՌՌՀԽՀՁԿՀԽՋՌՌԽՋՌ

ԻՇՆԻԽՆՌՊԹՌՁՇՆԹՌՄԽԹՋՌՏԽԽՃՄՑԦՆԹԼԼՁՌՁՇՆ

ԻՇՆՌՁՆՍՇՍՋՌԽՅՈԽՊԹՌՍՊԽՅՇՆՁՌՇՊՁՆԿՇԾԹՌՄԽԹՋՌ

ՇՆԽՌԹՆՃՁՋԽՆԻՇՍՊԹԿԽԼ

ԡ԰ՌՍԼՑԡԽՋՁԿՆԹՆԼԞԼԼՁՌՁՇՆԹՄԢՐՈԽՊՁՅԽՆՌԹՄԠՇՆԼՁՌՁՇՆՋ

ԱԹԺՄԽԯԹՆԿԽԣՁՆԼՁՆԿ԰ՌՍԼՑԠՇՆԼՁՌՁՇՆՋ
ՁԾԞՈՈՄՁԻԹԺՄԽ

ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ
Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ

ԴԹՋԹՊԹՆԿԽԾՁՆԼԽՊԻՇՆԼՍԻՌԽԼ! ԫՇ ԢԭԞՊԽԻՇՅՅԽՆԼՋԻՇՆԼՍԻՌՁՆԿԹՊԹՆԿԽԾՁՆԼԽՊՁԾ

ՀՇՍՊԩԠԼԹՌԹԾՇՊՌՀԽԾԹՌՀԽԹԼՅՁՆՆՇՏԹՊԽ

ՍՆԹՎԹՁՄԹԺՄԽ

ԦԾՑԽՋՏՀԹՌՏԹՋՌՀԽՅԽՌՀՇԼԾՇՊԼԽՌԽՊՅՁՆՁՆԿՌՀԽՀՁԿՀԽՋՌՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՁՆՌՀԽՊԹՆԿԽԾՁՆԼԽՊ!

ԫԞ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՀԽՀՁԿՀԽՋՌՌԽՋՌ

ԻՇՆԻԽՆՌՊԹՌՁՇՆԺԽՋԽՄԽԻՌԽԼԺԹՋԽԼՇՆՌՇՐՁԻՁՌՑԼԹՌԹ

ԾՇՊՇՌՀԽՊԾՁՋՀՋՌՍԼՁԽՋՇՊՋՈԽԻՁԽՋՁԾԹՎԹՁՄԹԺՄԽ

ԬՌՀԽՊՏՁՋԽԽՁՌՀԽՊ

ՌՀԽՋՇՄՍԺՁՄՁՌՑՄՁՅՁՌՇԾՌՀԽՌԽՋՌԻՇՅՈՇՍՆԼՇՊ

ՅԿԩ
ՏՀՁԻՀԽՎԽՊՁՋՄՇՏԽՊՁՋԹՈՈՊՇՈՊՁԹՌԽ

԰ՈԽԻՁԽՋ ԫԞ

Page 80 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ
Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ

ԩՁԾԽՋՌԹԿԽ ԫԞ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՊԹՆԿԽԾՁՆԼՁՆԿՌԽՋՌՋԺԽ

ՈԽՊԾՇՊՅԽԼՏՁՌՀԾՁՋՀՇԾՋՁՅՁՄԹՊԹԿԽԹՆԼՋՁՒԽՌՇ

ՌՀՇՋԽՌՀԹՌՏՇՍՄԼԺԽՍՌՁՄՁՒԽԼՁՆՌՀԽՌԽՋՌ

ԱԽՋՌԼՍՊԹՌՁՇՆ ԫԞ ԢԭԞՊԽԻՇՅՅԽՆԼՋԹՀՇՍՊԽՐՈՇՋՍՊԽ

ԞԼԼՁՌՁՇՆԹՄԼԽՌԹՁՄՋ ԫԞ ԢԭԞՊԽԻՇՅՅԽՆԼՋԻՇՆԼՍԻՌՁՆԿԹՊԹՆԿԽԾՁՆԼԽՊՏՁՌՀ

ԾՁՎԽՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՋՈՄՍՋԹԻՇՆՌՊՇՄ
ՋՁՐՌՇՌԹՄ

ՌՊԽԹՌՅԽՆՌՄԽՎԽՄՋՏՁՌՀԾՇՍՊԾԽՅԹՄԽՋԹՆԼՌՏՇՅԹՄԽՋ

ՈԽՊԽՐՈՇՋՍՊԽՌԹՆՃ
ԾՁՋՀՌՇՌԹՄԱՀԽՆՍՅԺԽՊՇԾ

ՅՇՊՌԹՄՁՌՁԽՋՌՀԹՌՇԻԻՍՊՅԹՑԺԽՍՋԽԼՌՇԼԽՎԽՄՇՈԹ

ԻՇՆԻԽՆՌՊԹՌՁՇՆՊԽՋՈՇՆՋԽԻՍՊՎԽ

ԟԹՋԽԼՍՈՇՆՌՀԽՊԽՋՍՄՌՋՌՀԽՀՁԿՀԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆ

ՌՀԹՌԼՇԽՋՆՇՌՊԽՋՍՄՌՁՆՁՆԻՊԽԹՋԽԼՅՇՊՌԹՄՁՌՑՇՊՋՁԿՆՋ

ՇԾ ՇՎԽՊՌ ՅՇՊԺՁԼՁՌՑ ԻՇՅՈԹՊԽԼ ՌՇ ԻՇՆՌՊՇՄՋ ՇՊ 

ՌՀԽԼԽՊՁՎԽԼՀՊԩԠՅԹՑԺԽՋԽՄԽԻՌԽԼԹՋՌՀԽ

ՀՁԿՀԽՋՌԽՐՈՇՋՍՊԽԻՇՆԻԽՆՌՊԹՌՁՇՆՁՆՌՀԽԼԹՑՌԽՋՌ

ԱԹԺՄԽԡԽԾՁՆՁՌՁՎԽ԰ՌՍԼՑԠՇՆԼՁՌՁՇՆՋ

ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ
Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ

ԱԽՋՌԼՍՊԹՌՁՇՆ ԼԹՑՋ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՀԽԼՍՊԹՌՁՇՆՇԾՌՀԽ

ԼԽԾՁՆՁՌՁՎԽՌԽՋՌԺԽԼԹՑՋ

ԪԽՌՀՇԼԾՇՊՋԽՄԽԻՌՁՆԿՌՀԽՀՁԿՀԽՋՌՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՁՆՌՀԽԼԽԾՁՆՁՌՁՎԽՌԽՋՌ

԰ԽՄԽԻՌԽԼԺՑՌՀԽ԰ՌՍԼՑ԰ՈՇՆՋՇՊԺԹՋԽԼՇՆՈՊԽՎՁՇՍՋՌՇՐՁԻՁՌՑՁՆԾՇՊՅԹՌՁՇՆՏՁՌՀԻԹՊԺԹՊՑՄ

ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՀԽՀՁԿՀԽՋՌՌԽՋՌ

ԻՇՆԻԽՆՌՊԹՌՁՇՆՁՋԽՁՌՀԽՊՌՀԽՋՇՄՍԺՁՄՁՌՑՄՁՅՁՌՇԾՌՀԽՌԽՋՌ

ԻՇՅՈՇՍՆԼՅԿԩՇՊԼԽՅՇՆՋՌՊԹՌԽՋԹԼԽՉՍԹՌԽ

ԽՎՁԼԽՆԻԽՇԾՌՇՐՁԻՁՌՑ
ԽԿՌՀԽՀՇՍՊԩԠ

ՏՀՁԻՀԽՎԽՊԻՇՆԻԽՆՌՊԹՌՁՇՆՁՋՄՇՏԽՋՌ

Page 81 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ
Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ

ԯԽԾԽՊԽՆԻԽՋՌՍԼՑԻՁՌԹՌՁՇՆ
ՁԾԹՈՈՄՁԻԹԺՄԽ

ԠԹՊՄՁՋՄԽ
  

԰ԽՈԹՊԹՌՁՇՆՇԾՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՋ 

ՅԿԩ

ԱՀԽՋՌՇԻՃՋՇՄՍՌՁՇՆՋՏԽՊԽՇԺՋԽՊՎԽԼՌՇԺԽԻՄԽԹՊԹՆԼԻՇՄՇՊՄԽՋՋՏՁՌՀՆՇՎՁՋՁԺՄԽՍՆԼՁՋՋՇՄՎԽԼՌԽՋՌՋՍԺՋՌԹՆԻԽԾՇՄՄՇՏՁՆԿՈՊԽՈԹՊԹՌՁՇՆ԰ՌՇԻՃՈՊԽՈԹՊԹՌՁՇՆՊԽՋՍՄՌԽԼՁՆԹՋՌՇԻՃՋՇՄՍՌՁՇՆԻՇՆԻԽՆՌՊԹՌՁՇՆԹՈՈՊՇՐՁՅԹՌԽՄՑՄՇՏԽՊՌՀԹՆՆՇՅՁՆԹՄԱՇԻՇՅՈԽՆՋԹՌԽԾՇՊՌՀՁՋՌՀԽՌՇՐՁԻԹՆՌԼԽՄՁՎԽՊՑՏԹՋՁՆԻՊԽԹՋԽԼԾՊՇՅՅԩԻՑԻՄԽՌՇՅԩԻՑԻՄԽ

ԢԭԞՋՍԿԿԽՋՌՋՌՀԹՌԹԻՇՆԻԽՆՌՊԹՌՁՇՆՋԽՈԹՊԹՌՁՇՆՇԾ

ԺԽՌՏԽԽՆ
ՇՊՌՀՊԽԽԾՇՄԼԹՆԼ
ՇՊՌԽՆԾՇՄԼ

ՁՋՋԻՁԽՆՌՁԾՁԻԹՄՄՑԹԻԻԽՈՌԹԺՄԽ



ԫՍՅԺԽՊՇԾՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՋ  ԢԭԞՊԽԻՇՅՅԽՆԼՋԹՅՁՆՁՅՍՅՇԾԻՇՆԻԽՆՌՊԹՌՁՇՆՋ

ԹՆԼԹԻՇՆՌՊՇՄՈՄՍՋՋՇՄՎԽՆՌԻՇՆՌՊՇՄՁԾԹՈՈՊՇՈՊՁԹՌԽ

ԞՊԽՆՇՅՁՆԹՄԻՇՆԻԽՆՌՊԹՌՁՇՆՋԹԼՂՍՋՌԽԼ

ԾՇՊՈՍՊՁՌՑ!

ԶԽՋ  

ԦՆԼՁԻԹՌԽՌՀԽՌՑՈԽՇԾՎԹՄՍԽՋՈՊԽՋԽՆՌԽԼԾՇՊՅԽԹՋՍՊԽԼԻՇՆԻԽՆՌՊԹՌՁՇՆՋ

ԪԽԹՆՅԽԹՋՍՊԽԼ  

ԩՁՅՁՌՇԾՉՍԹՆՌՁԾՁԻԹՌՁՇՆ
ԩԬԮ ՅԿԹՁԩ  ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌԾՇՊԻՀԽՅՁԻԹՄՌԽՋՌ

ԻՇՆԻԽՆՌՊԹՌՁՇՆՋԺԽՄՇՏՌՀԽԩԬԮԹՆԹՄՑՋԽՋԺԽ

ԻՇՆԼՍԻՌԽԼՇՆՌՀԽՋՌՇԻՃՋՇՄՍՌՁՇՆՋ

ԩԽՎԽՄՇԾԼԽՌԽԻՌՁՇՆ
ԩԬԡ ԫՇՌԯԽՈՇՊՌԽԼԣՊԽՉՍԽՆԻՑՇԾՅԽԹՋՍՊԽՅԽՆՌ ԰ԹՅՈՄԽՋՏԽՊԽԻՇՄՄԽԻՌԽԼԾՇՊ

ԹՆԹՄՑՋՁՋՇՆԡԹՑՋԹՆԼՁՆՊԽՈՄՁԻԹՌԽՋԾՇՊԽԹԻՀՌՊԽԹՌՅԽՆՌԹՌԽԹԻՀՌՁՅԽՈՇՁՆՌ

ԦՌՁՋՊԽԻՇՅՅԽՆԼԽԼՌՀԹՌՌԽՋՌՁՌԽՅԻՇՆԻԽՆՌՊԹՌՁՇՆԺԽ

ՅԽԹՋՍՊԽԼՈՊՁՇՊՌՇՌՀԽԹԼԼՁՌՁՇՆՇԾԾՁՋՀՁՆԹՄՄՌԹՆՃՋ

ԹՆԼԹՌՄԽԹՋՌՏԽԽՃՄՑՌՀԽՊԽԹԾՌԽՊՁՆՌՏՇՊԽՈՄՁԻԹՌԽՋՈԽՊ

ՌՊԽԹՌՅԽՆՌՄԽՎԽՄ

Page 82 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ
Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ

ԴԹՋՌՀԽՊԹՆԼՇՅՁՒԽԼԻՇՅՈՄԽՌԽԺՄՇԻՃԼԽՋՁԿՆՍՋԽԼ!

ԶԽՋ  ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌԹՄՄԾՁՋՀԺԽՊԹՆԼՇՅՄՑ

ԹՋՋՁԿՆԽԼՌՇՌԹՆՃՋԼՍՊՁՆԿՈՊԽԽՐՈՇՋՍՊԽԱԹՆՃՋԹՊԽ

ՌՀԽՆՊԹՆՃԽԼԹԻԻՇՊԼՁՆԿՌՇՈՊԽԽՐՈՇՋՍՊԽԾԽԻՍՆԼՁՌՑ

ԹՆԼՌՀԽՌԹՆՃՋՏՁՌՀՌՀԽՀՁԿՀԽՋՌԾԽԻՍՆԼՁՌՑԹՊԽ

ՊԹՆԼՇՅՄՑԹՋՋՁԿՆԽԼՌՇԹԼԽԾՁՆՁՌՁՎԽՌԽՋՌՌՊԽԹՌՅԽՆՌ

ԹՆԼԺՄՇԻՃԾՁՊՋՌԢԹԻՀԺՄՇԻՃԻՇՆՌԹՁՆՋՇՆԽՊԽՈՄՁԻԹՌԽ

ՇԾԽԹԻՀՌՊԽԹՌՅԽՆՌՁՆԻՄՍԼՁՆԿԻՇՆՌՊՇՄՋ

ԫՍՅԺԽՊՇԾՊԽՈՄՁԻԹՌԽՋՁՆԻՇՆՌՊՇՄ  ԢԭԞՊԽԻՇՅՅԽՆԼՋՊԽՈՄՁԻԹՌԽՋԫՍՅԺԽՊՇԾՊԽՈՄՁԻԹՌԽՋՁՆՋՇՄՎԽՆՌԻՇՆՌՊՇՄ
ՁԾԹՈՈՄՁԻԹԺՄԽ

ԫԞ  ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԽՍՋԽՇԾԹԻՇՆԻՍՊՊԽՆՌՋՇՄՎԽՆՌ

ԻՇՆՌՊՇՄՏՀԽՆԹՋՇՄՍԺՁՄՁՒՁՆԿԹԿԽՆՌՁՋՍՋԽԼԢԭԞ

ՊԽԻՇՅՅԽՆԼՋՊԽՈՄՁԻԹՌԽՋ

ԫՍՅԺԽՊՇԾՊԽՈՄՁԻԹՌԽՋՈԽՊՌԽՋՌՁՌԽՅՌՊԽԹՌՅԽՆՌՄԽՎԽՄ

  ԢԭԞՊԽԻՇՅՅԽՆԼՋՊԽՈՄՁԻԹՌԽՋ

ԫՍՅԺԽՊՇԾՅԹՄԽԾՁՋՀՈԽՊՊԽՈՄՁԻԹՌԽԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ

  ԢԭԞՊԽԻՇՅՅԽՆԼՋՅԹՄԽՋՈԽՊՊԽՈՄՁԻԹՌԽ

ԫՍՅԺԽՊՇԾԾԽՅԹՄԽԾՁՋՀՈԽՊՊԽՈՄՁԻԹՌԽԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ

  ԢԭԞՊԽԻՇՅՅԽՆԼՋԾԽՅԹՄԽՋՈԽՊՊԽՈՄՁԻԹՌԽ

ԴԹՋԹՋՇՄՎԽՆՌՍՋԽԼ! ԫՇ

Page 83 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԭԹՊԹՅԽՌԽՊ ԳԹՄՍԽ
Ջ ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ

԰ՇՄՎԽՆՌՌՑՈԽ
ՁԾԹՈՈՄՁԻԹԺՄԽ ԫԞԪԹՐՁՅՍՅՋՇՄՎԽՆՌԻՇՆԻԽՆՌՊԹՌՁՇՆ
ՁԾԹՈՈՄՁԻԹԺՄԽ

ԫԞ  ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՀԽՋՇՄՎԽՆՌՆՇՌԽՐԻԽԽԼ

ՅՄԩ ԬԢԠԡՊԽԻՇՅՅԽՆԼՋՌՀԹՌՋՇՄՎԽՆՌ

ՀԹՎԽՆՇԽԾԾԽԻՌՇՆՋՍՊՎՁՎԹՄՆՇՊՈՊՇԼՍԻԽԹՆՑՇՌՀԽՊ

ԹԼՎԽՊՋԽԽԾԾԽԻՌՋԹՆԼՌՀԹՌԻՇՆԻԽՆՌՊԹՌՁՇՆՆՇՌԺԽ

ԿՊԽԹՌԽՊՌՀԹՆ

ՅՄԩ 

ԴԹՋԹՈՇՋՁՌՁՎԽԻՇՆՌՊՇՄՍՋԽԼ! ԫՇԭՇՋՁՌՁՎԽԻՇՆՌՊՇՄ
ՁԾԹՈՈՄՁԻԹԺՄԽ ԫԞԭՇՋՁՌՁՎԽԻՇՆՌՊՇՄԻՇՆԻԽՆՌՊԹՌՁՇՆ
Ջ
ՁԾԹՈՈՄՁԻԹԺՄԽ

ԫԞ  

ԭՀՇՌՇՈԽՊՁՇԼ ՀՊՋՄՁԿՀՌ

ՀՊՋԼԹՊՃ

ԢԭԞՊԽԻՇՅՅԽՆԼՋՈՀՇՌՇՈԽՊՁՇԼ
ՄՁԿՀՌԼԹՊՃ

ԩՁԿՀՌՁՆՌԽՆՋՁՌՑԹՌՏԹՌԽՊ0ՋՋՍՊԾԹԻԽ ՄՍՐ ԢԭԞՊԽԻՇՅՅԽՆԼՋՄՁԿՀՌՁՆՌԽՆՋՁՌՑ-ՄՍՐ


ԹՌՏԹՌԽՊ0ՋՋՍՊԾԹԻԽ

ԞԼԼՁՌՁՇՆԹՄԼԽՌԹՁՄՋ ԞՄՄԽՐՈՇՋՍՊԽՋՇՄՍՌՁՇՆՋՏԽՊԽՇԺՋԽՊՎԽԼՌՇԺԽ

ԻՄԽԹՊԹՆԼԻՇՄՇՊՄԽՋՋ

ϯ ԥՍՌԻՀՁՆՋՇՆԱԥ԰ՀՁՄՄԹԺԽԽՊԫԴՁՆՌԽՊԪԧԭՁԻՃԾՇՊԼԡԟ
ԞԻՍՌԽԹՆԼԻՀՊՇՆՁԻԽԾԾԽԻՌՋՇԾԻԹՊՊՁԽՊՋՇՄՎԽՆՌՋՁՆԹՉՍԹՌՁԻՇՊԿԹՆՁՋՅՋԞԻՊՁՌՁԻԹՄՊԽՎՁԽՏԯԽՎՁԽՏԞՉՍԹՌՁԻ

ԱՇՐՁԻՇՄՇԿՑՈՈ-

κ ԬԢԠԡ
ԤՍՁԼԹՆԻԽԡՇԻՍՅԽՆՌՇՆԞՉՍԹՌՁԻԱՇՐՁԻՁՌՑԱԽՋՌՁՆԿՇԾԡՁԾԾՁԻՍՄՌ԰ՍԺՋՌԹՆԻԽՋԹՆԼԪՁՐՌՍՊԽՋԢՆՎՁՊՇՆՅԽՆՌԹՄԥԽԹՄՌՀԹՆԼ԰ԹԾԽՌՑԭՍԺՄՁԻԹՌՁՇՆՋ԰ԽՊՁԽՋՇՆԱԽՋՌՁՆԿԹՆԼ

ԞՋՋԽՋՋՅԽՆՌԫՇԭԹՊՁՋԣՊԹՆԻԽ

Page 84 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԱԹԺՄԽ԰ՍՅՅԹՊՑՇԾԱՊԽԹՌՅԽՆՌԠՇՆԻԽՆՌՊԹՌՁՇՆՋՁՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԱՊԽԹՌՅԽՆՌԦԡ

ԫՇՅՁՆԹՄ

ԠՇՆԻԽՆՌՊԹՌՁՇՆ
ՅԿ

ԹՁԩ

ԪԽԹՋՍՊԽԼ

ԠՇՆԻԽՆՌՊԹՌՁՇՆ
ՅԿ

ԹՁԩ

ԪԽԹՆԠԳ
 ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋ ԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ

ԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄ  ԩԬԮ ԫԞ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌԽՋՌՁՌԽՅԻՇՆԻԽՆՌՊԹՌՁՇՆՋԺԽ

ՅԹՁՆՌԹՁՆԽԼԹՌԹԻՇԽԾԾՁԻՁԽՆՌՇԾՎԹՊՁԹՌՁՇՆ
ԠԳ ԱՊԽԹՌՅԽՆՌ   

ԱՊԽԹՌՅԽՆՌ   ԱՊԽԹՌՅԽՆՌ   

ԞԺԺՊԽՎՁԹՌՁՇՆՋԠԳԠՇԽԾԾՁԻՁԽՆՌՇԾՎԹՊՁԹՌՁՇՆ

ԩԬԮՅԿԹՁԩ

ԢԬԺՋԽՊՎԹՌՁՇՆՋ

ԟՁՇՄՇԿՁԻԹՄԢՆԼՈՇՁՆՌՋՋՍՊՎՁՎԹՄԾԽՊՌՁՄՁՒԹՌՁՇՆՋՍԻԻԽՋՋԾԽԻՍՆԼՁՌՑՅԽԼՁԹՆՌՍԺԽՊԻՄԽՋԻՇՊԽԺՇԼՑՏԽՁԿՀՌՄԽՆԿՌՀԤ԰ԦԳԱԤԻՇՆԻԽՆՌՊԹՌՁՇՆԿՇՆԹԼ

ՀՁՋՌՇՈԹՌՀՇՄՇԿՑՋԽԻՇՆԼԹՊՑՋԽՐԻՀԹՊԹԻՌԽՊՁՋՌՁԻՋԻՄՁՆՁԻԹՄՇԺՋԽՊՎԹՌՁՇՆՋ

ԴԽՊԽՊԹՏ
ՁՆԼՁՎՁԼՍԹՄԼԹՌԹՈՊՇՎՁԼԽԼ!ԶԽՋ

ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՇԺՋԽՊՎԹՌՁՇՆՋՇԾՋՍՊՎՁՎԹՄԾԽԻՍՆԼՁՌՑԾԽՊՌՁՄՁՒԹՌՁՇՆՋՍԻԻԽՋՋՋԽԻՇՆԼԹՊՑՋԽՐԻՀԹՊԹԻՌԽՊՁՋՌՁԻՋԹՆԼՇՌՀԽՊԻՄՁՆՁԻԹՄՋՁԿՆՋՇԻԻՍՊԹՌՄԽԹՋՌԼԹՁՄՑ

ԞՌՌԽՋՌՌԽՊՅՁՆԹՌՁՇՆ
ԡԹՑԹԼԼՁՌՁՇՆԹՄՇԺՋԽՊՎԹՌՁՇՆՋՁՆԻՄՍԼԽԺՇԼՑՏԽՁԿՀՌԹՆԼՄԽՆԿՌՀՆՍՈՌՁԹՄՌՍԺԽՊԻՄԽՋԻՇՊԽԿՇՆԹԼԹՄՋՌԹԿՁՆԿԹՆԼՀՁՋՌՇՈԹՌՀՇՄՇԿՑՈՄԹՋՅԹ

ՎՁՌԽՄՄՇԿԽՆՁՆԹՆԼՈՄԹՋՅԹՋԽՐՋՌԽՊՇՁԼՋ
ՌԽՋՌՇՋՌԽՊՇՆԽԹՆԼȕԽՋՌՊԹԼՁՇՄՁԾՅԽԹՋՍՊԽԼԤՇՆԹԼՇՋՇՅԹՌՁԻՁՆԼԽՐ
Ԥ԰ԦՁՋԻԹՄԻՍՄԹՌԽԼՍՋՁՆԿԹՊԹՌՁՇՇԾԿՇՆԹԼ

ՏԽՁԿՀՌՌՇԺՇԼՑՏԽՁԿՀՌ
ԿՇՆԹԼՏԽՁԿՀՌՌՇՌՀԽՆԽԹՊԽՋՌՅԿԺՇԼՑՏԽՁԿՀՌՁՆՅԿՐԹՌՌԽՋՌՌԽՊՅՁՆԹՌՁՇՆ

Page 85 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԠՄՁՆՁԻԹՄՋՁԿՆՋՇԾՇՎԽՊՌՌՇՐՁԻՁՌՑՅԹՑՁՆԻՄՍԼԽ
ԺՍՌԹՊԽՆՇՌՄՁՅՁՌԽԼՌՇՀԽՅՇՊՊՀԹԿԽԻԽՋՋԹՌՁՇՆՇԾԾԽԽԼՁՆԿԹՆԼՇՌՀԽՊԹԺՆՇՊՅԹՄԺԽՀԹՎՁՇՊ

ԦԦԯԢ԰ԲԩԱ԰ԞԫԡԡԦ԰ԠԲ԰԰ԦԬԫ

ԞԯԽՋՍՄՌՋ

ԟՑՌԽՋՌՌԽՊՅՁՆԹՌՁՇՆԹՋՁՆԿՄԽՅԹՄԽՅՇՊՌԹՄՁՌՑՇԻԻՍՊՊԽԼՁՆՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈ
ԱԹԺՄԽԣԽՅԹՄԽՋՍՊՎՁՎԹՄԹՎԽՊԹԿԽԼ

ՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՆԼԹՆԼՁՆՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼԹՆԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՊԽՋՈԽԻՌՁՎԽՄՑԬՎԽՊԹՄՄՋՍՊՎՁՎԹՄՏԹՋԹՆԼՁՆՌՀԽԻՇՆՌՊՇՄԹՆԼԹՆԼՅԿԹՁԩԿՊՇՍՈՋՊԽՋՈԽԻՌՁՎԽՄՑ

ԱԹԺՄԽԞԼՍՄՌԣՁՋՀ԰ՍՊՎՁՎԹՄՁՆԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ

ԱՊԽԹՌՅԽՆՌ


ՅԿԹՁԩ

#ՅԽԹՆՅԽԹՋՍՊԽԼ%

ԪԹՄԽՋ ԣԽՅԹՄԽՋ

Ն ԰ՍՊՎՁՎՁՆԿ ԰ՍՊՎՁՎԹՄ Ն ԰ՍՊՎՁՎՁՆԿ ԰ՍՊՎՁՎԹՄ

ԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄ
ԩԬԮ                        

ԞԺԺՊԽՎՁԹՌՁՇՆՋԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽ

ԩԬԮՅԿԹՁԩ

Page 86 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԪԹՄԽՏԽՁԿՀՌՊԹՆԿԽԼԾՊՇՅՌՇԿՁՆՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼԹՆԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՊԽՋՈԽԻՌՁՎԽՄՑ
ԱԹԺՄԽԪԹՄԽՄԽՆԿՌՀ

ՏԹՋՄՇՏԽՋՌՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄ
ՅՅԹՆԼԿՊԽԹՌԽՋՌՁՆՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈ
ՅՅԣԽՅԹՄԽՏԽՁԿՀՌՊԹՆԿԽԼԾՊՇՅԿՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄՌՇԿՁՆՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈԣԽՅԹՄԽՄԽՆԿՌՀՊԹՆԿԽԼԾՊՇՅՅՅՁՆՌՀԽ

ՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄՌՇՅՅՁՆՌՀԽՌՏՇՄՇՏԽՋՌՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋ

ԱԹԺՄԽ԰ՁՒԽԹՌԱԽՋՌԱԽՊՅՁՆԹՌՁՇՆՁՆԣԹՌՀԽԹԼԪՁՆՆՇՏ
ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ

ԱՊԽԹՌՅԽՆՌ


ՅԿԹՁԩ

#ՅԽԹՆՅԽԹՋՍՊԽԼ%

ԟՇԼՑԴԽՁԿՀՌ ԩԽՆԿՌՀ

ԪԹՄԽՋ ԣԽՅԹՄԽՋ ԪԹՄԽՋ ԣԽՅԹՄԽՋ

Ն

ԪԽԹՆ


Կ

չ԰ԡ Ն

ԪԽԹՆ


Կ

չ԰ԡ Ն

ԪԽԹՆ


ՅՅ

չ԰ԡ Ն

ԪԽԹՆ


ՅՅ

չ԰ԡ

ԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄ
ԩԬԮ                                                

ԞԺԺՊԽՎՁԹՌՁՇՆՋԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽԫԡԫՇՌԼԽՌԽՊՅՁՆԽԼ԰ԡ԰ՌԹՆԼԹՊԼԼԽՎՁԹՌՁՇՆ

ԩԬԮՅԿԹՁԩ

Page 87 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԣԽԻՍՆԼՁՌՑՊԹՆԿԽԼԾՊՇՅՌՇԽԿԿՋԾԽՅԹՄԽԼԹՑՁՆՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼՅԿԹՁԩԹՆԼՆԽԿԹՌՁՎԽ

ԻՇՆՌՊՇՄՊԽՋՈԽԻՌՁՎԽՄՑ
ԱԹԺՄԽԪԽԹՆԾԽՊՌՁՄՁՒԹՌՁՇՆՋՋՍԻԻԽՋՋՏԹՋՁՆՌՀԽԻՇՆՌՊՇՄԹՆԼԹՄՄՌՊԽԹՌՅԽՆՌ

ԿՊՇՍՈՋ

ԱԹԺՄԽԣԽԻՍՆԼՁՌՑԹՆԼԣԽՊՌՁՄՁՒԹՌՁՇՆ԰ՍԻԻԽՋՋՁՆԣԹՌՀԽԹԼԪՁՆՆՇՏ
ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ

ԱՊԽԹՌՅԽՆՌ


ՅԿԹՁԩ

#ՅԽԹՆՅԽԹՋՍՊԽԼ%

ԣԽԻՍՆԼՁՌՑ ԣԽՊՌՁՄՁՒԹՌՁՇՆ԰ՍԻԻԽՋՋ


ԪԽԹՆ ԰ԡ ԪԽԹՆ ԰ԡ

ԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄ
ԩԬԮ    

    

    

    

ԞԺԺՊԽՎՁԹՌՁՇՆՋԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽԫԡԫՇՌԼԽՌԽՊՅՁՆԽԼ԰ԡ԰ՌԹՆԼԹՊԼԡԽՎՁԹՌՁՇՆ



 ԣԽԻՍՆԼՁՌՑՁՋԻԹՄԻՍՄԹՌԽԼԹՋՌՀԽՆՍՅԺԽՊՇԾԽԿԿՋՈԽՊՋՍՊՎՁՎՁՆԿԾԽՅԹՄԽՈԽՊՊԽՈՊՇԼՍԻՌՁՎԽԼԹՑՈԽՊՊԽՈՄՁԻԹՌԽ



 ԣԽՊՌՁՄՁՒԹՌՁՇՆՋՍԻԻԽՋՋ
ՁՋԻԹՄԻՍՄԹՌԽԼԹՋՌՀԽՆՍՅԺԽՊՇԾԽՅԺՊՑՇՋԼՁՎՁԼԽԼԺՑՌՀԽՆՍՅԺԽՊՇԾԽԿԿՋՅՍՄՌՁՈՄՁԽԼԺՑ

ԩԬԮՅԿԹՁԩ

ԪԽԼՁԹՆՅԹՄԽՌՍԺԽՊԻՄԽՋԻՇՊԽՏԹՋՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՆԼԹՆԼՁՆՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼ

ԹՆԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՊԽՋՈԽԻՌՁՎԽՄՑ
ԱԹԺՄԽԫՇՆՍՈՌՁԹՄՌՍԺԽՊԻՄԽՋՏԽՊԽ

ՇԺՋԽՊՎԽԼՁՆԾԽՅԹՄԽՋ

ԱԹԺՄԽԫՍՈՌՁԹՄԱՍԺԽՊԻՄԽ԰ԻՇՊԽՁՆԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ

ԱՊԽԹՌՅԽՆՌ


ՅԿԹՁԩ

#ՅԽԹՆՅԽԹՋՍՊԽԼ%

ԪԹՄԽՋ ԣԽՅԹՄԽՋ

Ն ԪԽԼՁԹՆԱՍԺԽՊԻՄԽ

԰ԻՇՊԽ Ն ԪԽԼՁԹՆԱՍԺԽՊԻՄԽ԰ԻՇՊԽ

ԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄ
ԩԬԮ    

    

    

    

ԞԺԺՊԽՎՁԹՌՁՇՆՋԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽԫԡԫՇՌԼԽՌԽՊՅՁՆԽԼ԰ԡ԰ՌԹՆԼԹՊԼԼԽՎՁԹՌՁՇՆ

ԩԬԮՅԿԹՁԩ

Page 88 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԪԽԹՆՅԹՄԽԤ԰ԦՊԹՆԿԽԼԾՊՇՅՌՇՁՆՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼԹՆԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌ

ԿՊՇՍՈՋՊԽՋՈԽԻՌՁՎԽՄՑ
ԱԹԺՄԽԪԽԹՆԾԽՅԹՄԽԤ԰ԦՊԹՆԿԽԼԾՊՇՅՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄՌՇ

ՁՆՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈ

ԱԹԺՄԽԤՇՆԹԼՇ԰ՇՅԹՌՁԻԦՆԼԽՐ
Ԥ԰ԦՁՆԣԹՌՀԽԹԼԪՁՆՆՇՏ
ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ

ԱՊԽԹՌՅԽՆՌ


ՅԿԹՁԩ

#ՅԽԹՆՅԽԹՋՍՊԽԼ%

ԪԹՄԽՋ ԣԽՅԹՄԽՋ

Ն ԪԽԹՆԤ԰Ԧ




չ԰ԡ Ն ԪԽԹՆԤ԰Ԧ




չ԰ԡ

ԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄ
ԩԬԮ      

      

      

      

ԞԺԺՊԽՎՁԹՌՁՇՆՋԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽ



 ԤՇՆԹԼՇՋՇՅԹՌՁԻՁՆԼԽՐ
ՁՋԻԹՄԻՍՄԹՌԽԼԹՋԿՇՆԹԼՏԽՁԿՀՌ
ՌՇՌՀԽՆԽԹՊԽՋՌՅԿԺՇԼՑՏԽՁԿՀՌ
ՅԿՐ

ԩԬԮՅԿԹՁԩ

ԪԽԼՁԹՆԿՇՆԹԼՋՌԹԿԽՏԹՋԹՆԼՁՆՌՀԽԻՇՆՌՊՇՄԹՆԼԹՄՄՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋԾՇՊՅԹՄԽՋԹՆԼԾԽՅԹՄԽՋ

ՊԽՋՈԽԻՌՁՎԽՄՑ
ԱԹԺՄԽ

ԱԹԺՄԽԤՇՆԹԼԹՄ԰ՌԹԿՁՆԿՁՆԣԹՌՀԽԹԼԪՁՆՆՇՏ
ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ

ԱՊԽԹՌՅԽՆՌ


ՅԿԹՁԩ

#ՅԽԹՆՅԽԹՋՍՊԽԼ%

ԪԹՄԽՋ ԣԽՅԹՄԽՋ

Ն ԪԽԼՁԹՆ԰ՌԹԿԽ Ն ԪԽԼՁԹՆ԰ՌԹԿԽ

ԫԽԿԹՌՁՎԽ ԠՇՆՌՊՇՄ 
ԩԬԮ    

    

    

    

ԞԺԺՊԽՎՁԹՌՁՇՆՋԧԧՍՎԽՆՁՄԽԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽԫԡԫՇՌԼԽՌԽՊՅՁՆԽԼԲԱ԰ԲՆԹԺՄԽՌՇՋՌԹԿԽ



 ԱՀԽԿՍՁԼԽՄՁՆԽՊԽԻՇՅՅԽՆԼՋՌՀԽԾՇՄՄՇՏՁՆԿԿՇՆԹԼԹՄՋՌԹԿՁՆԿՋԻԹՄԽԾՇՊՅԹՄԽԾԹՌՀԽԹԼՅՁՆՆՇՏՍՆԼԽՎԽՄՇՈԽԼԽԹՊՄՑ

ՋՈԽՊՅԹՌՇԿԽՆՁԻՅՁԼՋՈԽՊՅԹՌՇԿԽՆՁԻՄԹՌԽՋՈԽՊՅԹՌՇԿԽՆՁԻՋՈԽՆՌ



 ԱՀԽԿՍՁԼԽՄՁՆԽՊԽԻՇՅՅԽՆԼՋՌՀԽԾՇՄՄՇՏՁՆԿԿՇՆԹԼԹՄՋՌԹԿՁՆԿՋԻԹՄԽԾՇՊԾԽՅԹՄԽԾԹՌՀԽԹԼՅՁՆՆՇՏՍՆԼԽՎԽՄՇՈԽԼԽԹՊՄՑ

ԼԽՎԽՄՇՈՅԽՆՌՅՁԼԼԽՎԽՄՇՈՅԽՆՌՄԹՌԽԼԽՎԽՄՇՈՅԽՆՌՄԹՌԽԼԽՎԽՄՇՈՅԽՆՌՀՑԼՊԹՌԽԼՈՇՋՌՇՎՍՄԹՌՇՊՑ

ԩԬԮՅԿԹՁԩ

Page 89 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԥՁՋՌՇՈԹՌՀՇՄՇԿՑՋԻՇՊԽՋԾՇՊԿՇՆԹԼԽՆԼՈՇՁՆՌՋՏԽՊԽԼԽՌԽՊՅՁՆԽԼԺԹՋԽԼՇՆՌՀԽՈԹՌՀՇՄՇԿՁՋՌ0ՋԽՐՈԽՊՁԽՆԻԽԦՆ

ՅԹՄԽՋՌՀԽՊԽՏԹՋԹՆՁՆԻՊԽԹՋԽԼՁՆԻՁԼԽՆԻԽՇԾՅՁՆՁՅԹՄՌԽՋՌՁԻՍՄԹՊԼԽԿԽՆԽՊԹՌՁՇՆԹՆԼՅՁՆՁՅԹՄՁՆԻՊԽԹՋԽՁՆՌՀԽ

ՈԽՊԻԽՆՌԹԿԽՇԾՋՈԽՊՅԹՌՇԿՇՆՁԹՁՆՌՀԽՀՁԿՀԽՋՌՌՊԽԹՌՅԽՆՌԿՊՇՍՈՆՇՇՌՀԽՊՌՊԽԹՌՅԽՆՌՊԽՄԹՌԽԼԽԾԾԽԻՌՋՏԽՊԽՆՇՌԽԼ

ՁՆՅԹՄԽՋ
ԱԹԺՄԽՋԹՆԼԱՀԽՊԽՏԽՊԽՆՇՌՊԽԹՌՅԽՆՌՊԽՄԹՌԽԼԿՇՆԹԼՇԺՋԽՊՎԹՌՁՇՆՋԾՇՊԾԽՅԹՄԽՋ
ԱԹԺՄԽՋ

ԹՆԼ

ԱԹԺՄԽԤՇՆԹԼԹՄԥՁՋՌՇՈԹՌՀՇՄՇԿՑՁՆԪԹՄԽԣԹՌՀԽԹԼԪՁՆՆՇՏ
ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ

ԱՊԽԹՌՅԽՆՌ


ՅԿԹՁԩ

#ՅԽԹՆՅԽԹՋՍՊԽԼ%

ԡՁԹԿՆՇՋՌՁԻԬԺՋԽՊՎԹՌՁՇՆՋ

԰ԽՎԽՊՁՌՑ

ԦՆԻՊԽԹՋԽԼ

ԭՊՇՈՇՊՌՁՇՆՇԾ

԰ՈԽՊՅԹՌՇԿՇՆՁԹ

ԭՊԽՋԽՆԻԽՇԾ

ԱԽՋՌՁՋԬՎԹ

ԦՆԻՊԽԹՋԽԼ

ԱԽՋՌՁԻՍՄԹՊ

ԡԽԿԽՆԽՊԹՌՁՇՆ

ԦՆՌԽՊՋՌՁՌՁԹՄԠԽՄՄ

ԥՑՈԽՊՌՊՇՈՀՑ

ԥՑՈԽՊՈՄԹՋՁԹ

Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ

ԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄ


ԩԬԮ

        

        

        

        

        



        

        

        

        

        



        

        

        

        

        

Page 90 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԱՊԽԹՌՅԽՆՌ


ՅԿԹՁԩ

#ՅԽԹՆՅԽԹՋՍՊԽԼ%

ԡՁԹԿՆՇՋՌՁԻԬԺՋԽՊՎԹՌՁՇՆՋ

԰ԽՎԽՊՁՌՑ

ԦՆԻՊԽԹՋԽԼ

ԭՊՇՈՇՊՌՁՇՆՇԾ

԰ՈԽՊՅԹՌՇԿՇՆՁԹ

ԭՊԽՋԽՆԻԽՇԾ

ԱԽՋՌՁՋԬՎԹ

ԦՆԻՊԽԹՋԽԼ

ԱԽՋՌՁԻՍՄԹՊ

ԡԽԿԽՆԽՊԹՌՁՇՆ

ԦՆՌԽՊՋՌՁՌՁԹՄԠԽՄՄ

ԥՑՈԽՊՌՊՇՈՀՑ

ԥՑՈԽՊՈՄԹՋՁԹ

Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ



        

        

        

        

        



 ԤՇՆԹԼԹՄՀՁՋՌՇՈԹՌՀՇՄՇԿՑԼՁԹԿՆՇՋՌՁԻՇԺՋԽՊՎԹՌՁՇՆՋԹՊԽԿՊԹԼԽԼ-ԺԹՋԽԼՇՆՋԽՎԽՊՁՌՑԫՇՌՊԽՅԹՊՃԹԺՄԽԪՁՆՁՅԹՄ

ԪՁՄԼԪՇԼԽՊԹՌԽ԰ԽՎԽՊԽ԰ԽԽԞՈՈԽՆԼՁՐԢՇԾՌՀԽՌԽՋՌԿՍՁԼԽՄՁՆԽԾՇՊՊԽԾԽՊԽՆԻԽ

ԩԬԮՅԿԹՁԩ

Page 91 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԱԹԺՄԽԞԼԼՁՌՁՇՆԹՄԤՇՆԹԼԹՄԥՁՋՌՇՈԹՌՀՇՄՇԿՑԬԺՋԽՊՎԹՌՁՇՆՋՁՆԪԹՄԽԣԹՌՀԽԹԼԪՁՆՆՇՏ
ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ

ԱՊԽԹՌՅԽՆՌ


ՅԿԹՁԩ

#ՅԽԹՆՅԽԹՋՍՊԽԼ%

ԞԼԼՁՌՁՇՆԹՄԡՁԹԿՆՇՋՌՁԻԬԺՋԽՊՎԹՌՁՇՆՋ

԰ԽՎԽՊՁՌՑ

ԡԽԻՊԽԹՋԽԼԭՊՇՈՇՊՌՁՇՆ

ՇԾ԰ՈԽՊՅԹՌՇԿՇՆՁԹ

ԦՆԻՊԽԹՋԽԼԳԹՋԻՍՄԹՊՇՊ

ԦՆՌԽՊՋՌՁՌՁԹՄ

ԭՊՇՌԽՁՆԹԻԽՇՍՋԣՄՍՁԼ

ԞՋՑՆԻՀՊՇՆՇՍՋ

ԤՇՆԹԼ

ԡԽՎԽՄՇՈՅԽՆՌ

ԞՄՌԽՊԽԼԭՊՇՈՇՊՌՁՇՆՋՇԾ

԰ՈԽՊՅԹՌՇԻՑՌԽՋՇՊ

԰ՈԽՊՅԹՌՁԼՋ

ԤՊԹՆՍՄՇՅԹՌՇՍՋ

ԦՆԾՄԹՅՅԹՌՁՇՆ

Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ

ԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄ
ԩԬԮ

          

          

                              

           

          

                              

           

          

                              

Page 92 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԱՊԽԹՌՅԽՆՌ


ՅԿԹՁԩ

#ՅԽԹՆՅԽԹՋՍՊԽԼ%

ԞԼԼՁՌՁՇՆԹՄԡՁԹԿՆՇՋՌՁԻԬԺՋԽՊՎԹՌՁՇՆՋ

԰ԽՎԽՊՁՌՑ

ԡԽԻՊԽԹՋԽԼԭՊՇՈՇՊՌՁՇՆ

ՇԾ԰ՈԽՊՅԹՌՇԿՇՆՁԹ

ԦՆԻՊԽԹՋԽԼԳԹՋԻՍՄԹՊՇՊ

ԦՆՌԽՊՋՌՁՌՁԹՄ

ԭՊՇՌԽՁՆԹԻԽՇՍՋԣՄՍՁԼ

ԞՋՑՆԻՀՊՇՆՇՍՋ

ԤՇՆԹԼ

ԡԽՎԽՄՇՈՅԽՆՌ

ԞՄՌԽՊԽԼԭՊՇՈՇՊՌՁՇՆՋՇԾ

԰ՈԽՊՅԹՌՇԻՑՌԽՋՇՊ

԰ՈԽՊՅԹՌՁԼՋ

ԤՊԹՆՍՄՇՅԹՌՇՍՋ

ԦՆԾՄԹՅՅԹՌՁՇՆ

Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ

           

          

                              

 ԤՇՆԹԼԹՄՀՁՋՌՇՈԹՌՀՇՄՇԿՑԼՁԹԿՆՇՋՌՁԻՇԺՋԽՊՎԹՌՁՇՆՋԹՊԽԿՊԹԼԽԼ-ԺԹՋԽԼՇՆՋԽՎԽՊՁՌՑԫՇՌՊԽՅԹՊՃԹԺՄԽԪՁՆՁՅԹՄԪՁՄԼԪՇԼԽՊԹՌԽ԰ԽՎԽՊԽ԰ԽԽԞՈՈԽՆԼՁՐԢՇԾ

ՌՀԽՌԽՋՌԿՍՁԼԽՄՁՆԽԾՇՊՊԽԾԽՊԽՆԻԽ

ԩԬԮՅԿԹՁԩ

Page 93 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԱԹԺՄԽԤՇՆԹԼԹՄԥՁՋՌՇՈԹՌՀՇՄՇԿՑՁՆԣԽՅԹՄԽԣԹՌՀԽԹԼԪՁՆՆՇՏ
ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ

ԱՊԽԹՌՅԽՆՌ


ՅԿԹՁԩ

#ՅԽԹՆՅԽԹՋՍՊԽԼ%

ԞԼԼՁՌՁՇՆԹՄԡՁԹԿՆՇՋՌՁԻԬԺՋԽՊՎԹՌՁՇՆՋ

԰ԽՎԽՊՁՌՑ

ԦՆԻՊԽԹՋԽԼԬՇԻՑՌԽԞՌՊԽՋՁԹ

ԭԽՊՁԾՇՄՄՁԻՍՄԹՊԠԽՄՄ

ԥՑՈԽՊՈՄԹՋՁԹԥՑՈԽՊՌՊՇՈՀՑ

ԡԽԻՊԽԹՋԽԼԶՇՄՃ

ԣՇՊՅԹՌՁՇՆ

Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ

ԫԽԿԹՌՁՎԽԻՇՆՌՊՇՄ


ԩԬԮ

      

      

      

      

      

       

      

      

      

      

       

      

      

      

      

       

      

      

      

      



 ԤՇՆԹԼԹՄՀՁՋՌՇՈԹՌՀՇՄՇԿՑԼՁԹԿՆՇՋՌՁԻՇԺՋԽՊՎԹՌՁՇՆՋԹՊԽԿՊԹԼԽԼ-ԺԹՋԽԼՇՆՋԽՎԽՊՁՌՑԫՇՌՊԽՅԹՊՃԹԺՄԽԪՁՆՁՅԹՄ

ԪՁՄԼԪՇԼԽՊԹՌԽ԰ԽՎԽՊԽ԰ԽԽԞՈՈԽՆԼՁՐԢՇԾՌՀԽՌԽՋՌԿՍՁԼԽՄՁՆԽԾՇՊՊԽԾԽՊԽՆԻԽ

ԩԬԮՅԿԹՁԩ

Page 94 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԱԹԺՄԽԞԼԼՁՌՁՇՆԹՄԤՇՆԹԼԹՄԥՁՋՌՇՈԹՌՀՇՄՇԿՑԬԺՋԽՊՎԹՌՁՇՆՋՁՆԣԽՅԹՄԽԣԹՌՀԽԹԼԪՁՆՆՇՏ
ԭՁՅԽՈՀԹՄԽՋ

ՈՊՇՅԽՄԹՋ

ԱՊԽԹՌՅԽՆՌ


ՅԿԹՁԩ

#ՅԽԹՆ

ՅԽԹՋՍՊԽԼ%

ԞԼԼՁՌՁՇՆԹՄԡՁԹԿՆՇՋՌՁԻԬԺՋԽՊՎԹՌՁՇՆՋ

԰ԽՎԽՊՁՌՑ

ԦՆՌԽՊՋՌՁՌՁԹՄ

ԣՁԺՊՇՋՁՋ

ԢԿԿԡԽԺՊՁՋՁՆ

ԬՎՁԼՍԻՌ

ԤՊԹՆՍՄՇՅԹՌՇՍՋ

ԦՆԾՄԹՅՅԹՌՁՇՆ

ԡԽԻՊԽԹՋԽԼԭՇՋՌ

ԬՎՍՄԹՌՇՊՑԣՇՄՄՁԻՄԽՋ

Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ

ԫԽԿԹՌՁՎԽ

ԠՇՆՌՊՇՄ
ԩԬԮ

        

        

        

        

        

         

        

        

        

        

         

        

        

        

        

         

        

        

        

        



 ԤՇՆԹԼԹՄՀՁՋՌՇՈԹՌՀՇՄՇԿՑԼՁԹԿՆՇՋՌՁԻՇԺՋԽՊՎԹՌՁՇՆՋԹՊԽԿՊԹԼԽԼ-ԺԹՋԽԼՇՆՋԽՎԽՊՁՌՑԫՇՌՊԽՅԹՊՃԹԺՄԽԪՁՆՁՅԹՄ

ԪՁՄԼԪՇԼԽՊԹՌԽ԰ԽՎԽՊԽ԰ԽԽԞՈՈԽՆԼՁՐԢՇԾՌՀԽՌԽՋՌԿՍՁԼԽՄՁՆԽԾՇՊՊԽԾԽՊԽՆԻԽ

ԩԬԮՅԿԹՁԩ

Page 95 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԪԽԹՆՈՄԹՋՅԹՎՁՌԽՄՄՇԿԽՆՁՆԻՇՆԻԽՆՌՊԹՌՁՇՆՋՏԽՊԽՀՁԿՀՄՑՎԹՊՁԹԺՄԽՁՆԺՇՌՀՅԹՄԽՋԹՆԼԾԽՅԹՄԽՋՁՆԹՄՄՌՊԽԹՌՅԽՆՌ

ԿՊՇՍՈՋ
ԱԹԺՄԽ

ԱԹԺՄԽԭՄԹՋՅԹԳՁՌԽՄՄՇԿԽՆՁՆՁՆԣԹՌՀԽԹԼԪՁՆՆՇՏ
ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ

ԱՊԽԹՌՅԽՆՌ


ՅԿԹՁԩ

#ՅԽԹՆՅԽԹՋՍՊԽԼ%

ԭՄԹՋՅԹԳՁՌԽՄՄՇԿԽՆՁՆ
ԳԱԤ

ԪԹՄԽՋ ԣԽՅԹՄԽՋ

Ն ԪԽԹՆ


ՆԿՅԩՈՄԹՋՅԹ

չ԰ԡ Ն ԪԽԹՆ


ՆԿՅԩՈՄԹՋՅԹ

չ԰ԡ

ԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄ
ԩԬԮ     Ր Ր

     Ր Ր

     Ր Ր

     Ր Ր

ԞԺԺՊԽՎՁԹՌՁՇՆՋԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽԫԡԫՇՌԼԽՌԽՊՅՁՆԽԼ԰ԡ԰ՌԹՆԼԹՊԼԼԽՎՁԹՌՁՇՆ

ԩԬԮՅԿԹՁԩ

Page 96 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԱԹԺՄԽԭՄԹՋՅԹ԰ԽՐ԰ՌԽՊՇՁԼՋՁՆԣԹՌՀԽԹԼԪՁՆՆՇՏ
ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ

ԱՀՁՋՌԹԺՄԽՁՋՇՅՁՌՌԽԼԺԽԻԹՍՋԽՈՄԹՋՅԹՋԽՐՋՌԽՊՇՁԼՋՏԽՊԽՆՇՌՅԽԹՋՍՊԽԼԱՀԽՊԽՏԽՊԽՆՇՆՇՌԹԺՄԽՇԺՋԽՊՎԹՌՁՇՆՋՏՁՌՀՊԽՋՈԽԻՌՌՇԺԽՀԹՎՁՇՊԻՇՄՇՊԹՌՁՇՆԺԹՆԼՁՆԿԻՀԹՆԿԽՋՁՆՇՎՁՈՇՋՁՌՇՊԹՈՈԽԹՊԹՆԻԽՇՊՋՁՒԽՇԾԼՇՊՋԹՄՆԹՈԽՈԹԼ
ԱԹԺՄԽ

ԱԹԺՄԽ԰ԽԻՇՆԼԹՊՑ԰ԽՐԠՀԹՊԹԻՌԽՊՁՋՌՁԻՋԹՆԼԠՄՁՆՁԻԹՄ԰ՁԿՆՋՁՆԣԹՌՀԽԹԼԪՁՆՆՇՏ
ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ

ԱՊԽԹՌՅԽՆՌ


ՅԿԹՁԩ

#ՅԽԹՆՅԽԹՋՍՊԽԼ%

԰ԽԻՇՆԼԹՊՑ԰ԽՐԠՀԹՊԹԻՌԽՊՁՋՌՁԻՋԹՆԼԠՄՁՆՁԻԹՄ԰ՁԿՆՋ

ԪԹՄԽՋ ԣԽՅԹՄԽՋ

ԱՑՈԽ Ն ԦՆԻՁԼԽՆԻԽ ԱՑՈԽ Ն ԦՆԻՁԼԽՆԻԽ

ԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄ
ԩԬԮ ԫՇՆԽ   ԫՇՆԽ   ԫՇՆԽ   ԫՇՆԽ   ԫՇՆԽ   ԫՇՆԽ   ԫՇՆԽ   ԫՇՆԽ  

ԩԬԮՅԿԹՁԩ

Page 97 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԟ԰ՌՍԼՑԞՍՌՀՇՊ0ՋԞՆԹՄՑՋՁՋԹՆԼԠՇՆԻՄՍՋՁՇՆՋ

ԞՄՄՋՌԹՌՁՋՌՁԻԹՄԹՆԹՄՑՋԽՋՏԽՊԽԻՇՆԼՍԻՌԽԼԹՌՌՀԽՄԽՎԽՄՇԾԻԽՊՌԹՁՆՌՑԽՐԻԽՈՌՁՆՌՀԽԻԹՋԽՇԾ԰ՀԹՈՁՊՇԴՁՄՃՋ0

ԹՆԼ ԩԽՎԽՆԽ0Ջ ԱԽՋՌՋ ՁՆ ՏՀՁԻՀ ՌՀԽ  ՄԽՎԽՄ ՇԾ ԻԽՊՌԹՁՆՌՑ ՏԹՋ ԹՈՈՄՁԽԼ ԰ՁԿՆՁԾՁԻԹՆՌ ԼՁԾԾԽՊԽՆԻԽՋ ՁՆ ՌՀԽ

ՈԽՊԻԽՆՌՋՍՊՎՁՎԹՄՏԽՊԽԼԽՌԽՊՅՁՆԽԼՍՋՁՆԿՌՀԽԠՇԻՀՊԹՆԞՊՅՁՌԹԿԽԱՊԽՆԼ԰ՌԽՈԡՇՏՆԱԽՋՌԣՇՊԽՆԼՈՇՁՆՌՋՌՀԹՌ

ՅԽՌ ՌՀԽ ԹՋՋՍՅՈՌՁՇՆՋ ՇԾ ՆՇՊՅԹՄՁՌՑ ԹՆԼ ՀՇՅՇԿԽՆԽՁՌՑ ՌՀԽ ՈԽՊԾՇՊՅԹՆԻԽ ՇԾ ՇՊԿԹՆՁՋՅՋ ԽՐՈՇՋԽԼ ՌՇ ԽԹԻՀ

ՌՊԽԹՌՅԽՆՌՄԽՎԽՄՏԹՋԻՇՅՈԹՊԽԼՏՁՌՀՌՀԽՈԽՊԾՇՊՅԹՆԻԽՇԾՌՀԽԻՇՆՌՊՇՄՍՋՁՆԿԡՍՆՆԽՌՌ0ՋԪՍՄՌՁՈՄԽԠՇՅՈԹՊՁՋՇՆ

ԱԽՋՌ ԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹ ԰ՌԽՈԡՇՏՆ ԱԽՋՌ 
ՇՆ ՅԽԼՁԹՆ ՎԹՄՍԽՋՏԹՋ ՍՋԽԼ ԾՇՊ ԻՇՅՈԹՊՁՋՇՆ ՇԾ ՌՀԽ ՅԹՄԽ

ՌՍԺԽՊԻՄԽ ՋԻՇՊԽ ԽՆԼՈՇՁՆՌ ԳՁՌԽՄՄՇԿԽՆՁՆ ԼԹՌԹ ՏԽՊԽ ԹՆԹՄՑՒԽԼ ԾՇՊ ՇՍՌՄՁԽՊՋ ԹՆԼ ՋՌԹՌՁՋՌՁԻԹՄ ԹՆԹՄՑՋՁՋ ՏԹՋ

ԻՇՆԼՍԻՌԽԼԺՇՌՀՏՁՌՀԹՆԼՏՁՌՀՇՍՌՋՍՋՈԽԻՌԽԼՇՍՌՄՁԽՊՋԬՍՌՄՁԽՊՋԾՇՊՌՀԽՅԹՄԽԼԹՌԹՏԽՊԽՁԼԽՆՌՁԾՁԽԼԺՑՎԹՄՍԽՋ

ՏՀՁԻՀ ԽՐԻԽԽԼԽԼ ՌՀԽ ՅԽԼՁԹՆ ՈՄՍՋ ՌՀՊԽԽ ՌՁՅԽՋ ՌՀԽ ՁՆՌԽՊՉՍԹՊՌՁՄԽ ՊԹՆԿԽ 
ՁԽ ՌՀԽ ԼՁԾԾԽՊԽՆԻԽ ԺԽՌՏԽԽՆ ՌՀԽ

ՌՀԹՆԼՌՀՈԽՊԻԽՆՌՁՄԽՋԣԽՅԹՄԽԼԹՌԹՏԽՊԽՄՇԿՌՊԹՆՋԾՇՊՅԽԼՈՊՁՇՊՌՇՇՍՌՄՁԽՊԹՆԹՄՑՋՁՋԬՍՌՄՁԽՊՋԾՇՊՌՀԽ

ԾԽՅԹՄԽԼԹՌԹՏԽՊԽՁԼԽՆՌՁԾՁԽԼԺՑՄՇԿՌՊԹՆՋԾՇՊՅԽԼՎԹՄՍԽՋՏՀՁԻՀՏԽՊԽԺԽՄՇՏՌՀԽՅԽԼՁԹՆՅՁՆՍՋՌՀՊԽԽՌՁՅԽՋ

ՌՀԽՁՆՌԽՊՉՍԹՊՌՁՄԽ ՊԹՆԿԽԞԼԼՁՌՁՇՆԹՄՇՍՌՄՁԽՊՋՏԽՊԽՁԼԽՆՌՁԾՁԽԼԺԹՋԽԼՇՆԻՇՆՋՍՄՌԹՌՁՇՆՏՁՌՀՌՀԽ԰ՌՍԼՑ԰ՈՇՆՋՇՊ

ԽՐՈԽՊՌՂՍԼԿՅԽՆՌՈՊՁՅԹՊՁՄՑԺԹՋԽԼՇՆՎԹՄՍԽՋԺԽՄՇՏՌՀԽԼԽՌԽԻՌՁՇՆՄՁՅՁՌՇՊՇՍՌՋՁԼԽՇԾՌՀԽՆՇՊՅԹՄՎՁՌԽՄՄՇԿԽՆՁՆ

ՊԹՆԿԽԦԾՌՀԽՊԽՏԽՊԽՆՇԼՁԾԾԽՊԽՆԻԽՋՁՆՌՀԽՊԽՋՍՄՌՋՇԾՌՀԽՋՌԹՌՁՋՌՁԻԹՄԹՆԹՄՑՋԽՋԺԽՌՏԽԽՆՌՀԽՌՏՇՋԽՌՋՇԾԼԹՌԹ

ՌՀԽՆՌՀԽ ԹՆԹՄՑՋՁՋ ՁՆԻՄՍԼՁՆԿՌՀԽ ՇՍՌՄՁԽՊՋՏԹՋ ՈՊԽՋԽՆՌԽԼԦԾ ԼՁԾԾԽՊԽՆԻԽՋՏԽՊԽ ՇԺՋԽՊՎԽԼՌՀԽՆ ԺՇՌՀՋԽՌՋ ՇԾ

ԼԹՌԹ ՏԽՊԽ ՈՊԽՋԽՆՌԽԼ ԠԢԱԦ԰ԱԪ ԳԽՊՋՁՇՆ  ՏԹՋ ՍՋԽԼ ՌՇ ՈԽՊԾՇՊՅ ԹՄՄ ՋՌԹՌՁՋՌՁԻԹՄ ԻՇՅՈՍՌԹՌՁՇՆՋ

ԥՁՋՌՇՈԹՌՀՇՄՇԿՑՊԽՋՍՄՌՋՏԽՊԽԽՎԹՄՍԹՌԽԼՉՍԹՄՁՌԹՌՁՎԽՄՑ

ԱՀԽՋՌՍԼՑԹՍՌՀՇՊԾՇՍՆԼԹՋՁԿՆՁԾՁԻԹՆՌԼԽԻՊԽԹՋԽՁՆՅԹՄԽՆՍՈՌՁԹՄՌՍԺԽՊԻՄԽՋԻՇՊԽԹՆԼԹՉՍԹՄՁՌԹՌՁՎԽՁՆԻՊԽԹՋԽՁՆ

ՅԹՄԽ ՀՁՋՌՇՈԹՌՀՇՄՇԿՁԻԹՄ ԾՁՆԼՁՆԿՋ ՁՆ ՌՀԽ ՅԽԹՆՅԽԹՋՍՊԽԼ  ՅԿ ԹՁԩ ՌՊԽԹՌՅԽՆՌ ԿՊՇՍՈ ԫՇ ՇՌՀԽՊ

ՋՁԿՆՁԾՁԻԹՆՌԽԾԾԽԻՌՋՏԽՊԽՆՇՌԽԼ

ԠԯԽՎՁԽՏԽՊ0ՋԞՆԹՄՑՋՁՋԹՆԼԠՇՆԻՄՍՋՁՇՆՋ

԰ՌԹՌՁՋՌՁԻԹՄԪԽՌՀՇԼՋԣԽՏՁՆԻՁԼԽՆՌԹՄՅՇՊՌԹՄՁՌՁԽՋՇԻԻՍՊՊԽԼԹՆԼԼՁԼՆՇՌԽՐՀՁԺՁՌԹԻՇՆԻԽՆՌՊԹՌՁՇՆԼԽՈԽՆԼԽՆՌ

ՊԽՋՈՇՆՋԽՌՀԽՊԽԾՇՊԽՋՍՊՎՁՎԹՄՏԹՋԹՆԹՄՑՒԽԼԽՅՈՁՊՁԻԹՄՄՑԫՇԽՆԼՈՇՁՆՌՋԽՐՀՁԺՁՌԽԼԹԻՄԽԹՊՅՇՆՇՌՇՆՁԻ

ՊԽՋՈՇՆՋԽԹՆԼԹՄՄԽՆԼՈՇՁՆՌՋԽՐԻԽՈՌՅԹՄԽԳԱԤՋԻՇՊԽՏԽՊԽՆՇՊՅԹՄՄՑԼՁՋՌՊՁԺՍՌԽԼ
԰ՀԹՈՁՊՇԴՁՄՃՋՈ 

ՏՁՌՀԽՉՍԹՄՎԹՊՁԹՆԻԽ
ԟԹՊՌՄԽՌՌ0ՋՌԽՋՌՈ ԹՆԼՏԽՊԽՌՀԽՊԽԾՇՊԽԹՆԹՄՑՒԽԼՍՋՁՆԿԡՍՆՆԽՌՌ0ՋԪՍՄՌՁՈՄԽ

ԠՇՅՈԹՊՁՋՇՆԱԽՋՌԪԹՄԽԳԱԤՏԹՋԹՆԹՄՑՒԽԼՍՋՁՆԿՌՀԽԪԹՆՆԴՀՁՌՆԽՑԲԱՏՇ԰ԹՅՈՄԽՌԽՋՌԥՇՏԽՎԽՊՅԹՄԽ

ՌՍԺԽՊԻՄԽՋԻՇՊԽՏԹՋԹՄՋՇԹՆԹՄՑՒԽԼՍՋՁՆԿՌՀԽԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹ԰ՌԽՈԡՇՏՆՌԽՋՌԞՄՄԹՆԹՄՑՋԽՋՏԽՊԽ

ԻՇՆԼՍԻՌԽԼՍՋՁՆԿԠԢԱԦ԰ՎՍՋՁՆԿԺԹԻՃԽՆԼՋԽՌՌՁՆԿՋԼԽՋՁԿՆԽԼԺՑԢԣԢԡԾՇՊԹՈՈՊՇՎԽԼՍՋԽՇՆ

ԲՆՄԽՋՋՇՌՀԽՊՏՁՋԽՁՆԼՁԻԹՌԽԼԽԾԾԽԻՌՋՏԽՊԽԻՇՆՋՁԼԽՊԽԼՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌԹՌՈ

Page 98 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԠՇՆԻՄՍՋՁՇՆՋԪԹՄԽՄԽՆԿՌՀՏԹՋՋՁԿՆՁԾՁԻԹՆՌՄՑՊԽԼՍԻԽԼ
ՊԽԼՍԻՌՁՇՆՊԽՄԹՌՁՎԽՌՇՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՌ

ՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌՄԽՎԽՄ
ԡՍՆՆԽՌՌ0ՋԪՍՄՌՁՈՄԽԠՇՅՈԹՊՁՋՇՆԱԽՋՌՈ

ԣՍՊՌՀԽՊՅԽԼՁԹՆՅԹՄԽՌՍԺԽՊԻՄԽՋԻՇՊԽՏԹՋՋՁԿՆՁԾՁԻԹՆՌՄՑՊԽԼՍԻԽԼԹՌՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼՅԿԹՁԩ

ՌՊԽԹՌՅԽՆՌԿՊՇՍՈ
ԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹ԰ՌԽՈԡՇՏՆԱԽՋՌՈԱՀԽՋՁԿՆՁԾՁԻԹՆՌՊԽԼՍԻՌՁՇՆՁՆՅԹՄԽ

ՌՍԺԽՊԻՄԽՋԻՇՊԽԹՌՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈՇԻԻՍՊՁՆԻՇՆԻԽՊՌՏՁՌՀԹՆՁՆԻՊԽԹՋԽ

ՁՆՁՆՌԽՆՋՁՌՑԹՆԼՋԽՎԽՊՁՌՑՇԾՅԹՄԽՀՁՋՌՇՈԹՌՀՇՄՇԿՁԻԹՄԾՁՆԼՁՆԿՋ
ԽԿՁՆԻՊԽԹՋԽԼՁՆԻՁԼԽՆԻԽՇԾՅՁՆՁՅԹՄՌԽՋՌՁԻՍՄԹՊ

ԼԽԿԽՆԽՊԹՌՁՇՆԹՆԼՅՁՆՁՅԹՄՁՆԻՊԽԹՋԽՁՆՌՀԽՈԽՊԻԽՆՌԹԿԽՇԾՋՈԽՊՅԹՌՇԿՇՆՁԹ

Page 99 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԱԹԺՄԽԯԽՈՊՇԼՍԻՌՁՎԽԹՆԼԥԭԤԢՆԼՈՇՁՆՌՋԾՇՊԪԹՄԽԣԹՌՀԽԹԼԪՁՆՆՇՏ
ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋՁՆՌՀԽԣ԰ԱԯԞՏՁՌՀԠԹՊԺԹՊՑՄ

ԱՊԽԹՌՅԽՆՌ


ՅԿԹՁԩ

#ՅԽԹՆՅԽԹՋՍՊԽԼ%

ԱՍԺԽՊԻՄԽ԰ԻՇՊԽ Ԥ԰Ԧ

ԤՇՆԹԼԹՄ԰ՌԹԿՁՆԿ

ԹՆԼԥՁՋՌՇ

ԭՄԹՋՅԹԳԱԤ ԭՄԹՋՅԹԱ ԭՄԹՋՅԹԢ

ԪԽԼՁԹՆ

Ո ԡՁԾԾ

Ո ԢԾԾԽԻՌ!
ԶԽՋԫՇ ԡՁԾԾ

Ո ԡՁԾԾ

Ո ԡՁԾԾ

Ո

ԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄ
ԩԬԮ

    ԫՇ      

     ԫՇ           ԫՇ           ԶԽՋ      ԰ՌԹՌՁՋՌՁԻԹՄԱԽՋՌ ԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹ ԡՍՆՆԽՌՌ ԫԞ ԪԹՆՆԴՀՁՌՆԽՑ  

ԞԺԺՊԽՎՁԹՌՁՇՆՋԠՇՆԻԠՇՆԻԽՆՌՊԹՌՁՇՆԡՁԾԾԡՁԾԾԽՊԽՆԻԽԢȕԽՋՌՊԹԼՁՇՄԤ԰ԦԤՇՆԹԼՇ԰ՇՅԹՌՁԻԦՆԼԽՐԥՁՋՌՇԥՁՋՌՇՈԹՌՀՇՄՇԿՑԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽԱԱԽՋՌՇՋՌԽՊՇՆԽԳԱԤԳՁՌԽՄՄՇԿԽՆՁՆ

 ԲՆՄԽՋՋՇՌՀԽՊՏՁՋԽՁՆԼՁԻԹՌԽԼԽԾԾԽԻՌՋԹՆԼՈԽՊԻԽՆՌ
ԼՁԾԾԽՊԽՆԻԽՋԹՊԽՊԽՈՇՊՌԽԼԺԹՋԽԼՇՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽՆԽԿԹՌՁՎԽ
ԻՄԽԹՆՏԹՌԽՊԻՇՆՌՊՇՄԠՇՆԻՄՍՋՁՇՆՋՊԽԿԹՊԼՁՆԿ

ՀՁՋՌՇՈԹՌՀՇՄՇԿՑՅԹՑԺԽՀԽԹՎՁՄՑՏԽՁԿՀՌԽԼԺՑՌՀԽԽՐՈԽՊՌՇՈՁՆՁՇՆՇԾԹԺՇԹՊԼԻԽՊՌՁԾՁԽԼՈԹՌՀՇՄՇԿՁՋՌ

 ԲՆՄԽՋՋՇՌՀԽՊՏՁՋԽՋՈԽԻՁԾՁԽԼԽԾԾԽԻՌՋԹՊԽԻՇՆՋՁԼԽՊԽԼՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌԹՌՈ

ԩԬԮՅԿԹՁԩ

Page 100 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԱԹԺՄԽԯԽՈՊՇԼՍԻՌՁՎԽԹՆԼԥԭԤԢՆԼՈՇՁՆՌՋԾՇՊԣԽՅԹՄԽԣԹՌՀԽԹԼԪՁՆՆՇՏ
ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋՁՆՌՀԽԣ԰ԱԯԞՏՁՌՀԠԹՊԺԹՊՑՄ

ԱՊԽԹՌՅԽՆՌ


ՅԿԹՁԩ

#ՅԽԹՆՅԽԹՋՍՊԽԼ%

ԣԽԻՍՆԼՁՌՑ ԣԽՊՌ԰ՍԻԻԽՋՋ ԱՍԺԽՊԻՄԽ԰ԻՇՊԽ Ԥ԰Ԧ ԤՇՆԹԼԹՄ԰ՌԹԿՁՆԿ

ԹՆԼԥՁՋՌՇ

ԭՄԹՋՅԹԳԱԤ ԭՄԹՋՅԹԱ ԭՄԹՋՅԹԢ

ԡՁԾԾ

Ո ԡՁԾԾ

Ո ԪԽԼՁԹՆ

Ո ԡՁԾԾ

Ո ԢԾԾԽԻՌ!
ԶԽՋԫՇ ԡՁԾԾ

Ո ԡՁԾԾ

Ո ԡՁԾԾ

Ո

ԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄ
ԩԬԮ

        ԫՇ      

         ԫՇ               ԫՇ               ԫՇ      ԰ՌԹՌՁՋՌՁԻԹՄԱԽՋՌ ԡՍՆՆԽՌՌ ԡՍՆՆԽՌՌ  ԡՍՆՆԽՌՌ ԫԞ ԡՍՆՆԽՌՌ  

ԞԺԺՊԽՎՁԹՌՁՇՆՋԠՇՆԻԠՇՆԻԽՆՌՊԹՌՁՇՆԡՁԾԾԡՁԾԾԽՊԽՆԻԽԢȕԽՋՌՊԹԼՁՇՄԣԽՊՌԣԽՊՌՁՄՁՒԹՌՁՇՆԤ԰ԦԤՇՆԹԼՇ԰ՇՅԹՌՁԻԦՆԼԽՐԥՁՋՌՇԥՁՋՌՇՈԹՌՀՇՄՇԿՑ

ԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽԱԱԽՋՌՇՋՌԽՊՇՆԽԳԱԤԳՁՌԽՄՄՇԿԽՆՁՆ ԲՆՄԽՋՋՇՌՀԽՊՏՁՋԽՁՆԼՁԻԹՌԽԼԽԾԾԽԻՌՋԹՆԼՈԽՊԻԽՆՌ
ԼՁԾԾԽՊԽՆԻԽՋԹՊԽՊԽՈՇՊՌԽԼԺԹՋԽԼՇՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽՆԽԿԹՌՁՎԽ
ԻՄԽԹՆՏԹՌԽՊԻՇՆՌՊՇՄԠՇՆԻՄՍՋՁՇՆՋՊԽԿԹՊԼՁՆԿ

ՀՁՋՌՇՈԹՌՀՇՄՇԿՑՅԹՑԺԽՀԽԹՎՁՄՑՏԽՁԿՀՌԽԼԺՑՌՀԽԽՐՈԽՊՌՇՈՁՆՁՇՆՇԾԹԺՇԹՊԼԻԽՊՌՁԾՁԽԼՈԹՌՀՇՄՇԿՁՋՌ

 ԲՆՄԽՋՋՇՌՀԽՊՏՁՋԽՋՈԽԻՁԾՁԽԼԽԾԾԽԻՌՋԹՊԽԻՇՆՋՁԼԽՊԽԼՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌԹՌՈ

ԩԬԮՅԿԹՁԩ

Page 101 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԱԹԺՄԽԤՊՇՏՌՀԢՆԼՈՇՁՆՌՋ ՁՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑ
ԣ԰ԱԯԞՏՁՌՀԠԹՊԺԹՊՑՄ

ԱՊԽԹՌՅԽՆՌ


ՅԿԹՁԩ

#ՅԽԹՆՅԽԹՋՍՊԽԼ%

ԟՇԼՑԴԽՁԿՀՌ ԩԽՆԿՌՀ

ԪԹՄԽՋ ԣԽՅԹՄԽՋ ԪԹՄԽՋ ԣԽՅԹՄԽՋ

ԡՁԾԾ Ո ԡՁԾԾ Ո ԡՁԾԾ Ո ԡՁԾԾ Ո

ԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄ
ԩԬԮ        

        

        

        

԰ՌԹՌՁՋՌՁԻԹՄԱԽՋՌ ԡՍՆՆԽՌՌ ԡՍՆՆԽՌՌ ԡՍՆՆԽՌՌ ԡՍՆՆԽՌՌ

ԞԺԺՊԽՎՁԹՌՁՇՆՋԡՁԾԾԡՁԾԾԽՊԽՆԻԽԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽԫԡԫՇՌԼԽՌԽՊՅՁՆԽԼ



 ԲՆՄԽՋՋՇՌՀԽՊՏՁՋԽՁՆԼՁԻԹՌԽԼՈԽՊԻԽՆՌ
ԼՁԾԾԽՊԽՆԻԽՋԹՊԽՊԽՈՇՊՌԽԼԺԹՋԽԼՇՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽՆԽԿԹՌՁՎԽ
ԻՄԽԹՆՏԹՌԽՊ

ԻՇՆՌՊՇՄ



 ԲՆՄԽՋՋՇՌՀԽՊՏՁՋԽՋՈԽԻՁԾՁԽԼԽԾԾԽԻՌՋԹՊԽԻՇՆՋՁԼԽՊԽԼՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌԹՌՈ

ԩԬԮՅԿԹՁԩ

Ԣ԰ՌՍԼՑԡԽԾՁԻՁԽՆԻՁԽՋ

ԞՄՄՈԽՊԾՇՊՅԹՆԻԽԻՊՁՌԽՊՁԹՏԽՊԽՅԽՌՁՆՌՀՁՋՋՌՍԼՑԽՐԻԽՈՌՌՀԹՌԾՇՊԻՇՆՌՊՇՄՊԽՈՄՁԻԹՌԽԡՋՈԹՏՆՁՆԿԼՁԼՆՇՌՇԻԻՍՊ

ԹՌ ՄԽԹՋՌ ԽՎԽՊՑ ԾՇՍՊ ԼԹՑՋ ՆՇՊ ՏԹՋ ՌՀԽ ԹՎԽՊԹԿԽ ԾԽԻՍՆԼՁՌՑ  ԽԿԿՋԾԽՅԹՄԽԼԹՑ ՇՊ ԿՊԽԹՌԽՊ ԞՄՌՀՇՍԿՀ ՌՀԽ

ԹՎԽՊԹԿԽԾԽԻՍՆԼՁՌՑԾՇՊՌՀԹՌՊԽՈՄՁԻԹՌԽՏԹՋԽԿԿՋԾԽՅԹՄԽԼԹՑԾԽԻՍՆԼՁՌՑՏԹՋՄՁՅՁՌԽԼԼՍՊՁՆԿՌՀԽԾՁՊՋՌՏԽԽՃ

ՇԾ ԽՐՈՇՋՍՊԽ ԺՍՌ ՁՆԻՊԽԹՋԽԼ ՇՎԽՊ ՌՀԽ ԼՍՊԹՌՁՇՆ ՇԾ ՌԽՋՌՁՆԿ ԞԼԼՁՌՁՇՆԹՄՄՑ ՌՀԽ ԾԽԻՍՆԼՁՌՑ ՊԹՌԽՋ ՁՆ ՌՀԽ ՇՌՀԽՊ

ՊԽՈՄՁԻԹՌԽՋ ՊԹՆԿԽԼԾՊՇՅ ՌՇ ԽԿԿՋԾԽՅԹՄԽԼԹՑ ՁՆԼՁԻԹՌՁՆԿՌՀԹՌԾԽԻՍՆԼՁՌՑՏԹՋ ԹԼԽՉՍԹՌԽ ՁՆՌՀԽԻՇՆՌՊՇՄ

ՌՊԽԹՌՅԽՆՌ ԞԼԼՁՌՁՇՆԹՄՄՑ  ՇԾՌՀԽ  ՋՈԹՏՆՁՆԿ ԿՊՇՍՈՋ ՋԽՄԽԻՌԽԼԾՇՊ ՌՀԽ ԼԽԾՁՆՁՌՁՎԽ ՋՌՍԼՑ ՀԹԼ ՈՊԽԽՐՈՇՋՍՊԽ

ԾԽԻՍՆԼՁՌՑ ՇԾ ՌՇ  ԽԿԿՋԾԽՅԹՄԽԼԹՑ ԿՍՁԼԹՆԻԽ ՊԽԻՇՅՅԽՆԼՋ ԹՌ ՄԽԹՋՌ  ԽԿԿՋԾԽՅԹՄԽԼԹՑԥՇՏԽՎԽՊ

ԽԹԻՀ ՇԾ ՌՀԽՋԽ ԿՊՇՍՈՋ ԼՁԼ ՋՈԹՏՆ ԹՌ ՄԽԹՋՌ ՇՆԽ ՌՁՅԽ ԽՎԽՊՑ  ԼԹՑՋԣՁՆԹՄՄՑ ՌՀԽ ՍՆՁՇՆՁՒԽԼ ԹՅՅՇՆՁԹ ԹՆԼ

ՊԽՋՁԼՍԹՄԻՀՄՇՊՁՆԽԻՇՆԻԽՆՌՊԹՌՁՇՆՋՁՆՌՀԽԼՁՄՍՌՁՇՆՏԹՌԽՊՏԽՊԽՆՇՌՋՈԽԻՁԾՁԽԼԞՄՄՇՌՀԽՊՈԽՊԾՇՊՅԹՆԻԽԹՆԼՎԹՄՁԼՁՌՑ

ԻՊՁՌԽՊՁԹՋՈԽԻՁԾՁԽԼՁՆԬԭԭԱ԰ՏԽՊԽՅԽՌ

ԣԯԽՎՁԽՏԽՊ0ՋԠՇՅՅԽՆՌՋ

ԱՀԽՊԽՎՁԽՏԽՊ0ՋՊԽՋՍՄՌՋՏԽՊԽՁՆԹԿՊԽԽՅԽՆՌՏՁՌՀՌՀՇՋԽՇԾՌՀԽՋՌՍԼՑԹՍՌՀՇՊ

ԱՀԽՁՆՄՁԾԽՈՇՊՌՁՇՆՇԾՌՀԽԼԽԾՁՆՁՌՁՎԽՌՇՐՁԻՁՌՑՌԽՋՌՏԹՋԻՇՆԼՍԻՌԽԼԾՊՇՅԣԽԺՊՍԹՊՑՌՇԪԹՊԻՀ

Page 102 of 149
ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ

ԭԹԿԽՇԾ

ԦԦԦԯԢԣԢԯԢԫԠԢ԰

ԞՆՃՄԽՑԤԱԨԪԧԽՆՋԽՆԪԡԨԹՀՄԧԧԨՇՊՌԽԹՆԼԢԞԪԹՃՑՆԽՆԡԽՋԻՊՁՈՌՁՇՆԹՆԼԽՎԹՄՍԹՌՁՇՆՇԾ

ԹՋՀՇՊՌՌԽՊՅՊԽՈՊՇԼՍԻՌՁՇՆՌԽՋՌՏՁՌՀՌՀԽԾԹՌՀԽԹԼՅՁՆՆՇՏ
ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋԢՆՎՁՊՇՆԱՇՐՁԻՇՄ

ԠՀԽՅ

ԠԹՊՄՁՋՄԽԧԠԱՀԽՌՇՐՁԻՁՌՑՇԾԠԹՊԺԹՊՑՄ
ԟԞԶԩԢԱԬԫՌՇՊԹՁՆԺՇՏՌՊՇՍՌԽԹՊՄՑՄՁԾԽՋՌԹԿԽՋ

ԟԹՑԽՊԠՊՇՈ԰ԻՁԽՆԻԽՍՆՈՍԺՄՁՋՀԽԼՊԽՈՇՊՌՋՌՍԼՑՆՍՅԺԽՊ

ԪՇՍՆՌԡԦԹՆԼԴԞԟՊՍՆԿՋԞՋՁՅՈՄՁԾՁԽԼԼՇՋՁՆԿԹՈՈԹՊԹՌՍՋԾՇՊԾՁՋՀՌՇՐՁԻՇՄՇԿՁԻԹՄՋՌՍԼՁԽՋԴԹՌԽՊ

ԯԽՋԽԹՊԻՀ԰ՈՊԹԿՍԽԧԟԪԽԹՋՍՊԽՅԽՆՌՇԾՈՇՄՄՍՌԹՆՌՌՇՐՁԻՁՌՑՌՇԾՁՋՀԟՁՇԹՋՋԹՑ

ՅԽՌՀՇԼՋԾՇՊԹԻՍՌԽՌՇՐՁԻՁՌՑԴԹՌԽՊԯԽՋԽԹՊԻՀ

Page 103 of 149
CETIS Summary Report Report Date: 22 Feb-13 12:56 (p 1 of 5)

Test Code: 056801 48669601 | 19-5522-1595

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Smithers Viscient

Batch ID: 03-8602-7723

Start Date: 10 Feb-12

Ending Date: 02 Mar-12

Test Type: EDSP FSTRA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Well Water

Species: Pimephales promelas Brine: Not Applicable

Source: Lab In-House Culture

Analyst:

Age: 21 w

Sample ID: 02-8887-2430

Sample Date: 10 Feb-12

Receive Date: 02 Mar-12

Code: 056801

Sample Age: NA

Source: Bayer CropScience AG

Station:

Client: CDM Smith

Material: Carbaryl Project: Insecticide

flow-through conditions 056801 48669601

056801 48669601

Batch Note:

Sample Note:

Comparison Summary

Analysis ID Endpoint NOEL LOEL TOEL PMSD TU Method

17-7492-0444 Fecundity 0.82 >0.82 NA 62.2% Dunnett Multiple Comparison Test

19-7179-0409 FemaleBodyWt 0.82 >0.82 NA 25.2% Dunnett Multiple Comparison Test

09-9665-4766 FemaleGSI 0.82 >0.82 NA 35.5% Dunnett Multiple Comparison Test

16-0630-4230 FemaleLength 0.82 >0.82 NA 4.99% Dunnett Multiple Comparison Test

08-5523-1336 FemaleVTG 0.82 >0.82 NA 110.0% Dunnett Multiple Comparison Test

04-2459-4489 Fertility 0.82 >0.82 NA 2.1% Dunnett Multiple Comparison Test

01-9575-7095 MaleBodyWt 0.82 >0.82 NA 23.2% Dunnett Multiple Comparison Test

07-2686-9313 MaleGSI 0.82 >0.82 NA 37.3% Dunnett Multiple Comparison Test

04-1932-5220 MaleLength 0.15 0.82 0.3507 8.33% Dunnett Multiple Comparison Test

03-3505-1637 MaleMedianTubercleScore 0.82 >0.82 NA 22.5% Dunnett Multiple Comparison Test

19-6138-7031 MaleVTG 0.82 >0.82 NA 382.0% Mann-Whitney U Two-Sample Test

000-503-186-3 CETISTM v1.8.7.4 Analyst:________ QA:________

Page 104 of 149
CETIS Summary Report Report Date: 22 Feb-13 12:56 (p 2 of 5)

Test Code: 056801 48669601 | 19-5522-1595

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Smithers Viscient

C-mg ai/L Control Type Count Mean Min Max Std Err Std Dev CV%

Fecundity Summary

95% LCL 95% UCL %Effect

0 Negative Control 4 18.3 10.6 25.9 12 22 2.39 4.79 26.2% 0.0%

0.031 4 12.8 8.18 17.3 11 17 1.44 2.87 22.5% 30.1%

0.15 4 15.5 -1.08 32.1 4.1 29 5.22 10.4 67.2% 14.9%

0.82 4 13.8 11 16.5 12 16 0.854 1.71 12.4% 24.7%

C-mg ai/L Control Type Count Mean Min Max Std Err Std Dev CV%

FemaleBodyWt Summary

95% LCL 95% UCL %Effect

0 Negative Control 4 1.3 1.17 1.44 1.25 1.43 0.042 0.084 6.44% 0.0%

0.031 4 1.46 1.26 1.65 1.34 1.57 0.0615 0.123 8.45% -11.7%

0.15 4 1.56 1.08 2.04 1.29 1.99 0.151 0.302 19.4% -19.4%

0.82 4 1.34 1.21 1.47 1.25 1.44 0.0397 0.0795 5.93% -2.63%

C-mg ai/L Control Type Count Mean Min Max Std Err Std Dev CV%

FemaleGSI Summary

95% LCL 95% UCL %Effect

0 Negative Control 4 11.7 10.8 12.6 11 12.3 0.285 0.57 4.85% 0.0%

0.031 4 14 8.4 19.6 9.3 17.8 1.76 3.52 25.1% -19.3%

0.15 4 13.3 11.4 15.2 12.3 14.9 0.609 1.22 9.16% -13.3%

0.82 4 13.6 10 17.2 12.2 17 1.13 2.25 16.5% -16.0%

C-mg ai/L Control Type Count Mean Min Max Std Err Std Dev CV%

FemaleLength Summary

95% LCL 95% UCL %Effect

0 Negative Control 4 40.6 38.8 42.3 39.1 41.8 0.557 1.11 2.75% 0.0%

0.031 4 41.7 40.9 42.6 41.1 42.3 0.26 0.52 1.24% -2.9%

0.15 4 41.9 39.5 44.2 40.2 43.7 0.724 1.45 3.46% -3.14%

0.82 4 40.7 39.1 42.2 39.9 42.1 0.487 0.974 2.4% -0.25%

C-mg ai/L Control Type Count Mean Min Max Std Err Std Dev CV%

FemaleVTG Summary

95% LCL 95% UCL %Effect

0 Negative Control 4 4.16E+6 1.27E+6 7.05E+6 2.64E+6 6.46E+6 9.08E+5 1.82E+6 43.7% 0.0%

0.031 4 5.89E+6 2.94E+6 8.84E+6 3.50E+6 7.70E+6 9.26E+5 1.85E+6 31.4% -41.7%

0.15 4 4.64E+6 4.83E+5 8.80E+6 2.28E+6 8.10E+6 1.31E+6 2.61E+6 56.3% -11.6%

0.82 4 4.62E+6 -3.69E+5 9.60E+6 2.16E+6 9.21E+6 1.57E+6 3.13E+6 67.9% -11.0%

C-mg ai/L Control Type Count Mean Min Max Std Err Std Dev CV%

Fertility Summary

95% LCL 95% UCL %Effect

0 Negative Control 4 99.5 98.6 100 99 100 0.289 0.577 0.58% 0.0%

0.031 4 98.8 96.7 101 97 100 0.629 1.26 1.27% 0.75%

0.15 4 98.3 95.5 101 96 100 0.854 1.71 1.74% 1.26%

0.82 4 99 99 99 99 99 0 0 0.0% 0.5%

C-mg ai/L Control Type Count Mean Min Max Std Err Std Dev CV%

MaleBodyWt Summary

95% LCL 95% UCL %Effect

0 Negative Control 4 2.9 2.37 3.43 2.57 3.29 0.166 0.333 11.5% 0.0%

0.031 4 2.93 2.31 3.54 2.4 3.31 0.194 0.387 13.2% -1.11%

0.15 4 2.76 2.14 3.39 2.5 3.34 0.196 0.392 14.2% 4.62%

0.82 4 2.87 2.4 3.34 2.49 3.15 0.147 0.293 10.2% 0.95%

C-mg ai/L Control Type Count Mean Min Max Std Err Std Dev CV%

MaleGSI Summary

95% LCL 95% UCL %Effect

0 Negative Control 4 1.33 0.794 1.86 0.96 1.74 0.168 0.335 25.3% 0.0%

0.031 4 1.23 1.04 1.42 1.07 1.35 0.0585 0.117 9.5% 7.34%

0.15 4 1.44 0.973 1.9 1.14 1.84 0.146 0.292 20.3% -8.29%

0.82 4 1.52 1.13 1.92 1.32 1.84 0.124 0.248 16.3% -14.7%

000-503-186-3 CETISTM v1.8.7.4 Analyst:________ QA:________

Page 105 of 149
CETIS Summary Report Report Date: 22 Feb-13 12:56 (p 3 of 5)

Test Code: 056801 48669601 | 19-5522-1595

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Smithers Viscient

C-mg ai/L Control Type Count Mean Min Max Std Err Std Dev CV%

MaleLength Summary

95% LCL 95% UCL %Effect

0 Negative Control 4 49.8 46.2 53.4 47.6 52.7 1.14 2.29 4.59% 0.0%

0.031 4 50.9 46.8 55.1 47.1 52.9 1.3 2.61 5.12% -2.26%

0.15 4 50.3 47 53.6 48.2 53.1 1.04 2.08 4.12% -1.05%

0.82 4 54.2 51.5 56.9 52.3 55.7 0.835 1.67 3.08% -8.84%

C-mg ai/L Control Type Count Mean Min Max Std Err Std Dev CV%

MaleMedianTubercleScore Summary

95% LCL 95% UCL %Effect

0 Negative Control 4 34 28.6 39.4 32 39 1.68 3.37 9.9% 0.0%

0.031 4 35 30.3 39.7 31 38 1.47 2.94 8.41% -2.94%

0.15 4 30.8 20.6 40.9 22 36 3.2 6.4 20.8% 9.56%

0.82 4 29.3 26 32.5 27 31 1.03 2.06 7.05% 14.0%

C-mg ai/L Control Type Count Mean Min Max Std Err Std Dev CV%

MaleVTG Summary

95% LCL 95% UCL %Effect

0 Negative Control 4 2.89E+3 -2.28E+3 8.05E+3 9.80E+1 6.23E+3 1.62E+3 3.24E+3 112.0% 0.0%

0.031 4 7.33E+2 -6.99E+1 1.54E+3 1.57E+2 1.36E+3 2.52E+2 5.05E+2 68.8% 74.6%

0.15 4 1.03E+4 -9.33E+3 2.98E+4 7.84E+2 2.67E+4 6.15E+3 1.23E+4 120.0% -255.0%

0.82 4 1.24E+3 2.51E+2 2.23E+3 3.44E+2 1.74E+3 3.12E+2 6.23E+2 50.2% 57.0%

000-503-186-3 CETISTM v1.8.7.4 Analyst:________ QA:________

Page 106 of 149
CETIS Summary Report Report Date: 22 Feb-13 12:56 (p 4 of 5)

Test Code: 056801 48669601 | 19-5522-1595

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Smithers Viscient

C-mg ai/L Control Type

Fecundity Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 22 22 17 12

0.031 12 17 11 11

0.15 17 29 4.1 12

0.82 13 16 14 12

C-mg ai/L Control Type

FemaleBodyWt Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 1.29 1.43 1.25 1.26

0.031 1.55 1.36 1.34 1.57

0.15 1.47 1.49 1.99 1.29

0.82 1.35 1.31 1.44 1.25

C-mg ai/L Control Type

FemaleGSI Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 12.3 11.9 11.8 11

0.031 14.8 9.3 14.1 17.8

0.15 12.4 14.9 12.3 13.6

0.82 12.4 17 12.2 12.9

C-mg ai/L Control Type

FemaleLength Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 40.6 41.8 39.1 40.8

0.031 42 42.3 41.1 41.6

0.15 41.5 42 43.7 40.2

0.82 40.4 39.9 42.1 40.3

C-mg ai/L Control Type

FemaleVTG Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 2.64E+6 4.76E+6 2.77E+6 6.46E+6

0.031 6.93E+6 3.50E+6 5.43E+6 7.70E+6

0.15 5.17E+6 8.10E+6 2.28E+6 3.01E+6

0.82 3.50E+6 3.59E+6 9.21E+6 2.16E+6

C-mg ai/L Control Type

Fertility Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 100 99 99 100

0.031 97 99 99 100

0.15 100 96 98 99

0.82 99 99 99 99

C-mg ai/L Control Type

MaleBodyWt Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 2.57 3.05 2.68 3.29

0.031 2.91 2.4 3.31 3.1

0.15 2.57 2.5 3.34 2.63

0.82 2.49 3.15 2.79 3.05

C-mg ai/L Control Type

MaleGSI Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 1.74 1.43 1.18 0.96

0.031 1.07 1.26 1.24 1.35

0.15 1.84 1.14 1.39 1.38

0.82 1.33 1.6 1.84 1.32

000-503-186-3 CETISTM v1.8.7.4 Analyst:________ QA:________

Page 107 of 149
CETIS Summary Report Report Date: 22 Feb-13 12:56 (p 5 of 5)

Test Code: 056801 48669601 | 19-5522-1595

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Smithers Viscient

C-mg ai/L Control Type

MaleLength Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 47.6 48.4 50.5 52.7

0.031 52.1 47.1 52.9 51.6

0.15 49.5 50.5 53.1 48.2

0.82 52.3 55.5 53.3 55.7

C-mg ai/L Control Type

MaleMedianTubercleScore Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 32 32 39 33

0.031 31 36 35 38

0.15 30 22 36 35

0.82 31 27 31 28

C-mg ai/L Control Type

MaleVTG Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 6.23E+3 5.10E+3 9.80E+1 1.13E+2

0.031 1.57E+2 8.46E+2 1.36E+3 5.70E+2

0.15 7.97E+2 1.28E+4 2.67E+4 7.84E+2

0.82 3.44E+2 1.57E+3 1.32E+3 1.74E+3

000-503-186-3 CETISTM v1.8.7.4 Analyst:________ QA:________

Page 108 of 149
CETIS Summary Report Report Date: 22 Feb-13 13:03 (p 1 of 1)

Test Code: 056801 48669601 | 19-5522-1595

OPPTS 890.1350 EDSP Fish Short-Term Reproduction Assay (FSTRA) Smithers Viscient

Batch ID: 03-8602-7723

Start Date: 10 Feb-12

Ending Date: 02 Mar-12

Test Type: EDSP FSTRA Tier 1

Duration: 21d 0h

Protocol: OCSPP 890.1350 Tier I FSTRA Diluent: Well Water

Species: Pimephales promelas Brine: Not Applicable

Source: Lab In-House Culture

Analyst:

Age: 21 w

Sample ID: 02-8887-2430

Sample Date: 10 Feb-12

Receive Date: 02 Mar-12

Code: 056801

Sample Age: NA

Source: Bayer CropScience AG

Station:

Client: CDM Smith

Material: Carbaryl Project: Insecticide

flow-through conditions 056801 48669601

056801 48669601

Batch Note:

Sample Note:

Comparison Summary

Analysis ID Endpoint NOEL LOEL TOEL PMSD TU Method

17-3444-4617 MaleMedianTubercleScore 0.15 0.82 0.3507 NA Jonckheere-Terpstra Step-Down Test

C-mg ai/L Control Type Count Mean Min Max Std Err Std Dev CV%

MaleMedianTubercleScore Summary

95% LCL 95% UCL %Effect

0 Negative Control 4 34 28.6 39.4 32 39 1.68 3.37 9.9% 0.0%

0.031 4 35 30.3 39.7 31 38 1.47 2.94 8.41% -2.94%

0.15 4 30.8 20.6 40.9 22 36 3.2 6.4 20.8% 9.56%

0.82 4 29.3 26 32.5 27 31 1.03 2.06 7.05% 14.0%

C-mg ai/L Control Type

MaleMedianTubercleScore Detail

Rep 1 Rep 2 Rep 3 Rep 4

0 Negative Control 32 32 39 33

0.031 31 36 35 38

0.15 30 22 36 35

0.82 31 27 31 28

000-503-186-3 CETISTM v1.8.7.4 Analyst:________ QA:________

Page 109 of 149
DATA EVALUATION RECORD

CARBARYL

Study Type: OCSPP 890.1400, In vivo Hershberger Assay

EPA Contract No. EP10H001452 

Task Assignment No. 3-07-2012 (MRID 47987701) 

Prepared for

Health Effects Division

Office of Pesticide Programs

U.S. Environmental Protection Agency 

2777 South Crystal Drive 

Arlington, VA 22202 

Prepared by

CSS-Dynamac Corporation

1910 Sedwick Road, 

Building 100, Suite B 

Durham, NC 27713 

Primary Reviewer: Signature:

Kelly Luck, M.S. Date: 7/31/2012

Secondary Reviewer: Signature:

David A. McEwen, B.S. Date: 8/8/2012

Program Manager: Signature:

Jack D. Early, M.S. Date: 8/10/2012

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 8/10/2012

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

signing by CSS-Dynamac Corporation personnel. 

Page 110 of 149
The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is 

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

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

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

assay to identify potential endocrine bioactivity with complementary endpoints within the 

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

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

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

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

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

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

Document).

Page 111 of 149
CARBARYL I 056801 

Primary Reviewer: =Kh==in=-=Oc..::o'-'--"-M-=-·=D:....:.. _______ _ 

Health Effects Division 

Secondary Reviewer: Greg Akerman, Ph.D. 

Health Effects Division 

In vivo Hershberger Assay (2003) I Page 1 of 13 

OCSPP 890.1400/ OECD 441 

Signature: _QN:....., 1......,: ........ • _· ,-----

Date: lil. 9 / 2. c 1 SSignature: .4 /2. -

Date: ·~ fuJer-: 

Template version 10/2011 

DATA EVALUATION RECORD 

STUDY TYPE: In Vivo Hershberger Assay (Rat); OCSPP 890.1400; OECD 441 

PC CODE: 05680 l DP BARCODE: D404352 

TXR#: 0056268 CAS#: 63-25-2 

TEST MATERIAL (PURITY): Carbary! (99.1% a.i.) 

SYNONYMS: 1-naphthalenyl N-methylcarbamate 

CIT A TI ON: Kennel, P. (2003). Hershberger assay to assess the potential antiandrogenic 

activity of carbaryl in rat. Bayer CropScience, Sophia Antipolis Cedex, France. 

Laboratory Report No.: SA 02261, January 15, 2003. MRID 47987701. 

Unpublished. 

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

TEST ORDER#: EDSP-056801-57 

EXECUTIVE SUMMARY: In a Hershberger Assay (MRID 47987701) screening for potential 

anti-androgenic activity, carbaryl (99. l % a.i., Batch #21114920) in 0.5% aqueous methyl 

cellulose 400 was administered daily via oral gavage to groups of six 8- to 9-week old, castrated 

male Sprague Dawley rats at dose levels of 0 (vehicle), 5, 15, or 50 mg/kg/day in conjunction 

with a daily dose of testosterone propionate (TP) at 0.3 mg/kg/day by subcutaneous (s.c.) 

injection. The negative control group consisted of six castrated rats dosed daily for 10 days with 

TP at 0.3 mg/kg/day with the aqueous solution of methylcellulose 400 at 0.5% daily by gavage. 

The anti-androgenic positive control group consisted of six castrated rats exposed to 0.3 

mg/kg/day TP (s.c. injection) plus 25 mg/kg/day flutamide (FT, via oral gavage). A final group 

of six castrated rats was dosed with control vehicles (methylcellulose and corn oil) via gavage 

and by the subcutaneous route to serve as a control check for androgenic activity of TP. 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 and liver were 

collected and weighed. 

All animals survived until scheduled termination. In the 50 mg/kg/day + TP group, treatmentrelated clinical signs consisted of increased salivation, reduced motor activity, tremors and 

piloerection were found in all animals throughout the study; chewing was observed in 4/6 

animals between Days 8 and 10 and soiled nose was observed in 1/6 animals between Days 3 

and 6. In the 15 mg/kg/day+ TP group, treatment-related clinical signs were noted between 

Days 1 and 4 and consisted of reduced motor activity and/or tremors in 5/6 animals, associated 

Page 112 of 149
In vivo Hershberger Assay (2003) / Page 2 of 13

CARBARYL / 056801 OCSPP 890.1400/ OECD 441

with increased salivation and piloerection in one animal. No clinical signs of toxicity were noted 

in animals in the 5 mg/kg/day+TP group, or any of the control groups. 

In the 50 mg/kg/day +TP group, there was a 12% decrease in final body weights, a 49% decrease 

in overall body weight gains, and a 20-32% decrease in food consumption (each p<0.01) 

compared to the TP control; body weights in this group were also decreased (not statistically 

analyzed) by 11% and 13% on Days 4 and 7, respectively. In the 15 mg/kg/day +TP group, 

decreases (p<0.05) of 6%, 20%, and 15-18% were observed in the final body weights, overall 

body weight gains, and food consumption, respectively. Final body weights, body weights gains, 

and food consumption in the 5 mg/kg/day +TP group were comparable to the TP control. Body 

weight gains in the positive control (FT + TP) group were decreased (p<0.05) by 13%, but final 

body weights and food consumption in this group were comparable to the TP control. There 

were no treatment-related effects on the weights of seminal vesicles, levator ani-bulbocavernosus 

muscle complex (LABC), Cowper’s glands, ventral prostate or glans penis for any carbaryl 

treatment group. Liver weights were decreased (p<0.05) by 12% at 15 mg/kg/day and by 18% at 

50 mg/kg/day compared to the TP control. Liver weights in the 5 m/kg/day and positive 

(TP+FT) control groups were comparable to the TP control. Animals in the positive (TP+FT) 

control group had the expected decreases (p<0.01) in weights of accessory sex organs as follows: 

93% in seminal vesicles; 94% in ventral prostate; 75% in LABC; 86% in Cowper’s gland; and 

38% in glans penis. 

The androgen agonist activity of carbaryl was not evaluated in this study. However, the 

androgenic activity of TP was validated. In the TP control group, final body weight, overall 

body weight gains, and food consumption were increased (p<0.05) by 9%, 57%, and 18-23%, 

respectively, compared to the vehicle control group. Animals in the TP control group had the 

expected increases (p<0.05) in weights of accessory sex organ weights as follows: 796% in 

seminal vesicles; 832% in ventral prostate; 252% in LABC; 600% in Cowper’s gland; and 14% 

in glans penis. In addition, liver weight was increased (p<0.05) by 14%. 

Percent CVs for the vehicle control, TP control, and 50 mg/kg/day +TP groups were compared to 

the performance criteria in the Guidelines. All %CV values were less than the maximum 

permissible values, with the exception of ventral prostate (CV = 57%; maximum permissible CV 

= 45%) and the glans penis (CV = 23%; maximum permissible CV = 22%) in the vehicle control 

group. 

The dosing for this study (high dose of 50 mg/kg/day) was considered adequate based on adverse 

clinical signs (increased salivation, reduced motor activity, tremors and piloerection) and the 

12% decrease final body weights observed at this dose level. 

No statistically significant decreases were seen in the weights of the five androgen sensitive 

tissues. Carbaryl was negative for anti-androgenicity in the Hershberger assay. 

The assay satisfies the EDSP Tier 1 Test Order requirements for a Hershberger assay (OCSPP 

890.1400). Only the antiandrogenic part of the Hershberger assay was conducted. 

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

Assurance statements were provided. 

Page 113 of 149
In vivo Hershberger Assay (2003) / Page 3 of 13

CARBARYL / 056801 OCSPP 890.1400/ OECD 441

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Bayer CropScience

Location: Sophia Antipolis Cedex, France

Study Director: P. Kennel

Other Personnel: J.P. Kocwin (Laboratory Animal Resources), B. Bonnafous (Toxicology 

Supervisor), G. Gaugey-Couette (Responsible Technician), A. Schlexer 

(Responsible Technician for Formulation), J.P. Tassel (Senior Chemist), C. 

Pallen (Statistician), M. Vagner (Report Unit Assistant)

Study Period: August 28, 2002 - January 15, 2003

2. Test Substance: Carbaryl

Description: Technical; white powder

Source: Aventis CropScience (Frankfurt am Main, Germany)

Batch #: 21114920 (expiration date 1/18/2003)

Purity: 99.1%

Stability: Stable for 28 days in aqueous methylcellulose under conditions similar to the 

storage conditions used in the current study

CAS #: 63-25-2

Structure:

3. Reference Androgen: Testosterone propionate (TP)

Supplier: Sigma Aldrich (St Quentin-Fallavier, France)

Batch #: 77H0387 (no expiration date provided)

Purity: >99%%

CAS #: 57-85-2

4. Reference Anti-androgen: Flutamide (FT)

Supplier: Sigma Aldrich (St Quentin-Fallavier, France)

Lot/Batch #: 41K1120 (no expiration date provided)

Purity: >99%%

CAS #: 13311-84-7

5. Solvent/Vehicle Control 

(carbaryl and FT):

Aqueous solution of methylcellulose 400

Supplier: Not reported

Lot/Batch #: Not reported

Rationale (if other than water): Not provided

Final concentration: 0.5%

Solvent/Vehicle Control 

(TP):

Corn oil

Supplier: Not reported

Lot/Batch #: Not reported

Rationale (if other than water): Not applicable

Final concentration: Not applicable

O

O

N

H

CH3

Page 114 of 149
In vivo Hershberger Assay (2003) / Page 4 of 13

CARBARYL / 056801 OCSPP 890.1400/ OECD 441

6. Test Animals:

Species:

Rat (males only)

Strain: Sprague Dawley [OFA-SD (IOPS Caw)]

Age/weight at dose initiation:

8 to 9 weeks (exact age not reported)/ 255-287 g 

Source: Iffa-Credo (St Germain-sur-L’Arbresle, France)

Housing: Individually housed in suspended stainless steel wire mesh cages with no 

bedding material

Diet: Certified rodent pelleted and irradiated diet A04C-10 (Usine d’Alimentation 

Rationnelle, Villenoisson-sur-Orge, France), ad libitum

Phytoestrogen content of diet was not reported

Water: Filtered and softened tap water, ad libitum

Environmental conditions: Temperature:

Humidity:

Air changes:

Photoperiod:

20-24 oC

40-70% 

10-15/hr (average; not monitored)

12 hrs light/12 hrs dark

Acclimation period: 9-10 days at facility

B. STUDY DESIGN

1. In-life Dates: Start: October 4, 2002 End: October 15, 2002 

2. Study Design: In a Hershberger Assay conducted to screen only for potential 

anti-androgenic activity, the test substance was administered daily via oral gavage to 

castrated male rats in conjunction with a daily dose of TP (0.3 mg/kg/day) by s.c. injection. 

A positive control group received FT plus TP. Anti-androgenic activity is indicated by a 

statistically significant decrease in two or more target organ weights of the treated groups 

(test substance + TP) compared to the TP only control group. The animals were dosed for 

10 consecutive days and necropsied approximately 24 hours after the final dose 

administration for organ weight measurements. 

3. Study Schedule: Male rats were castrated by the supplier at 6 weeks of age. The rats were 

acclimated to laboratory conditions for 9-10 days and were 8-9 weeks old at the initiation of 

dosing; exact age of animals was not reported. Rats were dosed for 10 consecutive days, 

and then euthanized 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 an 

automatic randomization procedure which selected animals from the middle of the weight 

range of the available animals. The body weight of each animal was within 20% of the 

overall mean on the days of animal assignment. 

Page 115 of 149
In vivo Hershberger Assay (2003) / Page 5 of 13

CARBARYL / 056801 OCSPP 890.1400/ OECD 441

TABLE 1. Study Designa

Test group Dose (mg/kg/day) # of Males

Anti-Androgen Assay

Vehicle controlb 0 6

Testosterone propionate (TP) control 0 + 0.3 6

Low (Carbaryl + TP) 5 + 0.3 6

Mid (Carbaryl + TP) 15 + 0.3 6

High (Carbaryl + TP) 50 + 0.3 6

Flutamide + TP, positive control 25 + 0.3 6

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

b Rats in this group were dosed with 0.5% methylcellulose 400 by oral gavage and corn oil by s.c. injection.

5. Dose Selection Rationale: The dose levels were reportedly selected based on studies from 

the toxicology database of carbaryl and a dose range-finding study;1 no results of the rangefinding study were reported. The dose levels for TP and FT were selected based on data 

from the literature and from in-house data based on the first step of the Hershberger OECD 

standardization exercise. 

6. (a) Dose Preparation: Dose formulations were prepared once for carbaryl and FT and 

daily for TP by mixing appropriate amounts of test substance with 0.5% aqueous 

methylcellulose (carbaryl and FT) or corn oil (TP). Stability was tested previously2

 under 

conditions similar to the storage conditions used in the current study. Homogeneity was 

tested in the current study in all three carbaryl formulations by collecting samples from the 

surface and bottom of the mixture; the results of the homogeneity analyses were used to 

demonstrate achieved concentration.

(b) Dose Analysis: 

Results

Homogeneity (%CV): 0.5-2.5% 

Stability: Reportedly stable for 28 days in aqueous methylcellulose at 0.5 and 200 g/L 

under conditions similar to the storage conditions used in the current study 

Concentration (% of nominal): 94-100% 

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. 

7. Dosage Administration: Carbaryl formulations were administered to the animals daily via 

oral gavage (5 mL/kg) for 10 days. TP was given via s.c. injection at 0.5 mL/kg, and FT

1

 P. Kennel (2002): Carbaryl: Range-finding assay in the rat by gavage (SA 02269), Bayer CropScience, Sophia 

Antipolis, France.

2

 M. Repetto-Larsay (1997): Carbaryl: Range-finding study for developmental toxicology in the rat by gavage (SA 

97479), Rh6ne-Poulenc, Sophia Antipolis, France.

 

Page 116 of 149
In vivo Hershberger Assay (2003) / Page 6 of 13

CARBARYL / 056801 OCSPP 890.1400/ OECD 441

was administered via oral gavage at 5 mL/kg. Dose volumes were adjusted daily based on 

the concurrent body weight measurement. 

It was noted that due to a technician error, three of the six animals in the 50 mg/kg/day 

group were not dosed on Day 10. 

8. Statistics: Statistical analyses were conducted using SAS programs (Version 8.2). 

Statistical comparisons were performed at the 5% and 1% levels. The following parameters

were statistically analyzed: food consumption, body weight parameters, sex accessory 

tissue weights, and liver weight. 

For comparing carbaryl +TP treatment groups with the TP control, the Bartlett test (twosided) was performed to compare group variances. When the Bartlett test was not significant 

(p=0.05), means were compared using the analysis of variance (ANOVA). When the 

ANOVA was not significant (p=0.05), group means were considered to be homogeneous. 

When the ANOVA was significant (p=0.05), group means were compared using the Dunnett 

test (two-sided). When the Bartlett test was significant, means were compared using the 

non-parametric analysis of variance of Kruskal-Wallis test. When the Kruskal-Wallis test 

was not significant, (p=0.05), group means were considered to be homogeneous. When the 

Kruskal-Wallis test was significant, group means were compared using the Mann-Whitney 

test (two-sided). 

For comparison of the positive control with the TP control, the F test (two-sided) was 

performed to compare group variances. When the F test was not significant (p=0.05), means 

were compared using the t-test (two-sided). When the F test was significant, means were 

compared using the modified t-test (two-sided). 

The statistical analyses were considered adequate. 

C. METHODS

1. Clinical Examinations: Cage-side checks for mortality and moribundity were conducted 

twice daily (once daily on weekends). Clinical observations were recorded at least once 

daily throughout the study, approximately 0.5-1 hours after dosing. 

2. Body Weight: Animals were weighed at randomization, daily throughout the dosing 

period, and prior to necropsy. 

3. Food Consumption: Food consumption was measured for each animal by subtracting the 

amount of food remaining in the cage from the amount supplied. Food consumption was 

records for two intervals, Days 1-6 and Days 7-11, and values were reported as group mean 

daily food consumption (g/animal/day). 

4. Serum Hormone Measurements (Optional): Serum hormone measurements were not 

conducted. 

5. Dissection and Measurement of Tissue and Organ Weights: Twenty-four hours after the 

final administration of the test substance, all surviving animals were anesthetized, 

Page 117 of 149
In vivo Hershberger Assay (2003) / Page 7 of 13

CARBARYL / 056801 OCSPP 890.1400/ OECD 441

euthanized by exsanguination, and subjected to a gross necropsy. The five mandatory 

androgen-dependent organs (ventral prostate, seminal vesicles with coagulating glands, 

LABC, Cowper’s gland, and glans penis) were excised, trimmed free of adhering fat, and 

weighed to the nearest milligram. Additionally, the liver was collected and weighed.

II. RESULTS

A. OBSERVATIONS

1. Mortality: All animals survived until scheduled termination. 

2. Clinical Signs of Toxicity: The incidence of clinical observations in rats is reported in 

Table 2. At 50 mg/kg/day, treatment-related clinical signs consisted of increased salivation, 

reduced motor activity, tremors, and piloerection in all animals throughout the study. In 

addition, chewing was observed in 4/6 animals between Days 8 and 10 and soiled nose was 

observed in 1/6 animals between Days 3 and 6. At 15 mg/kg/day, treatment-related clinical 

signs were noted between Days 1 and 4 and consisted of reduced motor activity and/or 

tremors in 5/6 animals, associated with increased salivation and piloerection in one animal. 

At 5 mg/kg/day, no clinical signs of toxicity were noted. No clinical signs were observed in 

control animals.

TABLE 2. Incidence of Clinical Observations in the Anti-Androgen Assaya

Observation

Dose (mg/kg/day)

TP Control

(0.3)

Positive Control 

FT+ TP 

(25 + 0.3)

Carbaryl + TP 

(5 + 0.3)

Carbaryl + TP 

(15 + 0.3)

Carbaryl + TP 

(50 + 0.3)

# 

Observed

# 

Examined

# 

Observed

# 

Examined

# 

Observed

# 

Examined

# 

Observed

# 

Examined

# 

Observed

# 

Examined

Piloerection 0 6 0 6 0 6 1 6 6 6

Reduced 

motor activity 0 6 0 6 0 6 4 6 6 6 

Tremors 0 6 0 6 0 6 4 6 6 6

Soiled nose 0 6 0 6 0 6 0 6 1 6

Increased 

salivation 0 6 0 6 0 6 1 6 6 6 

Chewing 0 6 0 6 0 6 0 6 4 6

a Data were obtained from Table 1 on page 31 of the study report. Results for the group in which rats were dosed 

with vehicle alone (0.5% methylcellulose + corn oil) are not included in this table; no clinical observations were 

noted.

Page 118 of 149
In vivo Hershberger Assay (2003) / Page 8 of 13

CARBARYL / 056801 OCSPP 890.1400/ OECD 441

B. BODY WEIGHT AND WEIGHT GAIN: Selected body weight and body weight gain 

data for the vehicle control and TP control groups are presented in Table 3, for the purposes 

of validating androgen agonist activity. Final body weights and overall body weight gains 

were increased (p<0.01) in the TP group (↑9% and 57%, respectively) compared to the 

vehicle control. 

TABLE 3. Selected Group Mean Body Weights and Cumulative Body Weight Gains (g) for Validation of 

Androgen Agonist Activitya

Study Day #

Dose (mg/kg/day)

Vehicle Control TP Control

(0.3)

N Mean SD N Mean SD

1 6 275 11 6 275 11

4 6 292 12 6 302 10

7 6 308 16 6 326 12

11 6 326.8 4.5b 6 356.2** (↑9) 5.4b

Body Weight Gain 

(Days 1-11) 6 51.8 1.7b 6 81.2** (↑57) 2.7b 

a Data were obtained from Tables 2A, 3A, and 5A on pages 33, 37, and 41 of the study report. Percent 

differences from vehicle controls, calculated by the reviewers, are included in parentheses. Statistical analyses 

were only conducted for final body weight (Day 11) and body weight gains (Days 1-11). 

b Standard error

N Number of animals in the group

SD Standard Deviation

** Significantly different from controls at p<0.01

Selected body weight and body weight gain data for the anti-androgen assay are presented in 

Table 4. In the 50 mg/kg/day +TP group, final body weights and overall body weight gains 

were decreased (p<0.01) compared to the TP control (↓12% and 49%, respectively); body 

weights in this group were also decreased (not statistically analyzed) by 11% and 13% on 

Days 4 and 7, respectively. In the 15 mg/kg/day +TP group, final body weights and overall 

body weight gains were decreased (p<0.05) by 6% and 20% respectively. It was reported 

that mean body weight gains in the 50 and 15 mg/kg/day groups were significantly 

decreased in the interval Days 1-6 (p<0.05; ↓81% and ↓33%, respectively) but were 

comparable to the TP control in the interval Days 6-11 (statistical analyses not included in 

the report). Final body weights and body weights gains in the 5 mg/kg/day +TP group were 

comparable to the TP control. Body weight gains in the positive (FT+TP) control group 

were decreased (p<0.05) by 13%, but the final body weights in this group were comparable 

to the TP control. 

Page 119 of 149
In vivo Hershberger Assay (2003) / Page 9 of 13

CARBARYL / 056801 OCSPP 890.1400/ OECD 441

TABLE 4. Selected Group Mean Body Weights and Cumulative Body Weight Gains (g) in the Anti-Androgen 

Assaya

Study Day #

Dose (mg/kg/day)

TP Control

(0.3)

Positive Control 

FT+ TP (25 + 0.3)

Carbaryl + TP 

(5 + 0.3)

Carbaryl + TP 

(15 + 0.3)

Carbaryl + TP 

(50 + 0.3)

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD

1 6 275 11 6 273 9 6 276 7 6 271 10 6 271 8

4 6 302 10 6 295 10 6 298 8 6 289 7 6 270

(↓11b) 10

7 6 326 12 6 316 12 6 324 8 6 309 6 6 284

(↓13b) 9 

11 6 356.2 5.4c 6 345.0 5.3c 6 355.7 6.0c 6 336.0* 

(↓6) 3.2c 6 312.3**(↓12) 3.8b 

Body Weight 

Gain (Days #-#) 6 81.2 2.7c 6 71.7* 

(↓12) 3.1c 6 79.3 5.5c 6 64.8* (↓20) 3.9c 6 41.2** (↓49) 2.4b 

a Data were obtained from Tables 2B, 2C, 3B, 3C, 5B, and 5C on pages 34, 35, 37, and 41 of the study report. Percent 

differences from negative controls are included in parentheses. Statistical analyses were only conducted for final body weight 

(Day 11) and body weight gains (Days 1-11).

b Calculated by the reviewers

c Standard error

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: Food consumption data for the anti-androgen assay are 

presented in Table 5. Food consumption was decreased (p<0.01) by 15-18% at 15 

mg/kg/day and by 20-32% at 50 mg/kg/day compared to the TP control. Food consumption 

in the 5 mg/kg/day and positive (FT+TP) control groups was comparable to the TP control. 

Data comparing the TP control group to the vehicle control group were included in the study 

report (not presented below). Compared to the vehicle control, food consumption in the TP 

control group was significantly increased (p<0.05; ↑18-23%).

TABLE 5. Food Consumption (g/animal/day) in the Anti-Androgen Assaya

Study Interval

Dose (mg/kg/day)

TP Control

(0.3)

Positive Control 

FT+ TP (25 + 0.3)

Carbaryl + TP 

(5 + 0.3)

Carbaryl + TP 

(15 + 0.3)

Carbaryl + TP 

(50 + 0.3)

N Mean SE N Mean SE N Mean SE N Mean SE N Mean SE

Days 1-6 6 32.7 0.8 6 30.8 0.8 6 30.3 0.8 6 26.9** 

(↓18) 0.5 6 22.3** (↓32) 0.8

Days 6-11 6 33.3 0.8 6 31.3 1.1 6 32.1 1.0 6 28.3** 

(↓15) 0.8 6 26.5** (↓20) 0.5

a Data were obtained from Table 4A and 4B on page 39 of the study report. Percent differences from negative control are 

included in parentheses.

N Number of animals in the group

SE Standard Error 

** Significantly different from controls at p<0.01

D. SERUM HORMONE CONCENTRATIONS (Optional): Serum hormone concentrations 

were not measured. 

Page 120 of 149
In vivo Hershberger Assay (2003) / Page 10 of 13

CARBARYL / 056801 OCSPP 890.1400/ OECD 441

E. ORGAN WEIGHTS: Accessory sex organ and liver weights for the vehicle control and 

TP control groups are presented in Table 6, for the purposes of validating androgen agonist 

activity. Compared to the vehicle control group, animals in the TP control group had 

increased (p<0.05) accessory sex organ weights as follows: 796% in seminal vesicles; 

832% in ventral prostate; 252% in LABC; 600% in Cowper’s gland; and 14% in glans 

penis. In addition, liver weight was increased (p<0.05) by 14%. 

Percent CVs for the vehicle control group were compared to the performance criteria in the 

Guidelines. The %CV values for seminal vesicle, LABC, and Cowper’s gland were less 

than the maximum permissible values; the maximum permissible values were exceeded for 

ventral prostate (CV = 57%; maximum = 45%) and glans penis (CV = 23%; maximum = 

22%). 

TABLE 6. Accessory Sex Organ and Liver Weights for Validation of from Androgen Agonist Activity in 

Sprague Dawley Ratsa

Organ

Dose (mg/kg/day)

Vehicle Control TP Control

(0.3)

N Mean SE CV (%) N Mean SE CV (%)

Seminal 

vesicles (mg) 6 41 4 27 6 366** 

(↑796) 25 17

Ventral 

prostate (mg) 6 16 4 57 6 151** 

(↑832) 11 18

LABC (mg) 6 95 4 10 6 334** 

(↑252) 18 13

Cowper’s 

glands (mg) 6 5 1 54 6 36**

(↑600) 3 20

Glans penis

(mg) 6 49 44 23 6 69**

(↑43) 2 8.1

Liver (g) 6 12.5 0.4 7.7 6 14.2*

(↑14) 0.5 14

a Data were obtained from Tables 6A and 7A on pages 43 and 45 of the study report. Percent differences from vehicle 

controls, calculated by the reviewers, are included in parentheses.

N Number of animals in the group

SE Standard Error 

CV Coefficient of Variation; calculated by the reviewers

* Significantly different from controls at p<0.05

** Significantly different from controls at p<0.01

Accessory sex organ and liver weights for the anti-androgen assay are presented in Table 7. 

There were no treatment-related effects in accessory sex organs for any carbaryl-treatment 

group compared to the TP control. Liver weights were decreased (p<0.05) at 15 mg/kg/day 

(↓12%) and 50 mg/kg/day (↓18%) compared to the TP control. Liver weights in the 5 

m/kg/day and positive control (FT + TP) groups were comparable to the TP control. 

Animals in the positive control group had decreased (p<0.01) accessory sex organ weights 

as follows: 93% in seminal vesicles; 94% in ventral prostate; 75% in LABC; 86% in 

Cowper’s gland; and 38% in glans penis. 

Page 121 of 149
In vivo Hershberger Assay (2003) / Page 11 of 13

CARBARYL / 056801 OCSPP 890.1400/ OECD 441

Percent CVs for the TP control and the 50 mg/kg/day treatment groups were compared to 

the performance criteria in the Guidelines. All %CV values were less than the maximum 

permissible values.

TABLE 7. Accessory Sex Organ and Liver Weights from Anti-Androgen Assay in Sprague Dawley Ratsa

Organ

Dose (mg/kg/day)

TP Control

(0.3)

Positive Control 

FT + TP (25 + 0.3) Carbaryl + TP (5 + 0.3) Carbaryl + TP (15 + 0.3)

Carbaryl + TP 

(50 + 0.3)

N Mean SE

CV 

(%) N Mean SE

CV 

(%) N Mean SE

CV 

(%) N Mean SE

CV 

(%) N Mean SE

CV 

(%)

Seminal 

vesicles 

(mg)

6 366 25 17 6 27** 

(↓93) 4 37 6 428 58 33 6 509 73 35 6 404 37 23

Ventral 

prostate 

(mg)

6 151 11 18 6 10** 

(↓94) 3 72 6 145 10 17 6 159 14 21 6 123 10 19

LABC (mg) 6 334 18 13 5b 84** 

(↓75) 4 12 6 367 8 5.6 6 386 14 9.2 6 340 21 15

Cowper’s 

glands (mg) 6 36 3 20 6 5** 

(↓86) 1 28 6 31 2 17 6 34 2 14 6 27 3 25

Glans penis 

(mg) 6 69 2 8 6 43** 

(↓38) 5 27 6 76 3 9.6 6 75 6 19 6 79 5 16

Liver (g) 6 14.2 0.5 7.7 6 14.8 0.5 7.6 6 14.2 0.4 7.1 5c 12.5* 

(↓12) 0.4 6.8 6 11.7** (↓18) 0.4 7.3

a Data were obtained from Tables 6B, 6C, 7B, and 7C on pages 43 and 45 of the study report. Percent differences from negative 

controls, calculated by the reviewers, are included in parentheses.

b Weight of LABC from one rat excluded at necropsy due to abnormal tissue shape.

c Weight of liver from one rat not collected due to technician error.

N Number of animals in the group

SD Standard Error 

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. MACROSCOPIC PATHOLOGY: All animals in all treatment groups achieved preputial 

separation by study termination with the exception of 5/6 rats in the vehicle control group 

and 2/6 rats in the FT + TP group. The study author noted that in the absence of preputial 

separation for some animals in the vehicle control and FT + TP groups, comparison of glans 

penis weight with data derived from these groups should be considered with caution. 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: The control for androgenic activity, TP at 0.3 

mg/kg/day, produced a significant increase in mean body weight gains, terminal body 

weight, food consumption, and sex accessory tissue weights, when compared to the vehicletreated animals. The positive control for anti-androgenic activity, FT + TP, produced a 

significant decrease in overall mean body weight gain and sex accessory tissue weights, 

when compared to the animals treated with TP alone. Carbaryl tested at 5, 15, or 50 

mg/kg/day in conjunction with TP was devoid of anti-androgenic activity in the rat 

Hershberger assay.

Page 122 of 149
In vivo Hershberger Assay (2003) / Page 12 of 13

CARBARYL / 056801 OCSPP 890.1400/ OECD 441

B. AGENCY COMMENTS: All animals survived until scheduled termination. At 50 

mg/kg/day carbaryl, treatment-related clinical signs consisted of increased salivation, 

reduced motor activity, tremors and piloerection in all animals throughout the study; 

chewing was observed in 4/6 animals between Days 8 and 10 and soiled nose was observed 

in 1/6 animal between Days 3 and 6. At 15 mg/kg/day, treatment-related clinical signs were 

noted between Days 1 and 4 and consisted of reduced motor activity and/or tremors in 5/6 

animals, associated with increased salivation and piloerection in one animal. No clinical 

signs of toxicity were noted in animals in the 5 mg/kg/day or control groups. 

In the anti-androgen assay, final body weights, overall body weight gains, and food 

consumption were decreased at 50 mg/kg/day +TP compared to the TP control; body 

weights in this group were also decreased (not statistically analyzed) on Days 4 and 7. At 

15 mg/kg/day +TP, final body weights, overall body weight gains, and food consumption 

were decreased. Final body weights, body weights gains, and food consumption in the 5 

mg/kg/day group were comparable to the TP control. Body weight gains in the positive 

control (FT + TP) group were decreased; final body weights and food consumption in this 

group were comparable to the TP control. There were no treatment-related effects on the 

weights of seminal vesicles, ventral prostate, LABC, Cowper’s glands, or glans penis for 

any carbaryl-treated group. Liver weights were decreased at 15 and 50 mg/kg/day carbaryl 

compared to the TP control. Liver weights in the 5 m/kg/day and positive control groups 

were comparable to the TP control. Animals in the positive (FT+TP) control group had the 

expected decreases in accessory sex organ weights. 

The androgen agonist activity of carbaryl was not evaluated in this study. However, the 

androgenic activity of TP was validated. In the TP control group, final body weight, overall 

body weight gains, and food consumption were increased compared to the vehicle only 

control group. In addition, animals in the TP control group had the expected increases in 

accessory sex organ weights; mean liver weights were also significantly increased. 

Percent CVs for the vehicle control, TP control, and 50 mg/kg/day +TP groups were 

compared to the performance criteria in the Guidelines. All %CV values were less than the 

maximum permissible values, with the exception of ventral prostate (CV = 57%; maximum 

permissible CV = 45%) and glans penis in the vehicle control group (CV = 23%; maximum 

permissible CV = 22%). 

The dosing for this study (high dose of 50 mg/kg/day) was considered adequate based on 

adverse clinical signs (increased salivation, reduced motor activity, tremors and 

piloerection) and the 12% decrease final body weights observed at this dose level. 

No statistically significant decreases in tissue weights were seen in two or more of the five 

androgen sensitive tissues. Carbaryl was negative for 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: 

• Three of the 6 animals in the 50 mg/kg/day were inadvertently not dosed on Day 10. 

Page 123 of 149
In vivo Hershberger Assay (2003) / Page 13 of 13

CARBARYL / 056801 OCSPP 890.1400/ OECD 441

• The weights of accessory sex organs were reported to the nearest milligram; the 

guidelines specify that sex organ weights should be recorded to the nearest 0.1 

milligram.

Page 124 of 149
DATA EVALUATION RECORD

CARBARYL

Study Type: OCSPP 890.1550, Steroidogenesis Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 2-20-2012 (MRID 48615205) 

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: 3/02/2012

Secondary Reviewer Signature:

Scott D. Studenberg, Ph.D., D.A.B.T. Date: 3/06/2012

Program Manager: Signature:

Jack D. Early, M.S. Date: 3/06/2012

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 3/06/2012

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

signing by CSS-Dynamac Corporation personnel. 

Page 125 of 149
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 126 of 149
CARBARYL I 056801 

Primary Reviewer: _Kh_in_O~o~M_._D_. _______ _ 

Health Effects Division 

Secondary Reviewer: John Liccione, Ph.D. 

Health Effects Division 

Steroidogenesis Assay (2011) I Page I of 11 

OCSPP 890.1550/ OECD None 

Signature:-_Gt __ ~:-" __ 

0

·....,....----

Date: 6 / Z-°I J z..016.-

Signature: ~~;t=.:;===:;::::;;:z:::_ 

Date: ---1.~-=-~,__,_ __ 

DATA EVALUATION RECORD 

STUDY TYPE: Steroidogenesis Assay (H295R Cells); OCSPP 890.1550 

PC CODE: 056801 DP BARCODE: 0397990 

TXR#: 0056268 CAS No.: 63-25-2 

TEST MATERIAL (PURITY): Carbaryl (100%) 

SYNONYMS: 1-naphthalenyl N-methylcarbamate 

CITATION: Tinwell, H. (2011) Evaluation of carbaryl in the H295R steroidogenesis 

assay. Bayer S.A.S., Bayer CropScience, Sophia Antipolis Cedex, France. 

Laboratory Study No.: SA 11066, October 18, 2011. MRID 48615205. 

Unpublished. 

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

TEST ORDER#: EDSP-056801-57 

EXECUTIVE SUMMARY: In a steroidogenesis assay (MRID 48615205), H295R cells 

cultured in vitro in 24-well plates were incubated with carbaryl (100% purity, Lot# 

NN CAAX0025) at serial log concentrations of 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 %. 

Testosterone and estradiol levels were measured using EIA. The hormone assay interference test 

demonstrated interference with measuring estradiol using EIA kits in the presence of 10-4 and 

I 0-5 M carbaryl; therefore, these concentrations were not evaluated for estradiol in the assays. A 

Quality Control (QC) plate was run concurrently with each independent run of the 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. 

In the QC plate, the minimum basal hormone production levels (500 pg/mL for testosterone, 40 

pg/mL for estradiol) were met in both blank and solvent control (SC) wells for testosterone. 

Guideline requirements for basal hormone production, induction, and inhibition were met for 

testosterone and estradiol, except for induction of testosterone with I 0 μM forskolin in Runs I 

and 3 (1 .66 to 1.79-fold) which was below the Guideline criteria of2'.:2-fold induction. 

Evaluation of the intra- and interplate variability (%CV) for the SC in the test and QC plates 

Page 127 of 149
Steroidogenesis Assay (2011) / Page 2 of 11

CARBARYL / 056801 OCSPP 890.1550/ OECD None

indicated consistent performance of the assays, except that the between plate %CV for estradiol

(33%) in the QC plates exceeded the Guideline criteria (≤30%). 

Precipitation and cytotoxicity were not reported at any concentration tested. No effects of 

carbaryl on testosterone production were noted from 10-10 to 10-5 M with the % changes for the 

individual evaluations varying between +12% (1.12-fold) and -33% (0.67-fold). In contrast, 

marked decreases of 52-59% (0.48- to 0.41-fold) were noted in testosterone production at 10-4 M 

in each run, for an overall significant reduction of 55% (p≤0.01; 0.45-fold). No concentration 

dependent effects of carbaryl were noted on estradiol production, although there was a 1.35-fold 

increase (not significant) in estradiol at the highest concentration successfully tested, 10-6 M. 

Based on the hormone responses in each of three independent runs, carbaryl treatment resulted in 

statistically significant and reproducible decreases in testosterone production, but did not result 

in statistically significant and reproducible alterations in 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, Quality Assurance, and Data 

Confidentiality statements were provided. 

Page 128 of 149
Steroidogenesis Assay (2011) / Page 3 of 11

CARBARYL / 056801 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

D. De Barbeyrac, Clinical Pathology Laboratory

P. Almeras, Report Unit Assistant

Study Period: April 11, 2011 through July 18, 2011

2. Test Substance: Carbaryl

Description: Beige to pink powder

Lot # (expiration date): NNCAAX0025 (February 16, 2013)

Purity: 100%

Solubility: Soluble in DMSO at 10-4 M

Vapor pressure: 4.1 x 10-2 mPa @ 23.5°C

Stability: Not reported

Storage conditions: Air-tight, light-resistant container at room temperature

CAS #: 63-25-2

Molecular weight: 201

Structure:

3. Positive Control: Forskolin

Description (molecular weight): White powder (410.50)

Source: Sigma-Aldrich (St. Louis, MO)

Batch # (expiration date): 109K5057V (not provided)

Purity: 98%

Solubility (in solvent): Soluble in DMSO up to 0.01 M

Storage conditions: Air-tight, light-resistant container at room temperature

CAS #: 66575-29-9

4. Negative Control: Prochloraz

Description (molecular weight): Off-white powder (376.67)

Source: Sigma-Aldrich (St. Louis, MO)

Batch # (expiration date): SZE6220X (August 8, 2012)

Purity: 99.1%

Solubility (in solvent): Soluble in DMSO up to 0.001 M

Storage conditions: Air-tight, light-resistant container at room temperature

CAS #: 67747-09-5

Page 129 of 149
Steroidogenesis Assay (2011) / Page 4 of 11

CARBARYL / 056801 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)

Batch # (expiration date): 76396EM (not provided)

Purity: Not provided

Storage conditions: Not provided

CAS #: 67-68-5

Justification for choice of 

solvent:

DMSO is a Guideline recommended solvent in which carbaryl is 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 # 769927 and 847134.

7. Test Cells: H295R human adrenocortical carcinoma cells (ATCC CLR-2128; Batch # 

58660579) at passage 5.5 – 10.5 were incubated in the stock medium. Incubation conditions 

were at 37±1°C with 5% CO2 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 – 10.5

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 compared to DMSO was 38% at 10-10 M carbaryl, 54% at 

10-5 M carbaryl, and 174% at 10-4 M carbaryl. Although 38% variation may be indicative of 

interference, the relevance of this effect on the lowest concentration tested is equivocal. 

Conversely, interference at 10-4 and 10-5 M led to the exclusion of these concentrations from 

the regression analysis. Variation at other concentrations in the estradiol assays and all 

concentrations in the testosterone assays were less than 30%. 

b. Hormone Extraction: Hormone extraction was not reported. 

Page 130 of 149
Steroidogenesis Assay (2011) / Page 5 of 11

CARBARYL / 056801 OCSPP 890.1550/ OECD None

c. Laboratory Proficiency Test: Two laboratory proficiency determinations (SA 10203; nonGLP) 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 given below (Table 1): 

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 Data were obtained from page 15 of the study report. 

b The Guideline (890.1550) specified range for EC50s for forskolin are 0.2-2.0 μM for testosterone and 0.3-3.0 μM for 

estradiol, and the specified range for ED50s for prochloraz are 0.01-0.1 μM for testosterone and 0.03-0.3 μM for 

estradiol.

2. Test Solutions: Carbaryl, 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 to 10-10 M for carbaryl. 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 was not reported. H295R cells 

were grown for five passages, frozen in liquid nitrogen, then thawed and cultured for five to 

ten 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 carbaryl (or solvent control) in triplicate, according to the 

schematic presented in Table 2.

TABLE 2. Dosing Schematic for the Exposure of H295R Cells to Carbaryl (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 pages 13 and 16 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 

Page 131 of 149
Steroidogenesis Assay (2011) / Page 6 of 11

CARBARYL / 056801 OCSPP 890.1550/ OECD None

production). The QC plate was prepared and dosed in the same manner with either forskolin 

or prochloraz, according to the schematic presented in Table 3.

TABLE 3. Dosing Schematic for the QC Plate for Positive Controls (Final Concentrations in μM).a

1 2 3 4 5 6

A Blankb Blank Blank Blank 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 carbaryl, 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 carbaryl for 48 h 

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 the second and third evaluation, 

the treated cells were stained with trypan blue to evaluate cell viability, and the Sponsor 

stated that this assay demonstrated the viability of the cells following treatment with 

10-4 M – 10-10 M carbaryl. 

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, USA) for testosterone and Cayman 

Chemical Company (Ann Arbor, USA) for estradiol. The limit of detection for testosterone 

was 95.7 pg/ml and for estradiol it was 5.7 pg/ml. 

The following performance criteria were met (indicated by an “x”):

X Method detection limit (100 pg/mL testosterone; 10 pg/mL estradiol)

X Spiked sample recovery acceptable for two concentrations of testosterone and estradiol (mean measured amount 

from triplicate samples within 30% of nominal concentration)

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 solvent control (SC)

value for each assay and results were expressed as fold-change relative to the SC in each 

Page 132 of 149
Steroidogenesis Assay (2011) / Page 7 of 11

CARBARYL / 056801 OCSPP 890.1550/ OECD None

exposure plate. All data were expressed as mean ± standard deviation (SD). Relative 

changes were calculated as follows:

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, and cytotoxicity was not observed at any tested concentration. The 

variability (CV) between the runs for the solvent controls was within the guideline 

recommendation (≤30%) for both the testosterone (21%) and estradiol (25%) assays. The 

%CVs were 0.6–6.5% for absolute testosterone concentrations and 7.7–20.7% for absolute 

estradiol concentrations in the solvent control replicate wells within a plate. 

No effects of carbaryl on testosterone production were noted between 10-10 and 10-5 M with 

the % changes for the individual evaluations varying between +12% (1.12-fold) and -33% 

(0.67-fold; Table 4). In contrast, a marked decrease of -52% (0.48-fold), -53% (0.47-fold) 

and -59% (0.41-fold) in testosterone production was observed at 10-4 M for each of the runs 

leading to an overall significant reduction of -55% (p≤0.01; 0.45-fold). 

No concentration dependent effects of carbaryl were noted on estradiol production, although 

there was a 1.35-fold increase (NS) in estradiol at the highest concentration successfully 

tested, 10-6 M. The effects of carbaryl on testosterone and estradiol production are 

presented in Table 4 and illustrated in Figures 1 and 2. 

Page 133 of 149
Steroidogenesis Assay (2011) / Page 8 of 11

CARBARYL / 056801 OCSPP 890.1550/ OECD None

TABLE 4. Mean (±SD) Hormone Concentrations Following Treatment with Carbaryl 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 7879±501 7129±463 10,507±66 — — — — — —

10-10 7279±960

(-8%)

5487±171

(-23%)

9859±1137

(-6.2%) 0.92±0.12 0.77±0.02 0.94±0.11 0.89 0.24 — 

10-9 7915±393

(no change)

4749±335

(-33%)

8924±1619

(-15%) 1.00±0.05 0.67±0.05 0.85±0.15 0.85 0.24 — 

10-8 7492±192

(-5%)

4875±1570

(-32%)

9235±469

(-12%) 0.95±0.02 0.68±0.22 0.88±0.04 0.85 0.24 — 

10-7 8361±254

(+6%)

7957±998

(+12%)

10,936±1089

(+4%) 1.06±0.03 1.12±0.14 1.04±0.1 1.07 0.19 — 

10-6 8608±261

(+9%)

7869±520

(+10%)

11,029±323

(+5%) 1.09±0.03 1.10±0.07 1.05±0.03 1.08 0.17 — 

10-5 8648±642

(+10%)

7426±408

(+4%)

9608±1634

(-9%) 1.10±0.08 1.04±0.06 0.91±0.16 1.01 0.15 — 

10-4 3755±371

(-52%)

3343±192

(-53%)

4356±455

(-59%) 0.48±0.05 0.47±0.03 0.41±0.04 0.45 0.06 **

Estradiol (pg/mL) Fold Difference

DMSO 168.7±35 251.0±19.3 163.7±16 — — — — — —

10-10 172.7±30.4

(+2%)

291.3±46.7

(+16%)

157.0±18.5

(-4%) 1.02±0.18 1.16±0.19 0.96±0.11 1.06 0.36 — 

10-9 179.3±15.5

(+6%)

351.3±44.6

(+5%)

172.3±20.5

(+5%) 1.06±0.09 1.40±0.18 1.05±0.13 1.20 0.47 — 

10-8 162.7±33.6

(-4%)

263.0±41.6

(+5%)

179.3±23.2

(+10%) 0.96±0.2 1.05±0.17 1.10±0.14 1.04 0.28 — 

10-7 203.0±18.1

(+20%)

307.0±21

(+22%)

197.7±28.4

(+21%) 1.20±0.11 1.22±0.08 1.21±0.17 1.21 0.29 — 

10-6 194.7±30.1

(+15%)

333.7±23.1

(+33%)

261.7±20.5

(+60%) 1.15±0.18 1.33±0.09 1.60±0.13 1.35 0.33 — 

a Data (rounded up) were obtained from pages 19–21 of the study report. The lower limit of quantification (LLQ) was 95.7

pg/mL for testosterone and 5.7 pg/mL for estradiol. Estradiol assay interference was noted at 10-5 and 10-4; therefore, these 

concentrations are not reported. Statistical significance was determined for the mean of the three runs.

** p ≤0.01

Page 134 of 149
Steroidogenesis Assay (2011) / Page 9 of 11

CARBARYL / 056801 OCSPP 890.1550/ OECD None

FIGURE 1. Change in Testosterone Production Relative to Carbaryl Concentration. 

FIGURE 2. Change in Estradiol Production Relative to Carbaryl Concentration. 

B. CYTOTOXICITY: No cytotoxicity was observed for carbaryl at 10-4 and 10-5 M by the 

XTT method. In addition, at the end of the second and third evaluation, no toxicity was 

noted in any of the carbaryl treated cells (10-4 M – 10-10 M) when evaluated with the trypan 

blue exclusion method. Detailed data were not provided. 

Page 135 of 149
Steroidogenesis Assay (2011) / Page 10 of 11

CARBARYL / 056801 OCSPP 890.1550/ OECD None

C. QC PLATE: The hormone concentrations after exposure to the reference chemicals, SC, 

and blank samples, as well as the fold difference change relative to SC (individual and mean 

± SD) for the three assay runs are presented in Table 5. The minimum basal hormone 

production levels (500 pg/mL for testosterone, 40 pg/mL for estradiol) were met in both 

blank and SC wells for testosterone. Guideline requirements for basal hormone production, 

induction, and inhibition were met for testosterone and estradiol, except for the induction of 

testosterone with 10 μM forskolin in Runs 1 and 3 (1.66– and 1.79 –fold) which was below 

the Guideline criteria of ≥2-fold induction. 

The between plate variability (%CV), based on the absolute hormone concentrations, was 

20% for testosterone and 33% for estradiol; the %CV of the SC for estradiol exceeded the 

Guideline criteria (≤30%). The SC within plate % CVs were 3.5-6.3% for testosterone and 

15.3-17.8% for estradiol, and within the Guideline recommendation (≤30%). 

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 8994.7±822.2 7327.5±531.3 10,897.3±812.6 — — — — —

DMSO 8698.2±544.3 7427.3±262.4 11,021.8±556.6 — — — — —

1 μM Forskolin 16,782.3±1032.7

(+92.9%)

12,516.3±1347.2

(+68.5%)

18,903±1540.3

(+71.5%) 1.93±0.12 1.69±0.18 1.72±0.14 1.78 0.34

10 μM Forskolin 15,587.7±818.3

(+79.2%)

14,981±371

(+101.7%)

18,267±109.8

(+65.7%) 1.79±0.09 2.02±0.05 1.66±0.01 1.80 0.17

0.1 μM Prochloraz 1839.7±319.2

(-78.8%)

1141.3±185.9

(-84.6%)

2306.7±50.4

(-79.1%) 0.21±0.04 0.15±0.02 0.21±0.0 0.19 0.06

1 μM Prochloraz 295.0±4.6

(-96.6%)

233.0±21

(-96.9%)

374.3±54

(-96.6%) 0.03±0.0 0.03±0.0 0.03±0.0 0.03 0.01

Estradiol (pg/mL) Fold Difference (Relative to DMSO)

Blank 246.3±32.8 330.7±51.4 208.5±38.2 — — — — —

DMSO 208.8±32.7 330.2±58.6 181.3±27.8 — — — — —

1 μM Forskolin 5454.7±725.1

(+2512%)

5701.3±1000.0

(+1627%)

7614.3±670.1

(+4100%) 26.1±3.5 17.3±3.0 42.0±3.7 26.1 5.2

10 μM Forskolin 12,761.0±547.6

(+6012%)

10,330.3±611.2

(+3029%)

10,928.3±1682.4

(+5928%) 61.1±2.6 31.3±1.9 60.3±9.3 47.2 6.0

0.1 μM Prochloraz 150.0b ±19.8

(-28.2%)

398.0±90.8

(+20.5%)

194.7±37.5

(+7.4%) 0.72±0.1 1.21±0.3 1.05±0.3 1.08 0.53

1 μM Prochloraz I 141.0b I I 0.43 I 0.59 0.13

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. 

b n=2 instead of 3

I Complete inhibition (<100 pg/mL)

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: Overall carbaryl was not cytotoxic to the H295R 

adrenal cells at any of the concentrations tested. Furthermore, with the exception of a 

marked decrease in testosterone secretion at 10-4 M, there were no concentration-related 

effects on either testosterone or estradiol secretion when testing carbaryl in the H295R 

steroidogenesis assay. 

Page 136 of 149
Steroidogenesis Assay (2011) / Page 11 of 11

CARBARYL / 056801 OCSPP 890.1550/ OECD None

B. AGENCY COMMENTS: In the 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. Guideline requirements for basal hormone production, induction, and 

inhibition were met for testosterone and estradiol, except for induction of testosterone with 

10 μM forskolin in Runs 1 and 3 (1.66– and 1.79 –fold) which was below the Guideline 

criteria of ≥2-fold induction. Evaluation of the intra- and interplate variability (%CV) 

generally indicated consistent performance of the assays. The between plate variability 

(%CV), based on the absolute hormone concentrations, was 20% for testosterone and 33% 

for estradiol; the %CV of the SC for estradiol exceeded the Guideline criteria (≤30%). The 

SC within plate % CVs were 3.5-6.3% for testosterone and 15.3-17.8% for estradiol, and 

within the Guideline recommendation (≤30%). 

 

Precipitation of the test compound was not reported at any concentration. Cytotoxicity was 

not observed at any tested concentration. The inter- and intraplate variability for the SC in 

the test plates were within the Guideline recommendation (≤30%). No effects on 

testosterone production were noted between 10-10 and 10-5 M with the % changes for the 

individual evaluations varying between +12% (1.12-fold) and -33% (0.67-fold). In contrast, 

a marked decrease of -52% (0.48-fold), -53% (0.47-fold) and -59% (0.41-fold) in 

testosterone secretion was recorded at 10-4 M for each of the runs leading to an overall 

significant reduction of -55% (p≤0.01; 0.45-fold). 

No concentration dependent effects of carbaryl were noted on estradiol production, although 

there was a 1.35-fold increase (NS) in estradiol at the highest concentration successfully 

tested, 10-6 M. 

C. STUDY DEFICIENCIES: The following deficiency was noted that is not considered to 

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

• Stability of carbaryl in the test system was not reported.

• The EIA used for determination of estradiol showed interference with carbaryl at the 

two highest concentrations tested. 

• Induction of testosterone in Runs 1 and 3 was marginally low (1.66– and 1.79–fold 

induction was observed rather than ≥2-fold induction guideline value). 

• The SC between plate CV for estradiol (33%) in the QC plates exceeded the Guideline 

criteria (≤30%). 

Page 137 of 149
DATA EVALUATION RECORD

CARBARYL

Study Type: OCSPP 890.1600, In vivo Uterotrophic Assay 

EPA Contract No. EP10H001452 

Task Assignment No. 3-07-2012 (MRID 47987702) 

Prepared for

Health Effects Division

Office of Pesticide Programs

U.S. Environmental Protection Agency 

2777 South Crystal Drive 

Arlington, VA 22202 

Prepared by

CSS-Dynamac Corporation

1910 Sedwick Road, 

Building 100, Suite B 

Durham, NC 27713 

Primary Reviewer: Signature:

Kelly Luck, M.S. Date: 8/02/2012

Secondary Reviewer: Signature:

David A. McEwen, B.S. Date: 8/9/2012

Program Manager: Signature:

Jack D. Early, M.S. Date: 8/10/2012

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 8/10/2012

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

signing by CSS-Dynamac Corporation personnel. 

Page 138 of 149
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 139 of 149
CARBARYL I 056801 

In vivo Uterotrophic Assay (2003) I Page 1 of 10 

OCSPP 890.1600/ OECD 440 

Primary Reviewer: ---'Kh==in=--O=o'-'Mc.:...::..:..:.D=-=--. ________ Signature: __ Qt_,,' ;.,g,y:~ ...... --~--- Health Effects Division Date: ---=-=:..=..:...:...=~::..__ 12/27/2012 __ 

Secondary Reviewer: ~Je_s~s_R_o_w_I_an~d~ _________ Signature· 

Health Effects Division Date; 

Template 

8~ version 09/2011 

DATA EVALUATION RECORD 

STUDY TYPE: Uterotrophic Assay (Rat); OCSPP 890.1600; OECD 440 

PC CODE: 056801 DP BARCODE: D404353 

TXR#: 0056268 CAS#: 63-25-2 

TEST MATERIAL (PURITY): Carbary! (99.1% a.i.) 

SYNONYMS: 1-naphthalenyl N-methylcarbamate 

CITATION: Blanck, 0. (2003). Uterotrophic assay to assess the potential estrogenic activity 

of carbaryl in the rat. Bayer CropScience, Sophia Antipolis Cedex, France. 

Laboratory Report No.: SA 02282, January 16, 2003. MRID 47987702. 

Unpublished. 

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

TEST ORDER#: EDSP-056801-57 

EXECUTIVE SUMMARY: In a Uterotrophic Assay (MRID 47987702) conducted to screen 

for potential estrogenic activity, carbaryl (99.1 % a.i., Batch #21114920) in com oil was 

administered daily via oral gavage to groups of 10 immature female Sprague Dawley rats at dose 

levels of 0 (vehicle), 3, 10, or 30 mg/kg/day on post-natal days (PND) 19-21. A positive control 

group was treated with a daily dose of l 7a-ethynyl estradiol (EE) at 0.003 mg/kg/day by oral 

gavage and a negative control group was treated with a daily dose of dibutylphthalate (DBP) at 

1,000 mg/kg/day by oral gavage. Body weights were determined daily. All animals were 

terminated and necropsied on PND 22 approximately 24 hours after the final dose administration 

to determine wet and blotted uterine weights. 

All animals survived until scheduled termination. At 30 mg/kg/day, tremors were noted in 7/ 10 

animals and chewing in 9/ 10 animals. Piloerection was also noted in 4/10 animals on Day 1 or 

Day 2 (post-dosing) and one animal presented many clinical signs during the dosing period: 

reduced motor activity, prostration, wasted appearance, genital discharge and soiled anogenital 

region. At 10 mg/kg/day, piloerection was noted in 2/ 10 animals on Day 1, and chewing was 

observed in 3/ 10 animals on Days 2 and 3. At 3 mg/kg/day, 1/10 animals had piloerection on 

Day 1 only. 

Final body weights were decreased (not statistically analyzed) by 10% in the 30 mg/kg/day 

group compared to vehicle controls, resulting in a decrease (p<O.O 1) of 39% in the overall (Days 

1-4) body weight gain. Body weights in the 3 and 10 mg/kg/day groups were comparable to the 

Page 140 of 149
In vivo Uterotrophic Assay (2003) / Page 2 of 10

CARBARYL / 056801 OCSPP 890.1600/ OECD 440

control throughout the study; however, overall body weight gain was decreased (p<0.05) by 11% 

in the 3 mg/kg/day group. The decreased body weights indicate that the dose levels tested were 

adequate. Uterine weights in the carbaryl treatment groups were comparable to the vehicle 

controls. 

Body weights and overall body weight gain in the positive control (EE) group were comparable 

to the controls. In the negative control (DBP) group, final body weight was decreased by 10%, 

and overall body weight gain was decreased (p<0.01) by 40%. Absolute wet and blotted uterus 

weights for the EE group were increased (p<0.01) by 518% and 394%, respectively, as expected. 

Uterine weights in the DBP group were comparable to controls. 

No statistically significant changes were seen in uterine weight in this assay. Carbaryl is negative 

in the uterotrophic assay. 

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

890.1600). 

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

Assurance statements were provided. 

Page 141 of 149
In vivo Uterotrophic Assay (2003) / Page 3 of 10

CARBARYL / 056801 OCSPP 890.1600/ OECD 440

I. MATERIALS AND METHODS

A. MATERIALS

1. Test Facility: Bayer CropScience

Location: Sophia Antipolis Cedex, France

Study Director: O. Blanck

Other Personnel: J.P. Kocwin (Laboratory Animal Resources), B. Bonnafous (Toxicology 

Supervisor), H. Lormeau-Espinasse (Responsible Technician), C. Pallen 

(Statistician), M. Vagner (Report Unit Assistant)

Study Period: October 28, 2002 - January 16, 2003

2. Test Substance: Carbaryl

Description: Technical; white powder

Source: Aventis CropScience (Frankfurt am Main, Germany)

Batch #: 21114920 (expiration date 1/18/2003)

Purity: 99.1%

Stability: Stable for 4 days in corn oil at ambient temperature

CAS #: 63-25-2

Structure:

3. Reference Estrogen: 17α-ethynyl estradiol (EE)

Supplier: Not reported

Batch #: 57H1178 (no expiration date provided)

Purity: Not reported

CAS #: Not reported

Reference (Negative 

Control):

Dibutylphthalate (DBP)

Supplier: Not reported

Batch #: K201/00278 (no expiration date provided)

Purity: Not reported

CAS #: Not reported

4. Solvent/Vehicle Control: Corn oil

Supplier: Not reported

Lot/Batch #: Not reported

Rationale (if other than water): Not reported

Final concentration: Not applicable

O

O

N

H

CH3

Page 142 of 149
In vivo Uterotrophic Assay (2003) / Page 4 of 10

CARBARYL / 056801 OCSPP 890.1600/ OECD 440

5. Test Animals:

Species: Rat (female only)

Strain: Sprague Dawley [OFA-SD (IOPS Caw)]

Age/weight at dose initiation:

PND 19/ 31.4-41.3 g 

Source: Iffa-Credo (St Germain-sur-L’Arbresle, France)

Housing: Two per Makrolon cage with soft wood bedding

Diet: Certified rodent pelleted and irradiated diet A04C-10 (Usine d’Alimentation 

Rationnelle, Villenoisson-sur-Orge, France), ad libitum; phytoestrogen content of 

diet was not reported

Water: Filtered and softened tap water, ad libitum

Environmental conditions: Temperature:

Humidity:

Air changes:

Photoperiod:

20-24 oC

40-70% 

15/hr (average; not monitored)

12 hrs light/12 hrs dark

Acclimation period: 6 days at facility; pups were housed with their dam

B. STUDY DESIGN 

1. In-Life Dates: Start: November 5, 2002 End: November 8, 2002 

2. Study Design: Following an initial 6-day acclimation period, immature, intact female rats

were administered the test substance from PND 19-21. Rats were euthanized approximately 

24 hours after the last dose and necropsied for uterine weight measurements. The pups were 

housed with their dam prior to weaning. 

3. Animal Assignment: Animals were assigned to the test groups noted in Table 1 using a 

randomization procedure which selected animals from the middle of the weight range of the 

available animals. The body weight of each animal was within 20% of the overall mean on 

the day of randomization. 

TABLE 1. Study Designa

Test Group Dose (mg/kg/day) # of Females

Estrogen Agonist Assay

Vehicle Control 0 10

Low 3 10

Mid 10 10

High 30 10

17α-ethynyl estradiol (EE), Reference Estrogen 0.003 10

Dibutylphthalate (DBP), Negative control 1000 10

a Data were obtained from page 13 of the study report.

4. Dose Selection Rationale: The dose levels were reportedly selected based on appropriate 

studies from the toxicology database of carbaryl and a dose range-finding study1 in which 

immature females rats were dosed with carbaryl in corn oil at 3, 10, or 30 mg/kg/day; 

however, no results of the range-finding study were reported. 

1

 Blanck, O.: Range-finding assay in young immature female rats by gavage with carbaryl (SA 02273, ongoing), 

Bayer CropScience, Sophia Antipolis, France, August 2002.

 

Page 143 of 149
In vivo Uterotrophic Assay (2003) / Page 5 of 10

CARBARYL / 056801 OCSPP 890.1600/ OECD 440

5. Dose Preparation and Analysis: Dose formulations were prepared once prior to initiation 

of treatment by suspending the test substance in corn oil. Dosing formulations for EE were 

prepared daily in corn oil containing 0.6% ethanol, and formulations of DBP were prepared 

daily in corn oil. Stability was tested at two concentration levels, 0.6 and 6 g/L in corn oil, 

after 4 days of storage at ambient temperature. Homogeneity was tested in two formulations 

(0.6 and 6 g/L) by collecting samples from the surface and bottom of the mixture; the results 

of the homogeneity analyses were used to demonstrate achieved concentration. 

Results of Dose Analysis 

Homogeneity (%CV): 0.4% 

Stability (% of initial): 99% and 100% after four days of storage at ambient temperature 

Concentration (% of nominal): 107-108% 

The analytical data indicated that the mixing procedure was adequate and that the variation 

between nominal and actual dosage to the animals was acceptable. 

6. Dosage Administration: Animals were administered the test formulations of carbaryl, EE, 

DBP, or vehicle 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 most recent body 

weight measurement.

7. Statistics: Statistical analyses were conducted using SAS programs (version 8). Statistical 

comparisons were performed at the 5% and 1% levels. Body weight gains and wet and 

blotted uterus weight data were subjected to statistical analyses. 

For comparing carbaryl treatment groups with the vehicle control, the Bartlett test (twosided) was performed to compare group variances. When the Bartlett test was not 

significant (p=0.05), means were compared using the analysis of variance (ANOVA). When 

the ANOVA was not significant (p=0.05), group means were considered to be 

homogeneous. When the ANOVA was significant (p=0.05), group means were compared 

using the Dunnett’s test (two-sided). For body weight changes, when the Bartlett test was 

significant, means were compared using the non-parametric analysis of variance of KruskalWallis test. For uterus weights, when the Bartlett test was significant, data were 

transformed (log transformation followed by square-root transformation if needed); when 

the Bartlett’s test on the transformed data was not significant, means were compared using 

the Dunnett’s test (two-sided) and when Bartlett’s test was significant after transformation, 

means were compared using the non-parametric analysis of variance of Kruskal-Wallis. For 

both parameters, when the Kruskal-Wallis test was not significant (p=0.05), group means 

were considered to be homogeneous. When the Kruskal-Wallis test was significant, group 

means were compared using the Mann-Whitney test (two-sided). 

For comparison of the vehicle control with the EE and DBP groups, the F test (two-sided) 

was performed to compare group variances. When the F test was not significant (p=0.05), 

means were compared using the t-test (two-sided). When the F test was significant, body 

weight change means were compared using the modified t-test (two-sided). For uterus 

Page 144 of 149
In vivo Uterotrophic Assay (2003) / Page 6 of 10

CARBARYL / 056801 OCSPP 890.1600/ OECD 440

weights, when the F test was significant, data were transformed (log transformation 

followed by square-root transformation if needed). When the F test on the transformed data 

was not significant, means were compared using the t-test (two-sided); means were 

compared using the Mann-Whitney test (two-sided) when the F test remained significant 

after transformation.

The statistical analyses were considered adequate. 

C. METHODS

1. Clinical Examinations: Cage-side checks for mortality and moribundity were conducted 

twice daily (once daily on weekends). Clinical observations were recorded at least once 

daily throughout the study, approximately 0.5-1 hours after dosing. 

2. Body Weight: Animals were weighed at randomization, daily throughout the dosing 

period, and prior to necropsy. 

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 surviving animals were anesthetized by 

intraperitoneal injection of pentobarbital then euthanized by exsanguination and subjected to 

a gross necropsy. Dissection of the uterus was performed according to the U.S. EPA 

Guideline. Briefly, the vagina was removed just below the cervix in order to retain the 

luminal fluid in the uterus. The “wet” uterus (i.e., containing the luminal fluid) was 

weighed. Subsequently, the uterine horns slit open to allow luminal fluid to leak out, the 

uterus was gently blotted, 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. 

2. Clinical Signs of Toxicity: The incidence of clinical observations is reported in Table 2. 

At 30 mg/kg/day, tremors were noted in 7/10 animals and chewing in 9/10 animals. 

Piloerection was also noted in 4/10 animals on Day 1 or Day 2 (post-dosing) and one animal 

presented many clinical signs during the dosing period: reduced motor activity, prostration, 

wasted appearance, genital discharge and soiled anogenital region. At 10 mg/kg/day, 

piloerection was noted in 2/10 animals on Day 1, and chewing was observed in 3/10 animals 

on Days 2 and 3. At 3 mg/kg/day, 1/10 animals had piloerection on Day 1 only. All other 

observations were considered not to be related to carbaryl treatment. 

In the EE group, chewing was noted in 1/10 animals, and in the DBP group, soiling around 

the mouth was noted in 1/10 animals. 

Page 145 of 149
In vivo Uterotrophic Assay (2003) / Page 7 of 10

CARBARYL / 056801 OCSPP 890.1600/ OECD 440

TABLE 2. Incidence of Clinical Observations in the Estrogen Agonist Assaya

Observation

Dose (mg/kg/day)

Vehicle Control Carbaryl (3) Carbaryl (10) Carbaryl (30)

Reference 

Estrogen

EE (0.003)

DBP (1000)

# 

Observed

# 

Examined

# 

Observed

# 

Examined

# 

Observed

# 

Examined

# 

Observed

# 

Examined

# 

Observed

# 

Examined

# 

Observed

# 

Examined

Piloerection 0 10 1 10 2 10 4 10 0 10 0 10

Ocular 

discharge 0 10 2 10 0 10 0 10 0 10 0 10

Reduced motor 

activity 0 10 0 10 0 10 1 10 0 10 0 10

Prostration 0 10 0 10 0 10 1 10 0 10 0 10

Tremors 0 10 0 10 0 10 7 10 0 10 0 10

Soiling around 

the mouth 0 10 0 10 0 10 0 10 0 10 1 10

Chewing 0 10 0 10 3 10 9 10 1 10 0 10

Wasted 

appearance

0 10 0 10 0 10 1 10 0 10 0 10

Genital 

discharge 0 10 0 10 0 10 1 10 0 10 0 10

Soiled 

anogenital 

region

0 10 0 10 0 10 1 10 0 10 0 10

a Data were obtained from Table 2 on page 24 of the study report.

B. BODY WEIGHT AND WEIGHT GAIN: Body weight and overall body weight gain data 

are presented in Table 3. Final body weights were decreased (not statistically analyzed) by 

10% in the 30 mg/kg/day group compared to vehicle controls, resulting in decreased 

(p<0.01) overall (Days 1-4) body weight gain (↓39%). Body weights in the 3 and 10 

mg/kg/day groups were comparable to the control throughout the study; however, overall 

body weight gain was decreased (p<0.05) in the 3 mg/kg/day group (↓11%). 

Body weights and overall body weight gain in the EE group were comparable to the 

controls. In the DBP group, final body weight was decreased by 10%, and overall body 

weight gain was decreased (p<0.01) by 40%. 

Page 146 of 149
In vivo Uterotrophic Assay (2003) / Page 8 of 10

CARBARYL / 056801 OCSPP 890.1600/ OECD 440

TABLE 3. Group Body Weights and Cumulative Body Weight Gains (g) In the Estrogen Agonist Assaya

Study

Day #

Dose (mg/kg/day)

Vehicle Control Carbaryl (3) Carbaryl (10) Carbaryl (30)

Reference 

Estrogen

EE (0.003)

DBP (1000)

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD

1 10 35.3 2.6 10 36.2 2.1 10 36.5 1.7 10 35.7 2.1 10 36.1 2.2 10 35.8 2.4

2 10 38.5 3.7 10 38.7 3.2 10 39.8 2.8 10 36.9 4.7 10 39.2 3.3 10 35.3 5.7

3 10 44.2 3.0 10 43.1 3.4 10 43.5 3.0 10 39.8 7.7 10 44.2 3.1 10 37.0 9.7

4 10 48.2 3.2 10 47.6 2.9 10 48.2 3.3 10 43.5

(↓10) 8.1 10 48.1 2.8 10 43.5(↓10) 6.4

Body 

Weight 

Gain (1 - 4)

10 12.9 1.04 10 11.4*

(↓11) 1.59 10 11.7 2.36 10 7.8**

(↓39) 6.51 10 12.0 1.18 10 7.7**

(↓40) 4.36

a Data were obtained from Tables 2a and 2b on pages 26 and 27 of the study report. Percent differences from vehicle 

controls, calculated by the reviewers, are included in parentheses. Statistical analyses were only conducted for body 

weight gains.

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. Uterine Weights: Uterine weight data are presented in Table 4. Uterine weights in the 

carbaryl treatment groups and the negative control (DBP) group were comparable to the 

vehicle controls. Absolute wet and blotted uterus weights for the positive control (EE) 

group were increased (p<0.01) by 518% and 394%, respectively. The increased uterine 

weights were within the expected range. 

The mean blotted uterine weight for the vehicle control group was 0.031% of terminal body 

weight, which is within the performance criteria (<0.09%) of the test guideline.

Page 147 of 149
In vivo Uterotrophic Assay (2003) / Page 9 of 10

CARBARYL / 056801 OCSPP 890.1600/ OECD 440

TABLE 4. Uterine Weights from Estrogen Agonist Assay in SD Ratsa

Parameter

Dose (mg/kg/day)

Vehicle Control Carbaryl (3) Carbaryl (10) Carbaryl (30)

Reference 

Estrogen

EE (0.003)

DBP (1000)

N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD N Mean SD

Terminal 

BW (g) 10 48.2 3.21 10 47.6 2.94 10 48.2 3.27 10 43.5(↓10) 8.14 10 48.1 2.80 10 43.5(↓10) 6.39

Wet, 

absolute 

(mg)

10 17.7 1.65 10 18.4 2.01 10 18.9 1.99 10 18.1 2.15 10 109.2** 

(↑518) 18.0 10 18.6 4.56

Wet, 

relative (%)b 10 0.0367 0.0028 10 0.0388 0.0043 10 0.0393 0.0036 10 0.0431 0.0101 10 0.227 0.035 10 0.0425 0.0054

Blotted, 

absolute 

(mg)

10 15.0 2.05 10 16.1 1.59 10 15.9 1.89 10 15.3 1.76 10 74.2** 

(↑394) 11.5 10 15.9 3.36

Blotted, 

relative (%)b 10 0.0311 0.0039 10 0.0337 0.0031 10 0.0329 0.0028 10 0.0365 0.0088 10 0.154 0.022 10 0.0363 0.0034

a Data were obtained from Tables 3A, 3B, and 3C on page 29 of the study report. Percent differences from vehicle controls, calculated 

by the reviewers, are included in parentheses.

b Calculated by the reviewers using data provided in Appendices C and E on pages 38, 39, 44, and 45 of the study report.

BW Body weight

N Number of animals in the group

SD Standard Deviation

** Significantly different from controls at p<0.01

2. Microscopic Examination (Optional): Microscopic examinations were not conducted. 

III. DISCUSSION AND CONCLUSIONS

A. INVESTIGATOR’S CONCLUSIONS: The positive control for estrogenic activity EE 

produced no effect on body weight but a significant increase in both wet and blotted uterus 

weights. The negative control for estrogenic activity, DBP, induced a slight reduction of 

body weight gain, but did not induce any modification in either wet or blotted uterus weight. 

Carbaryl tested at 30, 10, and 3 mg/kg/day in immature young female rats did not show any 

estrogenic activity under the conditions of the study. 

B. AGENCY COMMENTS: All animals survived until scheduled termination. At 30 

mg/kg/day, tremors were noted in 7/10 animals and chewing in 9/10 animals. Piloerection 

was also noted in 4/10 animals on Day 1 or Day 2 (post-dosing) and one animal presented 

many clinical signs during the dosing period: reduced motor activity, prostration, wasted 

appearance, genital discharge and soiled anogenital region. At 10 mg/kg/day, piloerection 

was noted in 2/10 animals on Day 1, and chewing was observed in 3/10 animals on Days 2 

and 3. At 3 mg/kg/day, 1/10 animals had piloerection on Day 1 only. 

Final body weights were decreased (not statistically analyzed) in the 30 mg/kg/day group 

compared to vehicle controls, resulting in decreased overall (Days 1-4) body weight gain. 

Body weights in the 3 and 10 mg/kg/day groups were comparable to the control throughout 

the study; however, overall body weight gain was decreased in the 3 mg/kg/day group. The 

decreased body weights indicate that the dose levels tested were adequate. Uterine weights 

in the carbaryl treatment groups were comparable to the vehicle controls. 

Page 148 of 149
In vivo Uterotrophic Assay (2003) / Page 10 of 10

CARBARYL / 056801 OCSPP 890.1600/ OECD 440

Body weights and overall body weight gain in the positive control (EE) group were 

comparable to the controls. In the negative control (DBP) group, final body weight was 

decreased, and overall body weight gain was decreased. Absolute wet and blotted uterus 

weights for the EE group were increased as expected. Uterine weights in the DBP group 

were comparable to controls. No statistically significant changes were seen in uterine weight 

in this assay. Carbaryl is negative in the uterotrophic assay. 

C. STUDY DEFICIENCIES: None 

Page 149 of 149