### 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: Greg Akerman, Ph.D.

## ~**~** Immediate Office

Health Effects Division (7509P)



### THROUGH: Jess Rowland

# ~~ Deputy Director

Health Effects Division



### TO: Jolene Trujillo Biologist/Chemical Review Manager

Risk Management and Implementation Branch V

Pesticide Re-evaluation Division (7505P)



### I. ACTION REQUESTED Ver.Apr. 2010



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

Division (HED) review the Endocrine Disruptor Screening Program (EDSP) Tier 1 assays submitted in response to the agency's Test Order for carbaryl: Test Order# EDSP-056801-57.

Page I of2

### 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

# DATA EVALUATION RECORD

C ARBARYL

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

.

### 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).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

- 1 0 

### 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 (Bma x ) 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.Androgen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 2 of 14

CARBARYL / 056801 O CSPP 89 0.1150 / OECD None



### In the c ompetitive binding experiment, c arbaryl 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 w as 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.

T he estimated mean l og IC 50 

### 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, t he 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 st udy assay. All performance criteria were met, with the exception that ligand depletion and non-specific binding ( NSB ) were not reported, but the IC 50 

### s for R1881 and the weak positive control were within the expected ranges

. The se deviations are considere d minor; the performance criteria were met.

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

R eceptor Binding A ssay.

The assay satisfies the EDSP Tier 1 Test Order requirement s for an A ndrogen R eceptor Binding Assay (OCSPP 890.1150).

COMPLIANCE : Signed and dated GLP and Quality Assurance statements were provided.Androgen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 3 of 14

CARBARYL / 056801 O CSPP 89 0.1150 / 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 ( expir y 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 : D imethyl sulfoxide (D MSO ) , 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%Androgen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 4 of 14

CARBARYL / 056801 O CSPP 89 0.1150 / 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 (PM SF)] 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 e xperiment measuring total and non-specific binding of [ 3 

### H] -R1881 was performed in a previous study (SA 11021) for the cytosol used in the subject c ompetitive binding e xperiment to demonstrate that the AR was present in reasonable concentrations and had the appropriate affinity for the R1881 ligand. The de tails and results of the saturation binding e xperiment were included as an attachment to the subject study. The conditions for the saturation binding e xperiment 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 [ 3 

### H] -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 t otal 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 eachAndrogen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 5 of 14

CARBARYL / 056801 O CSPP 89 0.1150 / 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 [ 3 

### H] -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 concurre nt 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, [ 3 

### H] -R1881, and/or R1881, the tubes were dried, dissolved in diluted prostate cytosol (300 μL), and incubated in a rotor in a refrigerator (5 ± 3oC) for 20 hours. To separate bound from free R1881, an aliquot of each sample tube was incubated with hydroxyapatite (HAP) slurry in an ice water bath for 20 minutes. The samples were then centrifuged, and the supernatant was discarded. The samples were washed 3 times in 50 mM TRIS buffer. Following the last wash and decanting of the Tris buffer, pellets were then extracted with 2 mL ethanol. The radiation was quantified by liquid scintillation counting of the ethanol extract. A total of three runs were performed.

3. Competitive Binding Experiment : A summary of the assay conditions for the c ompetitive binding e xperiment is included in Table 3.Androgen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 6 of 14

CARBARYL / 056801 O CSPP 89 0.1150 / 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 page s 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 c ompetitive binding e xperiment was performed according to the protocol provided in

the EPA Test Guidelines OCSPP 890.1150. The c ompetitive binding e xperiment 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 precipitati on 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 c ompetitive binding e xperiments 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.Androgen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 7 of 14

CARBARYL / 056801 O CSPP 89 0.1150 / 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 (Hot Mix) DMSO R1881 Dexamethasone Carbaryl

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

buffe r 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 (B
max 

### ), dissociation constant (K d 

### ), and inhibition concentration (IC 50 

### ) 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, I nc. ).

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

CARBARYL / 056801 O CSPP 89 0.1150 / OECD None



### b. Descriptors for receptor binding

B max 

### : maximal binding capacity

K d 

### : dissociation constants

IC 50 

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

### Relative Binding Affinity (RBA) : IC 50 

### of R1881 × 100 ÷ IC 50 

### of test substance

II. RESULTS

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

### ) for [ 3 

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

### was 14 fmol/100μg protein (±1.5) for the single batch of prostate cytosol used for the assay. The goodness of fit (r 2 

### ) was not reported and t he K d 

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

### s from the other two runs were within the recommended range (0.685 to 1.57 nM) and the Scatchard plot for

the three s aturation 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 Cytosol a

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

- 3 c

r 2 NR NR NR NA

B
max (nM) 0.34 0.31 0.38 0.34 ± 0.035

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

K d (nM) 1.19 0.59 1.25 1.01 ± 0.365

a Data were obtained from page 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 [ 3 

### H] -R1881 to the androgen receptor. The data fit results in a linear plot.Androgen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 9 of 14

CARBARYL / 056801 O CSPP 89 0.1150 / OECD None



### F IGURE 1. Binding of [ 3 

### H] -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 BindingAndrogen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 10 of 14

CARBARYL / 056801 O CSPP 89 0.1150 / OECD None



### F IGURE 2. Scatchard Plots of the Binding of [ 3 

### H]

- R1881 to the Androgen Receptor.

B. COMPETITIVE BINDING EXPERIMENT : Competitive binding e xperiment 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 l og IC 50 

### was

− 3.5 M for carbaryl

, − 9.1 M for R1881 , and −4.4 M for the weak positive control. When compared to R1881, t he 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.Androgen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 11 of 14

CARBARYL / 056801 O CSPP 89 0.1150 / OECD None

TABLE 6 . Competitive Binding Assay of Carbaryl with A R 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 IC 50 (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

IC 50 ( 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 0.002 0 0.0022 0.002 0 Carbaryl 0.0005 2 0.00016 0.00020 0.0002 9

a Data were obtained from page s 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 5 0 % activity at the highest concentration (10

− 3 

### M) of carbar yl. A graph of the mean values of carbaryl across the three runs is provided in Figure 4.Androgen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 12 of 14

CARBARYL / 056801 O CSPP 89 0.1150 / OECD None



### F IGURE 3. Percentage R1881 Bound to the Androgen Receptor in the Presence of Test Compound s.Androgen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 13 of 14

CARBARYL / 056801 O CSPP 89 0.1150 / 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 c ompetitive binding a ssay 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 K d (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.Androgen Receptor Binding (Rat Prostate Cytosol) ( 2011 ) / Page 14 of 14

CARBARYL / 056801 O CSPP 89 0.1150 / 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 IC 50 

### 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 C OMMENTS : The saturation bindi ng experiment demonstrated that the androgen receptor was present in a reasonable concentration and functioning with appropriate affinity for the R1881 ligand. The mean K d 

### for [ 3 

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

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

### ) was not reported and t he K d 

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

### s from the other two runs were within the recommended range (0.685 to 1.57 nM) and the Scatchard plots for

the three s aturation 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 c ompetitive 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 bind er in the AR assay as specific binding was reduced to < 50% at the highest concentration

tested in all three runs.

T he mean l og IC 50 

### 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 we ak 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 perfo rmance criteria were met, with the exception that ligand depletion and NSB were not reported, but the IC 50 

### s for R1881 and the weak positive control were within the expected ranges demonstrating that the ligand was available at an appropriate level. The se 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 c ompetitive binding experiments.

• The NSB was not provided for the c ompetitive binding experiment.

• The adjusted specific activity values for the competitive assays were not reported.

# 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 / 0 2 /2012

Secondary Reviewer Signature:
Ronnie J. Bever Jr., Ph.D., D.A.B.T. Date: 3/ 0 5/2012

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

.

### 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 streng ths 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 bu t 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).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.Aromatase (Human Reco mbinant) Assay ( 2011 ) / Page 2 of 12

CARBARYL / 056801 OCSPP 890.1200 / OECD None



### For the positive control (4- OH A SDN), aromatase activity averaged 0.0849 ± 0.0216 nmol/mgprotein/min at the lowest tested concentration (10 −10 

### M) and 0.0027 ± 0.0008 nmol/mgp rotein/min at the highest tested concentration (10 −5 

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

### . 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 IC 50 

### 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 high est tested concentration (10

− 3 

### M) . Carbaryl reduced aromatase activity compared to controls, on average, to approximately 6 5% 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% ( l og IC 50 

### = −3.83 M), the average slope of the inhibition curve was steeper than –2 (-2.92). T herefore, carbaryl is classified as Equivocal for aromatase inhibition in this assay

.

The assay satisfies the EDSP Tier 1 Test Order requirement s for an Aromatase a ssay (OCSPP 890.1200).

COMPLIANCE : Signed and dated Data Confidentiality, GLP Compliance, and Quality Assurance statements were provided.Aromatase (Human Reco mbinant) Assay ( 2011 ) / Page 3 of 12

CARBARYL / 056801 OCSPP 890.1200 / OECD None



### I. MATERIALS AND METHODS

A. MATERIALS

1. Test S ubstance : Carbaryl Technical

Description: Beige to pink powder

Source: Bayer CropScience, Germany

Lot/Batch #: NNCAAX0025 (expires February 16, 2013) Purity: 100 % Volatility: Not provided Storage C onditions: In air

- tight, light

- resistant containers at r oom t emperature Stability: Not provided

Solvent : DMSO Solubility (in T est S olvent): Soluble up to 10

− 3 M Highest Concentration Tested: 10

− 3 M Stock Solution Preparation : Methodology: Serial dilutions

Molecular W eight: 201.22 g/mol

CAS #: 63

- 25

- 2

Structure:



### 2. Non

- L abeled S ubstrate : Androstenedione (ASDN)

CAS # : 63

- 05

- 8

Source: Molekula, Ltd. (product # 15237310)

Lot/Batch #: 63779 (expiry January 2013) Purity: 99.25%



### 3

. Radiolabeled S ubstrate : 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 A ctivity: 20.7 Ci/mmol (0.766 TBq/mmol) Radiochemical Purity (In

- lab D etermination): Not provided



### 4. Positive C ontrol : 4

- hydroxyandrostenedione (4

- OH ASDN ; Formestane )

CAS # : 566

- 48

- 3

Source: Sigma

- Aldrich (Catalog # F2552)

Lot/Batch #: 081K2133 Purity: 99.6 %



### 5. Solvent (V ehicle C ontrol ) : DMSO

Source: Sigma

- Aldrich ( Ref. # 154938 )

Lot/Batch #: 07796KK and 76396EM

Justification for Choice of S olvent : Acceptable vehicle by OCSPP guideline 890.1200
Concentration (% of T otal V olume in A ssays) : 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/minAromatase (Human Reco mbinant) Assay ( 2011 ) / Page 4 of 12

CARBARYL / 056801 OCSPP 890.1200 / OECD None



### B. METHODS

1. Assay C omponent s 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 wer e 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 sto red 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

S odium phosphate buffer (pH 7.4) 0.1 M

Microsomal Protein 0.004 mg/mL

NADPH 0.3 mM

[ 3 H]ASDN 100 nM Propylene Glycol 5%
Temperature 37°C

Incubation Time 15 min

a Data obtained from page 14 of the study report.



### 2. Suitability Ass es s ments : 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 A ssay : Each assay run contained four tubes for the full enzyme activity and background activity controls, respectively, and full concentration curves in the positiveAromatase (Human Reco mbinant) 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 th e procedures described in OCSPP 890.1200 (Section h, pp. 9-10).

The amount of 3 

### H 2 

### O in the aqueous fraction was quantified for each assay tube by LSC, and aromatase activity was reported in units of nmol/mg

- protein/min.

4. Demonstration of P roficiency : 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 a ssays. 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 L aboratory P roficiency : The positive control data met the following criteria:

• Mean aromatase activity in the absence of an inhibitor was at least 0.1 nmol/mg

protein/min.

• Mean background control activity was ≤15% of the full ac tivity 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 pref erred ranges.

Demonstration o f P roficiency of N ew T echnician for Conducting Assay (when applicable) : The positive control data met the criteria as listed in section (i) of OCSPP 890.1200, except

for t he log IC 50 

### 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 IC 50 (M) −7.3 −7.0 −6.7

a Data obtained from page 17 of the study report.



### b. Proficiency C hemicals : 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 reportedAromatase (Human Reco mbinant) Assay ( 2011 ) / Page 6 of 12

CARBARYL / 056801 OCSPP 890.1200 / OECD None



### 5. Determination of Aromatase A ctivity with T est C hemical(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 reach ed 65 % full activity, similar to the po sitive control.

TABLE 4. Test Chemical Study Design for each Test Run

Sample Type Repe titions (Tubes) Description Reference or Chemical (M)
Full Activity Control 4 All test components a plus solvent vehicle N/A Bkgd Activity Control 4 Same as above without NADPH N/A

4

- OH ASDN Conc 1 2 All test components plus 4

- OH ASDN 1×10 −5

4

- OH ASDN Conc 2 2 All test components plus 4

- OH ASDN 1×10 −6

4

- OH ASDN Conc 3 2 All test components plus 4

- OH ASDN 1×10 −6.5

4

- OH ASDN Conc 4 2 All test components plus 4

- OH ASDN 1×10 −7

4

- OH ASDN Conc 5 2 All test components plus 4

- OH ASDN 1×10 −7.5

4

- OH ASDN Conc 6 2 All test components plus 4

- OH ASDN 1×10 −8

4

- OH ASDN Conc 7 2 All test components plus 4

- OH ASDN 1×10 −9

4

- OH ASDN Conc 8 2 All test components plus 4

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

Carbaryl Conc 3 b 3 All test components plus carbaryl 1×10 −5
Carbaryl Conc 4 b 3 All test components plus carbaryl 1×10 −6
Carbaryl Conc 5 b 3 All test components plus carbaryl 1×10 −7
Carbaryl Conc 6 b 3 All test components plus carbaryl 1×10 −8

Carbaryl Conc 7 b 3 All test components plus carbaryl 1×10 −9
Carbaryl Conc 8 b 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. D ATA ANALYSIS

1. Raw D ata : 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 minuteAromatase (Human Reco mbinant) Assay ( 2011 ) / Page 7 of 12

CARBARYL / 056801 OCSPP 890.1200 / OECD None



### 2. S tatistical M ethods : 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 a long 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% activity b Equivocal
Average lowest portion of curves across runs is greater than 75% activity b Non

- inhibitor

Data do not fit model

---

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

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

− 2.0, the result is classified as equivocal.

b If the test compound was not soluble above 10

− 6 M and the inhibition curve does not 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 contr ol 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 substan ce (4

- OH A SD N), aromatase activity averaged 0. 0849 ± 0.0216 nmol/mg

- protein/min at the lowest tested concentrationAromatase (Human Reco mbinant) 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 IC 50 

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

4

- OH A S D N (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 8 3.3 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 VarianceAromatase (Human Reco mbinant) Assay ( 2011 ) / Page 9 of 12

CARBARYL / 056801 OCSPP 890.1200 / OECD None



### FIGURE 1. Inhibition R esponse C urve s 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. 6 5% 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. C arbaryl exhibited a concentration dependent inhibition of aromatase activity and decreased activity by more than 50% at 10 –3 

### M ( log IC 50 

### = −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

.Aromatase (Human Reco mbinant) Assay ( 2011 ) / Page 10 of 12

CARBARYL / 056801 OCSPP 890.1200 / OECD None



### FIGURE 2. Inhibition R esponse C urves for Carbaryl F rom E ach T est R un.

FIGURE 3. Mean I nhibition R esponse C urve for Carbaryl.Aromatase (Human Reco mbinant) 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 IC 50 

### 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 IC 50 

### 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 P ercent of C ontrol) F rom Independent Runs a

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

Log IC 50 (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 IC 50 

### values for the positive control, formestane were

− 6.63,

− 6.60,

− 7.25 for each of the three independent evaluations with an overall log IC 50 

### of

− 6.73. Although the log IC 50 

### values were slightly outside the range (

− 7.3 to

− 7.0) proposed in the guideline all other criteria were met. The log IC 50 

### for carbaryl was between

− 3.7 and

− 3.947 with an overall l og IC 50 

### of approximately

− 3.84, which equates to an IC 50 

### 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 -protei n/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 IC 50 

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

− 10 

### to 10

− 6Aromatase (Human Reco mbinant) 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 th e highest tested concentration (10

− 3 

### M) . Carbaryl reduced aromatase activity compared to controls, on average, to approximately 6 5% at 10 −4 

### M and 42 % at 10 −3 

### M. The estimated log IC 50 

### 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 IC 50 

### 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 carbar yl 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 IC 50 

### = −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

IC 50 

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

• For the positive control, the log IC 50 

### 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.

# DATA EVALUATION RECORD

C ARBARYL

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

.

### 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 strengt hs 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).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 E R 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

- 1 0 

### to 10

- 3 

### M).

DMSO was used as the vehicle at a final concentration of S 2%. 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 Bm ax 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 negativ e 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 

# --Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 2 of 18

CARBARYL / 056801 O CSPP 89 0.1250 / 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 IC 50 

### 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, c arbaryl is classified as Equivocal in th e Estrogen Receptor Binding Assay.

The assay satisfies the EDSP Tier 1 Test Order requirement s for an E strogen R eceptor Binding

a ssay (OCSPP 890.1250).

COMPLIANCE : Signed and dated GLP and Quality Assurance statements were provided.Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 3 of 18

CARBARYL / 056801 O CSPP 89 0.1250 / 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 : 17 β

- estradiol

Supplier: Sigma

- Aldrich, St. Louis, MO Catalog # : E8875

Batch #: 010M0142 Purity: ≥98%

CAS #: 50

- 28

- 2



### 4

. Radioactive ligand : [ 3 

### H]

- 17 β

- estradiol

Supplier: Perkin Elmer, Boston, MA Catalog #: NET517

Batch#: 650702 Radiochemical purity: >97% Specific activity: 130.2 Ci/ m mol

Concentration of stock: 1 nM



### 5

. Positive control : 19

- n oreth i ndr one Supplier: Sigma

- Aldrich, St. Louis, MO Catalog # : N4128

Batch #: 019K1735 Purity: ≥98%

CAS # : 66

- 22

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

CARBARYL / 056801 O CSPP 89 0.1250 / 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 R at U terine 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 e xperiment s (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 c ytosol was divided into aliquots (volume not reported) for storage at

− 74 ± 10 oC until use.

2. Saturation (radioligand) B inding E xperiment : Three independent saturation binding

e xperiment s measuring total and non-specific binding of [ 3 

### H] -17 β

- estradiol were performed in a previous study (SA 11020) for the cytosol used in the subject c ompetitive binding

e xperiment 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 s aturation binding

e xperiment were included as an attachment to the subject study. The conditions for the saturation binding e xperiment 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.Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 5 of 18

CARBARYL / 056801 O CSPP 89 0.1250 / OECD None



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

### H] -17 β

- 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 17 β

- estradiol were prepared in a similar manner to achieve concentrations that

were 100

- fold greate r 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 -1 30 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 al one 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] -17 β

- estradiol . An aliquot of supernatant was radioassayed by scintillation counting. The temperature was maintained at 4 oC throughout

th e assay prior to extraction with ethanol.

3. Competitive B inding E xperiment : A summary of the experiment conditions for the

c ompetitive bind ing e xperiment is included in Table 3.Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 6 of 18

CARBARYL / 056801 O CSPP 89 0.1250 / OECD None

TABLE 3 . Summary of Conditions for Competitive Binding Experiment a
Source of receptor Rat U terine C ytosol
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 page s 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] -17 β -estradiol was adjusted for

de cay 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 comm ercially available ), the negative control (octyltriethoxysilane), and the reference material (non- labeled 17β

- estradiol) were prepared to achieve the concentrations shown in Table 4. E ach assay consisted of three runs, and each run contained three replica tes 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.Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 7 of 18

CARBARYL / 056801 O CSPP 89 0.1250 / 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 non

labeled 17β

- estradiol; 390 μL of TEDG + PMSF buffer with [ 3 H]

- 17β

- estradiol; and 100 μL of uterine cytosol (with ER), for a total of 500 μL.

c Each concentration of each chemical was run in triplicate, for a total of 105 tubes per run. Tube numbers were not provided, but were assigned by the reviewer based on data spreadsheets on pages 3 6-46.

d Solvent was DMSO (2%) in Tubes 1

- 3 and 100

- 102.



### The procedures for assay incubation, separation of bound from free estradiol ( wash ing with buffer and extraction with ethanol), followed by scintillation counting of bound [ 3 

### H] -17 β

estradiol were similar to those used in the s aturation binding e xperiment

.

C. D ATA ANALYSIS : For both the competitive binding and saturation binding assays, GraphPad Prism was used to generate nonlinear regression used to fit a curve (for 17β

estradiol, the positive control, and the test substance) to the Hill equation formula which incorporated IC 50 

### as a parameter to be estimated. For parameters reported from the saturation binding e xperiment ( K d 

### and B
max 

### ) and c ompetitive binding e xperiment (log IC 50 

### and RBA), the m eans 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 wa s 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 fitEstrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 8 of 18

CARBARYL / 056801 O CSPP 89 0.1250 / 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 :

B max 

### : maximum specific binding number (fmol ER/100 μg cytosol protein) measures the concentration of active receptor sites

K d 

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

IC 50 

### : concentration of the test substance at which 50% of the radioligand is displaced from the receptor Relative Binding Affinity ( RBA % ): IC 50 

### of 17 β

- estradiol ÷ IC 50 

### 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 s aturation binding a ssays are summarized in Table 5 and shown graphically in Figures

1-9. The mean K d 

### for [ 3 

### H]

- 17β

- estradiol was 0.070 nM, and the mean estimated B
max 

### was 34.3 fmol/100 μg protein for the prepared rat uterine cytosol. The K d 

### and B
max 

### for 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 (r 2 

### ) was not reported.Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 9 of 18

CARBARYL / 056801 O CSPP 89 0.1250 / OECD None

TABLE 5. Saturation Binding Experiment of 17 β

- 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

B
max (nM) 0.027 0.037 0.039 0.034 ± 0.011

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

K d (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



### F IGURE 1. Total and Non- specific binding Run 1.

FIGURE 2. Specific binding Run 1.Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 10 of 18

CARBARYL / 056801 O CSPP 89 0.1250 / OECD None



### F IGURE 3. Scatchard plot for Specific binding Run 1.

F IGURE 4. Total and Non- specific binding Run 2.Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 11 of 18

CARBARYL / 056801 O CSPP 89 0.1250 / OECD None



### F IGURE 5. Specific binding Run 2.

F IGURE 6. Scatchard plot for Specific binding Run 2.Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 12 of 18

CARBARYL / 056801 O CSPP 89 0.1250 / OECD None



### F IGURE 7. Total and Non- specific binding Run 3.

F IGURE 8. Specific binding Run 3.Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 13 of 18

CARBARYL / 056801 O CSPP 89 0.1250 / OECD None



### F IGURE 9. Scatchard plot for Specific binding Run 3.

B. COMPETITIVE BINDING EXPERIMENT : The results from the three c ompetitive binding experiments are summarized in Table 6 and presented graphically in Figures 10-13. Precipitat ion was not observed at any concentration. In two assay run s, c arbaryl 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 l og IC 50 

### was not calculate d for c arbaryl as the percent binding inhibition did not reach 50% for any run. The mean IC 50 

### 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 c arbaryl

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

CARBARYL / 056801 O CSPP 89 0.1250 / OECD None

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

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

r 2 (unweighted) 17 β

- estradiol NR NR NR NA

19

- norethindrone NR NR NR NA

C arbaryl NR NR NR NA Log IC 50 ( M) 17 β

- estradiol

− 8.75

− 8.74

− 8.76

− 8.75

19

- norethindrone

− 5.05

− 5.00

− 5.04

− 5.03

C arbaryl NA NA NA NA

IC 50 (M) 17 β

- 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

C arbaryl NA NA NA NA Log (RBA) c 19

- norethindrone

− 3 .70

− 3 .74

− 3 .72

− 3 .72

C arbaryl NA NA NA NA RBA (%) 19

- norethindrone 0.020 0.018 0.019 0.019

C arbaryl NA NA NA NA

a Data were obtained from page 1 7 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 C arbaryl 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 <0.5Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 15 of 18

CARBARYL / 056801 O CSPP 89 0.1250 / OECD None



### FIGURE 10. Percentage [ 3 

### H]

- E2 Bound to the Estrogen Receptor in the Presence of Carbaryl, Unlabeled E2, 19-Norethindrone or Octyltriethoxysilane. Run 1

FIGURE 11. Percentage [ 3 

### H]

- E2 Bound to the Estrogen Receptor in the Presence of Carbaryl, Unlabeled E2, 19- Norethindrone or Octyltriethoxysilane. Run 2Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 16 of 18

CARBARYL / 056801 O CSPP 89 0.1250 / OECD None



### FIGURE 12. Percentage [ 3 

### H]

- E2 Bound to the Estrogen Receptor in the Presence of Carbaryl, Unlabeled E2, 19-Norethindrone or Octyltriethoxysilane. Run 3

FIGURE 13. Mean Percentage [ 3 

### H]

- 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 function ed properly, each run was evaluated using the following criteria (Table 8) :Estrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 17 of 18

CARBARYL / 056801 O CSPP 89 0.1250 / OECD None

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

17 β

- estradiol fitted curve parameters
Log
e 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 (log 10 (M)

- 1 )

− 1.1 to

− 0.7

− 2.0 to

− 1.7 X

Weak Positive control ( 19

- noreth i ndr one ) fitted curve parameters b

Log
e residual SD NA NR Top (% binding) NA 103 to 110 X Bottom (% binding) NA

− 4.4 to 7.5 X Hill Slope (log 10 (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]

- 17 β

- estradiol from the ER on average

across all concentrations ≤25% 13.1

- 15.7% X

a Data were obtained from page s 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 s lope 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 IC 50 

### values for 17 β

- estradiol were

determined to be 1.8×10

− 9 

### , 1.8×10

− 9 

### and 1.7×10

− 9 

### M. The IC 50 

### 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 17 β -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 [ 3 

### H]

- 17β

estradiol

. The mean K d 

### for [ 3 

### H]

- 17β

- estradiol was 0.070 nM, and the mean estimated B
max



### was 34.3 fmol/100 μg protein for the prepared uterine cytosol. The K d 

### for each run wasEstrogen Receptor Binding (Rat Uterine Cytosol) ( 2011 ) / Page 18 of 18

CARBARYL / 056801 O CSPP 89 0.1250 / OECD None



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

Scatc hard plots. The goodness of fit (r 2 

### ) was not reported.

For the c ompetitive binding experiment, t he estimated mean IC 50 

### was not calculated for carbaryl as it did not achieve 50% binding. The mean IC 50 

### 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, c arbaryl 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. C arbaryl is classified as equivocal in this assay as the mean specific ER binding

was >5 0 % 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 17 β

- estradiol.

• The criteria of l og e 

### residual SD for the comp etitive 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 (m inor concern as it was a single animal).

# 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

.

### 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 bat tery 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).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

### .~

## Dat~e.**Date:~** 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. 

# ..Estrogen Receptor Transcriptional Activation ( 2011 ) / Page 2 of 10

CARBARYL /056801 O CSPP 89 0.1300 / 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 : N ot reported (NR) Volatility : NR Stability: NR Storage conditions: NR Vapor pressure : NR

CAS #: 63

- 25

- 2

Structure :

O O

N

H CH

3



### 2

. Reference substances

17 β

- estradiol (strong estrogen; positive control)

Supplier : Sigma

- Aldrich St. Louis, MO Catalogue and Batch #: Batch # 101M0142 Purity: 100%

CAS # : 50

- 28

- 2



### 17α

- estradiol ( w eak 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 conc e n tration ): 0.1%Estrogen Receptor Transcriptional Activation ( 2011 ) / Page 3 of 10

CARBARYL /056801 O CSPP 89 0.1300 / OECD 455



### B. METHODS

1. C ell C ulture : Stably

- transfected hER α

- HeLa -9903 cell s were obtained from the Japanes e 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 ATF 32351), in an incubator under 5% CO 2 

### at 37 oC. Upon reaching 75 -90% confluency, cells were subcultured 6 times prior to exposure to the test

material.

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



### cells /100 μL medium/well in a 96

- well plate and allowed to attach for 3 hours. 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 e ach concentration of carbaryl or reference chemical was tested in triplicate wells , except the positive control (1 nM 17 β

- 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 we lls, 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 wer e 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. P reliminary T est : 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 proficie ncy chemicals . The details of the proficiency test were not provided, only the

results.Estrogen Receptor Transcriptional Activation ( 2011 ) / Page 4 of 10

CARBARYL /056801 O CSPP 89 0.1300 / OECD 455

Compound CAS No. Concentration Range (M) Expected Response a Note s

Diethylstilbes t rol (DES) 56

- 53

- 1 10

− 14 to 10

− 8 Positive

--- 17 α

- Ethynyl estradiol (EE) 57

- 63

- 6 10

− 14 to 10

− 8 Positive

--- Hexestrol 84

- 16

- 2 10

− 13 to 10

− 7 Positive

--- Genistein 446

- 72

- 0 10

− 12 to 10

− 5 Positive Cytotoxic at 0.01 b , 0.1, and 1 mM

Estrone 53

- 16

- 7 10

− 12 to 10

− 6 Positive

--- Butyl paraben 94

- 26

- 8 10

− 11 to 10

− 4 Positive Cytotoxic at 0.1 b and 1 mM

1,3,5

- Tris(4

- hydroxyphenyl)benzene c 15797

- 52

- 1 10

− 12 to 10

− 5 Positive Cytotoxic at 100μM. PC max approx. 50% of PC. Binds to

hER α and has ER antagonistic activity
Dibutyl phthalate (DBP) 84

- 74

- 2 10

− 11 to 10

− 4 Negative d Cytotoxic at 1 mM

Atrazine 1912

- 24

- 9 10

− 11 to 10

− 4 Negative Cytotoxic at 1 mM b

Corticosterone 50

- 22

- 6 10

− 10 to 10

− 4 Negative If not cytotoxic at 1 mM, then that should be the highest tested

concentration

a Positive = RPC max ≥10% of the response of the positive control in at least 2 of 2 (or 2 of 3) runs Negative = RPC max fails to achieve at least 10% of the response of the positive control in 2 of 2 (or 2 of 3) runs

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

c Compound s e lected to challenge solubility and cytotoxicity

d DBP is negative for ER α mediated transcriptional activation, but may not be negative for non

- ER β mediated transcriptional activation. A positive result would indicate that the system is detecting activity other than that due to pure ER α, and is therefore unacceptable.



### c. Reference Chemicals : To ensure th e 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 R ange 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 t he 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 norm alize the data ; each

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

valu e 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 t he EC 50 

### , PC 10 

### , PC 50 

### , RPC Max 

### , and PC Max 

### for carbaryl when applicable

. T he test material was defined as negative for inducing estrogen receptor transcriptional

activation if the RPC Max 

### < PC 10 

### in at least 2 of 2 (or 2 of 3) runs. Log EC 50 

### and Hill slop e values are calculated only if a positive response is observed. Coefficients of variation (CV)

w ere calculated for the luminescence data triplicates. Concentrations showing >20% cytotoxicity or evidence of insolubility were excluded from analyses.

4. D efinitions

EC 50 

### = concentration of agonist that induces a response halfway between the baseline (bottom) and maximum (top) responseEstrogen Receptor Transcriptional Activation ( 2011 ) / Page 5 of 10

CARBARYL /056801 O CSPP 89 0.1300 / OECD 455



### PC 10 

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

PC 50 

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

RPC Max 

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

PC Max 

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



### II. RESULTS

A. PRELIMINARY TEST : In order to identify a suitable top concentration for use in the transcriptional activation assays, preliminary cytotoxicity and precipitation assays were conducted. Precipitation was observed in 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 Comments c

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 Tab le 2. In the first run, corticosterone produced an

RPC M ax 

### of 16%, indicati ng a weakly po sitive response. In the second run, the RPC M ax 

### was

− 14%, indicating a negative response.Estrogen Receptor Transcriptional Activation ( 2011 ) / Page 6 of 10

CARBARYL /056801 O CSPP 89 0.1300 / OECD 455

TABLE 2. Proficiency Chemicals a

Compound Expected Response Lab Response
Diethylstilbestrol Positive Positive

17 α

- 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 ., l og PC 50 

### , log PC 10 

### , log EC 50, 

### Hill slope) for the four concurrently run reference materials are

included in Table 3. Performance criteria were met for 17β

- estradiol and corticosterone.

The l og PC 10 

### for 17 α -estradiol was lower than the expected range in the first assay, as was the Hill Slope. Th e log PC 50 

### and log PC 10 

### for 17 α

- 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 l og PC 10 

### for 17 α

- estradiol was lower than l og PC 10 

### 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

- estradiol a
LogPC 50 −11.4 to −10.1 −10.4 −10.7 X
LogPC 10 <−11 −11.8 −11.4 X
LogEC 50 −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

- estradiol a
LogPC 50 −9.6 to −8.1 −8.9 −8.9 X
LogPC 10 −10.7 to −9.3 −12.3 −10.1 X
LogEC 50 −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

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

17α

- methyltestosterone b
LogPC 50 −6.0 to −5.1 −7.02 −5.99 X
LogPC 10 −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 page s 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

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

CARBARYL /056801 O CSPP 89 0.1300 / 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 value s , 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 PC 50 

### 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. T he PC 10 

### 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 t ested chemical was reported in the appendices, suggesting that raw data for only one plate was reported. M ean 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 17 β

- 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 Figure s 1 and

2 below. The mean RPC M ax 

### was 2.7% for the first run and 0.5% for the second run, and the

associated PC M ax 

### was 10

− 4 

### M for both runs. Because the RPC Max 

### < PC 10 

### in both runs, carbaryl is considered negative for estrogen receptor transcriptional activation in this test

system

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

CARBARYL /056801 O CSPP 89 0.1300 / OECD 455

TABLE 5. Relative Transcriptional Activation (RTA) of Carbaryl a

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 −1 0

− 5.9 4.1

− 5.1 3.1

LogEC 50 b 1.987

− 3.841
Hill Slope b 0.7105 7.528

RPC M ax 2.7 0.5

PC M ax 10

− 4 10

− 4

PC 50 1.987

− 3.841

PC 10 0.7024

− 4.187

a Data were calculated by the reviewer, using data obtained from pages 5 2 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.Estrogen Receptor Transcriptional Activation ( 2011 ) / Page 9 of 10

CARBARYL /056801 O CSPP 89 0.1300 / OECD 455



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

3. Performance C riteria : 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 PC 10 

### for 17 α

- estradiol was lower than the acceptable range in the first assay, as was the Hill Slope. The log PC 50 

### and log PC 10 

### for 17 α

- 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 l og PC 10 

### for 17 α

- estradiol was less than

l og PC 10 

### for 17β

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

l uciferase activity was 4.2-fold for the first run and 4.1-fold for the second run. The foldinduction corresponding to the PC 10 

### of the concurrent PC was greater than 1+2 standard deviations of the vehicle control on all plates. Variability was minimal and results were reproducible among runs. For 17α

- methyltestosterone, the mean RPC Max 

### was 83.5 % for the

first run and 69.5% for the second run. For corticosterone

, the apparent RPC Max 

### 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, RPC Ma x 

### 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 17 β -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 compoundsEstrogen Receptor Transcriptional Activation ( 2011 ) / Page 10 of 10

CARBARYL /056801 O CSPP 89 0.1300 / OECD 455



### included lower Hill slope and l og PC 10 

### values for 17 α -estradiol in one run, and l og PC 10 

### for

17 α

- estradiol was less than Log PC 10 

### for 17β -estradiol in run 1. In addition, the log PC 50 

### and log PC 10 

### for 17 α

- methyltestosterone were lower than the acceptable range for both

assays , indicating poten tial increased sensitivity to weak agonists

. The mean RPC M ax 

### for carbaryl was 2.7% for the first run and 0.5% for the second run, and the associated PC M ax 

### was 10

− 4 

### M for both runs. Because the RPC Max 

### < PC 10 

### in both runs , carbaryl was considered negative for estrogen receptor transcriptional activation in this test system.

C. STUDY DEFICIENCIES : No deficiencies were noted.

### ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀ

ԡԹՌԹԯԽՉՍՁՊԽՅԽՆՌ Ԣ

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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'

### ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋ Հ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹ ՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ 

ԭԹԿԽՇԾ ԠԦԱԞԱԦԬԫ ԶՇՊՃԡԬԠԹՊԺԹՊՑՄ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀ ԣԹՌՀԽԹԼԪՁՆՆՇՏ ԭՁՅԽՈՀԹՄԽՋ

ՈՊՇՅԽՄԹՋ ԣՇՄՄՇՏՁՆԿԬԭԭԱ԰ԹՆԼԬԢԠԡԤՍՁԼԽՄՁՆԽՋԲՆՈՍԺՄՁՋՀԽԼՋՌՍԼՑՈԽՊԾՇՊՅԽԼԺՑ

԰ՅՁՌՀԽՊՋ ԳՁՋԻՁԽՆՌԴԹՊԽՀԹՅ ԪԹՋՋԹԻՀՍՋԽՌՌՋ  ԩԹԺՇՊԹՌՇՊՑ ՊԽՈՇՊՌ ՆՍՅԺԽՊ  ԰ՌՍԼՑ

ՋՈՇՆՋՇՊԽԼԺՑԟԹՑԽՊԠՊՇՈ԰ԻՁԽՆԻԽԯԽՋԽԹՊԻՀԱՊՁԹՆԿՄԽԭԹՊՃԫՇՊՌՀԠ ԹՊՇՄՁՆԹ԰ՌՍԼՑԻՇՅՈՄԽՌԽԼ

ԧՍՆԽ

ԫՇՌԽ ԱՀԽԲ԰ԢԭԞԢՆԼՇԻՊՁՆԽԡՁՋՊՍՈՌՇՊ԰ԻՊԽԽՆՁՆԿԭՊՇԿՊԹՅ
Ԣԡ԰ԭԱՁԽՊ ՋԻՊԽԽՆՁՆԿԺԹՌՌԽՊՑՁՋԻՇՅՈՊՁՋԽԼՇԾ

ԽՄԽՎԽՆՋԻՊԽԽՆՁՆԿԹՋՋԹՑՋՁՆՌԽՆԼԽԼՌՇՁԼԽՆՌՁԾՑԹԻՀԽՅՁԻԹՄ0ՋՄՁՃԽՄ ՑԽՆԼՇԻՊՁՆԽԺՁՇԹԻՌՁՎՁՌՑՁԽՁՌՋՈՇՌԽՆՌՁԹՄՌՇ

ՁՆՌԽՊԹԻՌՏՁՌՀՌՀԽԽՋՌՊՇԿԽՆԹՆԼՊՇԿԽՆՇՊՌՀՑՊՇՁԼ
ԢԞՇՊԱՈ ԹՌՀՏԹՑՋԱՀԽՊՇԺՍՋՌՆԽՋՋՇԾՌՀԽԱՁԽՊ

ԺԹՌՌԽՊՑՁՋԺԹՋԽԼՇՆՌՀԽՋՌՊԽՆԿՌՀՋՇԾԽԹԻՀՁՆԼՁՎՁԼՍԹՄԹՋՋԹՑՌՇՁ ԼԽՆՌՁԾՑՈՇՌԽՆՌՁԹՄԽՆԼՇԻՊՁՆԽԺՁՇԹԻՌՁՎՁՌՑՏՁՌՀ

ԻՇՅՈՄԽՅԽՆՌԹՊՑԽՆԼՈՇՁՆՌՋՏՁՌՀՁՆՌՀԽԹՋՋԹՑՏՀԽՊԽԹՎԹՁՄԹԺՄԽԹՆԼ ՊԽԼՍՆԼԹՆԻՑԹԻՊՇՋՋՌՀԽԺԹՌՌԽՊՑԱՀՍՋ

ՌՀԽՊԽՋՍՄՌՋՇԾԽԹԻՀՁՆԼՁՎՁԼՍԹՄԹՋՋԹՑՋՀՇՍՄԼՆՇՌԺԽԻՇՆՋՁԼԽՊԽԼՁՆՁՋՇՄԹՌՁՇՆԺՍՌՊԹՌՀԽՊՋՀՇՍՄԼԺԽԻՇՆՋՁԼԽՊԽԼ

ՁՆՌՀԽԻՇՆՌԽՐՌՇԾՇՌՀԽՊԹՋՋԹՑՋՁՆՌՀԽԺԹՌՌԽՊՑԹՋՏԽՄՄԹՋԬՌՀԽՊ ԰ԻՁԽՆՌՁԾՁԻԹՄՄՑԯԽՄԽՎԹՆՌԦՆԾՇՊՅԹՌՁՇՆ
Ԭ԰ԯԦ

ԦՆՇՊԼԽՊՌՇԼԽՌԽՊՅՁՆԽՁԾԹԻՀԽՅՁԻԹՄՀԹՋՌՀԽՈՇՌԽՆՌՁԹՄՌՇՁՆՌԽՊԹ ԻՌՏՁՌՀՌՀԽԢԞՇՊԱՈԹՌՀՏԹՑՋԹԴԽՁԿՀՌՇԾ

ԢՎՁԼԽՆԻԽ
ԴՇԢԽՎԹՄՍԹՌՁՇՆՇԾԱՁԽՊԹՋՋԹՑՊԽՋՍՄՌՋՁՆԻՇՅԺՁՆԹՌՁ ՇՆՏՁՌՀՌՀԽԾՁՆԼՁՆԿՋՁՆՌՀԽԬ԰ԯԦՋՀՇՍՄԼ

ԺԽՍՆԼԽՊՌԹՃԽՆ
ՊԽԾԽՊՌՇՌՀԽԴՇԢԡՇԻՍՅԽՆՌ

ԢԵԢԠԲԱԦԳԢ԰ԲԪԪԞԯԶ

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

ԻՇՆԼՍԻՌԽԼՍՆԼԽՊԾՄՇՏՌՀՊՇՍԿՀԻՇՆԼՁՌՁՇՆՋԞԼՍՄՌԾՁՋՀ
ՏԽԽՃՋՇ ՄԼՅԹՄԽՋԹՆԼԾԽՅԹՄԽՋՁՆԽԹԻՀԿՊՇՍՈ

ԿՊՇՍՈՋՈԽՊՌՊԽԹՌՅԽՆՌՏԽՊԽԽՐՈՇՋԽԼՌՇԻԹՊԺԹՊՑՄ
ՈՍՊՁՌՑ ԹՌՆՇՅՁՆԹՄԻՇՆԻԽՆՌՊԹՌՁՇՆՋՇԾ
ՆԽԿԹՌՁՎԽ

ԻՇՆՌՊՇՄ   ԹՆԼ  ՅԿ ԹՁԩ ՅԽԹՆՅԽԹՋՍՊԽԼ ԻՇՆԻԽՆ ՌՊԹՌՁՇՆՋ ՏԽՊԽ  
ԩԬԮ

ԻՇՆՌՊՇՄԹՆԼՅԿԹՁԩԱՀԽՌԽՋՌՋՑՋՌԽՅՏԹՋ ՅԹՁՆՌԹՁՆԽԼԹՌՌՇ ոԠԹՆԼԹՈԥՇԾ

ՌՇ

ԞՄՄԽԾԾԽԻՌՋԹՊԽՊԽՈՇՊՌԽԼԺԹՋԽԼՇՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽՆԽԿԹՌՁՎԽԻՇ ՆՌՊՇՄԪԽԹՆՋՍՊՎՁՎԹՄՁՆՅԹՄԽՋՏԹՋՁՆ

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


ԣԽՅԹՄԽՋՍՊՎՁՎԹՄՏԹՋ


ԹՆԼ
ՁՆՌՀԽԻՇՆՌՊՇՄ

 ԹՆԼՅԿ ԹՁԩԿՊՇՍՈՋ ՊԽՋՈԽԻՌՁՎԽՄՑԠՇՅԺՁՆԽԼՋԽՐՋՍՊ ՎՁՎԹՄՏԹՋ 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

### ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋ Հ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹ ՑՏՁՌՀԠԹՊԺԹՊՑՄ

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 ՌՊԽԹՌՅԽ ՆՌ ԿՊՇՍՈ ԞԼԼՁՌՁՇՆԹՄՄՑ ՈՄԹՋՅԹ ՎՁՌԽՄՄՇԿԽՆՁՆ

ԳԱԤԻՇՆԻԽՆՌՊԹՌՁՇՆՋՏԽՊԽՍՆԹԾԾԽ ԻՌԽԼՁՆՅԹՄԽՋԹՆԼԾԽՅԹՄԽՋԹՆԼՌ ՀԽՊԽՏԽՊԽՆՇՌՊԽԹՌՅԽՆՌՊԽՄԹՌԽԼԽԾԾԽԻՌՋՇՆ

Ԥ԰ԦՁՆԽՁՌՀԽՊՋԽՐԥՇՏԽՎԽՊՅԽԼՁԹՆՅԹՄԽՆՍՈՌՁԹՄՌՍԺԽՊԻՄԽՋԻՇՊԽ ՏԹՋՄՇՏԽՊՁՆՌՀԽՅԹՁԩՌՊԽԹՌՅԽՆՌ

ԿՊՇՍՈ
ՌՀԹՆՁՆԻՇՆՌՊՇՄԾՁՋՀ

ՈԥՁՋՌՇՈԹՌՀՇՄՇԿՁԻԹ ՄՇԺՋԽՊՎԹՌՁՇՆՋՁՆՅԹՄԽՋՁՆԻՄՍԼԽԼՁՆԻՊԽԹՋԽՋ

ՁՆ ՅՁՆՁՅԹՄ ՌԽՋՌՁԻՍՄԹՊ ԼԽԿԽՆԽՊԹՌՁՇՆ ԹՆԼ ՌՀԽ ՈԽՊԻԽՆՌԹԿԽ ՇԾ ՋՈԽՊՅԹ ՌՇԿՇՆՁԹ ԹՌ ՌՀԽ ՅԽԹՆՅԽԹՋՍՊԽԼ  ՅԿ

ԹՁԩՌՊԽԹՌՅԽՆՌՄԽՎԽՄԫՇՊԽՅԹՊՃԹԺՄԽՀՁՋՌՇՈԹՌՀՇՄՇԿՁԻԹՄՇԺՋԽՊՎԹ ՌՁՇՆՋՏԽՊԽՆՇՌԽԼՁՆՅԹՄԽՋԹՌՇՌՀԽՊՌՊԽԹՌՅԽՆՌ

ՄԽՎԽՄՋՇՊՁՆԾԽՅԹՄԽՋԭՄԹՋՅԹՋԽՐՋՌԽՊՇՁԼՋՏԽՊԽՆՇՌՅԽԹՋՍՊԽԼ

ԞՄՄՈԽՊԾՇՊՅԹՆԻԽԻՊՁՌԽՊՁԹՏԽՊԽՅԽՌՁՆՌՀՁՋՋՌՍԼՑԽՐԻԽՈՌՌՀԹՌԾՇ ՊԻՇՆՌՊՇՄՊԽՈՄՁԻԹՌԽԡՋՈԹՏՆՁՆԿԼՁԼՆՇՌՇԻԻՍՊԹՌ

ՄԽԹՋՌԽՎԽՊՑԾՇՍՊԼԹՑՋՆՇՊՏԹՋՌՀԽԹՎԽՊԹԿԽԾԽԻՍՆԼՁՌՑԽԿԿՋԾԽՅ ԹՄԽԼԹՑՇՊԿՊԽԹՌԽՊԥՇՏԽՎԽՊՌՀԽԹՎԽՊԹԿԽ

ԾԽԻՍՆԼՁՌՑԾՇՊՌՀԹՌ ՊԽՈՄՁԻԹՌԽՏԹՋԽԿԿՋԾԽՅԹՄԽԼԹՑԼՍՊՁՆԿՌՀԽԾՁՊՋՌՏԽԽՃՇԾԽՐՈՇՋՍՊԽԹՆԼԾԽԻՍՆԼՁՌՑՏԹՋ

ՁՆԻՊԽԹՋԽԼ ՇՎԽՊՌՀԽ ԼՍՊԹՌՁՇՆ ՇԾՌԽՋՌՁՆԿԞԼԼՁՌՁՇՆԹՄՄՑՌՀԽԾԽԻՍՆԼՁՌՑ ՊԹՌԽՋ ՁՆՌՀԽ ՇՌՀԽՊ ՊԽՈՄՁԻԹՌԽՋՏԽՊԽ 

ԽԿԿՋԾԽՅԹՄԽԼԹՑՁՆԼՁԻԹՌՁՆԿՌՀԹՌԾԽԻՍՆԼՁՌՑՏԹՋԹԼԽՉՍԹՌԽՁՆՌՀԽ ԻՇՆՌՊՇՄԿՊՇՍՈ

ԱՀՁՋԹՋՋԹՑ ՋԹՌՁՋԾՁԽՋ ՌՀԽԢԡ԰ԭԱՁԽՊԱԽՋՌԬՊԼԽՊ ՊԽՉՍՁՊԽՅԽՆՌՋԾՇՊԹԣՁՋՀ԰ՀՇՊՌԱԽՊՅ ԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑ

ԬԠ԰ԭԭԤՍՁԼԽՄՁՆԽ

ԯԽՋՍՄՌՋ԰ՑՆՇՈՋՁՋ

ԱԽՋՌՇՊԿԹՆՁՋՅԹԿԽԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆՏԽԽՃՋ

ԪԽԹՆԺՇԼՑՏԽՁԿՀՌԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ
ԿԾՇՊՅԹՄԽՋ 
ԿԾՇՊԾԽՅԹՄԽՋ

ԪԽԹՆՄԽՆԿՌՀԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆԫՇՌԯԽՈՇՊՌԽԼ

ԱԽՋՌՌՑՈԽԾՄՇՏՌՀՊՇՍԿՀԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽՇԾԱԹԺՄԽ԰ՍՅՅԹՊՑՇԾԯԽՈՊՇԼՍԻՌՁՎԽԹՆԼԥԭԤԢԾԾԽԻՌՋՁՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑ
ԣ԰ԱԯԞՏՁՌՀԠԹՊԺԹՊՑՄԱՊԽԹՌՅԽՆՌՅԿԹՁԩ#ՅԽԹՆՅԽԹՋՍՊԽԼ%ԣԽԻՍՆԼՁՌՑԣԽՊՌ԰ՍԻԻԽՋՋԱՍԺԽՊԻՄԽ԰ԻՇՊԽԤ԰ԦԤՇՆԹԼԹՄԥՁՋՌՇԭՄԹՋՅԹԳԱԤԭՄԹՋՅԹԱԭՄԹՋՅԹԢԪԣԪԣԪԣԪԣԪԣԪԣԫՇ ԫՇ ԫՇ ԫՇ ԫՇ ԫՇ ԫՇ ԫՇ ԫՇ ԫՇ    ԫՇ ԫՇ ԫՇ ԫՇ ԫՇ ԫՇ ԫՇ ԫՇ ԫՇ ԫՇ    ԫՇ ԫՇԶԽՋԫՇ ԫՇ ԫՇԶԽՋԫՇ ԫՇ ԫՇ    ԞԺԺՊԽՎՁԹՌՁՇՆՋԠՇՆԻԠՇՆԻԽՆՌՊԹՌՁՇՆԡՁԾԾԡՁԾԾԽՊԽՆԻԽԢ֦ԽՋՌՊԹԼՁՇՄԣԣԽՅԹՄԽԣԽՊՌԣԽՊՌՁՄՁՒԹՌՁՇՆԤ԰ԦԤՇՆԹԼՇ԰ՇՅԹՌՁԻԦՆԼԽՐԥՁՋՌՇԥՁՋՌՇՈԹՌՀՇՄՇԿՑԪԪԹՄԽԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽԱԱԽՋՌՇՋՌԽՊՇՆԽԳԱԤԳՁՌԽՄՄՇԿԽՆՁՆ Ԟ՟ՑԽՋՠՁՆԼՁԻԹՌԽՋԹՋՁԿՆՁԾՁԻԹՆՌԼՁԾԾԽՊԽՆԻԽԺԹՋԽԼՇՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽՆԽԿԹՌՁՎԽ
ԻՄԽԹՆՏԹՌԽՊԻՇՆՌՊՇՄՍՆՄԽՋՋՇՌՀԽՊՏՁՋԽՋՈԽԻՁԾՁԽԼ ԱՀԽԻՊՁՌԽՊՁԹԾՇՊՋՁԿՆՁԾՁԻԹՆԻԽԹՊԽԼԽՋԻՊՁԺԽԼՁՆՌՀԽԯԽՎՁԽՏԽՊ0ՋԞՆԹՄՑՋՁՋԹՆԼ԰ՌԹՌՁՋՌՁԻԹՄԳԽՊՁԾՁԻԹՌՁՇՆՋԽԻՌՁՇՆՋՇԾՌՀԽԡԢԯԠՇՆԻՄՍՋՁՇՆՋՊԽԿԹՊԼՁՆԿՀՁՋՌՇՈԹՌՀՇՄՇԿՑՅԹՑԺԽՀԽԹՎՁՄՑՏԽՁԿՀՌԽԼԺՑՌՀԽԽՐՈԽՊՌՇՈՁՆՁՇՆՇԾԹԺՇԹՊԼԻԽՊՌՁԾՁԽԼՈԹՌՀՇՄՇԿՁՋՌ

### ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋ Հ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹ ՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ 

ԭԹԿԽՇԾ Ԧ ԪԞԱԢԯԦԞԩ԰ԞԫԡԪԢԱԥԬԡ԰

ԤՍՁԼԽՄՁՆԽԣՇՄՄՇՏԽԼ ԱՀՁՋՋՌՍԼՑՏԹՋԻՇՆԼՍԻՌԽԼԾՇՄՄՇՏՁՆԿՌՀԽՈՊՇՌՇԻՇՄԼԽՎԽՄՇՈԽԼԺՑԞՆ ՃՄԽՑԽՌԹՄ

ԹՆԼՅԽԽՌՋՌՀԽՊԽՉՍՁՊԽՅԽՆՌՋՇԾԬԠ԰ԭԭԤՍՁԼԽՄՁՆԽԹՆԼ ԬԢԠԡ

ԤՍՁԼԽՄՁՆԽԾՇՊՌՀԽԾՁՋՀՋՀՇՊՌՌԽՊՅՊԽՈՊՇԼՍԻՌՁՇՆԹՋՋԹՑԱՀԽԾ ՇՄՄՇՏՁՆԿ

ԼԽՎՁԹՌՁՇՆՋՏԽՊԽՆՇՌԽԼ

 ԱՀՊԽԽՇԾՌՀԽՋՈԹՏՆՁՆԿԿՊՇՍՈՋՀԹԼՈՊԽԽՐՈՇՋՍՊԽԾԽԻՍՆԼՁՌՑՇ ԾՌՇ

ԽԿԿՋԾԽՅԹՄԽԼԹՑԿՍՁԼԹՆԻԽՊԽԻՇՅՅԽՆԼՋԹՌՄԽԹՋՌԽԿԿՋԾԽՅԹՄԽԼԹ Ց

ԥՇՏԽՎԽՊԽԹԻՀՇԾՌՀԽՋԽԿՊՇՍՈՋՋՈԹՏՆԽԼԹՌՄԽԹՋՌԽՎԽՊՑԼԹՑՋ

 ԠՇՆԻԽՆՌՊԹՌՁՇՆՋՇԾՍՆՁՇՆՁՒԽԼԹՅՅՇՆՁԹԹՆԼՊԽՋՁԼՍԹՄԻՀՄՇՊՁՆԽՁՆ ՌՀԽԼՁՄՍՌՁՇՆ

ՏԹՌԽՊՏԽՊԽՆՇՌՊԽՈՇՊՌԽԼ

 ԦՆՊԽՈՄՁԻԹՌԽԡՇԾՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄՋՈԹՏՆՁՆԿԼՁԼՆՇՌՇԻԻՍՊԹՌՄԽԹՋՌԽՎԽՊՑ

ԾՇՍՊԼԹՑՋՆՇՊՏԹՋՌՀԽԹՎԽՊԹԿԽԾԽԻՍՆԼՁՌՑԽԿԿՋԾԽՅԹՄԽԼԹՑՇՊԿ ՊԽԹՌԽՊ

ԥՇՏԽՎԽՊՌՀԽԹՎԽՊԹԿԽԾԽԻՍՆԼՁՌՑԾՇՊՌՀԹՌՊԽՈՄՁԻԹՌԽՏԹՋԽԿԿՋԾ ԽՅԹՄԽԼԹՑ

ՏՀՁԻՀՏԹՋՋՄՁԿՀՌՄՑՄԽՋՋՌՀԹՆՌՀԽՊԽԻՇՅՅԽՆԼԽԼՊԹՌԽԹՆԼՌՀԽԾԽԻՍ ՆԼՁՌՑՊԹՌԽՋՁՆ

ՌՀԽՇՌՀԽՊՊԽՈՄՁԻԹՌԽՋՊԹՆԿԽԼԾՊՇՅՌՇԽԿԿՋԾԽՅԹՄԽԼԹՑՁՆԼՁԻ ԹՌՁՆԿՌՀԹՌ

ԾԽԻՍՆԼՁՌՑՏԹՋԹԼԽՉՍԹՌԽՁՆՌՀԽԻՇՆՌՊՇՄՌՊԽԹՌՅԽՆՌ

ԱՀԽՋԽԼԽՎՁԹՌՁՇՆՋԼՇՆՇՌՁՅՈԹԻՌՌՀԽԹԻԻԽՈՌԹԺՁՄՁՌՑՇՊՊԽՋՍՄՌՋՇԾ ՌՀԽՋՌՍԼՑ

ԠՇՅՈՄՁԹՆԻԽ ԰ՁԿՆԽԼԹՆԼԼԹՌԽԼԫՇԡԹՌԹԠՇՆԾՁԼԽՆՌՁԹՄՁՌՑԤԩԭԹՆԼԮՍԹՄՁՌՑԞՋՋՍ ՊԹՆԻԽ

ՋՌԹՌԽՅԽՆՌՋՏԽՊԽՈՊՇՎՁԼԽԼԱՀՁՋՋՌՍԼՑՏԹՋԻՇՆԼՍԻՌԽԼՁՆԻՇՅՈՄՁԹՆԻ ԽՏՁՌՀ

ԹՄՄՈԽՊՌՁՆԽՆՌԲ԰ԢԭԞ
ԠԣԯԭԹՊՌԤՇՇԼԩԹԺՇՊԹՌՇՊՑԭՊԹԻՌ ՁԻԽ

ՊԽԿՍՄԹՌՁՇՆՋՏՁՌՀՌՀԽԾՇՄՄՇՏՁՆԿԽՐԻԽՈՌՁՇՆՋՊՇՍՌՁՆԽԾՇՇԼԹՆԼՏԹՌ ԽՊՋԻՊԽԽՆՁՆԿ

ԹՆԹՄՑՋԽՋՏԽՊԽԻՇՆԼՍԻՌԽԼԹՌԤԽՇԩԹԺՋԦՆԻԟՊԹՁՆՌՊԽԽԪԹՋՋԹԻՀՍՋԽ ՌՌՋ

ՍՋՁՆԿՋՌԹՆԼԹՊԼԲ԰ԢԭԞՈՊՇԻԽԼՍՊԽՋԹՆԼԹՊԽԻՇՆՋՁԼԽՊԽԼԾԹԻՁՄՁՌՑ ՊԽԻՇՊԼՋ

ՍՆԼԽՊ԰ՅՁՌՀԽՊՋԳՁՋԻՁԽՆՌ0Ջ԰ՌԹՆԼԹՊԼԬՈԽՊԹՌՁՆԿԭՊՇԻԽԼՍՊԽՋ

ԞԱԽՋՌԪԹՌԽՊՁԹՄ ԠԹՊԺԹՊՑՄ

ԡԽՋԻՊՁՈՌՁՇՆ ԞՈՀՑՋՁԻԹՄԼԽՋԻՊՁՈՌՁՇՆՏԹՋՆՇՌՈՊՇՎՁԼԽԼ

### ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋ Հ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹ ՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ 

ԭԹԿԽՇԾ ԬԢԠԡՊԽԻՇՅՅԽՆԼՋԼԽՋԻՊՁԺՁՆԿՏԹՌԽՊՋՇՄՍԺՁՄՁՌՑՅԽՄՌՁՆԿԺՇ ՁՄՁՆԿՈՇՁՆՌՋՌԹԺՁՄՁՌՑՁՆՏԹՌԽՊԹՆԼՄՁԿՀՌ

ՈԨԹԭՇՏՇՊԨՇՏՎԹՈՇՊՈՊԽՋՋՍՊԽՇԾՌԽՋՌԻՇՅՈՇՍՆԼԽՐՈՁՊԹՌՁՇՆԼԹ ՌԽ

ԩՇՌԫՇԟԹՌԻՀԫՇ ԡԠ
ԩՇՌԫՇ԰ԪԳԫՇ

ԭՍՊՁՌՑ 

ԦՅՈՍՊՁՌՁԽՋ ԫՇՆԽԯԽՈՇՊՌԽԼ

԰ՌԹԺՁՄՁՌՑՇԾԠՇՅՈՇՍՆԼ ԰ՌԹԺՄԽԪԽԹՆՅԽԹՋՍՊԽԼԻՇՆԻԽՆՌՊԹՌՁՇՆՋՑՁԽՄԼԽԼՊԽԻՇՎԽՊՁԽՋՇԾՌՇ

ՇԾՆՇՅՁՆԹՄՏՁՌՀԻՇԽԾԾՁԻՁԽՆՌՋՇԾՎԹՊՁԹՌՁՇՆ
ԠԳՇԾՌՇ

԰ՌՇՊԹԿԽԠՇՆԼՁՌՁՇՆՋՇԾ

ԱԽՋՌԠՀԽՅՁԻԹՄՋ ԰ՌՇՊԽԼԹՌՊՇՇՅՌԽՅՈԽՊԹՌՍՊԽՁՆՌՀԽՇՊՁԿՁՆԹՄԻՇՆՌԹՁՆԽՊՁՆԹԼԹՊՃ ՎԽՆՌՁՄԹՌԽԼ

ԻԹԺՁՆԽՌԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽՇԾԟԱԽՋՌԬՊԿԹՆՁՋՅԱԹԺՄԽԤԽՆԽՊԹՄԦՆԾՇՊՅԹՌՁՇՆԞԺՇՍՌՌՀԽԱԽՋՌ԰ՈԽԻՁԽՋԹՆԼԞԻԻՄՁՅԹՌՁՇՆԭԹՊԹՅԽՌԽՊԳԹՄՍԽ
ՋԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ԰ՈԽԻՁԽՋԻՇՅՅՇՆՆԹՅԽԣԹՌՀԽԹԼԪՁՆՆՇՏ ԢԭԞՊԽԻՇՅՅԽՆԼՋԾԹՌՀԽԹԼՅՁՆՆՇՏ
ԭՁՅԽՈՀԹՄԽՋ

ՈՊՇՅԽՄԹՋ԰ՈԽԻՁԽՋՋԻՁԽՆՌՁԾՁԻՆԹՅԽ ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ԰ՈԽԻՁԽՋՋՌՊԹՁՆ
ՁԾՋՌԹՌԽԼԫՇՌԯԽՈՇՊՌԽԼ԰ԪԳԩՇՌԫՇԞԴԽՊԽԾՁՋՀՇԺՌԹՁՆԽԼԾՊՇՅԹՋՁՆԿՄԽՄԹԺՇՊԹՌՇՊՑՋՌՇԻՃ!ԶԽՋ ԰ՌՇԻՃԾՁՋՀՏԽՊԽՊԽՈՊՇԼՍԻՌՁՎԽՄՑՅԹՌՍՊԽԹՆՁՅԹՄՋԹՈՈՊՇՐՁՅԹՌԽՄՑՏԽԽՃՋՇՄԼԹՆԼՁՆՋՈԹՏՆՁՆԿԻՇՆԼՁՌՁՇՆ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌԾՁՋՀԺԽԾՊՇՅԹ ՋՁՆԿՄԽՄԹԺՇՊԹՌՇՊՑՋՌՇԻՃԴԽՊԽԹԻԻՄՁՅԹՌՁՇՆԻՇՆԼՁՌՁՇՆՋՋԹՅԽԹՋԼԽԾՁՆՁՌՁՎԽՌԽՋՌ!ԶԽՋ  ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌԾՁՋՀԺԽԹԻԻՄՁՅԹՌԽԼՍՆԼԽՊ ՏԹՌԽՊՉՍԹՄՁՌՑԹՆԼՁՄՄՍՅՁՆԹՌՁՇՆԻՇՆԼՁՌՁՇՆՋՌՀԹՌԹՊԽ ՋՁՅՁՄԹՊՌՇՌՀԽԼԽԾՁՆՁՌՁՎԽՌԽՋՌԞԻԻՄՁՅԹՌՁՇՆՈԽՊՁՇԼՏԽԽՃՋ ԢԭԞ ՊԽԻՇՅՅԽՆԼՋ Թ ՅՁՆՁՅՍՅ ՌՏՇՏԽԽՃ ԹԻԻՄՁՅԹՌՁՇՆ ՈԽՊՁՇԼ ԫՇՌԽ ՌՀԹՌ ՌՀԽ ԹԻԻՄՁՅԹՌՁՇՆ ՈԽՊՁՇԼ ՁՋ ԼՁԾԾԽՊԽՆՌ ԾՊՇՅ ՌՀԽ ՋՍԺՋԽՉՍԽՆՌ ՁՆ ՋՁՌՍ
ՈՊԽԽՐՈՇՋՍՊԽՈՀԹՋԽԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽՇԾԭԹՊԹՅԽՌԽՊԳԹՄՍԽ
ՋԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋԡԽՌԹՁՄՋՇՆՀԽԹՄՌՀԫՇՋՁԿՆՁԾՁԻԹՆՌՅՇՊՌԹՄՁՌՑՏԹՋՇԺՋԽՊՎԽԼՁՆՌՀՁՋՈՇՈՍՄԹՌՁՇՆՁՆՌՀԽՋԽՎԽՆԼԹՑՋՈՊՁՇՊՌՇՌՀԽՈՊԽԽՐՈՇՋՍՊԽՈԽՊՁՇԼԣՁՋՀԼՁԼՆՇՌՊԽԻԽՁՎԽՌՊԽԹՌՅԽՆՌԾՇՊԼՁՋԽԹՋԽՁՆՌՀԽՌՏՇՏԽԽՃՋՈՊՁՇՊՌՇՌՀԽՋՈԹՏՆՁՆԿՉՍԹՄՁԾՁԻԹՌՁՇՆՈԽՊՁՇԼՈՊԽԻԽԼՁՆԿՌՀԽՌԽՋՌՇՊԼՍՊՁՆԿՌՀԽԽՐՈՇՋՍՊԽՈԽՊՁՇԼ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՅՇՊՌԹՄՁՌՑԼՍՊՁՆԿՌՀԽԼԹՑՋ ՈՊՁՇՊՌՇՌՀԽՈՊԽԽՐՈՇՋՍՊԽՈՀԹՋԽԺԽՄԽՋՋՌՀԹՆ ՇԾՌՀԽԻՍՄՌՍՊԽՈՇՈՍՄԹՌՁՇՆԦԾՅՇՊՌԹՄՁՌՑԼՍՊՁՆԿՌՀԽՋԽ ԼԹՑՋՁՋԿՊԽԹՌԽՊՌՀԹՆԢԭԞՊԽԻՇՅՅԽՆԼՋ ՌՀԹՌՌՀԽԾՁՋՀԺԽՊԽՂԽԻՌԽԼԦԾՅՇՊՌԹՄՁՌՑՁՋԺԽՌՏԽԽՆ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌԾՁՋՀԺԽՀԽՄԼԹՆՇՌՀԽՊ ԼԹՑՋԦԾՅՇՊՌԹՄՁՌՁԽՋԿՊԽԹՌԽՊՌՀԹՆՇԻԻՍՊ ԼՍՊՁՆԿՌՀՁՋԽՐՌԽՆԼԽԼԹԻԻՄՁՅԹՌՁՇՆՈԽՊՁՇԼԢԭԞ ՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՀԽԾՁՋՀՆՇՌԺԽՍՋԽԼԱՑՈԽՇԾԾՇՇԼԩՁՎԽԺՊՁՆԽՋՀՊՁՅՈՆԹՍՈՄՁՁ  ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌԾՁՋՀԺԽԾԽԼԾՊՇՒԽՆԺՊՁՆԽ ՋՀՊՁՅՈՌՏՁԻԽՈԽՊԼԹՑՌՇՈՊՇՅՇՌԽԹԻՌՁՎԽ ՊԽՈՊՇԼՍԻՌՁՇՆԹՆԼՅԹՁՆՌԹՁՆԺՇԼՑԻՇՆԼՁՌՁՇՆ԰ՇՍՊԻԽՇԾԾՇՇԼԫՇՌՋՈԽԻՁԾՁԽԼԩՇՌԫՇԣՊԽՉՍԽՆԻՑՇԾԾԽԽԼՁՆԿՌՇՌՁՅԽՋԼԹՑ ԡԽՌԹՁՄՋՇՆԾԽԽԼՁՆԿԣՁՋՀՏԽՊԽԾԽԼԹՅԽԹՋՍՊԽԼԹՅՇՍՆՌՇԾՄՁՎԽԺՊՁՆԽՋՀՊՁՅՈՆԹՍՈՄՁՁԣՁՋՀՏԽՊԽԾԽԼՌՏՁԻԽԼԹՁՄՑԼՍՊՁՆԿՌՀԽՈՊԽԽՐՈՇՋՍՊԽՍՈՌՇԼԹՑՆՁՆԽԹԾՌԽՊՏՀՁԻՀԾԽԽԼՁՆԿՏԹՋՁՆԻՊԽԹՋԽԼՌՇՌՀՊԽԽՌՁՅԽՋԼԹՁՄՑԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽՇԾԱԹԺՄԽԣՁՋՀ԰ԽՄԽԻՌՁՇՆԹՆԼԭՊԽԢՐՈՇՋՍՊԽԭԽՊԾՇՊՅԹՆԻԽԭԹՊԹՅԽՌԽՊԳԹՄՍԽ
ՋԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋԞԿԽԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆՏԽԽՃՋ ԢԭԞՊԽԻՇՅՅԽՆԼՋՊԽՈՊՇԼՍԻՌՁՎԽՄՑՅԹՌՍՊԽ
ՋԽՐՍԹՄՄՑ ԼՁՅՇՊՈՀՁԻԾՁՋՀՅՇՆՌՀՋՇՄԼԪԽԹՆՏԽՁԿՀՌՇԾՅԹՄԽՋԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ
ՁԾԼԽՌԽՊՅՁՆԽԼԿ ԯԽՈՊԽՋԽՆՌԹՌՁՎԽՋԹՅՈՄԽ
ԫԾՊՇՅՌՀԽՌԽՋՌՈՇՈՍՄԹՌՁՇՆ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌԹՋՍԺՋԹՅՈՄԽՇԾԾՁՋՀԺԽ ՏԽՁԿՀԽԼԺԽԾՇՊԽՌՀԽՌԽՋՌՌՇԽՋՌՁՅԹՌԽՌՀԽՅԽԹՆ ՏԽՁԿՀՌԾՇՊԽԹԻՀՋԽՐԦՌՁՋՊԽԻՇՅՅԽՆԼԽԼՌՀԹՌ ՌՀԽՁՆԼՁՎՁԼՍԹՄՏԽՁԿՀՌՇԾԽԹԻՀԾՁՋՀՋԽՄԽԻՌԽԼԾՇՊՌՀԽ ՌԽՋՌԺԽՏՁՌՀՁՆչՇԾՌՀԽԽՋՌՁՅԹՌԽԼՅԽԹՆԾՇՊ

ԽԹԻՀՋԽՐԯԹՆԿԽՇԾՁՆԼՁՎՁԼՍԹՄՏԽՁԿՀՌՋ
ՅԹՄԽՋԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ
ՁԾԼԽՌԽՊՅՁՆԽԼԿԿ
ՏՁՌՀՁՆՇԾՌՀԽՅԽԹՆԪԽԹՆՏԽՁԿՀՌՇԾԾԽՅԹՄԽՋԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ
ՁԾԼԽՌԽՊՅՁՆԽԼԿ ԯԽՈՊԽՋԽՆՌԹՌՁՎԽՋԹՅՈՄԽ
ԫԾՊՇՅՌՀԽՌԽՋՌՈՇՈՍՄԹՌՁՇՆԯԹՆԿԽՇԾՁՆԼՁՎՁԼՍԹՄՏԽՁԿՀՌՋԾԽՅԹՄԽՋԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ
ՁԾԼԽՌԽՊՅՁՆԽԼԿԿ
ՏՁՌՀՁՆՇԾՌՀԽՅԽԹՆԪԽԹՆՄԽՆԿՌՀՇԾՅԹՄԽՋԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆՁԾԼԽՌԽՊՅՁՆԽԼԫՇՌՊԽՈՇՊՌԽԼԪԽԹՆՄԽՆԿՌՀՇԾԾԽՅԹՄԽՋԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ
ՁԾԼԽՌԽՊՅՁՆԽԼԫՇՌՊԽՈՇՊՌԽԼ  ԡՍՊԹՌՁՇՆՇԾՈՊԽԽՐՈՇՋՍՊԽՈՀԹՋԽ ԼԹՑՋ ԢԭԞՊԽԻՇՅՅԽՆԼՋԹՅՁՆՁՅՍՅՇԾԼԹՑՋԴԽՊԽՈՊԽԽՐՈՇՋՍՊԽԻՇՆԼՁՌՁՇՆՋՁԼԽՆՌՁԻԹՄՌՇՌՀԽԼԽԾՁՆՁՌՁՎԽՌԽՋՌ!ԶԽՋ  ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՈՊԽԽՐՈՇՋՍՊԽԻՇՆԼՁՌՁՇՆՋ ՁՆԻՄՍԼՁՆԿՌԽՅՈԽՊԹՌՍՊԽՈՀՇՌՇՈԽՊՁՇԼԾԽԽԼՁՆԿԽՌԻ ԺԽՁԼԽՆՌՁԻԹՄՌՇԼԽԾՁՆՁՌՁՎԽՌԽՋՌԻՇՆԼՁՌՁՇՆՋԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽՇԾԭԹՊԹՅԽՌԽՊԳԹՄՍԽ
ՋԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋԫՍՅԺԽՊՇԾՈՊԽԽՐՈՇՋՍՊԽՌԹՆՃՋ ԞԼԼՁՌՁՇՆԹՄԽՐՈՇՋՍՊԽԻՀԹՅԺԽՊՋՏԽՊԽՋԽՌՍՈԼՍՊՁՆԿՌՀԽՈՊԽԽՐՈՇՋՍՊԽՈՀԹՋԽՌՇԹԻԻՇՍՆՌԾՇՊԹՈՇՌԽՆՌՁԹՄՄԹԻՃՇԾՋՈԹՏՆՁՆԿՁՆՋՇՅԽԻՀԹՅԺԽՊՋԹՆԼՇՊՅՇՊՌԹՄՁՌՑԼՍՊՁՆԿՌՀԽՈՀԹՋԽ ԢԭԞ ՊԽԻՇՅՅԽՆԼՋ ՌՀԹՌ ԹԼԼՁՌՁՇՆԹՄ ՌԹՆՃՋ ՋԽՌ ՍՈ ԹՌ ՌՀԽ ԺԽԿՁՆՆՁՆԿ ՇԾ ՈՊԽԽՐՈՇՋՍՊԽ ՏՁՄՄ ԽՆՋՍՊԽ ՌՀԹՌ ՋՍԾԾՁԻՁԽՆՌ ՊԽՈՄՁԻԹՌԽՋ ՏՁՌՀ ՌՀԽ ԻՇՊՊԽԻՌ ՋԽՐ ՊԹՌՁՇ ԹՊԽ

ԹՎԹՁՄԹԺՄԽ ԾՇՊՌՀԽԼԽԾՁՆՁՌՁՎԽՌԽՋՌԫՍՅԺԽՊՇԾՅԹՄԽՋՈԽՊՌԹՆՃ ԫՍՅԺԽՊՇԾԾԽՅԹՄԽՋՈԽՊՌԹՆՃ ԭՊԽԽՐՈՇՋՍՊԽԾԽԻՍՆԼՁՌՑՇԾՌՀԽՋՈԹՏՆՁՆԿԿՊՇՍՈՋՍՋԽԼՁՆՌՀԽԼԽԾՁՆՁՌՁՎԽԽՐՈՇՋՍՊԽՀԹԼՈՊԽԽՐՈՇՋՍՊԽԾԽԻՍՆԼՁՌՑՇԾԽԿԿՋԾԽՅԹՄԽՊԽՈՊՇԼՍԻՌՁՎԽԼԹՑՊԽՈՄՁԻԹՌԽԱՀԽՊԽՅԹՁՆՁՆԿՌՀՊԽԽԿՊՇՍՈՋՀԹԼԾԽԻՍՆԼՁՌՑՊԹՆԿՁՆԿԾՊՇՅՌՇԽԿԿՋԾԽՅԹՄԽԼԹՑԹՄՌՀՇՍԿՀՌՀԽՋԽԿՊՇՍՈՋՋՈԹՏՆԽԼԹՌՄԽԹՋՌՇՆԻԽԽՎԽՊՑԼԹՑՋ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՈՊԽԽՐՈՇՋՍՊԽԾԽԻՍՆԼՁՌՑՁՆ ԽԹԻՀՊԽՈՄՁԻԹՌԽ
ՌԹՆՃՋԽՄԽԻՌԽԼԾՇՊՍՋԽՁՆՌՀԽ ԼԽԾՁՆՁՌՁՎԽՌԽՋՌԺԽԹՌՄԽԹՋՌ ԽԿԿՋԾԽՅԹՄԽՊԽՈՊՇԼՍԻՌՁՎԽԼԹՑՊԽՈՄՁԻԹՌԽԼՍՊՁՆԿ ՌՀԽԼԹՑՋՈՊՁՇՊՌՇՌՀԽԼԽԾՁՆՁՌՁՎԽՌԽՋՌԫՍՅԺԽՊՇԾՋՈԹՏՆՋԼՍՊՁՆԿՈՊԽԽՐՈՇՋՍՊԽՌՁՅԽՋՁՆԼԹՑՋ  ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՋՈԹՏՆՁՆԿՇԻԻՍՊԹՌՄԽԹՋՌ ՌՏՁԻԽՁՆՌՀԽԼԹՑՋՈՊՁՇՊՌՇՌՀԽԼԽԾՁՆՁՌՁՎԽՌԽՋՌԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽՇԾԭԹՊԹՅԽՌԽՊԳԹՄՍԽ
ՋԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋԡԽՌԹՁՄՋՇՆՈՊԽԽՐՈՇՋՍՊԽԡՍՊՁՆԿՌՀԽՈՊԽԽՐՈՇՋՍՊԽՈՀԹՋԽՋՍՁՌԹԺՁՄՁՌՑԾՇՊՌԽՋՌՁՆԿՏԹՋԽՋՌԹԺՄՁՋՀԽԼՏՀԽՆՊԽԿՍՄԹՊՋՈԹՏՆՁՆԿՇԻԻՍՊՊԽԼՁՆԽԹԻՀՊԽՈՄՁԻԹՌԽԻՀԹՅԺԽՊԹՌՄԽԹՋՌՌՏՁԻԽՁՆՌՀԽՁՅՅԽԼՁԹՌԽԼԹՑՈԽՊՁՇԼՈՊԽԻԽԼՁՆԿՌԽՋՌՁՆՁՌՁԹՌՁՇՆՇՊՏՀԽՆԹՆԽԿԿՈՊՇԼՍԻՌՁՇՆՊԹՌԽՇԾԽԿԿՋԾԽՅԹՄԽԼԹՑՏԹՋԹԻՀՁԽՎԽԼԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽՇԾԠԢՐՈՇՋՍՊԽ԰ՑՋՌԽՅԱԹԺՄԽ԰ՍՅՅԹՊՑՇԾԦՆԾՇՊՅԹՌՁՇՆՇՆՌՀԽԢՐՈՇՋՍՊԽ԰ՑՋՌԽՅԹՆԼԱԽՋՌԳԽՋՋԽՄԠՀԹՊԹԻՌԽՊՁՋՌՁԻՋԭԹՊԹՅԽՌԽՊԳԹՄՍԽ
ՋԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋԱՑՈԽՇԾԽՐՈՇՋՍՊԽԣՄՇՏՌՀՊՇՍԿՀ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԽՍՋԽՇԾԹԾՄՇՏՌՀՊՇՍԿՀ ՋՑՋՌԽՅԞՋՆՇՌԽԼՁՆՌՀԽԠՇՊՊԽԻՌՁՇՆՋԹՆԼ ԠՄԹՊՁԾՁԻԹՌՁՇՆՋԼՇԻՍՅԽՆՌ  ՌՀԽՍՋԽՇԾԹՋՌԹՌՁԻ ՊԽՆԽՏԹՄՋՑՋՌԽՅՁՋՆՇՌՊԽԻՇՅՅԽՆԼԽԼԾՇՊՌՀՁՋ

ԹՋՋԹՑԱՑՈԽՇԾԾՄՇՏՌՀՊՇՍԿՀԼՁՄՍՌՁՇՆՋՑՋՌԽՅԦՆՌԽՊՅՁՌՌԽՆՌԾՄՇՏՈՊՇՈՇՊՌՁՇՆԹՄԼՁՄՍՌԽՊ ԦՆՌԽՊՅՁՌՌԽՆՌԾՄՇՏՈՊՇՈՇՊՌՁՇՆԹՄԼՁՄՍՌԽՊՋՇՊ ԻՇՆՌՁՆՍՇՍՋԾՄՇՏՋԽՊՁԹՄԼՁՄՍՌԽՊՋԹՊԽ ՊԽԻՇՅՅԽՆԼԽԼ ԣՄՇՏՌՀՊՇՍԿՀՊԹՌԽ+ՅԩՅՁՆԱՀԽԾՄՇՏՊԹՌԽՎՇՄՍՅԽՊԽՈՄԹԻԽՅԽՆՌՋԽՎԽՊՑՀՇՍՊՋՏՁՌՀԹՊԽՈՄԹԻԽՅԽՆՌՌՁՅԽՇԾՀՇՍՊՋ ԯԽԻՇՅՅԽՆԼԽԼԾՄՇՏՌՀՊՇՍԿՀՊԹՌԽՁՋՅԩՅՁՆ  ԩՀՊ ՇՊ ԹՌ ՄԽԹՋՌ  ՌՇՌԹՄ ՎՇՄՍՅԽ
ԽՐԻՀԹՆԿԽՋՈԽՊԼԹՑԲ԰ԢՆՎՁՊՇՆՅԽՆՌԹՄԭՊՇՌԽԻՌՁՇՆԞԿԽՆԻՑ
ԢԭԞ
ԠՇՊՊԽԻՌՁՇՆՋԹՆԼԠՄԹՊՁԾՁԻԹՌՁՇՆՋՇՆԱԽԻՀՆՁԻԹՄԞՋՈԽԻՌՋՇԾՌՀԽԱԽՋՌԤՍՁԼԽՄՁՆԽՋԾՇՊՌՀԽԢՆԼՇԻՊՁՆԽԡՁՋՊՍՈՌՇՊ԰ԻՊԽԽՆՁՆԿԭՊՇԿՊԹՅԱՁԽՊԞՋՋԹՑՋ
ԬԠ԰ԭԭԱԽՋՌԤՍՁԼԽՄՁՆԽ԰ԽՊՁԽՋԪԹՊԻՀԬԾԾՁԻԽՇԾԠՀԽՅՁԻԹՄ԰ԹԾԽՌՑԹՆԼԭՇՄՄՍՌՁՇՆԭՊԽՎԽՆՌՁՇՆԬԠ԰ԭԭԴԹՋՀՁՆԿՌՇՆԡԠՀՌՌՈՏՏՏԽՈԹԿՇՎԽՆԼՇՈՍԺՋԹՋՋԹՑՎԹՄՁԼԹՌՁՇՆԻՄԹՊՁԾՁԻԹՌՁՇՆԼՇԻՈԼԾԞԼԼՁՌՁՇՆԹՄԿՍՁԼԹՆԻԽԾՇՊԹՉՍԹՌՁԻՌԽՋՌԼԽՋՁԿՆՁՋՄՇԻԹՌԽԼՁՆԬԠ԰ԭԭԤՍՁԼԽՄՁՆԽ԰ՈԽԻՁԹՄԠՇՆՋՁԼԽՊԹՌՁՇՆՋԾՇՊԠՇՆԼՍԻՌՁՆԿԞՉՍԹՌՁԻԩԹԺՇՊԹՌՇՊՑ԰ՌՍԼՁԽՋԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽՇԾԭԹՊԹՅԽՌԽՊԳԹՄՍԽ
ՋԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋԡԽՌԹՁՄՋՇՆՌՇՐՁԻԹՆՌՅՁՐՁՆԿԾՇՊԾՄՇՏՌՀՊՇՍԿՀՋՑՋՌԽՅՋԞՆԣԪԦՈՍՅՈՏԹՋՍՋԽԼՌՇԼԽՄՁՎԽՊՋՌՇԻՃՋՇՄՍՌՁՇՆՌՇՅՁՐՁՆԿԻՀԹՅԺԽՊՋՈՇՋՁՌՁՇՆԽԼՇՎԽՊԹՅԹԿՆԽՌՁԻՋՌՁՊՈՄԹՌԽԣՄՇՏՋՈՄՁՌՌՁՆԿԻՀԹՅԺԽՊՋՏԽՊԽՍՋԽԼՌՇՈՊՇՅՇՌԽԾՍՊՌՀԽՊՅՁՐՁՆԿԹՆԼՌՇԽՉՍԹՄՄՑԼՁՋՌՊՁԺՍՌԽՌՀԽՌԽՋՌՋՇՄՍՌՁՇՆՋՌՇԽՐՈՇՋՍՊԽԹՉՍԹՊՁԹԠԹՄՁԺՊԹՌՁՇՆՏԹՋԻՇՆԾՁՊՅԽԼՈՊՁՇՊՌՇՁՆՁՌՁԹՌՁՇՆԹՆԼՌՀԽԾՍՆԻՌՁՇՆՇԾՌՀԽԼՁՄՍՌԽՊՏԹՋՅՇՆՁՌՇՊԽԼԼԹՁՄՑԹՆԼԹՎՁՋՍԹՄԻՀԽԻՃՏԹՋՈԽՊԾՇՊՅԽԼՌՏՁԻԽԼԹՁՄՑ ԯԽԻՇՅՅԽՆԼԽԼՌՇՐՁԻԹՆՌՅՁՐՁՆԿԾՇՊԾՄՇՏՌՀՊՇՍԿՀ ՋՑՋՌԽՅՋԪՁՐՁՆԿԻՀԹՅԺԽՊՁՋՊԽԻՇՅՅԽՆԼԽԼ ԺՍՌՆՇՌՊԽՉՍՁՊԽԼԞԽՊԹՌՁՇՆՁՋՆՇՌ
ՊԽԻՇՅՅԽՆԼԽԼ ԾՇՊՅՁՐՁՆԿԞԼԽՅՇՆՋՌՊԹՌՁՇՆՌՀԹՌՌՀԽՌԽՋՌ ՋՇՄՍՌՁՇՆՁՋԻՇՅՈՄԽՌԽՄՑՅՁՐԽԼԺԽԾՇՊԽՁՆՌՊՇԼՍԻԽԼ ՁՆՌՇՌՀԽՌԽՋՌՋՑՋՌԽՅՁՋՊԽԻՇՅՅԽՆԼԽԼԱՀԽ ՊԽԻՇՅՅԽՆԼԽԼԾՄՇՏՋՈՄՁՌՌՁՆԿԹԻԻՍՊԹԻՑՁՋՏՁՌՀՁՆ

ԞԽՊԹՌՁՇՆ!ԫՇ ԢԭԞՊԽԻՇՅՅԽՆԼՋԹԽՊԹՌՁՇՆՁԾԼՁՋՋՇՄՎԽԼՇՐՑԿԽՆ

ՊԽԹԻՀԽՋ ՅԿԩ
 ՋԹՌՍՊԹՌՁՇՆ԰ՇՍՊԻԽՇԾԼՁՄՍՌՁՇՆՏԹՌԽՊԴԽՄՄՏԹՌԽՊ ԢԭԞՊԽԻՇՅՅԽՆԼՋՆԹՌՍՊԹՄՇՊՊԽԻՇՆՋՌՁՌՍՌԽԼ

ՏԹՌԽՊ ՁՌՁՋՊԽԻՇՅՅԽՆԼԽԼՌՀԹՌՆԹՌՍՊԹՄՏԹՌԽՊԺԽ ՋՌԽՊՁՄՁՒԽԼՏՁՌՀԲԳԹՆԼՌԽՋՌԽԼԾՇՊՈԽՋՌՁԻՁԼԽՋ ՀԽԹՎՑՅԽՌԹՄՋԹՆԼՇՌՀԽՊՈՇՋՋՁԺՄԽԻՇՆՌԹՅՁՆԹՆՌՋ ԬԢԠԡԹԻԻԽՈՌՋԹՆՑՏԹՌԽՊՁՆՏՀՁԻՀՌՀԽՌԽՋՌ ՋՈԽԻՁԽՋՋՀՇՏԻՇՆՌՊՇՄՋՍՊՎՁՎԹՄԹՌՄԽԹՋՌԹՋԿՇՇԼ ԹՋՁՆԼՁԻԹՌԽԼՁՆՌՀԽՌԽՋՌԿՍՁԼԽՄՁՆԽԴԹՋԼՁՄՍՌՁՇՆՏԹՌԽՊԹՆԹՄՑՒԽԼԾՇՊՈԽՋՌՁԻՁԼԽՋՀԽԹՎՑՅԽՌԹՄՋԹՆԼՇՌՀԽՊԻՇՆՌԹՅՁՆԹՆՌՋ!ԶԽՋԫՇՈԽՋՌՁԻՁԼԽՋԭԠԟՋՇՊՌՇՐՁԻՅԽՌԹՄՋՏԽՊԽԼԽՌԽԻՌԽԼԹՌԻՇՆԻԽՆՌՊԹՌՁՇՆՋԻՇՆՋՁԼԽՊԽԼՌՇՐՁԻԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽՇԾԭԹՊԹՅԽՌԽՊԳԹՄՍԽ
ՋԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋԱԽՋՌՎԽՋՋԽՄՌՑՈԽՅԹՌԽՊՁԹՄՋ ԿՄԹՋՋ ԢԭԞԹՆԼԬԢԠԡՊԽԻՇՅՅԽՆԼՌՀԹՌՏԹՌԽՊԻՇՆՌԹԻՌ ՈՇՊՌՁՇՆՋՇԾՌՀԽՋՑՋՌԽՅՆՇՌԻՇՅՈՊՇՅՁՋԽՌՀԽ

ՋՌՍԼՑ
ԽԿ ԹՄՄԿՄԹՋՋՎԽՋՋԽՄՋՇՊԿՄԹՋՋՎԽՋՋԽՄՋ ՏՁՌՀՋՌԹՁՆՄԽՋՋՋՌԽԽՄԾՊԹՅԽՋԹՊԽԹԻԻԽՈՌԹԺՄԽ

ԽՐԹՅՈՄԽՋԱԽՋՌՎԽՋՋԽՄՋՁՒԽԩ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԽՍՋԽՇԾԩՌԽՋՌ

ԻՀԹՅԺԽՊՋ ԽԿՐՐԻՅԣՁՄՄՎՇՄՍՅԽԩ ԢԭԞՊԽԻՇՅՅԽՆԼՋԩՋՇՄՍՌՁՇՆՈԽՊՌԹՆՃ԰ՈԹՏՆՁՆԿՋՍԺՋՌՊԹՌԽՅԹՌԽՊՁԹՄ ԞԿԽԼԭԳԠ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌԽԹԻՀՌԹՆՃԻՇՆՌԹՁՆՌՀՊԽԽ ՋԽՅՁԻՁՊԻՍՄԹՊՋՈԹՏՆՁՆԿՋՍԺՋՌՊԹՌԽՋԽԿԹԿԽԼ ԭԳԠՈՁՈԽԻՅՁՆՄԽՆԿՌՀՋՈՄՁՌ

ՄԽՆԿՌՀՏՁՋԽ԰ՈԹՏՆՁՆԿՋՍԺՋՌՊԹՌԽՋՁՒԽ ԻՅԞԼԼՁՌՁՇՆԹՄԼԽՌԹՁՄՋՇՆԽՐՈՇՋՍՊԽՋՑՋՌԽՅԫԞ  ԞԺԺՊԽՎՁԹՌՁՇՆՋԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽԱԹԺՄԽ԰ՍՅՅԹՊՑՇԾԴԹՌԽՊԮՍԹՄՁՌՑԠՀԹՊԹԻՌԽՊՁՋՌՁԻՋՁՆՌՀԽԱԽՋՌ԰ՑՋՌԽՅԭԹՊԹՅԽՌԽՊԪՁՆՁՅՍՅԪԹՐՁՅՍՅԪԽԹՆԪԽԹՋՍՊԽՅԽՆՌԦՆՌԽՊՎԹՄԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋԱԽՅՈԽՊԹՌՍՊԽ
ոԠ ԠՇՆՌՁՆՍՇՍՋՄՑ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌԽՅՈԽՊԹՌՍՊԽչՇԠՁՆՌԽՊ ՊԽՈՄՁԻԹՌԽԹՆԼՁՆՌԽՊՌՊԽԹՌՅԽՆՌԼՁԾԾԽՊԽՆՌՁԹՄՋՋՀՇՍՄԼ

ՆՇՌԽՐԻԽԽԼ° ԠԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽՇԾԭԹՊԹՅԽՌԽՊԪՁՆՁՅՍՅԪԹՐՁՅՍՅԪԽԹՆԪԽԹՋՍՊԽՅԽՆՌԦՆՌԽՊՎԹՄԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋՈԥ ԪԽԹՋՍՊԽԼՁՆՌԽՋՌՎԽՋՋԽՄԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆԹՆԼՁՆԹՄՌԽՊՆԹՌՁՆԿՊԽՈՄՁԻԹՌԽՋԼԹՁՄՑՌՀԽՊԽԹԾՌԽՊ ԢԭԞՊԽԻՇՅՅԽՆԼՋՈԥՌՇԡՁՋՋՇՄՎԽԼՇՐՑԿԽՆ
ՅԿԩ ԪԽԹՋՍՊԽԼՁՆՌԽՋՌՎԽՋՋԽՄԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆԹՆԼՁՆԹՄՌԽՊՆԹՌՁՆԿՊԽՈՄՁԻԹՌԽՋԼԹՁՄՑՌՀԽՊԽԹԾՌԽՊ ԢԭԞՊԽԻՇՅՅԽՆԼՋԼՁՋՋՇՄՎԽԼՇՐՑԿԽՆ
ԡԬ  ՅԿԩ
 ԹՁՊՋԹՌՍՊԹՌՁՇՆԱՇՌԹՄԹՄՃԹՄՁՆՁՌՑ
ՅԿԩԹՋԠԹԠԬ ԱԽՋՌՁՆՁՌՁԹՌՁՇՆԹՆԼՏԽԽՃՄՑՌՀԽՊԽԹԾՌԽՊ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՇՌԹՄԹՄՃԹՄՁՆՁՌՑ ՅԿԩԹՋ

ԠԹԠԬ  ԥԹՊԼՆԽՋՋ
ՅԿԩԹՋԠԹԠԬԱԽՋՌՁՆՁՌՁԹՌՁՇՆԹՆԼՏԽԽՃՄՑՌՀԽՊԽԹԾՌԽՊԱՇՌԹՄՇՊԿԹՆՁԻԻԹՊԺՇՆ
ՅԿԩԫԞ ԣԽԺՊՍԹՊՑԹՆԼԪԹՊԻՀ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՇՌԹՄՇՊԿԹՆՁԻԻԹՊԺՇՆՁՆ ԼՁՄՍՌՁՇՆՏԹՌԽՊԺԽ ՅԿԩԲՆՁՇՆՁՒԽԼԹՅՅՇՆՁԹ
սԿԩԫՇՌԯԽՈՇՊՌԽԼ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՍՆՁՇՆՁՒԽԼԹՅՅՇՆՁԹՁՆ ՌՀԽԼՁՄՍՌՁՇՆՏԹՌԽՊԺԽ ս ԿԩԯԽՋՁԼՍԹՄԻՀՄՇՊՁՆԽ
սԿԩԫՇՌԯԽՈՇՊՌԽԼ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՊԽՋՁԼՍԹՄԻՀՄՇՊՁՆԽՁՆ ԼՁՄՍՌՁՇՆՏԹՌԽՊԺԽս ԿԩԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽՇԾԭԹՊԹՅԽՌԽՊԪՁՆՁՅՍՅԪԹՐՁՅՍՅԪԽԹՆԪԽԹՋՍՊԽՅԽՆՌԦՆՌԽՊՎԹՄԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋԠՇՆԼՍԻՌՁՎՁՌՑ
ս԰ԻՅ ԱԽՋՌՁՆՁՌՁԹՌՁՇՆԹՆԼՏԽԽՃՄՑՌՀԽՊԽԹԾՌԽՊ ԤԽՆԽՊԹՄՊԽԻՇՅՅԽՆԼԹՌՁՇՆՋԾՇՊԾՊԽՉՍԽՆԻՑՇԾ ՅԽԹՋՍՊԽՅԽՆՌՋԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌ ՌԽՅՈԽՊԹՌՍՊԽՈԥԹՆԼԼՁՋՋՇՄՎԽԼՇՐՑԿԽՆԺԽ ՅԽԹՋՍՊԽԼՁՆԹՄՄՌԽՋՌՌԹՆՃՋԹՌՄԽԹՋՌՏԽԽՃՄՑԹՆԼ ՌՀԹՌՀԹՊԼՆԽՋՋԹՆԼԹՄՃԹՄՁՆՁՌՑԺԽՅԽԹՋՍՊԽԼՁՆ ԻՇՆՌՊՇՄՋԹՆԼՁՆՇՆԽՌԹՆՃԹՌՌՀԽՀՁԿՀԽՋՌՌԽՋՌ ԻՇՆԻԽՆՌՊԹՌՁՇՆԹՌՄԽԹՋՌՏԽԽՃՄՑԦՆԹԼԼՁՌՁՇՆ ԻՇՆՌՁՆՍՇՍՋՌԽՅՈԽՊԹՌՍՊԽՅՇՆՁՌՇՊՁՆԿՇԾԹՌՄԽԹՋՌ ՇՆԽՌԹՆՃՁՋԽՆԻՇՍՊԹԿԽԼԡ԰ՌՍԼՑԡԽՋՁԿՆԹՆԼԞԼԼՁՌՁՇՆԹՄԢՐՈԽՊՁՅԽՆՌԹՄԠՇՆԼՁՌՁՇՆՋԱԹԺՄԽԯԹՆԿԽԣՁՆԼՁՆԿ԰ՌՍԼՑԠՇՆԼՁՌՁՇՆՋ
ՁԾԞՈՈՄՁԻԹԺՄԽԭԹՊԹՅԽՌԽՊԳԹՄՍԽ
ՋԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋԴԹՋԹՊԹՆԿԽԾՁՆԼԽՊԻՇՆԼՍԻՌԽԼ! ԫՇ ԢԭԞՊԽԻՇՅՅԽՆԼՋԻՇՆԼՍԻՌՁՆԿԹՊԹՆԿԽԾՁՆԼԽՊՁԾ ՀՇՍՊԩԠԼԹՌԹԾՇՊՌՀԽԾԹՌՀԽԹԼՅՁՆՆՇՏԹՊԽ

ՍՆԹՎԹՁՄԹԺՄԽԦԾՑԽՋՏՀԹՌՏԹՋՌՀԽՅԽՌՀՇԼԾՇՊԼԽՌԽՊՅՁՆՁՆԿՌՀԽՀՁԿՀԽՋՌՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՁՆՌՀԽՊԹՆԿԽԾՁՆԼԽՊ!ԫԞ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՀԽՀՁԿՀԽՋՌՌԽՋՌ ԻՇՆԻԽՆՌՊԹՌՁՇՆԺԽՋԽՄԽԻՌԽԼԺԹՋԽԼՇՆՌՇՐՁԻՁՌՑԼԹՌԹ ԾՇՊՇՌՀԽՊԾՁՋՀՋՌՍԼՁԽՋՇՊՋՈԽԻՁԽՋՁԾԹՎԹՁՄԹԺՄԽ

ԬՌՀԽՊՏՁՋԽԽՁՌՀԽՊ ՌՀԽՋՇՄՍԺՁՄՁՌՑՄՁՅՁՌՇԾՌՀԽՌԽՋՌԻՇՅՈՇՍՆԼՇՊ ՅԿԩ
ՏՀՁԻՀԽՎԽՊՁՋՄՇՏԽՊՁՋԹՈՈՊՇՈՊՁԹՌԽ԰ՈԽԻՁԽՋԫԞԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽՇԾԭԹՊԹՅԽՌԽՊԳԹՄՍԽ
ՋԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋԩՁԾԽՋՌԹԿԽԫԞ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՊԹՆԿԽԾՁՆԼՁՆԿՌԽՋՌՋԺԽ ՈԽՊԾՇՊՅԽԼՏՁՌՀԾՁՋՀՇԾՋՁՅՁՄԹՊԹԿԽԹՆԼՋՁՒԽՌՇ ՌՀՇՋԽՌՀԹՌՏՇՍՄԼԺԽՍՌՁՄՁՒԽԼՁՆՌՀԽՌԽՋՌԱԽՋՌԼՍՊԹՌՁՇՆԫԞ ԢԭԞՊԽԻՇՅՅԽՆԼՋԹՀՇՍՊԽՐՈՇՋՍՊԽԞԼԼՁՌՁՇՆԹՄԼԽՌԹՁՄՋԫԞ ԢԭԞՊԽԻՇՅՅԽՆԼՋԻՇՆԼՍԻՌՁՆԿԹՊԹՆԿԽԾՁՆԼԽՊՏՁՌՀ ԾՁՎԽՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՋՈՄՍՋԹԻՇՆՌՊՇՄ
ՋՁՐՌՇՌԹՄ ՌՊԽԹՌՅԽՆՌՄԽՎԽՄՋՏՁՌՀԾՇՍՊԾԽՅԹՄԽՋԹՆԼՌՏՇՅԹՄԽՋ ՈԽՊԽՐՈՇՋՍՊԽՌԹՆՃ
ԾՁՋՀՌՇՌԹՄԱՀԽՆՍՅԺԽՊՇԾ ՅՇՊՌԹՄՁՌՁԽՋՌՀԹՌՇԻԻՍՊՅԹՑԺԽՍՋԽԼՌՇԼԽՎԽՄՇՈԹ ԻՇՆԻԽՆՌՊԹՌՁՇՆՊԽՋՈՇՆՋԽԻՍՊՎԽ ԟԹՋԽԼՍՈՇՆՌՀԽՊԽՋՍՄՌՋՌՀԽՀՁԿՀԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆ ՌՀԹՌԼՇԽՋՆՇՌՊԽՋՍՄՌՁՆՁՆԻՊԽԹՋԽԼՅՇՊՌԹՄՁՌՑՇՊՋՁԿՆՋ ՇԾ ՇՎԽՊՌ ՅՇՊԺՁԼՁՌՑ ԻՇՅՈԹՊԽԼ ՌՇ ԻՇՆՌՊՇՄՋ ՇՊ  ՌՀԽԼԽՊՁՎԽԼՀՊԩԠՅԹՑԺԽՋԽՄԽԻՌԽԼԹՋՌՀԽ ՀՁԿՀԽՋՌԽՐՈՇՋՍՊԽԻՇՆԻԽՆՌՊԹՌՁՇՆՁՆՌՀԽԼԹՑՌԽՋՌԱԹԺՄԽԡԽԾՁՆՁՌՁՎԽ԰ՌՍԼՑԠՇՆԼՁՌՁՇՆՋԭԹՊԹՅԽՌԽՊԳԹՄՍԽ
ՋԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋԱԽՋՌԼՍՊԹՌՁՇՆԼԹՑՋ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՀԽԼՍՊԹՌՁՇՆՇԾՌՀԽ ԼԽԾՁՆՁՌՁՎԽՌԽՋՌԺԽԼԹՑՋԪԽՌՀՇԼԾՇՊՋԽՄԽԻՌՁՆԿՌՀԽՀՁԿՀԽՋՌՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՁՆՌՀԽԼԽԾՁՆՁՌՁՎԽՌԽՋՌ԰ԽՄԽԻՌԽԼԺՑՌՀԽ԰ՌՍԼՑ԰ՈՇՆՋՇՊԺԹՋԽԼՇՆՈՊԽՎՁՇՍՋՌՇՐՁԻՁՌՑՁՆԾՇՊՅԹՌՁՇՆՏՁՌՀԻԹՊԺԹՊՑՄ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՀԽՀՁԿՀԽՋՌՌԽՋՌ ԻՇՆԻԽՆՌՊԹՌՁՇՆՁՋԽՁՌՀԽՊՌՀԽՋՇՄՍԺՁՄՁՌՑՄՁՅՁՌՇԾՌՀԽՌԽՋՌ ԻՇՅՈՇՍՆԼՅԿԩՇՊԼԽՅՇՆՋՌՊԹՌԽՋԹԼԽՉՍԹՌԽ ԽՎՁԼԽՆԻԽՇԾՌՇՐՁԻՁՌՑ
ԽԿՌՀԽՀՇՍՊԩԠ   ՏՀՁԻՀԽՎԽՊԻՇՆԻԽՆՌՊԹՌՁՇՆՁՋՄՇՏԽՋՌԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽՇԾԭԹՊԹՅԽՌԽՊԳԹՄՍԽ
ՋԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋԯԽԾԽՊԽՆԻԽՋՌՍԼՑԻՁՌԹՌՁՇՆ
ՁԾԹՈՈՄՁԻԹԺՄԽԠԹՊՄՁՋՄԽ
  ԰ԽՈԹՊԹՌՁՇՆՇԾՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՋ ՅԿԩԱՀԽՋՌՇԻՃՋՇՄՍՌՁՇՆՋՏԽՊԽՇԺՋԽՊՎԽԼՌՇԺԽԻՄԽԹՊԹՆԼԻՇՄՇՊՄԽՋՋՏՁՌՀՆՇՎՁՋՁԺՄԽՍՆԼՁՋՋՇՄՎԽԼՌԽՋՌՋՍԺՋՌԹՆԻԽԾՇՄՄՇՏՁՆԿՈՊԽՈԹՊԹՌՁՇՆ԰ՌՇԻՃՈՊԽՈԹՊԹՌՁՇՆՊԽՋՍՄՌԽԼՁՆԹՋՌՇԻՃՋՇՄՍՌՁՇՆԻՇՆԻԽՆՌՊԹՌՁՇՆԹՈՈՊՇՐՁՅԹՌԽՄՑՄՇՏԽՊՌՀԹՆՆՇՅՁՆԹՄԱՇԻՇՅՈԽՆՋԹՌԽԾՇՊՌՀՁՋՌՀԽՌՇՐՁԻԹՆՌԼԽՄՁՎԽՊՑՏԹՋՁՆԻՊԽԹՋԽԼԾՊՇՅՅԩԻՑԻՄԽՌՇՅԩԻՑԻՄԽ ԢԭԞՋՍԿԿԽՋՌՋՌՀԹՌԹԻՇՆԻԽՆՌՊԹՌՁՇՆՋԽՈԹՊԹՌՁՇՆՇԾ ԺԽՌՏԽԽՆ
ՇՊՌՀՊԽԽԾՇՄԼԹՆԼ
ՇՊՌԽՆԾՇՄԼ ՁՋՋԻՁԽՆՌՁԾՁԻԹՄՄՑԹԻԻԽՈՌԹԺՄԽ  ԫՍՅԺԽՊՇԾՌԽՋՌԻՇՆԻԽՆՌՊԹՌՁՇՆՋ  ԢԭԞՊԽԻՇՅՅԽՆԼՋԹՅՁՆՁՅՍՅՇԾԻՇՆԻԽՆՌՊԹՌՁՇՆՋ ԹՆԼԹԻՇՆՌՊՇՄՈՄՍՋՋՇՄՎԽՆՌԻՇՆՌՊՇՄՁԾԹՈՈՊՇՈՊՁԹՌԽԞՊԽՆՇՅՁՆԹՄԻՇՆԻԽՆՌՊԹՌՁՇՆՋԹԼՂՍՋՌԽԼԾՇՊՈՍՊՁՌՑ!ԶԽՋ  ԦՆԼՁԻԹՌԽՌՀԽՌՑՈԽՇԾՎԹՄՍԽՋՈՊԽՋԽՆՌԽԼԾՇՊՅԽԹՋՍՊԽԼԻՇՆԻԽՆՌՊԹՌՁՇՆՋԪԽԹՆՅԽԹՋՍՊԽԼ  ԩՁՅՁՌՇԾՉՍԹՆՌՁԾՁԻԹՌՁՇՆ
ԩԬԮ ՅԿԹՁԩ  ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌԾՇՊԻՀԽՅՁԻԹՄՌԽՋՌ ԻՇՆԻԽՆՌՊԹՌՁՇՆՋԺԽՄՇՏՌՀԽԩԬԮԹՆԹՄՑՋԽՋԺԽ ԻՇՆԼՍԻՌԽԼՇՆՌՀԽՋՌՇԻՃՋՇՄՍՌՁՇՆՋԩԽՎԽՄՇԾԼԽՌԽԻՌՁՇՆ
ԩԬԡԫՇՌԯԽՈՇՊՌԽԼԣՊԽՉՍԽՆԻՑՇԾՅԽԹՋՍՊԽՅԽՆՌ ԰ԹՅՈՄԽՋՏԽՊԽԻՇՄՄԽԻՌԽԼԾՇՊԹՆԹՄՑՋՁՋՇՆԡԹՑՋԹՆԼՁՆՊԽՈՄՁԻԹՌԽՋԾՇՊԽԹԻՀՌՊԽԹՌՅԽՆՌԹՌԽԹԻՀՌՁՅԽՈՇՁՆՌ ԦՌՁՋՊԽԻՇՅՅԽՆԼԽԼՌՀԹՌՌԽՋՌՁՌԽՅԻՇՆԻԽՆՌՊԹՌՁՇՆԺԽ ՅԽԹՋՍՊԽԼՈՊՁՇՊՌՇՌՀԽԹԼԼՁՌՁՇՆՇԾԾՁՋՀՁՆԹՄՄՌԹՆՃՋ ԹՆԼԹՌՄԽԹՋՌՏԽԽՃՄՑՌՀԽՊԽԹԾՌԽՊՁՆՌՏՇՊԽՈՄՁԻԹՌԽՋՈԽՊ

ՌՊԽԹՌՅԽՆՌՄԽՎԽՄԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽՇԾԭԹՊԹՅԽՌԽՊԳԹՄՍԽ
ՋԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋԴԹՋՌՀԽՊԹՆԼՇՅՁՒԽԼԻՇՅՈՄԽՌԽԺՄՇԻՃԼԽՋՁԿՆՍՋԽԼ!ԶԽՋ  ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌԹՄՄԾՁՋՀԺԽՊԹՆԼՇՅՄՑ ԹՋՋՁԿՆԽԼՌՇՌԹՆՃՋԼՍՊՁՆԿՈՊԽԽՐՈՇՋՍՊԽԱԹՆՃՋԹՊԽ ՌՀԽՆՊԹՆՃԽԼԹԻԻՇՊԼՁՆԿՌՇՈՊԽԽՐՈՇՋՍՊԽԾԽԻՍՆԼՁՌՑ ԹՆԼՌՀԽՌԹՆՃՋՏՁՌՀՌՀԽՀՁԿՀԽՋՌԾԽԻՍՆԼՁՌՑԹՊԽ ՊԹՆԼՇՅՄՑԹՋՋՁԿՆԽԼՌՇԹԼԽԾՁՆՁՌՁՎԽՌԽՋՌՌՊԽԹՌՅԽՆՌ ԹՆԼԺՄՇԻՃԾՁՊՋՌԢԹԻՀԺՄՇԻՃԻՇՆՌԹՁՆՋՇՆԽՊԽՈՄՁԻԹՌԽ ՇԾԽԹԻՀՌՊԽԹՌՅԽՆՌՁՆԻՄՍԼՁՆԿԻՇՆՌՊՇՄՋԫՍՅԺԽՊՇԾՊԽՈՄՁԻԹՌԽՋՁՆԻՇՆՌՊՇՄ  ԢԭԞՊԽԻՇՅՅԽՆԼՋՊԽՈՄՁԻԹՌԽՋԫՍՅԺԽՊՇԾՊԽՈՄՁԻԹՌԽՋՁՆՋՇՄՎԽՆՌԻՇՆՌՊՇՄ
ՁԾԹՈՈՄՁԻԹԺՄԽԫԞ  ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԽՍՋԽՇԾԹԻՇՆԻՍՊՊԽՆՌՋՇՄՎԽՆՌ ԻՇՆՌՊՇՄՏՀԽՆԹՋՇՄՍԺՁՄՁՒՁՆԿԹԿԽՆՌՁՋՍՋԽԼԢԭԞ ՊԽԻՇՅՅԽՆԼՋՊԽՈՄՁԻԹՌԽՋԫՍՅԺԽՊՇԾՊԽՈՄՁԻԹՌԽՋՈԽՊՌԽՋՌՁՌԽՅՌՊԽԹՌՅԽՆՌՄԽՎԽՄ  ԢԭԞՊԽԻՇՅՅԽՆԼՋՊԽՈՄՁԻԹՌԽՋԫՍՅԺԽՊՇԾՅԹՄԽԾՁՋՀՈԽՊՊԽՈՄՁԻԹՌԽԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ  ԢԭԞՊԽԻՇՅՅԽՆԼՋՅԹՄԽՋՈԽՊՊԽՈՄՁԻԹՌԽԫՍՅԺԽՊՇԾԾԽՅԹՄԽԾՁՋՀՈԽՊՊԽՈՄՁԻԹՌԽԹՌՌԽՋՌՁՆՁՌՁԹՌՁՇՆ  ԢԭԞՊԽԻՇՅՅԽՆԼՋԾԽՅԹՄԽՋՈԽՊՊԽՈՄՁԻԹՌԽԴԹՋԹՋՇՄՎԽՆՌՍՋԽԼ!ԫՇԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽՇԾԭԹՊԹՅԽՌԽՊԳԹՄՍԽ
ՋԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋ԰ՇՄՎԽՆՌՌՑՈԽ
ՁԾԹՈՈՄՁԻԹԺՄԽ ԫԞԪԹՐՁՅՍՅՋՇՄՎԽՆՌԻՇՆԻԽՆՌՊԹՌՁՇՆ
ՁԾԹՈՈՄՁԻԹԺՄԽԫԞ  ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌՀԽՋՇՄՎԽՆՌՆՇՌԽՐԻԽԽԼ

ՅՄԩ  ԬԢԠԡՊԽԻՇՅՅԽՆԼՋՌՀԹՌՋՇՄՎԽՆՌ ՀԹՎԽՆՇԽԾԾԽԻՌՇՆՋՍՊՎՁՎԹՄՆՇՊՈՊՇԼՍԻԽԹՆՑՇՌՀԽՊ ԹԼՎԽՊՋԽԽԾԾԽԻՌՋԹՆԼՌՀԹՌԻՇՆԻԽՆՌՊԹՌՁՇՆՆՇՌԺԽ

ԿՊԽԹՌԽՊՌՀԹՆ ՅՄԩ  ԴԹՋԹՈՇՋՁՌՁՎԽԻՇՆՌՊՇՄՍՋԽԼ! ԫՇԭՇՋՁՌՁՎԽԻՇՆՌՊՇՄ
ՁԾԹՈՈՄՁԻԹԺՄԽ ԫԞԭՇՋՁՌՁՎԽԻՇՆՌՊՇՄԻՇՆԻԽՆՌՊԹՌՁՇՆ
Ջ
ՁԾԹՈՈՄՁԻԹԺՄԽԫԞ  ԭՀՇՌՇՈԽՊՁՇԼՀՊՋՄՁԿՀՌՀՊՋԼԹՊՃ ԢԭԞՊԽԻՇՅՅԽՆԼՋՈՀՇՌՇՈԽՊՁՇԼ
ՄՁԿՀՌԼԹՊՃԩՁԿՀՌՁՆՌԽՆՋՁՌՑԹՌՏԹՌԽՊ0ՋՋՍՊԾԹԻԽ ՄՍՐ ԢԭԞՊԽԻՇՅՅԽՆԼՋՄՁԿՀՌՁՆՌԽՆՋՁՌՑ-ՄՍՐ ԹՌՏԹՌԽՊ0ՋՋՍՊԾԹԻԽԞԼԼՁՌՁՇՆԹՄԼԽՌԹՁՄՋԞՄՄԽՐՈՇՋՍՊԽՋՇՄՍՌՁՇՆՋՏԽՊԽՇԺՋԽՊՎԽԼՌՇԺԽԻՄԽԹՊԹՆԼԻՇՄՇՊՄԽՋՋϯԥՍՌԻՀՁՆՋՇՆԱԥ԰ՀՁՄՄԹԺԽԽՊԫԴՁՆՌԽՊԪԧԭՁԻՃԾՇՊԼԡԟ
ԞԻՍՌԽԹՆԼԻՀՊՇՆՁԻԽԾԾԽԻՌՋՇԾԻԹՊՊՁԽՊՋՇՄՎԽՆՌՋՁՆԹՉՍԹՌՁԻՇՊԿԹՆՁՋՅՋԞԻՊՁՌՁԻԹՄՊԽՎՁԽՏԯԽՎՁԽՏԞՉՍԹՌՁԻԱՇՐՁԻՇՄՇԿՑՈՈ-κԬԢԠԡ
ԤՍՁԼԹՆԻԽԡՇԻՍՅԽՆՌՇՆԞՉՍԹՌՁԻԱՇՐՁԻՁՌՑԱԽՋՌՁՆԿՇԾԡՁԾԾՁԻՍՄՌ԰ՍԺՋՌԹՆԻԽՋԹՆԼԪՁՐՌՍՊԽՋԢՆՎՁՊՇՆՅԽՆՌԹՄԥԽԹՄՌՀԹՆԼ԰ԹԾԽՌՑԭՍԺՄՁԻԹՌՁՇՆՋ԰ԽՊՁԽՋՇՆԱԽՋՌՁՆԿԹՆԼԞՋՋԽՋՋՅԽՆՌԫՇԭԹՊՁՋԣՊԹՆԻԽԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽՇԾԱԹԺՄԽ԰ՍՅՅԹՊՑՇԾԱՊԽԹՌՅԽՆՌԠՇՆԻԽՆՌՊԹՌՁՇՆՋՁՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄԱՊԽԹՌՅԽՆՌԦԡԫՇՅՁՆԹՄԠՇՆԻԽՆՌՊԹՌՁՇՆ
ՅԿԹՁԩԪԽԹՋՍՊԽԼԠՇՆԻԽՆՌՊԹՌՁՇՆ
ՅԿԹՁԩԪԽԹՆԠԳ
ԡԽՌԹՁՄՋՇՊԯԽՅԹՊՃՋԤՍՁԼԽՄՁՆԽԯԽԻՇՅՅԽՆԼԹՌՁՇՆՋԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄԩԬԮԫԞ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՌԽՋՌՁՌԽՅԻՇՆԻԽՆՌՊԹՌՁՇՆՋԺԽ ՅԹՁՆՌԹՁՆԽԼԹՌԹԻՇԽԾԾՁԻՁԽՆՌՇԾՎԹՊՁԹՌՁՇՆ
ԠԳ ԱՊԽԹՌՅԽՆՌԱՊԽԹՌՅԽՆՌԱՊԽԹՌՅԽՆՌԞԺԺՊԽՎՁԹՌՁՇՆՋԠԳԠՇԽԾԾՁԻՁԽՆՌՇԾՎԹՊՁԹՌՁՇՆԩԬԮՅԿԹՁԩԢԬԺՋԽՊՎԹՌՁՇՆՋԟՁՇՄՇԿՁԻԹՄԢՆԼՈՇՁՆՌՋՋՍՊՎՁՎԹՄԾԽՊՌՁՄՁՒԹՌՁՇՆՋՍԻԻԽՋՋԾԽԻՍՆԼՁՌՑՅԽԼՁԹՆՌՍԺԽՊԻՄԽՋԻՇՊԽԺՇԼՑՏԽՁԿՀՌՄԽՆԿՌՀԤ԰ԦԳԱԤԻՇՆԻԽՆՌՊԹՌՁՇՆԿՇՆԹԼՀՁՋՌՇՈԹՌՀՇՄՇԿՑՋԽԻՇՆԼԹՊՑՋԽՐԻՀԹՊԹԻՌԽՊՁՋՌՁԻՋԻՄՁՆՁԻԹՄՇԺՋԽՊՎԹՌՁՇՆՋԴԽՊԽՊԹՏ
ՁՆԼՁՎՁԼՍԹՄԼԹՌԹՈՊՇՎՁԼԽԼ!ԶԽՋ ԢԭԞՊԽԻՇՅՅԽՆԼՋՌՀԹՌՇԺՋԽՊՎԹՌՁՇՆՋՇԾՋՍՊՎՁՎԹՄԾԽԻՍՆԼՁՌՑԾԽՊՌՁՄ ՁՒԹՌՁՇՆՋՍԻԻԽՋՋՋԽԻՇՆԼԹՊՑՋԽՐԻՀԹՊԹԻՌԽՊՁՋՌՁԻՋԹՆԼՇՌՀԽՊԻՄՁՆՁ ԻԹՄՋՁԿՆՋՇԻԻՍՊԹՌՄԽԹՋՌԼԹՁՄՑ
ԞՌՌԽՋՌՌԽՊՅՁՆԹՌՁՇՆ
ԡԹՑԹԼԼՁՌՁՇՆԹՄՇԺՋԽՊՎԹՌՁՇՆՋՁՆԻՄՍԼԽԺ ՇԼՑՏԽՁԿՀՌԹՆԼՄԽՆԿՌՀՆՍՈՌՁԹՄՌՍԺԽՊԻՄԽՋԻՇՊԽԿՇՆԹԼԹՄՋՌԹԿՁՆԿ ԹՆԼՀՁՋՌՇՈԹՌՀՇՄՇԿՑՈՄԹՋՅԹ

ՎՁՌԽՄՄՇԿԽՆՁՆԹՆԼՈՄԹՋՅԹՋԽՐՋՌԽՊՇՁԼՋ
ՌԽՋՌՇՋՌԽՊՇՆԽԹՆԼȕ ԽՋՌՊԹԼՁՇՄՁԾՅԽԹՋՍՊԽԼԤՇՆԹԼՇՋՇՅԹՌՁԻՁՆԼԽՐ
Ԥ԰ԦՁՋԻԹՄԻՍՄ ԹՌԽԼՍՋՁՆԿԹՊԹՌՁՇՇԾԿՇՆԹԼ ՏԽՁԿՀՌՌՇԺՇԼՑՏԽՁԿՀՌ
ԿՇՆԹԼՏԽՁԿՀՌՌՇՌՀԽՆԽԹՊԽՋՌՅԿԺՇԼՑՏԽՁԿՀՌՁՆՅԿՐԹՌՌԽՋՌՌԽՊՅՁՆԹՌՁՇՆԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽՇԾ ԠՄՁՆՁԻԹՄՋՁԿՆՋՇԾՇՎԽՊՌՌՇՐՁԻՁՌՑՅԹՑՁՆԻՄՍԼԽ
ԺՍՌԹՊԽՆՇՌՄՁՅՁՌ ԽԼՌՇՀԽՅՇՊՊՀԹԿԽԻԽՋՋԹՌՁՇՆՇԾԾԽԽԼՁՆԿԹՆԼՇՌՀԽՊԹԺՆՇՊՅԹՄԺԽՀԹՎՁՇՊԦԦԯԢ԰ԲԩԱ԰ԞԫԡԡԦ԰ԠԲ԰԰ԦԬԫԞԯԽՋՍՄՌՋԟՑՌԽՋՌՌԽՊՅՁՆԹՌՁՇՆԹՋՁՆԿՄԽՅԹՄԽՅՇՊՌԹՄՁՌՑՇԻԻՍՊՊԽԼՁՆՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈ
ԱԹԺՄԽԣԽՅԹՄԽՋՍՊՎՁՎԹՄԹՎԽՊԹԿԽԼՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՆԼԹՆԼՁՆՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼԹՆԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՊԽՋՈԽԻՌՁՎԽՄՑԬՎԽՊԹՄՄՋՍՊՎՁՎԹՄՏԹՋԹՆԼՁՆՌՀԽԻՇՆՌՊՇՄԹՆԼԹՆԼՅԿԹՁԩԿՊՇՍՈՋՊԽՋՈԽԻՌՁՎԽՄՑԱԹԺՄԽԞԼՍՄՌԣՁՋՀ԰ՍՊՎՁՎԹՄՁՆ ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋԱՊԽԹՌՅԽՆՌՅԿԹՁԩ#ՅԽԹՆՅԽԹՋՍՊԽԼ%ԪԹՄԽՋԣԽՅԹՄԽՋՆ԰ՍՊՎՁՎՁՆԿ԰ՍՊՎՁՎԹՄՆ԰ՍՊՎՁՎՁՆԿ԰ՍՊՎՁՎԹՄԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄ
ԩԬԮԞԺԺՊԽՎՁԹՌՁՇՆՋԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽԩԬԮՅԿԹՁԩԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽՇԾԪԹՄԽՏԽՁԿՀՌՊԹՆԿԽԼԾՊՇՅՌՇԿՁՆՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼԹՆԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՊԽՋՈԽԻՌՁՎԽՄՑ
ԱԹԺՄԽԪԹՄԽՄԽՆԿՌՀՏԹՋՄՇՏԽՋՌՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄ
ՅՅԹՆԼԿՊԽԹՌԽՋՌՁՆՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈ
ՅՅԣԽՅԹՄԽՏԽՁԿՀՌՊԹՆԿԽԼԾՊՇՅԿՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄՌՇԿՁՆՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈԣԽՅԹՄԽՄԽՆԿՌՀՊԹՆԿԽԼԾՊՇՅՅՅՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄՌՇՅՅՁՆՌՀԽՌՏՇՄՇՏԽՋՌՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋԱԹԺՄԽ԰ՁՒԽԹՌԱԽՋՌԱԽՊՅՁՆԹՌՁՇՆՁՆԣԹՌՀԽԹԼԪՁՆՆՇՏ ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋԱՊԽԹՌՅԽՆՌ
ՅԿԹՁԩ#ՅԽԹՆՅԽԹՋՍՊԽԼ%ԟՇԼՑԴԽՁԿՀՌԩԽՆԿՌՀԪԹՄԽՋԣԽՅԹՄԽՋԪԹՄԽՋԣԽՅԹՄԽՋՆԪԽԹՆԿչ԰ԡՆԪԽԹՆԿչ԰ԡՆԪԽԹՆՅՅչ԰ԡՆԪԽԹՆՅՅչ԰ԡԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄ
ԩԬԮ                                            ԞԺԺՊԽՎՁԹՌՁՇՆՋԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽԫԡԫՇՌԼԽՌԽՊՅՁՆԽԼ԰ԡ԰ՌԹՆԼԹՊԼԼԽՎՁԹՌՁՇՆԩԬԮՅԿԹՁԩ

### ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋ Հ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹ ՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ 

ԭԹԿԽՇԾ ԣԽԻՍՆԼՁՌՑՊԹՆԿԽԼԾՊՇՅՌՇԽԿԿՋԾԽՅԹՄԽԼԹՑՁՆՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼՅԿԹՁԩԹՆԼՆԽԿԹՌՁՎԽ

ԻՇՆՌՊՇՄՊԽՋՈԽԻՌՁՎԽՄՑ
ԱԹԺՄԽԪԽԹՆԾԽՊՌՁՄՁՒԹՌՁՇՆՋՋՍԻԻԽՋՋՏ ԹՋ  ՁՆՌՀԽԻՇՆՌՊՇՄԹՆԼԹՄՄՌՊԽԹՌՅԽՆՌ

ԿՊՇՍՈՋ

ԱԹԺՄԽԣԽԻՍՆԼՁՌՑԹՆԼԣԽՊՌՁՄՁՒԹՌՁՇՆ԰ՍԻԻԽՋՋՁՆԣԹՌՀԽԹԼԪՁՆՆՇՏ  ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ 

ԱՊԽԹՌՅԽՆՌ

ՅԿԹՁԩ

#ՅԽԹՆՅԽԹՋՍՊԽԼ% ԣԽԻՍՆԼՁՌՑ  ԣԽՊՌՁՄՁՒԹՌՁՇՆ԰ՍԻԻԽՋՋ
 

ԪԽԹՆ ԰ԡ ԪԽԹՆ ԰ԡ

ԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄ
ԩԬԮ    

    

    

    

ԞԺԺՊԽՎՁԹՌՁՇՆՋ ԫԞ ԫՇՌԹՈՈՄՁԻԹԺՄԽ ԫԡ ԫՇՌԼԽՌԽՊՅՁՆԽԼ ԰ԡ ԰ՌԹՆԼԹՊԼԡԽՎՁԹՌՁՇՆ

  ԣԽԻՍՆԼՁՌՑՁՋԻԹՄԻՍՄԹՌԽԼԹՋՌՀԽՆՍՅԺԽՊՇԾԽԿԿՋՈԽՊՋՍՊՎՁՎՁՆԿԾԽ ՅԹՄԽՈԽՊՊԽՈՊՇԼՍԻՌՁՎԽԼԹՑՈԽՊՊԽՈՄՁԻԹՌԽ

  ԣԽՊՌՁՄՁՒԹՌՁՇՆՋՍԻԻԽՋՋ
ՁՋԻԹՄ ԻՍՄԹՌԽԼԹՋՌՀԽՆՍՅԺԽՊՇԾԽՅԺՊՑ ՇՋԼՁՎՁԼԽԼԺՑՌՀԽՆՍՅԺԽՊՇԾԽԿԿՋՅՍՄՌՁՈՄՁԽԼԺՑ

ԩԬԮՅԿԹՁԩ

ԪԽԼՁԹՆՅԹՄԽՌՍԺԽՊԻՄԽՋԻՇՊԽՏԹՋՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՆԼ ԹՆԼՁՆՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼ

ԹՆԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈՋՊԽՋՈԽԻՌՁՎԽՄՑ ԱԹԺՄԽԫՇՆՍՈՌՁԹՄՌՍԺԽՊԻՄԽՋՏԽՊԽ

ՇԺՋԽՊՎԽԼՁՆԾԽՅԹՄԽՋ

ԱԹԺՄԽԫՍՈՌՁԹՄԱՍԺԽՊԻՄԽ԰ԻՇՊԽՁՆ ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ



ԱՊԽԹՌՅԽՆՌ

ՅԿԹՁԩ

#ՅԽԹՆՅԽԹՋՍՊԽԼ% ԪԹՄԽՋ ԣԽՅԹՄԽՋ

Ն ԪԽԼՁԹՆԱՍԺԽՊԻՄԽ

԰ԻՇՊԽ Ն ԪԽԼՁԹՆԱՍԺԽՊԻՄԽ

԰ԻՇՊԽ

ԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄ
ԩԬԮ    

    

    

    

ԞԺԺՊԽՎՁԹՌՁՇՆՋԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽԫԡԫՇՌԼԽՌԽՊՅՁՆԽԼ԰ԡ԰ՌԹՆԼԹՊ ԼԼԽՎՁԹՌՁՇՆ

ԩԬԮՅԿԹՁԩ

### ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋ Հ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹ ՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ 

ԭԹԿԽՇԾ ԪԽԹՆՅԹՄԽԤ԰ԦՊԹՆԿԽԼԾՊՇՅՌՇՁՆՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼԹՆԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌ

ԿՊՇՍՈՋՊԽՋՈԽԻՌՁՎԽՄՑ
ԱԹԺՄԽԪԽԹՆԾԽՅԹՄԽԤ԰ԦՊԹՆԿԽԼԾՊՇՅ ՁՆՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄՌՇ

ՁՆՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊՇՍՈ

ԱԹԺՄԽԤՇՆԹԼՇ԰ՇՅԹՌՁԻԦՆԼԽՐ
Ԥ԰ԦՁՆԣԹՌՀԽԹԼԪՁՆՆՇՏ ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ 

ԱՊԽԹՌՅԽՆՌ

ՅԿԹՁԩ

#ՅԽԹՆՅԽԹՋՍՊԽԼ% ԪԹՄԽՋ ԣԽՅԹՄԽՋ

Ն ԪԽԹՆԤ԰Ԧ 

 չ԰ԡ Ն ԪԽԹՆԤ԰Ԧ 

 չ԰ԡ

ԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄ
ԩԬԮ      

      

      

      

ԞԺԺՊԽՎՁԹՌՁՇՆՋ ԫԞ ԫՇՌԹՈՈՄՁԻԹԺՄԽ

  ԤՇՆԹԼՇՋՇՅԹՌՁԻՁՆԼԽՐ
ՁՋԻԹՄԻՍՄԹՌԽԼԹՋԿՇՆԹԼՏԽՁԿՀՌ
ՌՇՌՀԽ ՆԽԹՊԽՋՌՅԿԺՇԼՑՏԽՁԿՀՌ
ՅԿՐ

ԩԬԮՅԿԹՁԩ

ԪԽԼՁԹՆԿՇՆԹԼՋՌԹԿԽՏԹՋԹՆԼՁՆՌՀԽԻՇՆՌՊՇՄԹՆԼԹՄՄՌՊԽԹՌՅԽՆՌ ԿՊՇՍՈՋԾՇՊՅԹՄԽՋԹՆԼԾԽՅԹՄԽՋ

ՊԽՋՈԽԻՌՁՎԽՄՑ
ԱԹԺՄԽ

ԱԹԺՄԽԤՇՆԹԼԹՄ԰ՌԹԿՁՆԿՁՆԣԹՌՀԽԹԼԪՁՆՆՇՏ ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ 

ԱՊԽԹՌՅԽՆՌ

ՅԿԹՁԩ

#ՅԽԹՆՅԽԹՋՍՊԽԼ% ԪԹՄԽՋ ԣԽՅԹՄԽՋ

Ն ԪԽԼՁԹՆ԰ՌԹԿԽ  Ն ԪԽԼՁԹՆ԰ՌԹԿԽ 

ԫ Խ Կ Թ Ռ Ձ Վ Խ Ԡ Շ Ն Ռ Պ Շ Մ  ԩ Ԭ Ԯ        

    



         

    

ԞԺԺՊԽՎՁԹՌՁՇՆՋ ԧ ԧՍՎԽՆՁՄԽ ԫԞ ԫՇՌԹՈՈՄՁԻԹԺՄԽ ԫԡ ԫՇՌԼԽՌԽՊՅՁՆԽԼ ԲԱ԰ ԲՆԹԺՄԽՌՇՋՌԹԿԽ

  ԱՀԽԿՍՁԼԽՄՁՆԽՊԽԻՇՅՅԽՆԼՋՌՀԽԾՇՄՄՇՏՁՆԿԿՇՆԹԼԹՄՋՌԹԿՁՆԿՋԻԹՄԽԾՇՊՅԹՄԽԾԹՌՀԽԹԼՅՁՆՆՇՏՍՆԼԽՎԽՄՇՈԽԼԽԹՊՄՑ

ՋՈԽՊՅԹՌՇԿԽՆՁԻՅՁԼՋՈԽՊՅԹՌՇԿԽՆՁԻՄԹՌԽՋՈԽՊՅԹՌՇԿԽՆՁԻՋՈԽ ՆՌ

  ԱՀԽԿՍՁԼԽՄՁՆԽՊԽԻՇՅՅԽՆԼՋՌՀԽԾՇՄՄՇՏՁՆԿԿՇՆԹԼԹՄՋՌԹԿՁՆԿՋԻԹՄԽԾՇՊԾԽՅԹՄԽԾԹՌՀԽԹԼՅՁՆՆՇՏՍՆԼԽՎԽՄՇՈԽԼԽԹՊՄՑ

ԼԽՎԽՄՇՈՅԽՆՌՅՁԼԼԽՎԽՄՇՈՅԽՆՌՄԹՌԽԼԽՎԽՄՇՈՅԽՆՌՄԹՌԽԼԽՎԽՄՇՈՅԽՆՌՀՑԼՊԹՌԽԼՈՇՋՌՇՎՍՄԹՌՇՊՑ

ԩԬԮՅԿԹՁԩ

### ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋ Հ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹ ՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ 

ԭԹԿԽՇԾ ԥՁՋՌՇՈԹՌՀՇՄՇԿՑՋԻՇՊԽՋԾՇՊԿՇՆԹԼԽՆԼՈՇՁՆՌՋՏԽՊԽԼԽՌԽՊՅՁՆԽԼԺԹՋԽԼՇՆՌՀԽՈԹՌՀՇՄՇԿՁՋՌ0ՋԽՐՈԽՊՁԽՆԻԽԦՆ

ՅԹՄԽՋՌՀԽՊԽՏԹՋԹՆՁՆԻՊԽԹՋԽԼՁՆԻՁԼԽՆԻԽՇԾՅՁՆՁՅԹՄՌԽՋՌՁԻՍՄԹՊԼ ԽԿԽՆԽՊԹՌՁՇՆԹՆԼՅՁՆՁՅԹՄՁՆԻՊԽԹՋԽՁՆՌՀԽ

ՈԽՊԻԽՆՌԹԿԽՇԾՋՈԽՊՅԹՌՇԿՇՆՁԹՁՆՌՀԽՀՁԿՀԽՋՌՌՊԽԹՌՅԽՆՌԿՊՇՍՈՆՇՇՌՀԽՊՌՊԽԹՌՅԽՆՌՊԽՄԹՌԽԼԽԾԾԽԻՌՋՏԽՊԽՆՇՌԽԼ

ՁՆՅԹՄԽՋ
ԱԹԺՄԽՋԹՆԼԱՀԽՊԽՏԽՊԽՆՇՌՊԽԹՌՅԽՆՌՊԽՄԹՌԽԼԿՇ ՆԹԼՇԺՋԽՊՎԹՌՁՇՆՋԾՇՊԾԽՅԹՄԽՋ
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         

        

        

        

        

         

        

        

        

        

         

        

        

        

        

### ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋ Հ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹ ՑՏՁՌՀԠԹՊԺԹՊՑՄ

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԰ԽԽԞՈՈԽՆԼՁՐԢՇԾՌՀԽՌԽՋՌԿՍՁԼԽ ՄՁՆԽԾՇՊՊԽԾԽՊԽՆԻԽ

ԩԬԮՅԿԹՁԩԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽՇԾԱԹԺՄԽԞԼԼՁՌՁՇՆԹՄԤՇՆԹԼԹՄԥՁՋՌՇՈԹՌՀՇՄՇԿՑԬԺՋԽՊՎԹՌՁՇՆՋՁՆԪԹՄԽԣԹՌՀԽԹԼԪՁՆՆՇՏ ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋԱՊԽԹՌՅԽՆՌՅԿԹՁԩ#ՅԽԹՆՅԽԹՋՍՊԽԼ%ԞԼԼՁՌՁՇՆԹՄԡՁԹԿՆՇՋՌՁԻԬԺՋԽՊՎԹՌՁՇՆՋ԰ԽՎԽՊՁՌՑԡԽԻՊԽԹՋԽԼԭՊՇՈՇՊՌՁՇՆՇԾ԰ՈԽՊՅԹՌՇԿՇՆՁԹԦՆԻՊԽԹՋԽԼԳԹՋԻՍՄԹՊՇՊԦՆՌԽՊՋՌՁՌՁԹՄԭՊՇՌԽՁՆԹԻԽՇՍՋԣՄՍՁԼԞՋՑՆԻՀՊՇՆՇՍՋԤՇՆԹԼԡԽՎԽՄՇՈՅԽՆՌԞՄՌԽՊԽԼԭՊՇՈՇՊՌՁՇՆՋՇԾ԰ՈԽՊՅԹՌՇԻՑՌԽՋՇՊ԰ՈԽՊՅԹՌՁԼՋԤՊԹՆՍՄՇՅԹՌՇՍՋԦՆԾՄԹՅՅԹՌՁՇՆՆԦՆԻՁԼԽՆԻԽՆԦՆԻՁԼԽՆԻԽՆԦՆԻՁԼԽՆԻԽՆԦՆԻՁԼԽՆԻԽՆԦՆԻՁԼԽՆԻԽԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄԩԬԮ                                                                                                                                                       ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽՇԾԱՊԽԹՌՅԽՆՌՅԿԹՁԩ#ՅԽԹՆՅԽԹՋՍՊԽԼ%ԞԼԼՁՌՁՇՆԹՄԡՁԹԿՆՇՋՌՁԻԬԺՋԽՊՎԹՌՁՇՆՋ԰ԽՎԽՊՁՌՑԡԽԻՊԽԹՋԽԼԭՊՇՈՇՊՌՁՇՆՇԾ԰ՈԽՊՅԹՌՇԿՇՆՁԹԦՆԻՊԽԹՋԽԼԳԹՋԻՍՄԹՊՇՊԦՆՌԽՊՋՌՁՌՁԹՄԭՊՇՌԽՁՆԹԻԽՇՍՋԣՄՍՁԼԞՋՑՆԻՀՊՇՆՇՍՋԤՇՆԹԼԡԽՎԽՄՇՈՅԽՆՌԞՄՌԽՊԽԼԭՊՇՈՇՊՌՁՇՆՋՇԾ԰ՈԽՊՅԹՌՇԻՑՌԽՋՇՊ԰ՈԽՊՅԹՌՁԼՋԤՊԹՆՍՄՇՅԹՌՇՍՋԦՆԾՄԹՅՅԹՌՁՇՆՆԦՆԻՁԼԽՆԻԽՆԦՆԻՁԼԽՆԻԽՆԦՆԻՁԼԽՆԻԽՆԦՆԻՁԼԽՆԻԽՆԦՆԻՁԼԽՆԻԽ                                                   ԤՇՆԹԼԹՄՀՁՋՌՇՈԹՌՀՇՄՇԿՑԼՁԹԿՆՇՋՌՁԻՇԺՋԽՊՎԹՌՁՇՆՋԹՊԽԿՊԹԼԽԼ-ԺԹՋԽԼՇՆՋԽՎԽՊՁՌՑԫՇՌՊԽՅԹՊՃԹԺՄԽԪՁՆՁՅԹՄԪՁՄԼԪՇԼԽՊԹՌԽ԰ԽՎԽՊԽ԰ԽԽԞՈՈԽՆԼՁՐԢՇԾՌՀԽՌԽՋՌԿՍՁԼԽՄՁՆԽԾՇՊՊԽԾԽՊԽՆԻԽԩԬԮՅԿԹՁԩ

### ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋ Հ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹ ՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ 

ԭԹԿԽՇԾ ԱԹԺՄԽԤՇՆԹԼԹՄԥՁՋՌՇՈԹՌՀՇՄՇԿՑՁՆԣԽՅԹՄԽԣԹՌՀԽԹԼԪՁՆՆՇՏ ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ 

ԱՊԽԹՌՅԽՆՌ

ՅԿԹՁԩ

#ՅԽԹՆՅԽԹՋՍՊԽԼ% ԞԼԼՁՌՁՇՆԹՄԡՁԹԿՆՇՋ ՌՁԻԬԺՋԽՊՎԹՌՁՇՆՋ 

԰ԽՎԽՊՁՌՑ ԦՆԻՊԽԹՋԽԼԬՇԻՑՌԽԞՌՊԽՋՁԹ ԭԽՊՁԾՇՄՄՁԻՍՄԹՊԠԽՄՄ

ԥՑՈԽՊՈՄԹՋՁԹԥՑՈԽՊՌՊՇՈՀՑ ԡԽԻՊԽԹՋԽԼԶՇՄՃ

ԣՇՊՅԹՌՁՇՆ

Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ Ն ԦՆԻՁԼԽՆԻԽ

ԫԽԿԹՌՁՎԽԻՇՆՌՊՇՄ

ԩԬԮ       

      

      

      

      

       

      

      

      

      

       

      

      

      

      

       

      

      

      

      

  ԤՇՆԹԼԹՄՀՁՋՌՇՈԹՌՀՇՄՇԿՑԼՁԹԿՆՇՋՌՁԻՇԺՋԽՊՎԹՌՁՇՆՋԹՊԽԿՊԹԼԽԼ- ԺԹՋԽԼՇՆՋԽՎԽՊՁՌՑԫՇՌՊԽՅԹՊՃԹԺՄԽԪՁՆՁՅԹՄ

ԪՁՄԼԪՇԼԽՊԹՌԽ԰ԽՎԽՊԽ԰ԽԽԞՈՈԽՆԼՁՐԢՇԾՌՀԽՌԽՋՌԿՍՁԼԽ ՄՁՆԽԾՇՊՊԽԾԽՊԽՆԻԽ

ԩԬԮՅԿԹՁԩ

### ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋ Հ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹ ՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ 

ԭԹԿԽՇԾ ԱԹԺՄԽԞԼԼՁՌՁՇՆԹՄԤՇՆԹԼԹՄԥՁՋՌՇՈԹՌՀՇՄՇԿՑԬԺՋԽՊՎԹՌՁՇՆՋՁՆԣԽՅ ԹՄԽԣԹՌՀԽԹԼԪՁՆՆՇՏ 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԰ԽԽԞՈՈԽՆԼՁՐԢՇԾՌՀԽՌԽՋՌԿՍՁԼԽ ՄՁՆԽԾՇՊՊԽԾԽՊԽՆԻԽ

ԩԬԮՅԿԹՁԩ

### ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋ Հ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹ ՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ 

ԭԹԿԽՇԾ ԪԽԹՆՈՄԹՋՅԹՎՁՌԽՄՄՇԿԽՆՁՆԻՇՆԻԽՆՌՊԹՌՁՇՆՋՏԽՊԽՀՁԿՀՄՑՎԹՊՁԹԺՄԽՁՆ ԺՇՌՀՅԹՄԽՋԹՆԼԾԽՅԹՄԽՋՁՆԹՄՄՌՊԽԹՌՅԽՆՌ

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 

ԞԺԺՊԽՎՁԹՌՁՇՆՋ ԫԞ ԫՇՌԹՈՈՄՁԻԹԺՄԽ ԫԡ ԫՇՌԼԽՌԽՊՅՁՆԽԼ ԰ԡ ԰ՌԹՆԼԹՊԼԼԽ ՎՁԹՌՁՇՆ

ԩԬԮՅԿԹՁԩԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽՇԾԱԹԺՄԽԭՄԹՋՅԹ԰ԽՐ԰ՌԽՊՇՁԼՋՁՆԣԹՌՀԽԹԼԪՁՆՆՇՏ ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋԱՀՁՋՌԹԺՄԽՁՋՇՅՁՌՌԽԼԺԽԻԹՍՋԽՈՄԹՋՅԹՋԽՐՋՌԽՊՇՁԼՋՏԽՊԽՆՇՌՅԽԹՋՍՊԽԼԱՀԽՊԽՏԽՊԽՆՇՆՇՌԹԺՄԽՇԺՋԽՊՎԹՌՁՇՆՋՏՁՌՀՊԽՋՈԽԻՌՌՇԺԽՀԹՎՁՇՊԻՇՄՇՊԹՌՁՇՆԺԹՆԼՁՆԿԻՀԹՆԿԽՋՁՆՇՎՁՈՇՋՁՌՇՊԹՈՈԽԹՊԹՆԻԽՇՊՋՁՒԽՇԾԼՇՊՋԹՄՆԹՈԽՈԹԼ
ԱԹԺՄԽԱԹԺՄԽ԰ԽԻՇՆԼԹՊՑ԰ԽՐԠՀԹՊԹԻՌԽՊՁՋՌՁԻՋԹՆԼԠՄՁՆՁԻԹՄ԰ՁԿՆՋՁՆԣԹՌՀԽԹԼԪՁՆՆՇՏ ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋԱՊԽԹՌՅԽՆՌՅԿԹՁԩ#ՅԽԹՆՅԽԹՋՍՊԽԼ%԰ԽԻՇՆԼԹՊՑ԰ԽՐԠՀԹՊԹԻՌԽՊՁՋՌՁԻՋԹՆԼԠՄՁՆՁԻԹՄ԰ՁԿՆՋԪԹՄԽՋԣԽՅԹՄԽՋԱՑՈԽՆԦՆԻՁԼԽՆԻԽԱՑՈԽՆԦՆԻՁԼԽՆԻԽԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄ
ԩԬԮԫՇՆԽ ԫՇՆԽ ԫՇՆԽ ԫՇՆԽ ԫՇՆԽ ԫՇՆԽ ԫՇՆԽ ԫՇՆԽ ԩԬԮՅԿԹՁԩ

### ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋ Հ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹ ՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ 

ԭԹԿԽՇԾ ԟ԰ՌՍԼՑԞՍՌՀՇՊ0ՋԞՆԹՄՑՋՁՋԹՆԼԠՇՆԻՄՍՋՁՇՆՋ

ԞՄՄՋՌԹՌՁՋՌՁԻԹՄԹՆԹՄՑՋԽՋՏԽՊԽԻՇՆԼՍԻՌԽԼԹՌՌՀԽՄԽՎԽՄՇԾԻԽՊ ՌԹՁՆՌՑԽՐԻԽՈՌՁՆՌՀԽԻԹՋԽՇԾ԰ՀԹՈՁՊՇԴՁՄՃՋ0

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

ՈԽՊԻԽՆՌՋՍՊՎՁՎԹՄՏԽՊԽԼԽՌԽՊՅՁՆԽԼՍՋՁՆԿՌՀԽԠՇԻՀՊԹՆԞՊՅՁՌԹԿԽԱՊԽ ՆԼ԰ՌԽՈԡՇՏՆԱԽՋՌԣՇՊԽՆԼՈՇՁՆՌՋՌՀԹՌ

ՅԽՌ ՌՀԽ ԹՋՋՍՅՈՌՁՇՆՋ ՇԾ ՆՇՊՅԹՄՁՌՑ ԹՆԼ ՀՇՅՇԿԽՆԽՁՌՑ ՌՀԽ ՈԽՊԾՇՊՅԹՆ ԻԽ ՇԾ ՇՊԿԹՆՁՋՅՋ ԽՐՈՇՋԽԼ ՌՇ ԽԹԻՀ

ՌՊԽԹՌՅԽՆՌՄԽՎԽՄՏԹՋԻՇՅՈԹՊԽԼՏՁՌՀՌՀԽՈԽՊԾՇՊՅԹՆԻԽՇԾՌՀԽԻՇՆՌՊՇ ՄՍՋՁՆԿԡՍՆՆԽՌՌ0ՋԪՍՄՌՁՈՄԽԠՇՅՈԹՊՁՋՇՆ

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

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

ԻՇՆԼՍԻՌԽԼԺՇՌՀՏՁՌՀԹՆԼՏՁՌՀՇՍՌՋՍՋՈԽԻՌԽԼՇՍՌՄՁԽՊՋԬՍՌՄՁԽՊՋԾՇ ՊՌՀԽՅԹՄԽԼԹՌԹՏԽՊԽՁԼԽՆՌՁԾՁԽԼԺՑՎԹՄՍԽՋ

ՏՀՁԻՀ ԽՐԻԽԽԼԽԼ ՌՀԽ ՅԽԼՁԹՆ ՈՄՍՋ ՌՀՊԽԽ ՌՁՅԽՋ ՌՀԽ ՁՆՌԽՊՉՍԹՊՌՁՄԽ ՊԹ ՆԿԽ 
ՁԽ ՌՀԽ ԼՁԾԾԽՊԽՆԻԽ ԺԽՌՏԽԽՆ ՌՀԽ

ՌՀԹՆԼՌՀՈԽՊԻԽՆՌՁՄԽՋԣԽՅԹՄԽԼԹՌԹՏԽՊԽՄՇԿՌՊԹՆՋԾՇՊՅԽԼՈ ՊՁՇՊՌՇՇՍՌՄՁԽՊԹՆԹՄՑՋՁՋԬՍՌՄՁԽՊՋԾՇՊՌՀԽ

ԾԽՅԹՄԽԼԹՌԹՏԽՊԽՁԼԽՆՌՁԾՁԽԼԺՑՄՇԿՌՊԹՆՋԾՇՊՅԽԼՎԹՄՍԽՋՏՀՁԻՀՏԽՊ ԽԺԽՄՇՏՌՀԽՅԽԼՁԹՆՅՁՆՍՋՌՀՊԽԽՌՁՅԽՋ

ՌՀԽՁՆՌԽՊՉՍԹՊՌՁՄԽ ՊԹՆԿԽԞԼԼՁՌՁՇՆԹՄՇՍՌՄՁԽՊՋՏԽՊԽՁԼԽՆՌՁԾՁԽԼԺԹ ՋԽԼՇՆԻՇՆՋՍՄՌԹՌՁՇՆՏՁՌՀՌՀԽ԰ՌՍԼՑ԰ՈՇՆՋՇՊ

ԽՐՈԽՊՌՂՍԼԿՅԽՆՌՈՊՁՅԹՊՁՄՑԺԹՋԽԼՇՆՎԹՄՍԽՋԺԽՄՇՏՌՀԽԼԽՌԽԻՌՁՇՆ ՄՁՅՁՌՇՊՇՍՌՋՁԼԽՇԾՌՀԽՆՇՊՅԹՄՎՁՌԽՄՄՇԿԽՆՁՆ

ՊԹՆԿԽԦԾՌՀԽՊԽՏԽՊԽՆՇԼՁԾԾԽՊԽՆԻԽՋՁՆՌՀԽՊԽՋՍՄՌՋՇԾՌՀԽՋՌԹՌՁ ՋՌՁԻԹՄԹՆԹՄՑՋԽՋԺԽՌՏԽԽՆՌՀԽՌՏՇՋԽՌՋՇԾԼԹՌԹ

ՌՀԽՆՌՀԽ ԹՆԹՄՑՋՁՋ ՁՆԻՄՍԼՁՆԿՌՀԽ ՇՍՌՄՁԽՊՋՏԹՋ ՈՊԽՋԽՆՌԽԼԦԾ ԼՁԾԾԽՊԽՆԻԽՋՏԽՊԽ ՇԺՋԽՊՎԽԼՌՀԽՆ ԺՇՌՀՋԽՌՋ ՇԾ

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

ԥՁՋՌՇՈԹՌՀՇՄՇԿՑՊԽՋՍՄՌՋՏԽՊԽԽՎԹՄՍԹՌԽԼՉՍԹՄՁՌԹՌՁՎԽՄՑ

ԱՀԽՋՌՍԼՑԹՍՌՀՇՊԾՇՍՆԼԹՋՁԿՆՁԾՁԻ ԹՆՌԼԽԻՊԽԹՋԽՁՆՅԹՄԽՆՍՈՌՁԹՄՌ ՍԺԽՊԻՄԽՋԻՇՊԽԹՆԼԹՉՍԹՄՁՌԹՌՁՎԽՁՆԻՊԽԹՋԽՁՆ

ՅԹՄԽ ՀՁՋՌՇՈԹՌՀՇՄՇԿՁԻԹՄ ԾՁՆԼՁՆԿՋ ՁՆ ՌՀԽ ՅԽԹՆՅԽԹՋՍՊԽԼ  ՅԿ Թ Ձԩ ՌՊԽԹՌՅԽՆՌ ԿՊՇՍՈ ԫՇ ՇՌՀԽՊ

ՋՁԿՆՁԾՁԻԹՆՌԽԾԾԽԻՌՋՏԽՊԽՆՇՌԽԼ

ԠԯԽՎՁԽՏԽՊ0ՋԞՆԹՄՑՋՁՋԹՆԼԠՇՆԻՄՍՋՁՇՆՋ

԰ՌԹՌՁՋՌՁԻԹՄԪԽՌՀՇԼՋ ԣԽՏՁՆԻՁԼԽՆՌԹՄՅՇՊՌԹՄՁՌՁԽՋՇԻԻՍՊՊԽԼԹՆԼԼՁԼՆՇՌԽՐՀՁԺՁՌԹԻՇՆԻԽ ՆՌՊԹՌՁՇՆԼԽՈԽՆԼԽՆՌ

ՊԽՋՈՇՆՋԽՌՀԽՊԽԾՇՊԽՋՍՊՎՁՎԹՄՏԹՋԹՆԹՄՑՒԽԼԽՅՈՁՊՁԻԹՄՄՑԫՇԽՆԼՈ ՇՁՆՌՋԽՐՀՁԺՁՌԽԼԹԻՄԽԹՊՅՇՆՇՌՇՆՁԻ

ՊԽՋՈՇՆՋԽԹՆԼԹՄՄԽՆԼՈՇՁՆՌՋԽՐԻԽՈՌՅԹՄԽԳԱԤՋԻՇՊԽՏԽՊԽՆՇՊՅԹՄՄՑ ԼՁՋՌՊՁԺՍՌԽԼ
԰ՀԹՈՁՊՇԴՁՄՃՋ Ո 

ՏՁՌՀԽՉՍԹՄՎԹՊՁԹՆԻԽ
ԟԹՊՌՄԽՌՌ0ՋՌԽՋՌ Ո ԹՆԼՏԽՊԽՌՀԽՊԽԾՇՊԽԹՆԹՄՑՒԽԼՍՋՁՆԿԡՍՆՆԽՌՌ0ՋԪՍՄՌՁՈՄԽ

ԠՇՅՈԹՊՁՋՇՆԱԽՋՌԪԹՄԽԳԱԤՏԹՋԹՆԹՄՑՒԽԼՍՋՁՆԿՌՀԽԪԹՆՆԴՀՁՌՆԽՑԲ ԱՏՇ԰ԹՅՈՄԽՌԽՋՌԥՇՏԽՎԽՊՅԹՄԽ

ՌՍԺԽՊԻՄԽՋԻՇՊԽՏԹՋԹՄՋՇԹՆԹՄՑՒԽԼՍՋՁՆԿՌՀԽԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹ ԰ՌԽՈԡՇՏՆՌԽՋՌԞՄՄԹՆԹՄՑՋԽՋՏԽՊԽ

ԻՇՆԼՍԻՌԽԼՍՋՁՆԿԠԢԱԦ԰ՎՍՋՁՆԿԺԹԻՃԽՆԼՋԽՌՌՁՆԿՋԼԽՋՁԿՆԽԼԺՑԢԣԢԡԾՇՊԹՈՈՊՇՎԽԼՍՋԽՇՆ

ԲՆՄԽՋՋՇՌՀԽՊՏՁՋԽՁՆԼՁԻԹՌԽԼԽԾԾԽԻՌՋՏԽՊԽԻՇՆՋՁԼԽՊԽԼՋՌ ԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌԹՌ Ո 

### ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋ Հ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹ ՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ 

ԭԹԿԽՇԾ ԠՇՆԻՄՍՋՁՇՆՋ ԪԹՄԽՄԽՆԿՌՀՏԹՋՋՁԿՆՁԾՁԻԹՆՌՄՑՊԽԼՍԻԽԼ
ՊԽԼՍԻՌՁՇՆՊԽՄԹՌՁՎԽ ՌՇՌՀԽՆԽԿԹՌՁՎԽԻՇՆՌՊՇՄԹՌ

ՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌՄԽՎԽՄ
ԡՍՆՆԽՌՌ0ՋԪՍՄ ՌՁՈՄԽԠՇՅՈԹՊՁՋՇՆԱԽՋՌ Ո 

ԣՍՊՌՀԽՊՅԽԼՁԹՆՅԹՄԽՌՍԺԽՊԻՄԽՋԻՇՊԽՏԹՋՋՁԿՆՁԾՁԻԹՆՌՄՑՊԽԼՍԻԽԼԹ ՌՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼՅԿԹՁԩ

ՌՊԽԹՌՅԽՆՌԿՊՇՍՈ
ԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹ԰ՌԽՈԡՇՏՆԱԽՋՌ Ո ԱՀԽՋՁԿՆՁԾՁԻԹՆՌՊԽԼՍԻՌՁՇՆՁՆՅԹՄԽ

ՌՍԺԽՊԻՄԽՋԻՇՊԽԹՌՌՀԽՅԽԹՆՅԽԹՋՍՊԽԼՅԿԹՁԩՌՊԽԹՌՅԽՆՌԿՊ ՇՍՈՇԻԻՍՊՁՆԻՇՆԻԽՊՌՏՁՌՀԹՆՁՆԻՊԽԹՋԽ

ՁՆՁՆՌԽՆՋՁՌՑԹՆԼՋԽՎԽՊՁՌՑՇԾՅԹՄԽՀՁՋՌՇՈԹՌՀՇՄՇԿՁԻԹՄԾՁՆԼՁՆԿՋ
Խ ԿՁՆԻՊԽԹՋԽԼՁՆԻՁԼԽՆԻԽՇԾՅՁՆՁՅԹՄՌԽՋՌՁԻՍՄԹՊ

ԼԽԿԽՆԽՊԹՌՁՇՆԹՆԼՅՁՆՁՅԹՄՁՆԻՊԽԹՋԽՁՆՌՀԽՈԽՊԻԽՆՌԹԿԽՇԾՋՈԽՊՅԹՌՇԿՇՆՁԹԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽՇԾԱԹԺՄԽԯԽՈՊՇԼՍԻՌՁՎԽԹՆԼԥԭԤԢՆԼՈՇՁՆՌՋԾՇՊԪԹՄԽԣԹՌՀԽԹԼԪՁՆՆՇՏ ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋՁՆՌՀԽԣ԰ԱԯԞՏՁՌՀԠԹՊԺԹՊՑՄԱՊԽԹՌՅԽՆՌՅԿԹՁԩ#ՅԽԹՆՅԽԹՋՍՊԽԼ%ԱՍԺԽՊԻՄԽ԰ԻՇՊԽԤ԰ԦԤՇՆԹԼԹՄ԰ՌԹԿՁՆԿԹՆԼԥՁՋՌՇԭՄԹՋՅԹԳԱԤԭՄԹՋՅԹԱԭՄԹՋՅԹԢԪԽԼՁԹՆ ՈԡՁԾԾ ՈԢԾԾԽԻՌ!
ԶԽՋԫՇԡՁԾԾ ՈԡՁԾԾ ՈԡՁԾԾ ՈԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄԩԬԮ   ԫՇ         ԫՇ          ԫՇ        ԶԽՋ     ԰ՌԹՌՁՋՌՁԻԹՄԱԽՋՌ ԧՇՆԻՃՀԽԽՊԽԱԽՊՈՋՌՊԹ ԡՍՆՆԽՌՌԫԞԪԹՆՆԴՀՁՌՆԽՑ ԞԺԺՊԽՎՁԹՌՁՇՆՋԠՇՆԻԠՇՆԻԽՆՌՊԹՌՁՇՆԡՁԾԾԡՁԾԾԽՊԽՆԻԽԢȕԽՋՌՊԹԼՁՇՄԤ԰ԦԤՇՆԹԼՇ԰ՇՅԹՌՁԻԦՆԼԽՐԥՁՋՌՇԥՁՋՌՇՈԹՌՀՇՄՇԿՑԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽԱԱԽՋՌՇՋՌԽՊՇՆԽԳԱԤԳՁՌԽՄՄՇԿԽՆՁՆ ԲՆՄԽՋՋՇՌՀԽՊՏՁՋԽՁՆԼՁԻԹՌԽԼԽԾԾԽԻՌՋԹՆԼՈԽՊԻԽՆՌ
ԼՁԾԾԽՊԽՆԻԽՋԹՊԽՊԽՈՇՊՌԽԼԺԹՋԽԼՇՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽՆԽԿԹՌՁՎԽ
ԻՄԽԹՆՏԹՌԽՊԻՇՆՌՊՇՄԠՇՆԻՄՍՋՁՇՆՋՊԽԿԹՊԼՁՆԿՀՁՋՌՇՈԹՌՀՇՄՇԿՑՅԹՑԺԽՀԽԹՎՁՄՑՏԽՁԿՀՌԽԼԺՑՌՀԽԽՐՈԽՊՌՇՈՁՆՁՇՆՇԾԹԺՇԹՊԼԻԽՊՌՁԾՁԽԼՈԹՌՀՇՄՇԿՁՋՌ ԲՆՄԽՋՋՇՌՀԽՊՏՁՋԽՋՈԽԻՁԾՁԽԼԽԾԾԽԻՌՋԹՊԽԻՇՆՋՁԼԽՊԽԼՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌԹՌ ՈԩԬԮՅԿԹՁԩԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋՀ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹՑՏՁՌՀԠԹՊԺԹՊՑՄԢԭԞԪԯԦԡԫՍՅԺԽՊԭԹԿԽՇԾԱԹԺՄԽԯԽՈՊՇԼՍԻՌՁՎԽԹՆԼԥԭԤԢՆԼՈՇՁՆՌՋԾՇՊԣԽՅԹՄԽԣԹՌՀԽԹԼԪՁՆՆՇՏ ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋՁՆՌՀԽԣ԰ԱԯԞՏՁՌՀԠԹՊԺԹՊՑՄԱՊԽԹՌՅԽՆՌՅԿԹՁԩ#ՅԽԹՆՅԽԹՋՍՊԽԼ%ԣԽԻՍՆԼՁՌՑԣԽՊՌ԰ՍԻԻԽՋՋԱՍԺԽՊԻՄԽ԰ԻՇՊԽԤ԰ԦԤՇՆԹԼԹՄ԰ՌԹԿՁՆԿԹՆԼԥՁՋՌՇԭՄԹՋՅԹԳԱԤԭՄԹՋՅԹԱԭՄԹՋՅԹԢԡՁԾԾ ՈԡՁԾԾ ՈԪԽԼՁԹՆ ՈԡՁԾԾ ՈԢԾԾԽԻՌ!
ԶԽՋԫՇԡՁԾԾ ՈԡՁԾԾ ՈԡՁԾԾ ՈԫԽԿԹՌՁՎԽԠՇՆՌՊՇՄԩԬԮ        ԫՇ              ԫՇ             ԫՇ              ԫՇ      ԰ՌԹՌՁՋՌՁԻԹՄԱԽՋՌ ԡՍՆՆԽՌՌ ԡՍՆՆԽՌՌԡՍՆՆԽՌՌԫԞԡՍՆՆԽՌՌԞԺԺՊԽՎՁԹՌՁՇՆՋԠՇՆԻԠՇՆԻԽՆՌՊԹՌՁՇՆԡՁԾԾԡՁԾԾԽՊԽՆԻԽԢȕԽՋՌՊԹԼՁՇՄԣԽՊՌԣԽՊՌՁՄՁՒԹՌՁՇՆԤ԰ԦԤՇՆԹԼՇ԰ՇՅԹՌՁԻԦՆԼԽՐԥՁՋՌՇԥՁՋՌՇՈԹՌՀՇՄՇԿՑԫԞԫՇՌԹՈՈՄՁԻԹԺՄԽԱԱԽՋՌՇՋՌԽՊՇՆԽԳԱԤԳՁՌԽՄՄՇԿԽՆՁՆ ԲՆՄԽՋՋՇՌՀԽՊՏՁՋԽՁՆԼՁԻԹՌԽԼԽԾԾԽԻՌՋԹՆԼՈԽՊԻԽՆՌ
ԼՁԾԾԽՊԽՆԻԽՋԹՊԽՊԽՈՇՊՌԽԼԺԹՋԽԼՇՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽՆԽԿԹՌՁՎԽ
ԻՄԽԹՆՏԹՌԽՊԻՇՆՌՊՇՄԠՇՆԻՄՍՋՁՇՆՋՊԽԿԹՊԼՁՆԿՀՁՋՌՇՈԹՌՀՇՄՇԿՑՅԹՑԺԽՀԽԹՎՁՄՑՏԽՁԿՀՌԽԼԺՑՌՀԽԽՐՈԽՊՌՇՈՁՆՁՇՆՇԾԹԺՇԹՊԼԻԽՊՌՁԾՁԽԼՈԹՌՀՇՄՇԿՁՋՌ ԲՆՄԽՋՋՇՌՀԽՊՏՁՋԽՋՈԽԻՁԾՁԽԼԽԾԾԽԻՌՋԹՊԽԻՇՆՋՁԼԽՊԽԼՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌԹՌ ՈԩԬԮՅԿԹՁԩ

### ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋ Հ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹ ՑՏՁՌՀԠԹՊԺԹՊՑՄ

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

ԞԺԺՊԽՎՁԹՌՁՇՆՋ ԡՁԾԾ ԡՁԾԾԽՊԽՆԻԽ ԫԞ ԫՇՌԹՈՈՄՁԻԹԺՄԽ ԫԡ ԫՇՌԼԽՌԽՊՅՁՆԽԼ

  ԲՆՄԽՋՋՇՌՀԽՊՏՁՋԽՁՆԼՁԻԹՌԽԼՈԽՊԻԽՆՌ
ԼՁԾԾԽՊԽՆԻԽՋԹՊԽՊԽՈՇՊՌ ԽԼԺԹՋԽԼՇՆԻՇՅՈԹՊՁՋՇՆՌՇՌՀԽՆԽԿԹՌՁՎԽ
ԻՄԽԹՆՏԹՌԽՊ

ԻՇՆՌՊՇՄ

  ԲՆՄԽՋՋՇՌՀԽՊՏՁՋԽՋՈԽԻՁԾՁԽԼԽԾԾԽԻՌՋԹՊԽԻՇՆՋՁԼԽՊԽԼՋՌԹՌՁՋՌՁԻԹՄՄՑՋՁԿՆՁԾՁԻԹՆՌԹՌ Ո 

ԩԬԮՅԿԹՁԩ

Ԣ԰ՌՍԼՑԡԽԾՁԻՁԽՆԻՁԽՋ

ԞՄՄՈԽՊԾՇՊՅԹՆԻԽԻՊՁՌԽՊՁԹՏԽՊԽՅԽՌՁՆՌՀՁՋՋՌՍԼՑԽՐԻԽՈՌՌՀԹՌԾՇ ՊԻՇՆՌՊՇՄՊԽՈՄՁԻԹՌԽԡՋՈԹՏՆՁՆԿԼՁԼՆՇՌՇԻԻՍՊ

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

ԹՎԽՊԹԿԽԾԽԻՍՆԼՁՌՑԾՇՊՌՀԹՌՊԽՈՄՁԻԹՌԽՏԹՋԽԿԿՋԾԽՅԹՄԽԼԹՑԾԽ ԻՍՆԼՁՌՑՏԹՋՄՁՅՁՌԽԼԼՍՊՁՆԿՌՀԽԾՁՊՋՌՏԽԽՃ

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

ՊԽՈՄՁԻԹՌԽՋ ՊԹՆԿԽԼԾՊՇՅ ՌՇ ԽԿԿՋԾԽՅԹՄԽԼԹՑ ՁՆԼՁԻԹՌՁՆԿՌՀԹՌ ԾԽԻՍՆԼՁՌՑՏԹՋ ԹԼԽՉՍԹՌԽ ՁՆՌՀԽԻՇՆՌՊՇՄ

ՌՊԽԹՌՅԽՆՌ ԞԼԼՁՌՁՇՆԹՄՄՑ  ՇԾՌՀԽ  ՋՈԹՏՆՁՆԿ ԿՊՇՍՈՋ ՋԽՄԽԻՌԽԼԾ ՇՊ ՌՀԽ ԼԽԾՁՆՁՌՁՎԽ ՋՌՍԼՑ ՀԹԼ ՈՊԽԽՐՈՇՋՍՊԽ

ԾԽԻՍՆԼՁՌՑ ՇԾ ՌՇ  ԽԿԿՋԾԽՅԹՄԽԼԹՑ ԿՍՁԼԹՆԻԽ ՊԽԻՇՅՅԽՆԼՋ ԹՌ Մ ԽԹՋՌ  ԽԿԿՋԾԽՅԹՄԽԼԹՑԥՇՏԽՎԽՊ

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

ՊԽՋՁԼՍԹՄԻՀՄՇՊՁՆԽԻՇՆԻԽՆՌՊԹՌՁՇՆՋՁՆՌՀԽԼՁՄՍՌՁՇՆՏԹՌԽՊՏԽՊԽՆՇՌՋՈԽԻՁԾՁԽԼԞՄՄՇՌՀԽՊՈԽՊԾՇՊՅԹՆԻԽԹՆԼՎԹՄՁԼՁՌՑ

ԻՊՁՌԽՊՁԹՋՈԽԻՁԾՁԽԼՁՆԬԭԭԱ԰ՏԽՊԽՅԽՌ

ԣԯԽՎՁԽՏԽՊ0ՋԠՇՅՅԽՆՌՋ

ԱՀԽՊԽՎՁԽՏԽՊ0ՋՊԽՋՍՄՌՋՏԽՊԽՁՆԹԿՊԽԽՅԽՆՌՏՁՌՀՌՀՇՋԽՇԾՌՀԽՋՌՍԼ ՑԹՍՌՀՇՊ

ԱՀԽՁՆՄՁԾԽՈՇՊՌՁՇՆՇԾՌՀԽԼԽԾՁՆՁՌՁՎԽՌՇՐՁԻՁՌՑՌԽՋՌՏԹՋԻՇՆԼՍԻՌ ԽԼԾՊՇՅԣԽԺՊՍԹՊՑՌՇԪԹՊԻՀ

### ԡԹՌԹԢՎԹՄՍԹՌՁՇՆԯԽԻՇՊԼՇՆՌՀԽԣՁՋ Հ԰ՀՇՊՌԱԽՊՅԯԽՈՊՇԼՍԻՌՁՇՆԞՋՋԹ ՑՏՁՌՀԠԹՊԺԹՊՑՄ

ԢԭԞԪԯԦԡԫՍՅԺԽՊ 

ԭԹԿԽՇԾ ԦԦԦԯԢԣԢԯԢԫԠԢ԰

ԞՆՃՄԽՑԤԱԨԪԧԽՆՋԽՆԪԡԨԹՀՄԧԧԨՇՊՌԽԹՆԼԢԞԪԹՃՑ ՆԽՆԡԽՋԻՊՁՈՌՁՇՆԹՆԼԽՎԹՄՍԹՌՁՇՆՇԾ

ԹՋՀՇՊՌՌԽՊՅՊԽՈՊՇԼՍԻՌՁՇՆՌԽՋՌՏՁՌՀՌՀԽԾԹՌՀԽԹԼՅՁՆՆՇՏ ԭՁՅԽՈՀԹՄԽՋՈՊՇՅԽՄԹՋ ԢՆՎՁՊՇՆԱՇՐՁԻՇՄ

ԠՀԽՅ 

ԠԹՊՄՁՋՄԽԧԠԱՀԽՌՇՐՁԻՁՌՑՇԾԠԹՊԺԹՊՑՄ
ԟԞԶԩԢԱԬԫ ՌՇՊԹՁՆԺՇՏՌՊՇՍՌԽԹՊՄՑՄՁԾԽՋՌԹԿԽՋ

ԟԹՑԽՊԠՊՇՈ԰ԻՁԽՆԻԽՍՆՈՍԺՄՁՋՀԽԼՊԽՈՇՊՌՋՌՍԼՑՆՍՅԺԽՊ

ԪՇՍՆՌԡԦԹՆԼԴԞԟՊՍՆԿՋԞՋՁՅՈՄՁԾՁԽԼԼՇՋՁՆԿԹՈՈԹՊԹՌ ՍՋԾՇՊԾՁՋՀՌՇՐՁԻՇՄՇԿՁԻԹՄՋՌՍԼՁԽՋԴԹՌԽՊ

ԯԽՋԽԹՊԻՀ԰ՈՊԹԿՍԽԧԟԪԽԹՋՍՊԽՅԽՆՌՇԾՈՇՄՄՍՌԹՆՌ ՌՇՐՁԻՁՌՑՌՇԾՁՋՀԟՁՇԹՋՋԹՑ

ՅԽՌՀՇԼՋԾՇՊԹԻՍՌԽՌՇՐՁԻՁՌՑԴԹՌԽՊԯԽՋԽԹՊԻՀ

### 

### 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

Brine: Not Applicable Species: Pimephales promelas

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

Project: Insecticide Material: Carbaryl

flow-through conditio ns 056801 48669601

056801 48669601 Batch Note:

Sample Note:

Comparison Summary

NOEL LOEL TOEL Endpoint Analysis ID Method PMSD TU

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

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

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

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

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

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

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

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

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

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

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

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

### 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

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

95% LCL 95% UCL %Effect

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

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

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

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

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

95% LCL 95% UCL %Effect

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

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

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

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

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

95% LCL 95% UCL %Effect

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

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

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

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

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

95% LCL 95% UCL %Effect

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

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

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

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

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

95% LCL 95% UCL %Effect

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

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

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

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

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

95% LCL 95% UCL %Effect

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

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

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

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

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

95% LCL 95% UCL %Effect

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

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

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

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

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

95% LCL 95% UCL %Effect

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

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

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

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

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

### 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

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

95% LCL 95% UCL %Effect

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

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

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

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

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

95% LCL 95% UCL %Effect

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

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

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

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

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

95% LCL 95% UCL %Effect

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

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

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

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

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

### 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

Control Type C-mg ai/L 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

Control Type C-mg ai/L 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

Control Type C-mg ai/L 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

Control Type C-mg ai/L 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

Control Type C-mg ai/L 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

Control Type C-mg ai/L 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

Control Type C-mg ai/L 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

Control Type C-mg ai/L 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

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

### 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

Control Type C-mg ai/L 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

Control Type C-mg ai/L 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

Control Type C-mg ai/L 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

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

### 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

Brine: Not Applicable Species: Pimephales promelas

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

Project: Insecticide Material: Carbaryl

flow-through conditio ns 056801 48669601

056801 48669601 Batch Note:

Sample Note:

Comparison Summary

NOEL LOEL TOEL Endpoint Analysis ID Method PMSD TU

0.15 0.82 0.3507 17-3444-4617 Jonckheere-Terpstra Step-Down Test NA MaleMedianTubercleScore

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

95% LCL 95% UCL %Effect

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

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

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

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

Control Type C-mg ai/L 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

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

# DATA EVALUATION RECORD

CARBARYL

Study Type: O CSPP 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.

### 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 strengt hs 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).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 

### S

### Signature: 

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### Date:

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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, associatedIn vivo Hershberger Assay (2003) / Page 2 of 13

CARBARYL / 056801 OCSPP 890.1400/ OECD 441



### with i ncreased 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 grou p 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 decrease s (p<0.01) in weights of accessory sex orga ns 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 validate d. 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 increase s (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 sa livation, 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 negati ve for anti-androgenicity in the Hershberger assay.

Th e assay satisf ies the EDSP Tier 1 Test Order requirement s 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.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 Un it 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 methylcellu l ose 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 CH 3In vivo Hershberger Assay (2003) / Page 4 of 13

CARBARYL / 056801 OCSPP 890.1400/ OECD 441



### 6

. Test Animals :

Species:

Rat (males only)

Strain: Sprague Da w ley [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 D ates : 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. T he animals were dosed for 10 consecutive days and necropsied approximately 24 hours after the final dose administration for organ weight measurements.

3. Study Schedule : M ale rats were castrated by the supp lier 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

.In vivo Hershberger Assay (2003) / Page 5 of 13

CARBARYL / 056801 OCSPP 890.1400/ OECD 441

TABLE 1. Study D esign a
Test group Dose (mg/kg/day) # of Males

Anti

- Androgen Assay

Vehicle control b 0 6

T estosterone propionate (TP) control 0 + 0.3 6
Low ( C arbaryl + TP) 5 + 0.3 6

Mid ( C arbaryl + TP) 15 + 0.3 6
High ( C arbaryl + 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 previously 2 

### 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. Dosa ge A dministration : 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.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 bas ed 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 we ights, and liver weight.

For comparing carbaryl +TP treatment groups with the TP control, the Bartlett test (two

sided) 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 mean s 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 t he 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 su rviving animals were anesthetized,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 vesic les with coagulating glands, LABC, Cowper’s gland, and glans penis) were excised, trimmed free of adhering fat , and weighed to the nearest milli gram . Additionally, the liver was collected and weighed.

II. RESULTS

A. OBSERVATIONS

1. Mortality : All animals survived until scheduled termination.

2. Clinical S igns 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 a ctivity, 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 animal s 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 w ere observed in

control animals.

TABLE 2

. Incidence of Clinical Observations in the Anti

- Androgen Assay a

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.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 Activity a

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 4.5 b 6 356.2** ( ↑ 9) 5.4 b

Body Weight Gain (Days 1

- 11) 6 51.8 1.7 b 6 81.2** ( ↑ 57) 2.7 b

a D ata 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 inter val 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% , bu t the final body weights in this group were comparable

to the TP control.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 Assay a

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
( ↓ 11 b ) 10

7 6 326 12 6 316 12 6 324 8 6 309 6 6 284
( ↓ 13 b ) 9

11 6 356.2 5.4 c 6 345.0 5.3 c 6 355.7 6.0 c 6 336.0*
( ↓ 6) 3.2 c 6 312.3**
( ↓ 12) 3.8 b

Body Weight Gain (Days #

- #) 6 81.2 2.7 c 6 71.7*
( ↓ 12 ) 3.1 c 6 79.3 5.5 c 6 64.8*
( ↓ 20) 3.9 c 6 41.2**
( ↓ 49) 2.4 b

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 a re 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+T P) 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 Assay a

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 Da ta 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.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, a nimals 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 A ctivit y in Sprague Dawley Rats a

Organ Dose (mg/kg/day)

Vehicle Control TP Control (0.3)

N Mean S E 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.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 permiss ible values.

TABLE 7

. Accessory Sex Organ and Liver Weights from Anti

- Androgen Assay in Sprague Dawley Rats a

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 5 b 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 5 c 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 Coeffic ient of Variation ; calculated by the reviewers

* Significantly different from controls at p<0.05

** Significantly different from controls at p<0.01



### F. MACROSCOPIC PATHOLOG Y : 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 vehicle

treated 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.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 decre ased; 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 gro up. 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 vehic le 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.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

milli gram.

# DATA EVALUATION RECORD

CARBARYL

Study Type: O CSPP 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/ 0 2 /201 2

Secondary Reviewer Signature: Scott D. Studenberg , Ph.D., D.A.B.T. Date: 3/ 0 6 /201 2

Program Manager: Signature: Jack D. Early, M.S. Date: 3 / 06 /201 2

Quality Assurance: Signature:

Jack D. Early, M.S. Date: 3 / 06 /201 2

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

.

### The US EPA Endocrine Disruptor Screening Program (EDSP) Tier 1 screening battery is comprised of eleven screening assays 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).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 platesSteroidogenesis Assay ( 2011 ) / Page 2 of 11

CARBARYL / 056801 O CSPP 89 0. 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 w ere 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 decrease s 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 reprod ucible decreases in testosterone production, but did not result in statistically significant and reproducible alterations in estradiol production.

Th e assay satisfies the EDSP Tier 1 Test Order requirement s for a Steroidogenesis a ssay (OCSPP 890.1550).

COMPLIANCE : Signed and dated GLP Compliance, Quality Assurance, and Data Confidentiality statements were provided.Steroidogenesis Assay ( 2011 ) / Page 3 of 11

CARBARYL / 056801 O CSPP 89 0. 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, R eplacement S tudy D irector
S. Colombel, R esponsible T echnician D. De Barbeyrac, Clinical P athology L aboratory
P. Almeras, Report U nit A ssistant Study Period: April 11, 2011 through July 18, 2011



### 2

. Test S ubstance : 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 C ontrol : Forskolin

Description (molecular weight) : White powder (410.50)

Source: Sigma

- Aldrich (St. Louis, MO)

Batch # ( expiration d ate) : 109K5057V ( not provided ) Purity: 98 % Solubility (in s olvent): Soluble in DMSO up to 0.01 M Storage conditions: Air

- tight, light

- resistant container at room temperature

CAS #: 66575

- 29

- 9



### 4. Negative C ontrol : Prochloraz

Description (molecular weight) : Off

- w hite powder (376.67)

Source: Sigma

- Aldrich (St. Louis, MO)

Batch # (expiration date) : SZE 6220X ( August 8, 2012 ) Purity: 99.1 % Solubility (in s olvent): Soluble in DMSO up to 0.0 0 1 M Storage conditions: Air

- tight, light

- resistant container at room temperature

CAS #: 6 7747

- 09

- 5Steroidogenesis Assay ( 2011 ) / Page 4 of 11

CARBARYL / 056801 O CSPP 89 0. 1550/ OECD None



### 5

. Solvent/ V ehicle C ontrol : Dimethyl Sulfoxide ( DMSO )

Description (molecular weight) : Clear liquid (78.13)

Source: Sigma

- Aldrich (St. Louis, MO)

Batch # (expiration date): 76396EM ( n ot 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 M edium : Dulbecco’s modified Eagle’s medium/ Ham’s F12 nutrient

mixture

Source: LGC ( Ref: 30

- 2006 )

Lot # (expiration date): 30001123 ( July 2011 )

Sodium bicarbonate : C omponent of stock medium

Nu

- Serum : 2.5%, BD Biosciences (Ref: 355100) ; L ot # 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% P enicillin/streptomycin ; GIBCO (15070

- 063 ) ; Lot # 769927 and 847134

.



### 7. Test C ells : 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% CO 2 

### 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 -T est Information

a. Hormone Assay I nterference T est : 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 test ed 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.Steroidogenesis Assay ( 2011 ) / Page 5 of 11

CARBARYL / 056801 O CSPP 89 0. 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 Pro chloraz. a Compound Concentrations tested ( μ M) Run Estimated EC 50 ( μ M) b

Testosterone Estradiol

Forskolin 0.03, 0.1, 0.3, 1, 3

and 10 1 st ~0.1 ~0.5

2 nd 0.7

- 0.8 ~1

Prochloraz 0.01, 0.03, 0.1, 0.3, 1, and 3 1 st 0.04

- 0. 0 5 ~0.2

2 nd ~0.05 0.1

- 0.2

a Data were obtained from page 1 5 of the study report.

b The Guideline (890.1550) specified range for EC 50 s for forskolin are 0.2

- 2.0 μ M for testosterone and 0.3

- 3.0 μ M for estradiol, and the specified range for ED 50 s for prochloraz are 0.01

- 0.1 μ M for te stosterone and 0.03

- 0.3 μ M for

estradiol.



### 2. Test Solutions : Carbaryl, f orskolin, and prochloraz were dissolved in DMSO and diluted

1:1000 in the final treatment medium. When added to the cell culture plates, these dilutions yie lded final concentrations of 1 μM and 10 μM for forskolin, 0.1 μM and 1 μ M for prochloraz, and 10 -4 

### to 10

- 10 

### M for carbaryl. The f inal 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 a n incubator (37 ±1 °C/ 5% CO 2 

### ) for approximately 24 hours prior to

chemical e xposure. 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 C oncentrations in M). a

1 2 3 4 5 6

A DMSO DMSO DMSO 10 -4 10 -4 10 -4

B 10 -5 10 -5 10 -5 10 -6 10 -6 10 -6

C 10 -7 10 -7 10 -7 10 -8 10 -8 10 -8

D 10 -9 10 -9 10 -9 10 -10 10 -10 10 -10

a Data were obtained from page s 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 e stradiolSteroidogenesis Assay ( 2011 ) / Page 6 of 11

CARBARYL / 056801 O CSPP 89 0. 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 Blank b Blank Blank Blank Blank Blank

B DMSO DMSO DMSO DMSO DMSO DMSO

C Forskolin

1 0 μM Forskolin

1 0 μM Forskolin

1 0 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 V iability/ Cytotoxicity A ssay : 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 cell s 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. N o 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 st ained 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 M easurement S ystem : 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 eachSteroidogenesis Assay ( 2011 ) / Page 7 of 11

CARBARYL / 056801 O CSPP 89 0. 1550/ OECD None



### exposure plate. All data were expressed as mean ± standard deviation (SD). Relative changes were calculated as follows:

Relative Cha nge = Hormone concentration in each well ÷ mean h ormone 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 s ignificant (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 th e 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 betw een 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 o n 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.Steroidogenesis Assay ( 2011 ) / Page 8 of 11

CARBARYL / 056801 O CSPP 89 0. 1550/ OECD None

TABLE 4. Mean (±SD) Hormone Concentrations Following Treatment with Carbaryl f or 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%) 3 33.7 ±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 page s 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 ≤ 0.01Steroidogenesis Assay ( 2011 ) / Page 9 of 11

CARBARYL / 056801 O CSPP 89 0. 1550/ OECD None



### F IGURE 1. Change in Testosterone P roduction Relative to Carbaryl C oncentration.

F IGURE 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.Steroidogenesis Assay ( 2011 ) / Page 10 of 11

CARBARYL / 056801 O CSPP 89 0. 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 hormo ne 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 o f 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 150.0 b ±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 141.0 b 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 estradio l.

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.Steroidogenesis Assay ( 2011 ) / Page 11 of 11

CARBARYL / 056801 O CSPP 89 0. 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 o f 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 concentrati on 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 w as marginally low (1.66– and 1.79–fold

induction was o bserved rather than ≥2 -fold induction guideline value).

• The SC between plate CV for estradiol (33%) in the QC plates exceeded the Guideline criteria (≤ 30%).

# DATA EVALUATION RECORD

CARBARYL

Study Type: O CSPP 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/ 0 2 /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.

### 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 strengt hs 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).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 /2 7 /2 012**---=-=:..=..:...:...=~::..__** __



### Secondary Reviewer:

# ~Je_s~s_R_o_w_I_an~d~ _________ 

### Signature·

Health Effects Division Date;

# 8~ Template 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
Da y 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 theIn 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 bo dy 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%, respecti vely, 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 O rder requirements for an uterotrophic assay (OCSPP 890.1600).

COMPLIANCE : Signed and dated Data Confidentiality, GLP Compliance, and Quality Assurance statements were provided.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 E strogen : 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 CH 3In vivo Uterotrophic Assay (2003) / Page 4 of 10

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### 5

. Test A nimals : 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 M akrolon 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 -L ife D ates : 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 A ssignment : 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.

T ABLE 1. Study Design a

Test G roup Dose (mg/kg/day) # of Females

Estrogen A gonist A ssay
Vehicle C ontrol 0 10

Low 3 10

Mid 10 10
High 30 10

17 α

- 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 S election Rationale : The dose levels were reportedly selected based on appropriate studies from the toxicology database of carbaryl and a dose range-finding study 1 

### 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.In vivo Uterotrophic Assay (2003) / Page 5 of 10

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### 5. Dose P reparation 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 u sed to demonstrate achieved concentration.

Results of D ose 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 A dministration : 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 (two

sided) 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, w hen the Bartlett test was significant, means were compared using the non- parametric analysis of variance of Kruskal

Wallis t est. 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

t he 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 DB P 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 u terusIn vivo Uterotrophic Assay (2003) / Page 6 of 10

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### 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); mea ns 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 W eight : Animals were weig hed 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 ho rns 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 S igns 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 ch ewing was observed in 3 /10 animals on Days 2 and 3. At 3 mg/kg/day, 1/10 animal s 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.In vivo Uterotrophic Assay (2003) / Page 7 of 10

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T ABLE 2. Incidence of Clinical Observations in the Estrogen Agonist Assay a

Observation Dose (mg/kg/day)

Vehicle Control Carbaryl (3) Carbaryl (10) Carbaryl (30) Reference Estrogen

EE (0.003) DB P (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 w eight 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%.In vivo Uterotrophic Assay (2003) / Page 8 of 10

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TABLE 3

. Group Body Weights and Cumulative Body Weight Gains (g) In the Estrogen Agonist Assay a

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 Table s 2a and 2b on page s 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 N umber 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 W eights : 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%, respective ly. 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.In vivo Uterotrophic Assay (2003) / Page 9 of 10

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T ABLE 4

. Uterine Weights from Estrogen Agonist Assay in SD Rats a

Parameter Dose (mg/kg/day)

Vehicle Control Carbaryl (3) Carbaryl (10) Carbaryl (30) Reference Estrogen

EE (0.003) DB P (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 Table s 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 B ody 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 acti vity 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.In vivo Uterotrophic Assay (2003) / Page 10 of 10

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### 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