# ~ DowAgroSciences

DowAgroSciences LLC 9330 Zionsville Road Indianapolis, IN 46268 USA
Bldg 308/2E 25 February, 2014
DocumentControl Office (DCO) (7407M) Office ofPollutionPrevention andToxice (OPPT) U. S. EnvironmentalProtectionAgency 1200PennsylvaniaAve., NW., Washington, DC 20460-0001 

## l\Ecc::..;c.o R

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Attention: 40CFR799.5089; DocketID NumberEPA-HQ-OPPT-2007-0531 CBIC Control Number



# II 358722

www.dowagro.com & 

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Scanned &Processed in CIS by: SHARON

Attached is the study plan for the testing ofphosphorochloridothiotic acid, 0,0-diethylester(CASRN 2524-04-1) (aka diethylchlorothiophosphate orDEPCT) complying with EPA ruling under section4 ofthe Toxic Substance ControlAct (TSCA) onJanuary7, 2011 (76FR 1067). In summary, DowChemical's testiniiifans for this substance are listed below:

• Studyreports are attached for melting point, boilingpoint, freezing point, and vapor pressure testing. • A reputable citation is providedfor the Kow value. • GLPcharacterizationofDEPCTtesting material willbeincluded with future studyreports • A hydrolysis study willbe conductedto account for the very shorthalf-life ofthis material in water. Theprotocol is attached and this studywill iilform the protocolsforthe water solubilitydetermination, ready biodegradation study, the aquatic tox studies. The hydrolysis studyresults andprotocols for each ofthese subsequent studies will be submitted to EPAby30September 2014 • Protocol is attached for the BacterialReverse Mutation study (Ames test) andit will besubmittedby30 September2014 • MammalianErythrocyteMicronucleusTestprotocolis attached andDowintends to conductthe study and submit the reportby 30September 2014. • Dow intends to submitto EPAa Reproduction IDevelopmentalToxicity ScreeningTestprotocol (OECD421) bySeptember30, 2014 and then submitthe studyreports for both studiesbyJune 30, 2015. Some preliminary testing regarding stability anddose range finding is required priorto initiating these two studies. Protocols for ,**~?** thesepreliminary tests are attached.
Ifyou have questions regarding this studyplan, please contactDarinLickfeldt at317-337-3774 ordwlickfeldt@dow.com. We lookforward to yourreview and feedback ofthe attached studyplan and studyreports. Respectfully,

### ~-**/~_,//~ ·-~~~~-~~~,**  DarinW. Lickfeldt, Ph.D Regulatory Manager DowAgroSciences LLC

®TMTrademark ofThe Dow Chemical Company ("Dow") oran affiliated company ofDow**··.~**

# (_

( ORPHANHPV TESTINGPROGRAM

StudyPlan for phosphorochloridothiotic acid, 0,0-diethyl ester (aka diethylchlorothiophosphate orDEPCT) CASRN 2524-04-1



### PREPARED BY: Dow AgroSciences LLC Indianapolis, IN 25 February 2014Manufacturing and Use Phosphorochloridothiotic acid, 0,0-diethyl ester (CASRN 2524-04-1) (aka diethylchlorothiophosphate or DEPCT) is a raw material that is typically imported to the U.S. and then used in the manufacturing of Chlorpyrifos (CASRN 2921-88-2). Chlorpyrifos is the active ingredient in insecticidal pesticides used primarily in agricultural crops such as field crops, fruit and nuttrees, and vegetable crops.
(i) Identity ofthe test rule. This study plan includes the detailed information and proposed plans for fulfilling requirements forthe EPA ruling under section 4 ofthe Toxic Substance Control Act (TSCA) on January 7, 2011 (76 FR 1067) and recorded in Docket: EPA-HQ-OPPT-2007-0531 relevant to 40 CFR 799.5089

(ii) The specific test requirements ofthat rule to be covered by the study plan Table 1

Test Study Study Testing Lab

Test Type Monitors 

### A1 Melting Point: ASTM E324-99 Joyce Battjes Harlan, Study (capillary tube) report attached in Appendix B 

### A2 Boiling Point: ASTM E1719-05 Joyce Battjes Harlan,Study report (ebulliometry) attached in Appendix B 

### A3 Vapor Pressure: ASTM E1782-Q3 Joyce Battjes Harlan,Study report (thermal analysis) attached in Appendix C Physical/ chemical Testing not properties n-Octanoi/Water Partition Joyce Battjes feasible. Calculated 

### A4 Coefficient (log 10 basis) or log value provided Kow: below Testing not feasible due to rapid hydrolysis. 

### AS Water Solubility: Joyce Battjes Estimated value provided. See Hydrolysis Study protocol in Appendix D 2 DEPCT**~** I \ )

## ( Environmental Fate and Need to first Environmental Pathways-Ready Biodegradation: conduct the fate and pathways 8 Laura hydrolysis protocol --ready Laughlin described in biodegradation OECD 301F Respirometry Test; Appendix D prior to developing the protocol for this study
Aquatic Toxicity: Need to first Test Grou12 1 for C1: conduct the 1. Acute Toxicity To Fish: ASTM E hydrolysis protocol Aquatic toxicity 

### Cl 729-96(2002); Nick Poletika described in 2. Acute Toxicity To Daphnia: ASTM Appendix D prior to developing the E729-96(2002); protocols for these 3. Toxicity To Plants (Algae): ASTM three studies E1218-Q4e1;


### El Bacterial Reverse Mutation Test (in Daland Dow protocol vitro): 40 CFR 799.9510 (note; same Juberg attached in Mammalian as OECD 471) Appendix E

toxicitygenotoxicity Mammalian Erythrocyte Daland 

### E2 Micronucleus Test: 40 CFR Juberg Dow protocol 799.9539 (note; same as OECD 474) attached in Appendix F Reproduction/Developmental Developmental Toxicity ScreeningTest: 40 CFR Toxicity/ 

### F2 799.9355 Daland Dow preliminary (Dow will conduct OECD 421- Juberg protocols attached Teratogenicity Reproduction 

### I Developmental in Appendix G&H Toxicity Screening Test)

\ 

### '- 3 DEPCT(iii) (A) All oral and written communication forthis test substance relative to testing under this ruling should be directed to this person below: Testing Sponsor Information Darin Lickfeldt, Ph.D. Dow AgroSciences LLC 9330 Zionsville Road Indianapolis, Indiana 46268 Phone: (317) 337-3774 Fax: (317) 337-7056 dwlickfeldt@dow.com (D) {1) The names and addresses ofthe testing facilities and the names, addresses, and telephone numbers ofthe testing facilities' administrative officials and project manager(s) responsible for the testing.

DEPCT Testing facilities and administrative officials and project manager(s) responsible for the testing Physical chem. testing Study Monitor: Joyce Battjes The Dow Chemical Co 1803 Building TERC Office 236 Midland, Ml 48674 (989) 636-4427 JEBattjes@dow.com Environmental Fate Testing Study Monitor: Laura Laughlin Dow AgroSciences LLC 9330 Zionsville rd. Indianapolis, IN 46268 (317) 337-3475 lalaughlin@dow.com Ecotox Testing: Study Monitor: Nick Poletika Dow AgroSciences LLC 9330 Zionsville rd. Indianapolis, IN 46268 (317) 337-3476 npoletika@dow.com Mammalian Tox Testing: Study Monitor: Daland Juberg Dow Agrosciences LLC 9330 Zionsville rd. Indianapolis, IN 46268 (317) 337-3787 DRJuberg@dow.com

All newfuture testing will be conducted at one ofthe following three testing facilities (Table 2) but communications should be directed through the Study Sponsor and Study Monitors listed above:



### - Ill

.. Ill 4

### ( Table 2 The Dow Chemical Co Harlan Laboratories Ltd. JAI Research Foundation 1803 Building Shardlow Business Park Dept ofToxicology TERC Office 236 Shardlow Valvada 396 108 Midland, Ml 48674 Derbyshire Dist Valsad DE72 2GD; UK Gujarat, India Contact: Joyce Battjes Anthony Burke, BSc (Hans) Phone: (989) 636-4427 PROGRAMME MANAGER Phone: 91-260-6540-242 Fax: (989) 638-9863 Phone:+44(0)1332792896 FAX: +44 (O) 1332 799018

{2) Briefsummaries ofthetraining and experience ofeach professional involved in the study, including study director, lead scientist and technical reviewer are attached inAppendix A with CVs of key personnel conductingtesting at Dow Chemical Co. or Harlan Laboratories Ltd.

(iv) Identityand data on the chemical substance(s) beingtested, including physical constants, spectral data, chemical analysis, and stability undertest and storage conditions, as appropriate. phosphorochloridothiotic acid, 0,0-diethyl ester CASRN 2524-04-1 (aka diethylchlorothiophosphate or DEPCT)

DEPCT 5Table 3- Physical Constants for DEPCT:

Property Value Method Reference Freeze Point* <-2o 

## ·c OECD Guideline 102: Harlan Laboratories Freezing Temperature Report ofStudy No. Method (GLP Compliant) 41304286 (2014), See Appendix B Boiling Point* 202 

## ·c OECD Guideline 103: Harlan Laboratories @ 98.3 kPa Differential Scanning Report ofStudy No. Calorimetry Method (GLP 41304286 (2014), See Compliant) Appendix B Vapor 93 Pascal OECD Guideline 104: Harlan Laboratories Pressure* @ 25 Vapor Pressure Balance Report ofStudy No. 

## ·c Method (GLP Compliant) 41304287{2014),See Appendix C Water Undefined, due to rapid hydrolysis. Furthertesting ofwater Solubility solubility should be waived, and information on hydrolysis and identity of primary hydrolysis product are to be determines as per the protocol in Appendix D. Estimated water solubility values for the substance, which are based on the structure-fragment contribution calculation (WATERNT vl.01) and on correlation of log Pawwith water solubility (WSKOW v1.42) methods ofthe U.S. EPA EPI Suite software are 4,296 and 1,249 mg/L Octanoi-Water 2.37 Testing should be US EPA EPI Suite software, Partition waived, due to reactivity v4.1. Coefficient (log ofsubstance with water Pow) and 1-octanol. A calculated value is provided, according to structure fragment constant method of KOWWIN v1.68 *properties determined fortest substance of99.13% purity

6 DEPCT
Ill**~** ISpectral data for DEPCF: •I •, •; t**:·~·-'/·~··:) :.~·=**  ./:

...:·. :-r.i : ...... :**··_(~::** )·i· ,·; .·: •. ··., •• ·:,.·\·:-**':::·:···-~:** \ .·.: •**~** ,_.; An infrared**sp~;!ctrum** was obtained forthe substan1=e (99.13% purity), An aliquot ofthe

..**_l.::··~·\::f;·~·:.~·:·.·.).:·:.:·.t<·:·t!~-** ..**--:!~:':~;-·:.:·~** ,·.·::· ·1
...1

..' ·**-~** '.:.' ·, ,·. •·.. ,**-~-.~·:=.i··':.-~.-=** ,·. •i:V·-'**~** ,_·;. ..::····. ..·1:

. . .**~1.1bstar,ce wc,~.s**  sc.anned .·directly ysipg .AtteniJatedJQtCI!**Refl~ctan~e** (ATR) overthe range ·, ,•·,-','.-;;.'},,·.,;**:.,,·'~:!•,\.,,.,'** :. ,;; •,l;'j.,**~·,·,** .,·o o'**'t(.~\''o~''•,,\** ', ')ol •,: I:'::**.·.~·:** ,. C • 1,·,,,-1,;**~-.'** of4000 to 600 cm-1. The**we~v.e** numbers and assignments ofthe moststructurally ·., ,·,.,;: ·::.**;.~** · ;n·;· \l"'**.~". ··-~·-**  .:,1· '\-1\.',: ·**~:;<··f ;·.-~:·**  -.:,:. ': ,.:.' · .:. ::v .. !- •·•.• :-:

... ,_ .·::•. • • ;' ·:1 ·• significant bands were'recorded, a'nd are consisteritwith the.expectedabsorbance**,·,;_•:--.~;~!·'t:·::· -~-·**  '**::··.~**.• ·,;· bands associated with the structural features ofthis substance

.

..· ..

i· ;._·.**100.7-~------**



### ... ' ·. .• :;;m:u,9UI
.. 80 '. ' .**293~~~~-90** l9i!S:06,87.33 '
. ' 1 • •

; : ·; l : 1 '**~:\** • •• .· ; : • ' l**~** . ' 70

._, .. '

60

.**~T** SO' '' ,.. ! ' 40
30 196.01.36.06
20 96S.29,2I.9II 651.01,16.U

.. .**~** ; .. ''**O.O.J-----..---.----~----,-----.---r----r---,-----.-_:·I=IIIM::::.l.;::9.1.65=.__-,------,**

.. 4000.o 2100 · 2400:... · 2000 lsoo ·· 1600 1400· ..• · 1200':··:· iooo · aoo 600.0 

## """'' '·:-· .·.

.**~** ' j ·'·:,:.

. ;_ 

## ... •
Chemical Analysis for DEPCTTest Material: Dowanticipatesthetestmaterial for newtestingwill be >99% pure DEPCT, butthis will be

, validated witha GLP characterization studies. The GLP characterization study will likely include

NMRand MS spectral information.

.. · · . ,· .' \ Stability u·ndertestconditions:. '!.· _.,· - '.**-~** The testmaterial (a diethyl esterofo-thiophosphoricacid chlorid.e or DEPCT) has reactivity similartothat ofcarboxylic acid chlorides (e.g., acetyl chloride). As such, the substance will readily react with water, proteins, alcohols, amines, etc. (i.e., any nucleophilic substance). This reactivity has significant influence on howtoxicological and ecological hazard testing is conducted on the substance, and makes certain physical and hazard property determinations

·. : '**:"".~ .,··-~~------~:.-**  ....... ·..**~.** ,.,,._{·,· ·t'**-~-· (_~'-**  ... · ··..• ·:·. •·.:. ': ,,. >:.: .: '·:···**·_:,·~** ·....-·''·**-~~** ,·)**·'~** .**~.** ',•;;' '-:'. ,\·.;'

. qifficult or irrelevant. These aspeq$ ofsubstance stability are summarized in more detail below:
', ; '' : , 0 ,<' • v; : •:. : ' • ! ' '· ', ·,,.: •·• : ·, ; •**~·,,** :• : • ' • : ... :· : •" ( ' • I ," • " ' ' ' I •0 • • ·! ·!', ,:·., : j < • , I • : ;' ' • .o'. ',. ;; ' '· '
,<'

.. '

DEPCT ":';!.·'··' ,-f.

. '·,·' The [)EPCT substance is stable in its pure form;when storedunder: appropriate" - conditions. For determinations offreeze point, boiling point, and;vapor pressure,.the
7substance evaluated was determined to be ofsuitable purity

### (99;13 %) prior: toor concurrently with performingthese measurements. The properties ofwater solubility,

### ~hJ'od:a,;o·I~:\J~t~'i**p~·ri:ltidrl!cd~fficie~t** 

## (ldd**Pow),tannot'bep~acticahy'determined** due

### t6'~~acti'vitVH~st~~iiitY)**bf.the!sobstahc~** iri'water aiid.in

### i~o'Cta;,oL Estimated values of**,_,:-.~** 'l· ·:···,_·.· :::··:···,:-,,**•,·~·:·•:i'.;i'·'.** :·.- ...·C ,-·1 ·**;:·~:~r·.'** ··::··'· ·.·•:- ' i=

. • ·:. •.• :· '>,:·

...;. ·• watersolubility ahd' log 

### Kow are provided (Table 3), as determined according to • '<·;..·:,**:·;,~!:** -.",;·•

.. ·.·,: · 1 ;··>: (i ):',\:**"T···.~··.'** ·.•.• .: ·:_'; ,· :·; . _'· ·J' :;. · accepted QSAR relationships.

)

# .. •'

Environmental Fate andPathways: For studieswhich determinethe fate of DEPCT in the environment, stability ofthe pure applied testsubstance is of importance/relevance; however, it is the purpose ofsuch studies to understand stability ofthis pure substance subsequent to its addition totest systems containing water, wastewater, soil, etc. The study of hydrolysis (protocol in Appendix D) would demonstratethe instability ofthat pure substance in water, also indicates relative stability ofthe primary hydrolysis product (0,0-diethyl hydrogen phosphorothioate; CASRN 2465-65-8). Aquatic Toxicity: In accordance with OECD guidance on aquatic toxicitytesting ofdifficultsubstances, the parent DEPCT substance would be allowed to hydrolyze tothe primary (and stable) hydrolysis product (0,0-diethyl hydrogen phosphorothioate) priorto exposure ofthe testorganisms. In each ofthese studies, the concentration and stabilityofthis hydrolysis productwould be determined overthe course ofthe aquatic exposures, with these measured concentrations as basis forthe reported E/LC50 and NOEC values. Mammalian Toxicitv-Genotoxicity ,, The stability ofDEPCT invarious vehicles/media is notknown and before protocolsfor : the Reproduction

### I Developmental·Toxicity Screen (OECD 421) can bewritten (with

knownvehicles to which DEPCTwould be added)j there is a needto get some ba'seline stability information in various media, including diet (food) and corn oil. This knowledge is needed as .it will influence the frequency with which DEPCT is mixed in whatever vehicle is deemed moststable foreach respective test. Because ofthis need, a protocol ' . .' . . . . . . .· .**-~** ' ·, .: 

### . : thatevaluates stability in food and corn oil, conducted underGLP, is attached in this study planas Appendix G- this work is essential todetermining'stability ofthetest material in the421 screen. ·, .:**~** . ' \ ·,,, '

.**~!** ' ··:.'**~** . ,. l**~** i

. .



### . (vlstudy prototol,.ihc:ludillg 1:'11e

### ~ational'e'tor any'combination of

### te~t protbcols;the rationale

.for'species/stra'in'selection; dose selectiotl (and supportil1gdata');

### ~o~te(s) tir

### method(~)of

exposure; description ofdietto be used and its source; inc;:ludi.ng**m•~r!ert~s** and:contaminants and theirconcentrations; for invitro testsystems, a description ofculture medium and its source; and a summaryofexpected spontaneous chronic diseases (includingtumors), genealogy, and lifespan; •· ·· ·· · '
8 DEPCT
IllI \ Each study to be conducted is addressed in the following appendices {Table 4) with either a protocol, a reference to previously conducted study, orjustification for preliminary studies that will be required in orderto conduct the EPA requested testing. Table 4 Study Appendix Study Study Plan Monitor Melting Point: Recently conducted study of Harlan Laboratories, attached ASTM E324-99{capillarytube) B Joyce Battjes in Appendix B Boiling Point: Recently conducted study of Harlan Laboratories, attached ASTME1719-05 (ebulliometry) B Joyce Battjes in Appendix B Vapor Pressure: Joyce Battjes Recently conducted study of 

## c Harlan Laboratories, attached ASTM E1782-Q3 thermal in Appendix C analysis n-Octanoi/Water Partition Testing is notfeasible. A Coefficient (log 10 basis) or calculated log Pow value of log Kow Joyce Battjes 2.37 is provided (USEPA EPI Suite, KOWWIN v1.68) Testing is notfeasible. Estimated values of4,296 and Water Solubility D 1,249 mg/L are provided Joyce Battjes {USEPA EPI Suite, 2010). Dow will conduct the hydrolysis study protocol in Appendix D Hydrolysis study in Appendix D Environmental Fate and is required priorto writing biodegradation protocol. Dow Pathways-Ready will conduct the hydrolysis Biodegradation: Preferred Laura Laughlin study and submit method notyet known biodegradation protocol by September 30, 2014 with study report submitted by 30 June 2015

9 DEPCTThe hydrolysis study described in Appendix D is required prior Aquatic Toxicity: to writing the three aquatic toxicity protocols. Dow will 1. Acute Toxicity To Fish: conduct the hydrolysis study ASTM E729-96{2002); and submit the three aquatic toxicity protocols by September 30, 2014 with the actual study 2. Acute Toxicity To Daphnia: Nick Poletika reports submitted by 30 June ASTM E729-96{2002); 2015 Analytical monitoring ofthe 3. Toxicity To Plants (Algae): concentrations of DEPCT and its ASTM E1218-04e1; primary acid degradation product shall be conducted during the dosing phase ofthe toxicitytest.

Protocol attached in Appendix Bacterial Reverse Mutation E. The study would be conducted at JAI Research Test (in vitro): 40 CFR Foundation and submitted to E 799.9510; OECD 471 D. Juberg EPA by 30 September 2014 Dow protocol attached in Mammalian Erythrocyte D. Juberg Appendix F. The study would Micronucleus Test. OECD 474 F be conducted and submitted by 30 September 2014 Protocols attached for preliminarytesting in Appendix G & H. Following the preliminarytesting, Dow Reproduction/Developmental intends to submit an OECD 421 Toxicity Screening Test: 40 CFR D. Juberg - Reproduction 

### I 799.9355 {OECD 421) G&H Developmental Toxicity Screening Test protocol by September 30, 2014 and submit the study report by June 30,2015.

10 DEPCT


### Ill**~** 

### I

### Summary

• Study reports are attached for melting point, boiling point, freezing point, and vapor pressure testing. • A reputable citation is provided for the Kow value. • GLP characterization of DEPCT testing material will be included with future study reports • A hydrolysis study is proposed to quantifythe challenges in testing DEPCT in aqueous media and will be more informative than the water solubility since DEPCT is highly reactive in water. • The hydrolysis study proposed here is needed priorto creating protocols for the Ready Biodegradation and the three Aquatic Toxicity studies. The hydrolysis study along with protocols forthese four subsequent studies will be submitted to EPA by 30 September 2014 followed by the study reports by 30 June 2015 • Protocols attached for Bacterial Reverse Mutation "Ames Test" and Mammalian Micronucleus testing. Both studies will be reported by 30 September 2014. • Dow intends to submit to EPA a Reproduction 

### I Developmental Toxicity Screening Test protocol (OECD 421)by September 30, 2014 and submit the study report by June 30, 2015. Some preliminarytesting regarding stability and dose range finding is required priorto initiatingthese two studies. Protocols forthese preliminarytests are attached.

11 DEPCTAppendices List Appendix A -Curriculum Vita for Study Directors, Lead Scientists, and Technical Reviewers Appendix B- Melting Point, Freezing Point, Boiling Point Study Report Appendix C- Vapor Pressure Study Report Appendix D -Water Hydrolysis Study Protocol Appendix E- Reverse Mutation Assay "Ames test" Study Protocol Appendix F- Mammalian Erythrocyte Micronucleus Proposed Protocol Appendix G- Stability in Rodent Feed and Corn Oil
- Preliminary Study Protocol Appendix H- Oral Gavage Range Finding Protocol for Mammalian Reproductivity Toxicity Study



### 12 DEPCT


### Ill**~** IAppendix A Curriculum Vita for Study Directors, Lead Scientists, and Technical Reviewers

13 DEPCTIll**~** I

### HARLAN LABRATORIES LTO Department of Physico-Chemical Properties 

### CURRICULUM VITAE
EMPLOYEE NAME DUNCAN TARRAN POSITION STUDY DIRECTOR SITE ADDRESS SHARDLOW BUSINESS PARK CURRENTHARLANEMPLOYMENT**~--·-·--.** -·------- ------·-------......._.., ................................... """"' "''"''"""''"''"''"''......... 

## I DATEFROM I POSITION & BRIEFDESCRIPTION**!o-ct~b~;**

## 2o1_1
__·**--~** 

## As 

## study Director 

## in ttie**Ptiysico~Chemical** Properties testing laboratories ifis my ! 

### ! responsibility to supervise all studies under my control in co-ordination with the 

## j i Manager of Physico-Chemical Properties. : Preparation of official documentation relating to Physico-Chemical testing, including

reports, Standard Operating Procedures (SOP's) and study plans etc, and anything pertaining to Good Laboratory Practice (GLP) is undertaken. This often necessitates liaising with clientele and interacting with the Quality Assurance Unit, Test Item Control and the Registration Department. Procuring the efficient utilization of staff,

.

. resources and time requires constant supervision of the Physico-Chemical scientific : 

### i team. Ensuring the training of new and existing staff with any techniques/procedures, 

### i 

### J is carried outasancj I,!Vhen required.

. .. . . .. .. .. .. _ f PREVIOUS EMPLOYMENT HISTORY

,,,,,..,,,,,,.,.,.,,,.,,..,........ V"""' 

# I

# ~:b:::~~**5** 

# I

# ::~ --

# [f:::~~f~:£~~:::~:::~0:.,**'~--·----·--** .. --- April2006 

# -·--·l'
.................................................................. November 2008

· November 2008 ' June 2009

June 2009 September 2011 1 ·------------------,- ....................................................................

' : LSafePharm Laboratories Limited Senior Technician Department of Physico-Chemical Properties : .. ?afePharm Laboratories Limited ' Senior Technician

Department of Physico-Chemical Properties

. Harlan Laboratories Ltd

....... ·•······················· Technician Department of Physico-Chemical Properties Harlan Laboratories Ltd As a Technician in the Physico-Chemical laboratories it is my 

### i responsibility, under the immediate supervision ofthe Laboratory 

## I Manager and Study Directors, to perform method development , 

# I Validation and analysis relating to Physico-Chemical testing. 

## i This necessitates adopting the requirements of Good Laboratory 

### ! Practice (GLP) and specifications of Standard Operating 

## I Procedures (SOPs).Some administrative duties are undertaken 

### i and reports prepared. Techniques used on a regular basis ' include high performance liquid chromatography (diode array and fluorescence), gas chromatography (FID, ECD and NPD),

, ultra-violet/visible spectroscopy and other classical wet ,

. _ ___ [_9hemistry and**P~y~i.c:>:G.h~.l!ljcal** techniques.

____________ Page 1 of 2EDUCATION/QUALIFICATIONS

---------------- -------------

- --- ,-------, -------·--·-··-··-···---·········- 

### 9J!I!LdE!9_!l!IQIJ§__._._._.
____ !DATE J LOCATION/PLACE OBTAINED !------; -------··-·····-···--·--------····· 3 A Levels Chemistry, Maths Computer Studies 

## ,i 1999-2001 

## !Bilborough Sixth Form College, Nottingham AS Levels Further Maths

·-··-··----·-·-··-·········-·····**~--------~-j** •**13~g__lH_c:>ns) 9he!DJ.~rY--·-···---·--**  2001-2004 .l,J_Qiversity of Nottingha_f!!

PROFESSIONAL TRAINING**_IRAIN~fi~--** ----·-------- -•r:'**D::...:Ac_:_:__:TE=--_,ir:L::...:O::..;C=-=A-'-'T<:...oi=O:...:N!J:..:_'P-=L:.:._:A=-=C=E'-'O=B=-:T.'-'-A=IN:...::E=D=---~-------i** Health and Safety Induction !.· 2006 

## !SafePharm Laboratories Limited Refresher Use of the Polymer Laboratories -+,--20_0
__ 9--+i -H-a-rla_n_L_a_b_o-ra-to-r-ie_s_L-td-.

### --------------1 G

### E_C_§y_§J~I1'1 ---·------ - !

EMPLOYEE SIGNATURE INITIALS DATE SIGNED

Page 2 of 2 Ill Ill

### CURRICULUM VITAE
NAME: Amanda K. 

### Andrus JOB TITLE: Study Director EDUCATION: 2004: Central Michigan University, Mount Pleasant, MI, Master ofScience in Applied Experimental Psychology 1998: Central Michigan University. Mount Pleasant, MI, Bachelors ofScience Major: Biology El\fPLOYMENT: 2003-Present: The Dow Chemical Company, Midland, MI. Study Director, Toxicology & 

### Environmental Research and Consulting 2000-2003: The Dow Chemical Company, Midland, MI, Technologist, Toxicology & 

### Environmental Research and Consulting 1999: The Dow Chemical Company, Midland, MI, Technologist, Health and Environmental Research Laboratory 1995-1999: The Dow Chemical Company, Midland, MI, Student Technologist, Health and Environmental Sciences, Oral/Dermal & 

### DART MEMBERSHIPS: 200!-Present: Neurobehavioral Teratology Society PUBLICATIONS: See Attached PRESENTATIONS: Sec Attached

Reviewed/Revised:

DOW RESTRICTED

### PUBLICATIONS 1) 

### Maurissen, J.P., B. R. 

### Marable, A. 

### K. Andrus and K. E. Stebbins. 2003. Factors affecting grip strength testing. Neurolo.ricolog,vand Teratolo[!,y 

### 25:543-553. 2) Andrus, A, K., 

### Marable, B. R., Dunbar, G. 

### L., Reilly, M.P., Maurissen, J.P. 

### J. 2007. Effects ofintensity and type ofprepulse stimulus on prepulse inhibition in scopolamine treated rats. 

### Pharmacol, Biochem, andBehavior87:481-488. 3) Dryzga MD, Yano BL, Andrus AK, Martsson JL. 

### 2007. Evaluation ofthe safety and nutritional equivalence ofa genetically modified cottonseed meal in a 90-day dietary toxicity study in rats. Food 

### Chern Toxicol. 

### 45( 10): 1994-2004 2



### 4) Marty, M.S., Zablotny, C.L., Stebbins, K.E., Woolhiscr, M., Andrus, A.K., Brzak, K.A., Kan, H.L., and Carney, E.W. An integrated study design to evaluate reproductive, immune and nervous system effects ofindole-3-carbinol (I3C) in CD rats exposed during gestation and lactation. 

### Teratology (in press).


### PRESENTATIONS 1) Andrus, A. 

### K., Marable, B. R., Dunbar, G. L., 

### Reilly, M.P. and Maurissen, J.P. Scopolamine and Prepulse Inhibition ofthe Auditory Startle Response: Effects of Prepulse Stimulus Duration, Frequency and Intensity. Posterpresented at the 29th Annual Meeting ofthe Neurobehavioral Teratology Society, 6/27/05 2) M.S. Marty, J.A. Hammond, A.J. Clark, D. Rick, J. Van Fleet, A.K. Andrus, and J.P. Maurissen. Dose Rate on Neurotoxicity Outcome in CD Rat Pups Given the N-Methyl-D-Aspartate Antagonist, MK-801. Posterpresented at the SOT44th Annual Meeting, March 6-10, 2005 3) Marable, B.R., A.K. 

### Andrus and J.P. Maurissen. 2001. Assessment ofthe Validity ofan Automated Auditory Stm1le Response System with orwithout Chemicals. Presented at the Annual Meeting ofthe Society.ofToxicology, March, 2001. (CDF#1024) 4) Maurisscn, J.P., Shankar, M.R., Marable, B., Andrus, A.K., Carney, E.W., Yano, B.L., and Holsapple, M.L. Are chemicals stealing ourchildren's future? Presented at the Dow Global Scientists' Organization meeting, Galveston, TX, Oclober 23-25, 2000. 5) A.K. 

### Andrus 2002. Resistance ofdifferent materials and cables to rodent attacks. 

### 6) Presented at the TERC Science Discussion Group, December 2, 2002. Andrus, A. 

### K. Scopolamine and Prepulse Inhibition ofthe Auditmy Startle Response: Effects ofPrepu1se Stimulus Duration, Frequency and intensity. Seminarpresented on 2115/05, TERC Science Seminar

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I Ill Ill

### CURRICULUM VITAE
NAME: 

### Carol L. 

### Zablotny JOB TITLE: 

### Study Director EDlJCATION: 

### 1989: Saginaw Valley State University, Immunohematology 1987: Saginaw Valley State University, Hematology 1987: The Dow Chemical Company, Assistant Lab Animal Tech. Course 1980-1985: Michigan State University, B.S., Animal Science 1979-1980: Adrian High School, Adrian, MI 1978- I

### 979: Notre Dame Academy, Toledo, OH 1976-1977: Nardin Academy, Buffalo, NY EMPLOYMENT: 

### 1999-present: The Dow Chemical Company, Midland, Ml, Reproductive Toxicology Group Study Director, Toxicology and Environmental Research & Consulting 1986-1999: The Dow Chemical Company, Midland, 

### MI, Reproductive Toxicology Group Study Director, Health and Environmental Research Laboratories PUBLICATIONS: 

### Sec Attached PRESENTATIONS: 

### See Attached

Reviewed/Revised: /(Signature) 7 

### (Date)

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### .PUBLICATIONS 

### I) Breslin, W. J., Ciezlak, 

### F. S., Zablotny, C. 

### L., Corley, R. A., Yano, 

### B. L. and Verschuuren, H. G. (1990). Developmental toxicity ofinhaled dipropylene glycol monomethyl ether (DPGME) in rabbits and rats. The Toxicologist 10, 

### 38. Abstract No. 154. 2) 

### Breslin, W. J., Vedula, U., 

### Zablotny, C. L. and Stebbins, K. E. 

### (1994). Developmental toxicity studies withpicloram triisopropanolamine salt and picloram 2-ethylhexyl esterin the rabbit. The Toxicologist 14, 162. Abstract No. 579. 3) Liberacki, A. B., Zablotny, C. L., Yano, B. 

### L. and Breslin, W. J. (1994). Developmental toxicity studies on a series of2,4-D salts and esters in rabbits. The Toxicologist 14, 162. AbstractNo. 580. 4) Zablotny, C. L., Breslin, W. J. and Kociba, R. 

### J. (1992). Developmental Toxicity ofOrthophcnylphenol (OPP) in NewZealand White Rabbits. The Toxicologist 12, 103. Abstracts No. 327. 5) Zablotny, C. 

### L., Carney, 

### E. W. and Dugard, 

### P. H. (2002). Evaluation of Trichloroethylene in a rat inhalation developmental toxicity study. The Toxicologist 66, Abstract No. 1161. 6) Marty, M.S., Zablotny, C.L., Stebbins, K.E., Woolhiser, M., Andrus, A. K., 

### Brzak, K. A.., Kan, 

### H.I., and Carney, E.W. (2003). An integrated study design to evaluate reproductive, immune and nervous system effects ofindole-3-carbinal (l3C) in CD Rats exposed during gestation and lactation. Birth Defects Res. 68:255. 7) Carney, E.W., Zablotny, C. L., Marty, M.S., Crissman, J.W., Anderson, P., utero and postnatal development in rats. Toxicol. Sci. 83:237-249. 8) Wilson, D. M., Carney, E. W., Zablotny, C. L., Yano, B. L., and Cawley, T. (2005). Assessment ofisobutyl heptyl ketone (IBHK) by oral gavage in an OECD 422 rat reproduction/developmental screening study with systemic and neurological endpoints. 44th Annual Society ofToxicology Meeting; New Orleans, LA. 9) Camey, E.W., Thorsrud, B.A., Dugard, P.H. and Zablotny, C.L. (2006). Developmental toxicity studies in Crl:CD (SO) rats following inhalation exposure to trichloroethylene and perchloroethylene. Birth Defects Res., Part B: Devel. Rcprod. Toxicol. 77:405-412.

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## ( CURRICULUM VITAE
NAME: 

### Melissa R. 

### Schisler .JOB TITLE: 

### Study Director EDUCATION: 

### 1996: Ferris State University, Big Rapids, Michigan, Bachelor ofScience Major: Biotechnology 1995: Ferris State University, Big Rapids, Michigan, Associates in Science EMPLOYMENT: 

### 2004-Present: The Dow Chemical Company, Midland, MI, Study Director Toxicology & 

### Environmental Research and Consulting 1999-2004: The Dow Chemical Company, Midland, MI, Technologist, Toxicology & 

### Environmental Research and Consulting 1997-1999: The Dow Chemical Company, Midland, MI, Technologist, Environmental Analytical 1996-1997: Kelly Scientific Resources, on assignment at The Dow Chemical Company, Midland, MI, Associate Technologist, Environmental Analytical 1996-96: Henry Ford Hospital, Neuro-oncology internship. PUBLICATIONS: 

### See Attached PRESENTATIONS: 

### See Attached

Reviewed/Revised: 

# CJ-jj4/;_!:)_

______ (Date)

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### PUBLICATIONS 

### I) Seidel, S.D., Kan, 

### I-LL., Stott, W.'f., Schisler, M.R. and GoJiapudi, 

### 13.13. (2003) Identification oftranscriptome profiles for the DNA-damaging agents bleomycin and hydrogen peroxide in L5178Y mouse lymphomacells, Environmental and Molecular Mutagenesis, 42(1), 19-25. 2) Seidel, S.D., Span·ow, B.R., Kan, H.L., 

### Stott, W.T., Schisler, M.R., Linscombe, V.A. and Gollapudi, B.B. (2004) Profiles ofgene expression changes in L5178Y mouse lymphoma cells treated with methyl methanesulfonate and sodium chloride, Mutagenesis, 19(3), 195-201. 3) Charles, G.D., Kan, H.L., Schisler, M., Gollapudi, B.B. and Marty, M.S. (2005). A comparison ofEDSTAC in vitro/in vivo test battery results for detecting antiandrogenic activity. Toxicol. Appl. Pharmacol. 202: I

### 08-120. 4) Charles, G.D., Spencer, P.J., Schisler, M.R., Cifone, M., Budinsky, R.A., and Gollapudi, B.B. (2005). Mode ofMutagenic Action for the Biocide BiobanTM CS-1246 in Mouse Lymphoma Cells and Implications for Its In Vivo Mutagenic Potential. Toxicol Sci. 84:73-80. 5) Zhang, F., Bartles, M.J., Pottenger, L.li., Gollapudi, B.B., and Schisler, M.R. (2005). Quantitation ofDNA adduct ofthymidylyl(3'-5')thymidine methyl phosphotriester by liquid chromatography/negative electrospraytandem mass spectrometry, Rapid Commun Mass Spectrom, 19(19):2767-72. 2



### 6) Zhang, F., Bartels, M. J., Pottenger, 

### L. H., Gol1apudi, B. B., and Schisler, M. R. (2006). Simultaneous quantitation of7-methyl- and 06-methylguanine adducts in DNA by liquid chromatography-positive electrospray tandem mass spectrometry. J 

### Chromatogr BAnalyt Techno! Biomed

### Life Sci, 833(2):141-8. 7) Zhang, F., Bartles, M.J., Pottenger, L.H., Gollapudi, B.B., and Schisler, M.R. (2007). Quantitation oflower levels ofDNA adduct ofThymidyly (3'-5') thymidine methyl phosphotriester by liquid chromatography/negative atmospheric pressure chemical ionization tandem mass spectrometry. Rapid Commun Mass Spectrom, 2I:I043-I048. 8) Zhang, F., Stott, W.T., Clark, A.J., Schisler, M.R.. Grundy, J.M., GoiJapudi, B.B., Bartels, M.J. (2007). Quantitation of8-hydroxydeoxyguanosine in DNA by liquid chromatography/positive atmospheric pressurephotoionization tandem mass spectrometry. Rapid Commun Mass Spectrom, 21 :3949-3955. 9) Zhang, F., Bartels, M.J., Geter, D.R., Jeong, Y.C., Schisler, M.R., Wood, A.J., Kan, H.L., and GolJapudi, B.B. (2008). Quantitation ofglutathione by liquid chromatography/positive electrospray ionization tandem mass spectrometry. Rapid Comrnun Mass Spectrom, 22(22):3608-3614.

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## " 10) Zhang, F., Bartels, M.J., Pottenger, L.H., Schisler, M.R., Grundy, J.J., and Gollapudi, B.B. (2008). Quantitation ofmethylated hemoglobin adducts in a signature peptide from rat blood by liquid chromatography/negative electrospray ionization tandem mass spectrometry. Rapid Commun Mass Spcctrom, 22( 1 0): 1455-1460. 11) Pottenger, L.H., Schisler, M.R., Zhang, F., Bartels, M.J., Fontaine, D.D., McFadden, L.G., and Gollapudi, B.B. (2009). Dose-response and operational thresholds/NOAELs 1nr 

### in vitro mutagenic effects from DNA-reactive mutagens, MMS and MNU. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 678(2): 138-47. 12) Geter, D.R., Zhang, F., Schisler, M.R., Wood, A.J., Kan, L.H., Jeong, Y.C., Bartels, M.J., McFadden, L., 

### and Gollapudi, B.B. 

### (2012). Genetic damage, but limited evidence ofoxidative stress markers in diethyl maleate-induced glutathione depleted mouse lymphoma L5178Y (TK(+/-) cell cultures. Toxiol Mech Methods 22(7):547-54. 13) Jeong, Y.C., Zhang, F., Geter, D.R., Wood, A.J., Schisler, M.R., Gollapudi, B.B., and Bartels, M.J. (2012). Measurement ofdeoxyinosine adduct: Can 

### it he a reliable tool to assess oxidative ornitrosative DNA damage? Toxicology Letter 1

7;214(2):226-33. 3



### 14) LeBaron, M.J., Schisler, M.R., Torous, O.K., Dertinger, S.D., and Gollapudi, B.B. (2013). Influence ofcounting methodology on erythrocyte ratios in the mouse micronucleus test. Environ Mol Mutagen 54(3):222-228.

DOW RESTRICJl:l)

### PRESENTATIONS 

### 1) DowMRDTG Poster Session (2000), Short-Term Tests for Identifying Chemical Mutagens and Carcinogens. 2) Dow MRDTG Poster Session (2004), Mode ofAction for the In Vitro 

### Mutagenicity ofBioban CS-1246 and Implications for its In Vitro Mutagenic Potential. 4



### 3) Schisler, M.R. and Gollapudi, B.

### B. (2005). A Search for a Positive Control for the In Vivo/In Vitro Rat Peripheral Blood Chromosomal Aberration Test. Presented at rnternational Conference ofEnvironmental Mutagens September 3-8, 2005. 4) Pottenger, L. H., Zhang, F., Schisler, M. R., Bartels, M. J. and Gollapudi, B. B. 

### Dose-Response and Apparent Thresholds for DNA Adducts and in vitro Mutagenicity in Mouse Lymphoma Cells Treated with MMS and MNU. Joint Industry DNA Adduct Research Review Meeting (May 16, 2006, Grenoble, France) and also presented at47th Annual EMS Meeting, Vancouver, Canada (September 2006). 5) Zhang, F., Bartels, M. J., Pottenger, L. 

### H., Gollapudi, 

### B. B. 

### and Schisler, M. R. Quantitation ofDNA Adduct**ofThymidyl~l(3'-5')thymidine** 

### Methyl Phosphotriesterby LC/APCr!MS-MS. 54n ASMS Conference on Mass Spectrometry and Allied Topics, Seatle, Washington, May 28- June I, 2006. 6) Pottenger, L.H., Zhang, F., Schisler, M.R., Bartels, M.J., Wiescinski, C.M., Charles, G.D., and Gollapudi, B.B. (2007). Dose response and apparent thresholds for induction ofDNA adducts and peripheral blood micronucleus in rats treated with MMS. Presented at 38th Annual Meeting ofthe EnvironmentalMutagen-Society, Atlanta, Georgia, Oct. 20-24(\ 2007. 7) Schisler, M.R., Wood, A.J., Kleinert, K.M., Geter, D.R., Seidel, S.S., and Gollapudi, B.B. (2007). Mutagenicity and loss ofheterozygosity (LOH) of Microsatellites markers in chromosome IIb ofmouse lymphomacells treated with taxol. Presented at 38111 

### Annual Meeting ofthe Environmental-MutagenSociety, Atlanta, Georgia, Oct. 20-24t11
, 

### 2007. 8) Zhang, F., Pottenger, L.H., Gollapudi, B.B., 

### Schisler, M.R., Grundy, J.M., and Bartels, M.J. (2007). Simultaneous quantitation ofmethylated hemoglobin adducts in signature peoptides from rate blood by liquid chromatography-negative electrospray tandem mass spectrometry. 55th ASMS Conference on Mass Spectrometry and Allied Topics, Indianapolis, Indiana, June, 2007. 9) Zhang, F., Stott, W.T., Clark, A.J., Grundy, .J.M., Schisler, M.R., Gollapudi, M.R., and Bartels, M.J. (2007). Quantitation of8-hydroxydeoxyguanosine in DNA 

### by liquid chromatography-positive atmospheric pressure. 55th ASMS

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### Ill5


### Conference on Mass Spectrometry and Allied Topics, Indianapolis, Indiana, June, ( 2007. I0) LeBaron, M.J., Kan, 

### H.L., Geter, D.R., Pottenger, L.H., Zhang, F., Schisler, M.R., Bartels, M.J., and Gollapudi, 

### B.B. (2008). Non-linear dose response in mcthylmethane sulfonate (MMS)-treated rats as measured by multiple endpoints. Presented at SOT Annual Meeting 

### & Tox ExpoTM, March 16-20,2008. 11) 

### Pottenger, L.H., Zhang, F., 

### SchislerM.R., Wiescinski, C.M., LeBaron, M.J., Bartels, M.J., and Gollapudi, 

### B.B. (2008). Dose-response and apparent thresholds for in vitro and in vivo mutagenic effects from DNA-reactive mutagens/carcinogens. Presented at ECETOC-sponsored JIG DNA Adduct Workshop, September 25-26, 2008. 12) Geter, D.R., Zhang, F., Schisler, M.R., Wood, A.J., Kan, 

### flL., Jeong, Y.C., Bartels, M.J., and Gollapudi, 

### B:B. (2009). Examination ofgenetic damage in glutathione (GSH)-depleted cell cultures using the micronucleus test, single cell electrophoresis assay (comet), and 8-hydroxy-deoxyguanosine**(8-0H-dG~** 

### levels in nuclear DNA (2009). Presented at SOT Annual Meeting & 

### Tox Expo M, March 15-19, 2009. 

### 13) LeBaron, 

### M.J., Kan, 

### H.L., Geter, D.R., Pottenger, 

### L.I-I., Zhang, 

### F., Schisler, M.R., Bartels, M.J., and GolJapudi, B.B. (2009). Biological thresholds and nonlinear dose-responses for multiple endpoints in methyl methanesulfonate (MMS)-treated rats. Presented at SOT Annual Meeting & 

### Tox ExpoTM, March 15-19,2009. 14) Schisler, M.R., Gollapudi, G.G., and Moore, M.M. (2010). Evaluation ofthe mouse lymphoma mutation assay (MLA) data ofthe U. 

### S. National Toxicology Program (NTP) using International Workshop on Genotoxicity Tests (IWGT) criteria. Presented at 41st Annual Meeting ofthe Environmental-MutagenSociety 2010. 

### I5) LeBaron, M.J., Schisler, M.R., Torous, 

### D.K., Dertinger, S.D., and Gollapudi, B.B. (20 1 

### 0) Reticulocyte rations estimated by manual and flow-based methods in the mammalian micronucleus test: need to update the guideline? Presented at 41st Annual Meeting ofthe Environmental-Mutagen-Society 20 I

### 0. 16) LeBaron, MJ, Schisler, MR, Kan, HL, Thomas, J, Eisenbrandt, DL and Gollapudi, BB (2011). Analysis ofMolecular, Cellular, and Biochemical Changes in the Liver ofMale B6C3F1 Mice Treated WithNitrapyrin- A Nitrogen Stabilizer. Presented at SOT 501 h

### Anniversary Annual Meeting & 

### Tox ExporM, March 6-10, 2011. 17) Schisler, M.R, Gollapudi, BB, Moore, NP and Pottenger, LH (2011). Evaluation ofPropylene Oxide (PO)-Induced Mutagenicity in a p53-Competent, HumanDerived Cell Line, TK6. Presented at 42nd Annual Meeting ofthe Environmental-Mutagen-Society October 15-19, 2011.

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### 18) Schisler, M.R., Gollapudi, 

### B.B., McDaniel, L.P., and Moore, M.M. (2012) Reevaluation ofthe mouse lymphomaforward mutation assay (MLA) results on 342 chemicals from the U.S. National Toxicology Programs (NIP)Database. Presented at 42nd Annual Meeting ofthe Environmental-Mutagen-Society September 8-12, 2012. 19) LeBaron, M.J., Schisler, M.R., Zhang, F., Jeong, Y.C., Bartels, M.J., Sura, R., Hotchkiss, J., Geter, D.R., Gollapudi, B.B., and Moore, N.P. (2012). Bioanalytical, molecular, and genetic alterations in**ethyl~ne** 

### oxide exposed male B6C3Fl mice. Presented at 42nd Annual Meeting ofthe EnvironmentalMutagen-Society September 8-12, 2012. 20) Gollapudi, B.B., Schisler, M.R., McDaniel, L.P., and Moore, M.M. (2012). 6



### Reevaluation ofthe U.S. National Toxicology program's (NTP) mouse lymphoma forward mutation assay (MLA) data using current standards reveals limitations of using the program's summary calls. Presented at SOT Annual Meeting & 

### Tox TM 

### Expo , 2012. 21) LeBaron, M.J., Schisler, M.R., Kan, H.L., Sura, R., Eisenbrandt, D.L., and Gollapudi, B.B. (2012). Analysis ofmolecular, cellular, and biochemical changes in the liver ofmale CD-I mice treated with the herbicide Pronamide. Presented at SOT Annual Meeting & 

### Tox ExpoTM. 2012.

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## RESUME

Name Dr. Rajendra M. Nagane
Home Address* A/P-Kem, Tal-Karmala, Dist-Solapur, Pin-413223, Maharashtra, INDIA Business Address Jai Research Foundation, Post Box No 30, GIDC, Vapi- 396 195, Gujarat, India Phone Number: Home(+91)9909900935 Office (+91) 260-6540-242 E-Mail Address: rajendra.nagane@ jrfonline.com dr_rajnagane@yahoo.co.in

Degree Received Major

MasterofVeterinary Veterinary Science (M.V.Sc.) Microbiology

BachelorofVeterinary All veterinary science Science & Animal Subjects Husbandry (B.V.Sc. & A. H.) (Equivalent to DVM)
12th Standard Physics, Chemistry, Higher Secondary Biology, English CertificateExamination Crop Science (H.S.C.) lOth Standard English, Hindi, Secondary School Marathi, Social CertificateExamination Science, Science (S.S.C.) Name and Address ofInstitution College: BombayVeterinary College, Mumbai, Pin- 400012, India University: Maharashtra Animal & Fishery Sciences University, Seminary Hills, Nagpur, Pin

- 440006, Maharashtra, India College: BombayVeterinary College, Mumbai, Pin -400 012, Maharashtra, India University: MaharashtraAnimal & Fishery Sciences University, Seminary Hills, Nagpur, Pin

- 440006, Maharashtra, India Malojiraje Sheti Vidyalaya, District- Satara, Maharashtra, India

Kern Prashala, Kern, District- Solapur, Pin

- 413223, Maharashtra, India Year of Passing

March 2006

July 2003

June 1998

June 1996Job Responsibilities: I. Working as a study director for following studies:**~** In vitro Bacterial reverse mutation test using Salmonella typhimurium and E. coli.**~** In vitro mammalian chromosome aberration test using CHO-Kl cell line**~** In vitro mammalian chromosome aberration test using human peripheral blood lymphocyte.**~** In vitro mammalian cell gene mutation test (HPRT assay) using CHO-Kl cell line.**~** In vivo chromosome aberration test in Rat and Mice.**~** In vitro Endocrine disruptor studies 2. Working as Head for Mutagenicity section in Department ofToxicology in OECD GLP accredited facility. Provide strategic and operational direction to designated study directors to ensure that the scientific, technical and regulatory aspects are conducted in-line with the specified requirements. 3. Planning, execution and interpretation of in vitro and in vivo genetic toxicology studies supporting IND and NDA of small molecules and agrochemicals as per GLP principles. Protocol design, study initiation and documentation, data analysis and interpretation, regulatory compliance, as well as reporting ofstudy results. 4. ConductedToxin Neutralization Assay for vaccines. 5. Responsibility as Institutional Animal Ethics Committee Member at Jai Research Foundation. 6. Responsibility as Institutional bio-safety committee member at Jai Research Foundation. 7. Responsibility as Biological Safety Officer at Jai Research Foundation. 8. Toprovide technical support for genetic toxicity studies in terms ofregulatory submission to the sponsors. 9. Tomonitor the genetic toxicity studies ofdifferent clients conducted at JRF. I0. To review the genetic toxicity study protocols and reports ofin-house validations conducted at Section Mutagenicity (Jai Research Foundation). TRAINING, WORKSHOP AND SEMINARATTENDED: 1. Attended 26th Annual Congress and National Symposium on "Recent trends in small animal surgery" held at Bombay Veterinary College, Mumbai, in November 2002. 2. Participated in "National seminar on Emerging Biotechnological frontiers in animal health and production Feb. 26 -27, 2004" held at Sardarkrishinaganr, Gujarat. 3. Attended 30th AICBC, All India Cell and MolecularBiology Conference at New Delhi. 4. Participated in National Seminar organized by Jai Research Foundation on"REACH" in 2006. 5. Under gone training on Moleculartechniques for 15 days at Indian Institute ofScience (liSe), Banglore. 

### 6. Completed GLP training organized at Jai Research Foundation. 7. Completed training on "Similarities and differences in OECD, EPA and FDA GLP" 8. Attended workshop on " Genetic toxicology" organized by National Center for Genetic Toxicology and Reproducti· Health, NIRRH, Mumbai, from ih- 14th Jan. 2008. 9. Attended workshop on "Animal welfare: ethics and enrichment on lOth May 2008 at Jai Research Foundation. 10. Actively Participated in conducting one day seminar Organized by Jai Research Foundation on "Changing trends in/ \ Animal Experimentation at NIPER, Hyderabad on12th September 2008. 11. Attended Conference on "Genetic toxicology" organized by Gorden Research Conferences at II Ciocco Hotel and Resort, Lucca (Barga), Italy, from lOth- 15th July 2011. Presented poster on "Historical Control Data for Vehicle Control and Positive Control Induced Micronucleus (MN) And P/E ratio in Mice" 12. Attended AgChem Forum "Chemical Industries Regulation 2012" Hotel Fira Palace, Barcelona, during September 05- 06, 2012. 13. Attended training on "General Handling of SkinEthic 3D Tissue models, focusing on ECVAM/ESAC SkinEthic Skin irritation Validated protocols" at SkinEthic Laboratories (Lyon, France), during September 10- 13, 2012. 14. Attended 52"ct, Society ofToxicology's Annual Meeting, at the Henry Gonzalez Convention Center San Antonio, Texas during March 10-14, 2013. LIFE MEMBERSHIP OFPROFESSIONAL SOCIETIES: 1. Life member ofIndian Journal ofEnvironment and Toxicology AWARDS: 1. Qualified ICAR Junior Research Fellowship examination (2003) 2. Awarded "National Merit Scholarship" (1998-2003)Name L.U. Sanghani, Ph.D., MRQA Birth Date April18, 1969 Job Title Dy. Director and Head QAU Education Qualification**~** M.Sc. in Agriculture Chemistry and Soil Science from Gujarat Agriculture University, Navsari**~** Ph.D. in Residue Chemistry from Bhavnagar University, Bhavnagar Job Career**~** Joined Jai research Foundation as Soil Chemist in 1993**~** Worked as Soil Chemist and StudyDirector for Residue and Persistence studies in Soil, Water and Crop from 1993 to 1996.**~** Worked as QA officer from 1996 to 2000**~** Heading QA ofJRF since 2001**~** Head ofGlobal QA ofJai Research Foundation since 2011 and co-coordinating & auditing of different sites ofJRF in US and UK Training/Competency**~** Trained Study Director for Residue field trials and GLP studies**~** Trained as GLP inspectorbyNational GLP Compliance Monitoring Authority, Government oflndia**~** Trained Laboratory Assessor ofNational Accreditation Board for Laboratories for auditing the laboratory operated as perIS0-17025**~** Development and implementation ofGLP quality system as per OECD, US-EPA andFDA GLP requirements**~** Attended 8SQA Annual meetings, 2 Global QA conferences, 5 RQA conference and various quality assurance trainings since 2006.**~** Development ofSOPs, Validationplans and policies under GLP. Auditing ofComputerized system**~** Qualifiedtrainer for training on various aspects ofGLP Total Experience 21 years Academic Society**~** MemberofSocietyofQualityAssurance, USA Membership**~** MemberofResearch QualityAssociation, UK**~** Member and founder Director ofIndian Chapter ofSociety ofQualityAssurance (ICSQA) Publication**~** Published 3 technical research papers and 7 _l)_osters on GLP quality system)

\ \\\

## RESUME

Name Ms. Kirti E. Tendulkar
HomeAddress 2/45, M.H.B. Colony, MithagarRoad, MulundEast, Mumbai- 400081. BusinessAddress Jai Research Foundation, Post BoxNo 30, GIDC, Vapi- 396 195, Gujarat, India PhoneNumber: Home (+91) 9687483386 Office (+91) 260-6540-242 E-Mail Address: kirti.tendulkar@jrfonline.com tendulkar.kirti@gmail.com
Degree Received Major

MasterofAppliedGenetics Applied Genetics (M.Sc.)

Bachelor ofBiotechnology Biotechnology (B.Sc.)

12th Standard Physics, Chemistry, Higher Secondary Maths, Biology, Certificate Examination (H.S.C.)
1Oth Standard English, Hindi, Secondary School Marathi, Social Certificate Examination Studies, Science, (S.S.C.) Mathematics Name and Address ofInstitution College: Acharya Institute ofLife Sciences University: Bangalore University, Karnataka, India
College: K.J SomaiyaIntitute ofScience and Commerce University: Mumbai University, Mumbai, Maharashtra, India

V.P.M JuniorCollege, Mulund East, Mumbai, Maharashtra, India

V.P.M English High School, Mulund East, Mumbai, Maharashtra, India Yearof Passing

March 2009

July 2007

June 2004

June 2002Job Responsibilities: 1. Working as study director for following studies:**~** In vitro Bacterial reverse mutation test using Salmonella typhimurium and E. coli.**~** In vivo Micronucleus Test inMice**~** In vitro mammalian chromosome aberration test using CHO-K1 cell line**~** In vitro mammalian chromosome aberration test using human peripheral blood lymphocyte.**~** In vitro mammalian cell gene mutation test (HPRT assay) using CHO-K1 cell line. 2. Working as study personnel for following study:**~** In vitro mammalian cell Micronucleus test using human peripheral blood lymphocyte.
3. Planning, execution and interpretation ofin vitro and in vivo genetic toxicology studies supporting IND and NDA of small molecules and agrochemicals as per GLP principles. Protocol design, study initiation and documentation, data analysis and interpretation, regulatory compliance, as well as reporting ofstudy results.

TRAINING ATTENDED: 1. Completed GLP training organized at Jai Research Foundation. 2. Attended 32"dAnnual Conference ofSociety ofToxicology (STOX) and International Symposium on ''New frontiers in Toxicology" held atLucknow, in December2012.

Ill

## CV- DR. T.G. MARVANIA Name Date ofBirth Qualification PresentDesignation Responsibility Tulsidas Gordhan Marvania 1



### ihJune, 1965 Ph.D., M.Sc. (Agri.) Deputy Director, JRF Project Coordination and Monitoring, JRF

TotalJob Experience Study Director for Field studies (Efficacy, Compatibility and Phytotoxicity) and Ecotox studies 24 years 1 Since 1993 till Date: JaiResearch Foundation, Joined as a Research Officer and presentposition is DeputyDirector
a. Current Designation smce 2012 : Dy. Director : Responsible for monitoring the Scientific Projects, looking after the administrative activities which involves liasoning with Govt. Officials, Vendors and execution ofthe projects. b. Assistant Director (2007-2012): Looking after the Sponsor's scientific projects i.e. Toxicity and Chemistry projects which includes the liasoning with Sponsor and Govt. Officials, link between Study Director and Sponsor, arrangement ofresources for Study Group and monitoring ofthe ongoingprojects with respect to contractualterms and conditions.
c. Principle Research Officer (2006-2007): Worked as study director for conducting toxicity tests of pesticides against beneficial arthropods i.e. Honey bee, earthworm etc. d. Senior Research Officer and Study Director (2000-2006) for conducting field trails i.e. Efficacy, Compatibility and Phytotoxicity for insecticides. Handling, Calibration and maintenance of Agricultural equipment (Plant Protection Equipment), Field lay out and adopting cropping practices. e. Research Officer (1993-2000) the job profile included the maintenance of Insect/pest colonies under laboratory conditions, Conducting Laboratory Bio-assay for screening ofnew formulations ofinsecticides including house hold insecticides. 2 1989-91 Workedwith United Phosphorus Limited, Mumbaifor one year as Sales Representative Thejobprofile includes the establishment ofProduct distribution chain (appointment ofDistributors and dealers), liasoning with State Agricultural Universities for product development /screening, field demonstration and farmers meeting and product promotion. 3 1991-93 Worked as Agricultural Officerfor GujaratLandDevelopment Corporation, A"bad The job profile includes the Water shed development project for water harvesting and water conservation. Farmers meeting for trailing and practical demonstration for Integrated Crop Management Practices developed by Agricultural Departments.

Page 1 of2Page 2 of2**I~--** 

### IllTOXICOLOGY

### AND ENVIRONMENTAL RESEARCH AND CONSULTING CURRICULUM VITAE
NAME: ChadA. Hales CURRENT JOB TITLE: Senior Environmental Technologist EDUCATION: 2010-2013: Saginaw Valley State University, University Center, Ml, Bachelors in Chemistry

EMPLOYMENT:

MEMBERSHIPS: PUBLICATIONS: PRESENTATIONS: 2001: DeltaCollege, University Center, MI, Associates ofScience in Chemical Technology 1997-1999: Michigan State University, East Lansing, Ml 1996-1997: Saginaw Valley State University, University Center, MI 2001-present: The Dow Chemical Company, Midland, Ml, Technologist, Toxicology and Environmental Research & Consulting 1999-2001: The Dow Chemical Company, Midland, MI, Technical Co-op 1998-1999: DTN Management, East Lansing, Ml, Leasing Agent/Maintenance Assistant 1998: Kelly Services Scientific Resources, The DowChemical Company, Midland, MI, Laboratory Technologist 1995-1997: The DowChemical Company, Midland, MI, Larkin Lab N/A N/A
See Attached
Employee Signature:

# -----'/Vt"'-__:..:.~.....:.....!.lQ/t__!........:~=---Date.__ 

## 2}'--l_f("--t-y
__ _IllPRESENTATIONS 1) Hancock, G. A., Marino, T. A., Najar, J.R., Staley, J. L., Yaroch, A.M., Hales, C. A., and McClymont, E. L. Aquatic Toxicity oftwo Propionic Estersto Freshwater Fish. 27th Annual Meeting ofthe Society for Environmental Toxicology and Chemistry, North America, held in Montreal, Quebec, November 5-9,2006. 2) West, R. J., Woodburn, K. B., Yaroch, A.M., Hales, C. A. and Henry, K. S. Application ofa Novel Flow-Through Exposure System in Evaluatingthe Chronic Toxicity of Chloroform to Sediment-Dwelling Organisms. 7th Annual Meeting ofthe Society for Environmental Toxicology and Chemistry, North America, held in Montreal, Quebec, November 5-9, 2006.

### )

I I Ill

### ClJRRIClJLtJM VIT**AI~** NAME: W. Brian Holzheuer .JOB TITLE: Study .Director/Sr. Chemist EOUCATlON: 2006 Eastern .Michigan University B.S. 

### in Chemistry, Minor in Mathematics 2002 Lansing Community College A.A.S in Chemical Technology 

### EMPLOYMENT: 20I0-present: The Dow Chemical Company, Study Director/Sr. Chemist, Toxicology and l:nvironmental Research & Consulting 2008-2010: TSRL 

### Inc. Ann Arbor, 

### MI, Associate Scientist, 2007-2008: Aerotek Scientific onAssignment at Roche Diagnostics, Indianapolis In, VaJidation Engineer 2002-2007: Pfizer Inc. Ann Arbor, Ml, Associate Scientist, Analytical Research and Development
PUBLICATIONS: See Attached 

### PRESENTATlONS: N/A

Reviewed/Revised: 

# ::2

.......

# ~?:-**--~~** --------- Signature)

## )

I I Illl'lJBUCATIONS An evaluation ofJ(mrcommercial HPLC chiral detectors: A comparison ofthree polarimeters and a circular dichroism detector, 

### Laila Kott*, W. Brian .H.olzheuer, Megan 

### M. Wong and Gregory 

### K. WebsterJournal ofPhammceutical and Biomedical Analysis (2005) Application ofa Low Cost Amperornetric Sensor f'iJr Detection ofAlcohols 

### in Process Related Headspace Applications;with Gregory K. 

### Webster, Tracy 

### l-lobson and 

### W. Brian Holzheucr, 

### submitted J. ofProc Anal Chem (2007) 2



### A 

### Comparison of

### IJPLC and SFC Chiral Method Development Screening Approaches for Compounds ofPharmaceutical Interest, Megan M Wong, 

### William B Holzheuer, and Gregory K Webster Curr Phann Anal 4(2): 1 01-l05 (2008) Reversed Phase Chiral Method Development Screening f(>r Compounds of Phannaceutical Interest, William B Holzheucr, Megan M Wong, and Gregory K Webster, CurrPharm Anal 5(4):346-357 (2009)

### CURRICULUMVITAE
NAME: Robert

### J. West JOB 

### TITLE: Group Leader EDUCATION: 1992: Central Michigan University, Bachelor ofScience, Chemistry 1989: St. ClairCountyCommunityCollege, Associate ofScience Degree 1987: Croswell-LexingtonHigh School 

### EMPLOYMENT: 2005-Present: The Dow Chemical Company, Midland, MI, SeniorResearch Specialist, Environmental ChemistryResearch Laboratory 2000-2005: The DowChemical Company, Midland, MI, Research Specialist, Environmental ChemistryResearch Laboratory 1997-2000: The Dow Chemical Company, Midland, MI, Senior Research Chemist, Environmental ChemistryResearch Laboratory 1994-1997: The Dow Chemical Company, Midland, Ml, Research Chemist, Environmental ChemistryResearch Laboratory 1992-1994: The Dow Chemical Company, Midland, MI, Chemist, Environmental Toxicology & ChemistryResearch 1990-1992: The Dow Chemical Company, Midland, MI, Student Technologist, Environmental Toxicology & ChemistryResearch Laboratory 1987-1989: Carpenter-Selfemployed, Croswell, MII! ill2 

### MEMBERSHIPS: Society ofEnvironmental Toxicology and Chemistry American Chemical Society-Environmental ChemistryDivision 

### PUBLICATIONS: See Attached 

### PRESENTATIONS: See Attached

Reviewed/Revised: (Signature) (Date)

### PUBLICATIONS 1) Schupp, T. and West, R. 

### J. (2007) Primary aromatic amines: Thecontribution ofsedimentorganisms to humanexposure. EXCLIJournal 6: 187-196. 2) West, 

### R. J., Davis,

### J. W., Pottenger, L. H., Banton, M. I., and Graham, C. (2007) Biodegradabilityrelationships amongpropylene glycol substances in the Organization for Economic Cooperation and Developmentready- and seawater biodegradabilitytests. Environ. Toxicol. Chern. 26:862-871. 3) Klecka, G. M., Gonsior, S. J., West, 

### R. 1., and Markham, D. A. (2002) Biodegradationofbisphenol A in aquatic environments- Riverdie-away. Environ. Toxicol. Chern. 20:2725-2735. 4) West, R. 

### J., Goodwin, P.A., and Klecka, G. M. (2001) Assessment ofthe ready biodegradabilityofbisphenol A. Bull. Environ. Contam. Toxicol. 67:106-112. 5) Davis,J. W., Odom, 

### J. M., DeWeerd, K. A., Stahl, D. A., Fishbain, S. S., West, 

### R. J., Klecka, G. M., andDecarolis,

### J. G. (2002) Natural attenuation of chlorinated solvents at Area 6, Dover AirForce Base: Part II. Characterization of microbial community structure. J. Contam. Hydro/. 57:41-59 6) Klier, N.J., West, R. J., and Donberg, P. A. (1999) Aerobic biodegradation of dichloroethylenes 

### in surface and subsurface soils. Chemosphere 38: 1175-1188. 7) Holmes, M. W., Morgan, P., Klecka, G. M., Klier, N. J., West, R. J., Davis, 

### J. W., Ellis, D. E., Lutz, E. J., Odom, J. M., Ei, T. A, Chapelle, F. H., Major, D. W., Salvo, 

### J. J., and Bell, M. 

### J. (1998) Thenatural attenuation ofchlorinatedethenes atDoverAirForce Base, Delaware, USA. Proceedings ofthe 6th International FZKITNO Conferenceon Contaminated Soil, Edinburgh, May 17-21, 1:143-152. 8) Davis, J. W., West, 

### R. J., and Klecka, G. M. (1997) Enhanced land treatment of petroleum-contaminated soils using solid peroxygen materials. Remediation 7:67-81. 9) West, R. J. and Gonsior, S. 

### J. (1996) Biodegradation oftriethanolamine. Environ. Toxicol. Chern. 15:472-480. lO)Gonsior, S. J. andWest, R. J. (1995) Biodegradation ofglycol ethers in soil. Environ. Toxicol. Chern. 14:1273-1279. 3Irl IllPRESENTATIONS I) Lowe, E. R., Rick, D. L., West, R. J., and Bartels, M. J. The Evaluation of Quantitative Structure PropertyRelationship Predictions ofTissue: Blood Partition Coefficients ofHighly Lipophilic Chemicals. 14th North American ISSX Meeting, International Society for the Study ofXenobiotics (ISSX), Rio Grande, Puerto Rico, October 22-26, 2006. 2) West, 

### R. J., Woodburn, 

### K. B., Yaroch, A.M.• Hales, C. 

### A. and Henry, K. S. 4

Application ofa Novel Flow-Through Exposure System in Evaluating the Chronic Toxicity ofChloroformto Sediment-Dwelling Organisms. 7th Annual Meeting of the Societyfor Environmental Toxicology and Chemistry, North America, held in Montreal, Quebec, November

### 5-9, 2006. 3) West, R. 

### J. and Mihaich, E. (2005). Overview ofthe Persistence, BioaccumulationPotential, and Inherent EcotoxicityofDiisocyanate Substances. Presentedto Existing Substances Assessment Division, Environment Canada, Montreal, Quebec. Jan. 17, 2005. 4) West, R. J; Henry, 

### K. S; and Davis, J. W. (2004). Examination offeeding modes and sediment equilibrationtimeused in evaluating chronic toxicity of toluenediamine (TDA) to sediment-dwelling organisms. Presented atFourth SETAC World Congress, Portland. Oregon. Nov. 17,2004. 5) Witt, M. E., West, R. J., Davis, J. W., and Klecka, G. M. (2001) Results of laboratory microcosm studies to investigate intrinsicbiodegradationofcarbon tetrachloride in afractured bassalt aquifer. In Leeson, 

### A. (Ed.) Proceedings ofthe 6th International In-Situ and On-Site Bioremediation Symposium, San Diego, CA June 4-7, 2:89-96I

### fl Ill( 1,

Appendix B Melting Point, Freezing Point, Boiling Point Study Report



### 14 

### DEPCTIll Ill -( I \ 

## REPORT
PHOSPHOROCHLORIDOTHIOTICACID, 0,0-DIETHYLESTER (DEPCT): Determination ofMelting/Freezing Temperature and Boiling Temperature

StudyDirector: Test Facility:

Sponsor:

Harlan StudyNumber: SponsorReferences: StudyCompletion Date: DATarran 

### HarlanLaboratories Ltd. ShardlowBusiness Park Shard!ow Derbyshire DE722GD UK 

### TheDow Chemical Company Building 1803 Midland Michigan48674 UNITED STATES OF AMERICA
41304286 130187 & 

### 130189 07 February 2014

Pal!e 1of 19Harlan StudyNumber: 41304286 SponsorReferences: 130187 & 130189 Report Page2



## CONTENTS CONTENTS
....................................................................................................................................2 STUDY DIRECTOR STATEMENT OF GLP COMPLIANCE
....................................................3 QUALITY ASSURANCE STATEMENT
......................................................................................4 SUMMARY
....................................................................................................................................5 GENERAL INFORMATION
.................;.......................................................................................6 Schedule
.................................................................................................................................6 Deviations from Study Plan
...................................................................................................6 Archiving
................................................................................................................................6 1 INTRODUCTIONAND PURPOSE
.....................................................................................7 1.1 Guidelines I Regulations
..........................................................................................7 2 TEST ITEM
........................................................................................................................... 8 3 TESTS
....................................................................................................................................9 3.1 Melting/Freezing temperature .................................................................................9 3.1.1 Test System
...............................................................................................9 3.1.2 Principle ofthe Test.
..................................................................................9 3.1.3 Performance oftheTest.
............................................................................9 3.1.4 DataHandling
............................................................................................9 3.1.5 Results
..................................................................................................... 10 3.1.6 Conclusion
............................................................................................... 13 3.2 Boiling Temperature
........................................................................................**~**

### ..... l4 3.2.1 Test System
............................................................................................. 14 3.2.2 Principle oftheTest.
................................................................................ 14 3.2.3 Performance oftheTest.
.......................................................................... 14 3.2.4 Data Handling
.......................................................................................... 15 3.2.5 Results
..................................................................................................... 16 3.2.6 Conclusion
............................................................................................... 17 APPENDICES
............................................................................................................................... 18 Appendix 1 Appendix 2 Certificate ofAnalysis
........................................................................................... 18 Monitoring Authority Statement ofGLP Compliance
.......................................... 19

Ill, IllHarlan StudyNumber: 41304286 SponsorReferences: 130187 & 130189 Report Page3

### ~ 

## STUDYDIRECTOR STATEMENT OFGLP COMPLIANCE \. 

### Harlan Laboratories Ltd., ShardlowBusiness Park, Shardlow, Derbyshire, DE72 2GD, UK
Harlan StudyNumber: Study Title: 41304286 PHOSPHOROCHLORIDOTHIOTIC ACID, 0,0-DIETHYLESTER (DEPCT): Determination of Melting/Freezing Temperature and Boiling Temperature This study was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)). These Regulations are in accordance with GLP standards published as OECD Principles on Good Laboratory Practice (revised 1997, ENV /MC/CHEM(98)17); and are in accordance with, and implement, the requirements ofDirectives 2004/9/EC and 2004/10/EC. These principles are compatible with Good Laboratory Practice regulations specified by regulatory authorities throughout the European Community, the United States (EPA and FDA), and Japan (MHLW, MAFF andMETI). This report fully and accurately reflects the procedures used and data generated. There were no circumstances consideredto have affected the integrity ofthe study orthe validity ofthe data. Study Director: D A 

### Tarran

Date: 

## 07 FEB 

## 20HHarlan StudyNumber: 41304286 Sponsor References: 130187 & 130189 Report Page4


## QUALITYASSURANCE STATEMENT Harlan StudyNumber: Study Title: 41304286 PHOSPHOROCHLORIDOTHIOTIC ACID, 0,0-DIETHYLESTER (DEPCT): Determination of Melting/Freezing Temperature and Boiling Temperature The general facilities and activities are inspected at least once a year and the results are reported to the relevant responsible person and management. Study-relatedprocedures conducted at the test facility were audited and inspected. The details of these audits and inspections are given below.
Reported tothe relevant Datesand Types ofQAInspections StudyDirector and Test FacilityManagement Date ofInspection Type ofInspection Phase Inspected ReportDate 05 December 2013 Study PlanVerification N/A 05 December2013 22 January 2014 Process

- based Melting/Freezing 22 January 2014 Temperature 14 January 2014 Study- based Boiling Temperature 14 January 2014 06 February2014 ReportAudit N/A 06 February 2014


### Verification ofstudyplan amendments for GLP compliance were performed during the course of the study. This statement confirms thatthis final report reflects the rawdata andthe procedures followed. Quality Assurance: 

# .L
.. 07FEB 

## 2014

Ill Ill( \ Harlan Study Number: 41304286 SponsorReferences: 130187 & 130189



## SUMMARY Report Page 5



### The melting/freezing temperature and boiling temperature of PHOSPHOROCHLORIDO THIOTIC ACID, 0,0-DIETHYL ESTER (DEPCT) have been determined. The results are summarizedbelow: Melting/Freezing Temperature. 

### Less than -20 °C (253 K) using a method designed to be compatible with Method Al Melting/Freezing Temperature ofCommission Regulation (EC) No 440/2008 of30 May 2008 and Method 102 ofthe OECD Guidelines for Testing ofChemicals, 27 July 1995. Boiling Temperature. 

### 202 ± 

### 0.5 °C (475 ± 

### 0.5 K) at 98.3 k:Pa by a differential scanning calorimetry method, designed to be compatible with Method A2 Boiling Temperature of Commission Regulation (EC) No 440/2008 of 30 May 2008 and Method 103 of the OECD Guidelines for Testing ofChemicals, 27 July 1995.Harlan StudyNumber: 41304286 SponsorReferences: 130187 & 130189 Report



## GENERALINFORMATION Schedule Experimental Starting Date: Experimental CompletionDate:


### Deviations from Study Plan 14 January 2014 22 January 2014

There were no deviations (unplanned changes) from the study plan. 

### Archiving Page6



### Initially, all original data and the final report will be retained in the Harlan Laboratories Ltd, Shardlow, UK archives. As soon as practicable the original data and final report will be transferred to The Dow Chemical Company, Building 1803, Midland, Michigan 48674, USA. Harlan Laboratories Ltd. holds no responsibility for the management of archiving at The Dow Chemical Company. A copy ofall original data and the final report will be retained in the Harlan Laboratories Ltd, Shardlow, UK archives for a minimum ofone monitoring authority inspection cycle, in accordance with current GLP requirements.

Ill· Ill( \. Harlan StudyNumber: 41304286 Sponsor References: 130187 & 130189 Report



## 1 

## INTRODUCTIONAND PURPOSE Page 7



### The purpose ofthis study is to evaluate the melting/freezingtemperature and boilingtemperature ofthe test item. 

## 1.1 Guidelines I

## Regulations The test methods described are designed to be compatible with the procedures indicated by the following internationally accepted guidelines andrecommendations:

• CommissionRegulation (EC) No 440/2008 (as amended) of30 May 2008. • OECD Guidelines for Testing ofChemicals.Harlan :Study Number: 413042!!6 SponsorReferences: 130187 & 130189



## 2 TESTITEM Report Page 8



### Information as provided by the Sponsor. The Certificate ofAnalysis provided by the Sponsor is presented as Appendix 1. Identification: Description: Batch: Purity: Expiry Date: Storage Conditions:

Ill Ill 

### PHOSPHOROCHLORIDOTHIOTIC ACID, 0,0-DIETHYLESTER (DEPCT) pale yellowliquid C032013081501 99.13% 14 August 2014 room temperature inthe dark undernitrogenHarlan StudyNumber: 41304286 SponsorReferences: 130187 & 130189



## 3 TESTS Report



## 3.1 Melting/Freezingtemperature 3.1.1 Test System Page9



### The determinationwas carried out using a procedure designed to be compatible with Method Al Melting/Freezing Temperature of Commission Regulation (EC) No 440/2008 of 30 May 2008 andMethod 102 ofthe OECD Guidelines for Testing ofChemicals, 27 July 1995.


### 3.1.2 Principle oftheTest An aliquot ofthe liquid test item is placed in a glass tube, which is then placed inside a glass jacket. The apparatus is then placed into a salt water/ice bath, an acetone/dry ice bath or liquid nitrogen, as appropriate to the freezing temperature of the test item, and cooled with gentle agitation of the test item. Temperatures and test item observations are recorded at regular intervals.


### 3.1.3 Performanceofthe Test An 

### aliquot**(~15** 

### mL) oftest item was placed into a test tube containing a thermometer. A wire loop with which the sample could be agitated was placed inside the tube to ensure sample homogeneity during cooling. The testtube was surroundedby a glassjacketand thenplaced into a large beaker containing a dry ice/acetone mixture. The temperature and observations ofthe test item were recorded every 30 seconds. The determinationwas carried outinduplicate.


### 3.1.4 DataHandling Measured temperatures were converted from °C to K using Equation 3.1.

where: 

### T 

### t 

## = temperature (K) temperature COC) T=t+273.15 

### Equation 3.1

Graphs of temperature (K) versus time (seconds) were constructed to obtain the freezing temperature (Figure 3.1 and Figure 3.2)..Hanan**~ruoy** Numoer: 4UU4LlSO SponsorReferences: 130187 & 130189



### 3.1.5 Results Keport



### Cooling datafor DeterminationsA and B are shown inthe following tables: 

### Table 3.1 Determination A Time(s) Temperature (K) Time(s) 0 295 600 30 293 630 60 290 660 90 288 690 120 286 720 150 284 750 180 282 780 210 280 810 240 279 840 270 277 870 300 276 900 330 274 930 360 273 960 390 271 990 420 270 1020 450 269 1050 480 268 1080 510 267 1110 540 266 1140 570 265 1170 

### Observations: There was no change inappearance during cooling. Result: Freezingtemperature <-20 °C (253 K).

I11 Ill Page lU

Temperature (K) 264 263 262 261 261 260 259 259 258 257 256 256 256 255 255 254 254 253 253 252Harlan StudyNumber: 41304286 SponsorReferences: 130187 & 130189



### Table 

### 3.2 Time(s) Temperature (K) 0 295 30 293 60 292 90 290 120 289 150 288 180 286 210 285 240 284 270 283 300 282 330 280 360 279 390 278 420 277 450 276 480 275 510 274 540 273 570 272 600 271 630 270 660 269 690 268 720 268 750 267 780 266 Report



### Determination B Time(s)

810 840 870 900 930 960 990 1020 1050 1080 1110 1140 1170 1200 1230 1260 1290 1320 1350 1380 1410 1440 1470 1500 1530 1560 1590 

### Observations: There was no change in appearance during cooling. Result: Freezing temperature <-20 °C (253 K). Page 11

Temperature (K) 265 265 264 264 263 263. 262 262 261 261 260 260 259 259 258 258 257 257 256 256 255 255 254 254 253 253 252



### Graphs of temperature (K) versus time (seconds) for Determinations A and B are shown in Figure 3.1 and Figure 3.2 respectively.Harlan StudyNumber: 41304286 SponsorReferences: 130187 & 130189 Report



### Figure 3.1 DeterminationA

g 297. ! 

# t 292.

### ~ 287. 282. 277.

262. 257. 252. Freezing**Temp~~n~ture**Detennlnatlon 1**247~----~----~----~----~------r-----r-­**

g 297. 

### e 

# t 292.

### ~ 287. 282.

272. 267. 262. 257. 252. o.o 200.0 400.0 600.0 800.0 1000.0 1200.0 Tlme(s) 

### Figure3.2 DeterminationB
FreezingTemperatureDetermination2

247
..9-t-----.---..----...---..,..---.---..---....,-----.---- o.o 200.0 400.0 600.0 600.0 1000.0 1200.0 1400.0 1600.0 Tlme(s)



### Ill: Ill Page 12Harlan StudyNumber: 41304286 SponsorReferences: 130187 & 130189 Report



### Theresults are summarizedinthe following table: 

### Table3.3 Freezing Points Freezing Point [OC] 1stdefmitive test <-20 2nd definitive test < -20 Mean value <-20 

### Both samples did not freeze underthe conditions ofthe test.


### 3.1.6 Conclusion Page 13

[K] <253 <253 <253



### The freezing temperature ofthe testitem has beendeterminedto be lessthan -20 °C (253 K).Harlan StudyNumber: 41304286 SponsorReferences: 130187 & 130189



## 3.2 Boiling Temperature 3.2.1 Test System Report Page 14



### The determination was carried out by differential scanning calorimetry using a procedure designed to be compatible with Method A2 Boiling Temperature of Commission Regulation (EC) No 440/2008 of 30 May 2008 and Method 103 of the OECD Guidelines for Testing of Chemicals, 27 July 1995. Instrument:
Sample crucibles: Reference crucible: Test conditions:


### 3.2.2 Principle ofthe Test Differential Scanning Calorimeter (DSC) DSC822e, Mettler Toledo incorporating ThermoHaake Cooler EK45/MT Aluminum crucibles 'with perforated lids. Empty aluminum crucible. The tests were performed in a nitrogen atmosphere.

A phase transformation, e.g. melting or evaporation, or a reaction, e.g. dissociation, is usually associated with a heat effect. In the calorimeter, two identical crucibles, one filled withthe test item and the other empty (used as a reference), are heated at a constant rate. During the experiment, the heat effect, i.e. the difference inheat flow betweenthe sample crucible and the reference crucible, is registered.


### 3.2.3 Performance ofthe Test 3.2.3.1 Calibration The temperature accuracy of the DSC was assessed using an indium reference- standard (purity* 100%). The melting temperature was determined to be 156.96 °C and withinthe defined tolerance (156.6 ± 

### 0.5 °C). The DSC was therefore considered acceptable for use. 3.2.3.2 Sample Aliquots (see following table) of test item were placed in pre-pierced aluminum crucibles. Before and afterthetest, the appearance ofthe samples was determined.

• Value quotedby supplier

Ill: IllHarlan StudyNumber: 41304286 SponsorReferences: 130187 & 130189

Determination 1 2 Report



### Table 3.4
Mass Taken(g) 0.0045 0.0058 

### The crucibles were heated up from 1 00 

# oc to 300

# oc at 20 °C/min.


### 3.2.4 Data Handling Page 15



### The standard boiling temperature is defined as the temperature at which the vapour pressure ofa liquidis the same as the standard pressure (101.325 kPa). During thermal analysis thepeakwidth ofan endothermic reaction also depends on sample amount and heating rate. Therefore, the onset point ofthe relevant endothermic peak is usually stated as the boiling temperature. Where the endothermicpeaks/effects are poorly defined, a range will be stated in the result section. Measured temperatures were convertedfrom °C to K using Equation 3.2:
Where: T 

### t = 

### temperature (K) temperature COC) T=t

### +273.15 

### Equation 3.2

The boiling temperature depends on the atmospheric pressure. A correction to normalized pressure can be made, but the correction factor is a specific parameter for each test item and has to be determined individually. As this determination is beyond the scope of this study, no correction will be applied to the boiling temperature observed. The boiling temperature is stated withregardto the ambientpressure during measurement.Harlan**~tudy** Number: 413042.86 Sponsor References: 130187 & 130189



### 3.2.5 Results Report Page 16



### Thermograms and thermographic data for determinations 1 and 2 are shown in Figure 3.3 and Figure 3.4 and in the following tables. 

### Figure3.3 Thermogram

- Determination 1 

# .-------------------------------------·-----------------------------

20 mW Method: PIJT640 eft 1.00s

100.o-300.00C 20.00°C/mln Synchronization enabled TI178946, 17.01.2014 11:05:04 TI178946, 4.5000 mg Residue: Novisible residue



### Table 3.5

ThermalEvent

Endotherm



### Ill: Ill Integral -619.00 mJ Onset 201.59 oc



### ThermographicData

- Determination 1
Interpretation Temperature 

## oc K Boilingtemperature 201.59 475 

### Atmospheric pressure: 98.3 kPa( \ Harlan StudyNumber: 41304286 SponsorReferences: 130187 & 130189



### Figure3.4 Report Page 17



### Thermogram

- Determination2**r---------------------------------------------------------------·---··----~**

20 mW Method: PIJT640 dt 1.(11)s

100.o-300.00C 20.00"C/mln Synchronization enabled TI178946, 17.01.2014 11:20:35 TI17f&t6, 5.8000 mg Residue: No visible residue 

### ....

# --

Imegral -795.55 ml Onset 201.90 °C

100.0 110.0 120.0 130.0 140.0 150.0 160.0 170.0 180.0 190.0 200.0 21 0 220.0 230.0 240.0 250.0 260.0 270.0 280.0 290.0 <>c I ill IIIII II II I II II IIIII 111111 I**I~** 0.0 0.5 1.0 1.5 2,0 2.5 3.0 3.5 4.0 4.5 5.0 5. 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 min ' 

### Table3.6 ThermographicData

- Determination2 Temperature Thermal Event Interpretation 

## oc K

Endotherm Boilingtemperature 201.90 475 

### Atmospheric pressure: 98.3 kPa Overall boiling temperature: 202 ± 

### 0.5 °C (475 ± 

### 0.5K)


### 3.2.6 Conclusion The boilingtemperature ofthe test item has been determined to be 202 

### ± 0.5 

# oc (475 

### ± 0.5K) at 98.3 kPa.Harlan StudyNumber: 41304286 SponsorReferences: 130187 & 130189


## APPENDICES Appendix 1 Certificate ofAnalysis Report

## .~J 

# mr 

# n:**~r ~**  

# -ft I 

# & .-m-**~** 

# lSI**~** a=J**~** 

### ZHEJIANG XINNONG CHEMICAL CO.,LTD. SANLIXI. XIANJU COUNTY, ZHEJIANG PROVINCE, CHINA. 

### CERTIFICATE OF ANALYSIS CXIXNTl I-03 N0.:2013081l

PRODUCT 0,0-DIETHYL PHOSPHOROCHLORIDOTH(8EPCT) BATCH NO. C032013081SOI TANK NO. EXFU 8104300 QUANTITY 20000.00kg SAMPLE 2000 TESTING DATE 20!3.8.15 INSPECTING STANDARD Q/XNJS0l(B)·20ll DATE OF EXPIRY 20!3..8.15-2014.814 NO SPClFICATION STANDARD RESULT 1 APPEARANCE COLOURLESS CLEAR LIQUID COLOURLESS CLEAR UQUID 2 0,0-Diethylphospborochloridothioate ;:.?>98.5% 99.!3% 3 MOISTURE (ppm)**~300** 284 4 CHROMA**~100** 70 5 0,0-diethylphosphorochloridate**~0.2%** 0.04% 6 0-ethyl phospborodichloridothioate**~0.30%** 0.16% 7 O,O,O..triethyl phosphorotbioate**~0.2%** 0.10% 8 O,O,S-t:riiethyl phospborothioate**~0.15%** 0.10% 9 0,0-dimetbyl phosphorochloridothioate ,.;o.t% N!A !0 0,0,0,0-tetraetbyl dithiodiphospbate ,;;;o.os% 0.03% 11 Ethyl mercaptan, diethylsulfide,**~0.10%** 0.08% dietbyl Disulfide, total 12 OTHER ..:;o.IO% 0.10% QUALIFIED CONCLUSION AUGt:ST IS, 2013

' .......**-~**....**~**..... 

### .. REMARK \: -:., .. ',,·· . .... "

.... ·.:..-;, ,,. .. ',\ ,;....

.**~** "'. ""\ ANALYZED BY.·Ql11;1.4N CHECKEDBY:HUJIN:.....
.. ......... -iXAMINEANDVERIFIED 8): Ql'l!Tl!VGCIII**~ ·':"~"**  """\ J.· :'•**/<,~** TN CASE OF SPILL, LEAK, EXPOSURE OR

### ACClDENT,cAuhMal:C;'ENC~**J;'~NE:INFOTRACT** AT 800-992-5994. DOW AGROSCIENCES LLC IND1ANA1o(ts•,**~N 462~8·**  i DOW AGROSCIENCES RAW MATERIAL SPECIFICATION 21126?.101. PURCHASE ORDER NO.: 16/45545329

Ill i Ill Page 18

## ( Harlan StudyNumber: 41304286 SponsorReferences: 130187 & 130189 Report



### Appendix 2 MonitoringAuthority Statement ofGLP Compliance


### j

# n'H) Department

### ~ otHealth
THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM GOOD LABORATORY PRACTICE

STATEMENT OF COMPLIANCE IN ACCORDANCE WITH DIRECTIVE 2004/9/EC
TEST FACILITY Harlan Laboratories Ltd Shardlow Business Park London Road Shardlow Derby DE722GD

DATE OF INSPECTION

10 July2012 TESTTYPE(S) Analytical/Clinical ChemistrY EnvironmentalToxicity Environmental Fate Mutagenicity Phys/Chem. Tests Toxicology

An inspection for compliance with the Principles of Good Laboratory Practice was carried out at the above test facility as part of the UK Good Laboratory Practice Compliance Monitoring Programme. This statement confirms that, on the date of issue, the UK Good Laboratory Practice Monitoring Authority were satisfied that the above test facility was operating in compliance with the OECD Principles of Good Laboratory Practice. This statement constitutes a Good Laboratory Practice Instrument (as defined in the UK Good Laboratory Practice Regulations 1999).

# ~~~ '30

# /o (tz_ Dr. Andrew J. Gray

### ~ Head, UK GLP Monitoring Authority

### ~ Page 19Ill; IllAppendix C Vapor Pressure Study Report



### 15 

### DEPCTIll: Ill \I J( 

## REPORT
PHOSPHOROCHLORIDOTHIOTICACID, 0,0-DIETHYLESTER(DEPCT): Determination ofVaporPressure

StudyDirector: TestFacility:

Sponsor:

Harlan StudyNumber: SponsorReference: StudyCompletion Date: DATarran 

### HarlanLaboratoriesLtd. ShardlowBusinessPark Shardlow Derbyshire DE722GD 

## UK TheDow Chemical Company Building 1803 Midland Michigan48674 UNITED STATES OF AMERICA


### 41304287 130188 18 February 2014

Pag:e 1of20Harlan StudyNumber: 41304287 SponsorReference: 130188


## CONTENTS Report Page2



### CONTENTS
....................................................................................................................................2 STUDY DIRECTOR STATEMENT OF GLP COMPLIANCE
....................................................3 QUALITY ASSURANCE STATEMENT
......................................................................................4 SUMMARY
....................................................................................................................................5 GENERAL INFORMATION
.........................................................................................................6 Schedule
.................................................................................................................................6 Deviations from Study Plan
...................................................................................................6 Archiving
...............................................................................................................................6 1 INTRODUCTION AND PURPOSE
.....................................................................................7 1.1 Guidelines I Regulations
..........................................................................................7 2 TEST ITEM
........................................................................................................................... 8 3 TESTING
...............................................................................................................................9 3.1 VaporPressure
.........................................................................................................9 3.1.1 Test System
...............................................................................................9 3.1.2 Principle oftheTest
...................................................................................9 3.1.3 Performance ofthe Test.
............................................................................9 3.1.4 Data Handling
.......................................................................................... II 3.1.5 Results
..................................................................................................... 12 3.1.6 Discussion
................................................................................................ 18 3.1.7 Conclusion
............................................................................................... 18 

### APPENDICES............................................................................................................................... l9 \1 

### Appendix 1 Appendix2 Certificate ofAnalysis
........................................................................................... 19 Monitoring Authority Statement ofGLP Compliance
..........................................20

Ill Ill \ j( ' Harlan :Study Number: 41JU42H7 SponsorReference: 130188 Report



## STUDYDIRECTOR STATEMENT OF GLP COMPLIANCE Harlan Laboratories Ltd., ShardlowBusiness Park, Shardlow, Derbyshire, DE72 2GD, UK
Harlan StudyNumber: Study Title: 41304287 PHOSPHOROCHLORIDOTHIOTIC ACID, 0,0-DIETHYLESTER (DEPCT): DeterminationofVapor Pressure Page 3



### This study was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)). These Regulations are in accordance with GLP standards published as OECD Principles on Good Laboratory Practice (revised 1997, ENV/MC/CHEM(98)17); and are in accordance with, and implement, the requirements ofDirectives 2004/9/EC and 2004/10/EC. These principles are compatible with Good Laboratory Practice regulations specified by regulatory authorities throughout the European Community, the United States (EPA and FDA), and Japan (MHLW, MAFF and METI). This report fully and accurately reflects the procedures used and data generated. There were no circumstances considered to have affectedthe integrity ofthe study orthe validity ofthe data. Study Director: D A Tarran

Date: 

## 1 8 FEB 2014Harlan StudyNumber: 41304287 SponsorReference: 130188 Report Page4


## QUALITYASSURANCE STATEMENT Harlan StudyNumber: StudyTitle: 41304287 PHOSPHOROCHLORIDOTHIOTIC ACID, 0,0-DIETHYL ESTER (DEPCT): DeterminationofVapor Pressure The general facilities and activities are inspected at least once a year and the results are reported to the relevantresponsible person andmanagement. Study-relatedprocedures conducted at the test facility were audited andinspected. The details of these audits and inspections are givenbelow.
Reported tothe relevant Dates and Types ofQA Inspections StudyDirector and Test FacilityManagement Date ofInspection Type ofInspection Phase Inspected ReportDate 05 December2013 Study Plan Verification N/A 05 December2013 13 January 2014 Process

- based VaporPressure 13 January 2014 11 February2014 ReportAudit N/A 11 February2014


### Verification ofstudyplan amendments for GLP compliance were performedduring the course of the study. This statement confirmsthatthis final report reflects the rawdata andtheprocedures followed. QualityAssurance:



## 1

## 7FEB 2014

Ill i 

### Ill( Harlan StudyNumber: 41304287 SponsorReference: 130188



## SUMMARY Report Page 5



### The vapor pressure of PHOSPHOROCHLORIDOTHIOTIC ACID, 0,0-DIETHYL ESTER (DEPCT) has been determined to be 93 Pa at 25 °C, using the vapor pressure balance method, designed to be compatible with Method A4 VapourPressure ofCommission Regulation (EC) No 440/2008 of30 May 2008 and Method 104 ofthe OECD Guidelines for Testing ofChemicals, 23 March 2006.Harlan StudyNumber: 41304287 Sponsor Reference: 130188 Report



## GENERALINFORMATION Schedule Experimental Starting Date: Experimental Completion Date:


### Deviations from Study Plan 23 January 2014 27 January 2014

There were no deviations (unplanned changes) from the study plan. 

### Archiving Page 6



### Initially, all original data and the final report will be retained in the Harlan Laboratories Ltd, Shardlow, UK archives. As soon as practicable the original data and final report will be transferred to The Dow Chemical Company, Building 1803, Midland, Michigan 48674, USA. Harlan Laboratories Ltd. holds no responsibility for the management ofarchiving at The Dow Chemical Company. A copy ofall original data and the final report will be retained inthe Harlan Laboratories Ltd, Shardlow, UK archives for a minimum ofone monitoring authority inspection cycle, in accordance with current GLP requirements.

Ill• Ill( Harlan :Study Number: 4 13042!!7 SponsorReference: 130188 Report



## 1 

## INTRODUCTIONAND PURPOSE The purpose ofthis study isto evaluate the vaporpressure ofthe test item. 

## 1.1 Guidelines I

## Regulations Page 7



### The test methods described are designed to be compatible with the procedures indicated by the following internationally accepted guidelines and recommendations:

• Commission Regulation (EC) No 440/2008 (as amended) of30May2008. • OECD Guidelines for Testing ofChemicals.Harlan StudyNumber: 41304287 Sponsor Reference: 130188



## 2 

## TEST ITEM Report Page 8



### Information as provided by the Sponsor. The Certificate ofAnalysis provided by the Sponsor is presented as Appendix 1. Identification: Description: Batch: Purity: Expiry Date: Storage Conditions:

Ill' 

### PHOSPHOROCHLORIDOTHIOTIC ACID, 0,0-DIETHYL ESTER (DEPCT) pale yellow liquid C032013081501 99.13% 14 August2014 roomtemperature inthe darkunder nitrogen' I Harlan Study Number: 41304287 SponsorReference: 130188



## 3 TESTING 3.1 VaporPressure 3.1.1 Test System Report Page9



### The determination was carried out using a procedure designed to be compatible with Method A4 VapourPressure ofCommissionRegulation (EC) No 440/2008 of30 May 2008 and Method 104 ofthe OECD Guidelines for Testing ofChemicals, 23 March 2006. Instrument: Vaporpressure balance.


### 3.1.2 Principle oftheTest The vapor pressure is determined using a vaporpressure balance with measurements being made at several temperatures and linear regression analysis used to calculate the vapor pressure at 25 °C.


### 3.1.3 Performance oftheTest The vapor pressure was determined using a vapor pressure balance. The temperature of the sample was controlled electronically. The mass and temperature readings were recorded automatically into a computer file. A diagram ofthe cross-section ofthe vapor pressure balance is represented in Figure 3.1. After evacuating the system, opening the shutter above the sample oven causes the escaping vaporjet to be directed at the scale pan. The difference in mass readings with the orifice covered and uncovered is proportional to the vaporpressure atthe given oventemperature. A preliminary run was undertaken over the temperature range -20 to 30 °C to provide information for a suitable temperature range for the determination. A final sequence ofthree runs was started after a sample oftest item had been under vacuum for approximately 17 hours. Temperature and pressure readings were taken between -25 and -15 

# oc with a one hour dwell at -25

# oc betweenruns.Hanan**~tuay** Numoer: 4UU4:LIS'/ SponsorReference: 130188 Report



### Figure3.1 SchematicDiagram oftheApparatus Used
3

# ~ 2 4 5

1 Microbalance 2 Oven 3 Glass viewing panel 4 Balance pan 5 Shutterwith orifice 6 Test sample



### Ill Ill Page 10Hartan ::stuay Number:**4UU42~/** Sponsor Reference: 130188 Keport



### 3.1.4 Data Handling The vaporpressure isrelatedto the observed mass difference byEquation 3.1.

where: Vp 

### om g A = = 

### V 

### om.g p=-- 

### A

vaporpressure (Pa) mass difference (kg) accelerationdue to gravity (9.813 m s"2) 

### area ofthe orifice (7.06858x10-6

### m2) 

### Vaporpressure is related to temperature byEquation

### 3.2.

where: Vp 

### T = = 

### . slope
. Log 10 

### [Vp]

## =-- +mtercept T

vaporpressure (Pa) temperature (K) Page 11



### Equation 

### 3.1

Equation 3.2

Therefore a plotoflog10 Vp versus reciprocal temperature gives a straightline graph. The vapor pressure of the sample was measured over a range of temperatures to enable extrapolationto 298.15 K.Harlan StudyNumber: 41304287 SponsorReference: 130188



### 3.1.5 Results Report Page 12



### Recorded temperatures, mass differences and the resulting calculated values ofvapor pressure are shownin the following tables and figures: 

### Runl Table 3.1 Vapor Pressure Data
Temperature Temperature Reciprocal Mass Mass Vapor Temperature Difference Difference Logw Vp COC) (K) (KI) (!!g) (kg) Pressure (Pa) -25 248.15 0.004029821 239.60 2.396E-07 0.332626185 -0.478043565 -24 249.15 0.004013646 281.45 2.815E-07 0.390724707 -0.408129126 -23 250.15 0.00399760I 313.60 3.136E-07 0.435357144 -0.361154325 -22 251.15 0.003981684 367.57 3.676E-07 0.510281331 -0.292190320 -21 252.15 0.003965893 404.13 4.041E-07 0.561035978 -0.251009288 -20 253.15 0.003950227 477.40 4.774E-07 0.662753509 -0.178647964 -19 254.15 0.003934684 548.43 5.484E-07 0.761361347 -0.118409175 -18 255.15 0.003919263 606.28 6.063E-07 0.841671968 -0.074857137 -17 256.15 0.003903963 700.20 7.002E-07 0.972056990**-0.01~308272** -16 257.15 0.003888781 779.48 7.795E-07 1.082117942 0.034274598 -15 258.15 0.003873717 894.14 8.941E-07 1.241295397 0.093875145 

### A plot oflog10 

### Vp versus reciprocal temperature for Run 

### 1 gives the following statistical data using an unweighted least squares treatment. Slope Standard error in slope Intercept Standarderrorinintercept -3.64 X 

### 103 

### 40.1 14.2 0.158 Extrapolationusing the slope and intercept above yields a vapor pressure (Pa) at 298.15 K with a commonlogarithm of 1.98.

Ill i Ill

### ( rtanan**~may** Numoer:**4UU4L~1** Sponsor Reference: 130188 Report Page l3



### Figure3.2 Run 1 - Graph ofLog10 VaporPressurevs Reciprocal Temperature

0.00384 0.00388 0.00392 0.00396 1/Temp(K) 0.00400 0.00404**0.2~--------~--------_.--------~~--------._--------~**

0.1

0.0

-0.1


# - - :. ! 

# = en en 

# e a. -0.2


# ... 0 Q, 

# "' > "Ci



# .. Cll 0 ...1 -0.3

-0.4

-0.5

-0.6 .._ __________________________ ....Harlan StudyNumber: 41304287 SponsorReference: 130188



### Run2

Temperature Temperature CCC) (K) -25 248.15 -24 249.15 -23 250.15 -22 251.15 -21 252.15 -20 253.15 -19 254.15 -18 255.15 -17 256.15 -16 257.15 -15 258.15 

### Table 3.2 Reciprocal Temperature (K"l) 0.004029821 0.004013646 0.003997601 0.003981684 0.003965893 0.003950227 0.003934684 0.003919263 0.003903963 0.003888781 0.003873717 Report Page 14



### Vapor Pressure Data

Mass Mass Vapor Difference Difference Logw Vp (I!g) (kg) Pressure (Pa) 245.30 2.453E-07 0.340539246 -0.467832831 281.84 2.818E-07 0.391266127 -0.407527749 318.65 3.187E-07 0.442367838 -0.354216456 366.50 3.665E-07 0.508795897 -0.293456400 409.65 4.097E-07 0.568699152 -0.245117420 471.91 4.719E-07 0.655131983 -0.183671198 545.01 5.450E-07 0.756613511 -0.121125908 600.70 6.007E-07 0.833925498 -0.078872747 703.94 7.039E-07 0.977249068 -0.009994735 792.85 7.929E-07 1.100678927 0.041660652 878.75 8.788E-07 1.219930134 0.086334959 

### A plot oflog10 Vp versus reciprocal temperature for Run 2 gives the following statistical data using anunweighted least squares treatment. Slope Standarderrorin slope Intercept Standard error in intercept -3.58x 103 

### 30.9 14.0 0.122 Extrapolationusing the slope and intercept above yields a vapor pressure (Pa) at298.15 K with a common logarithm of 1.95.

ITl I Ill( \ .ttartan ::stuay Number: 4UU42lr/ Sponsor Reference: 130188 Report Page 15



### Figure3.3 Run 2- Graph ofLogtoVaporPressurevs Reciprocal Temperature

0.00384 0.00388 0.00392 0.00396 1/Temp(K) 0.00400 0.00404**0.2+---------~--------~---------+--------~--------~**

0.1

0.0



# - :: e -o.1 :r Ill Ill 

### ! a. r.. 0 a.

### ~**~** -0.2 0) 

## ..9

-0.3

-0.4

-0.5 ...._ _______________________________________ __..ttanan ;:sruay Numoer: 4 UU4:LlS 1 SponsorReference: 130188



### Run3

Temperature Temperature 

### ec) (K) -25 248.15 -24 249.15 -23 250.15 -22 251.15 -21 252.15 -20 253.15 -19 254.15 -18 255.15 -17 256.15 -16 257.15 

### Table 

### 3.3 Reciprocal Temperature (Kl) 0.004029821 0.004013646 0.003997601 0.003981684 0.003965893 0.003950227 0.003934684 0.003919263 0.003903963 0.003888781 Keport Page 16



### VaporPressure Data

Mass Mass Vapor Difference Difference Log1o Vp (f.lg) (kg) Pressure (Pa) 238.41 2.384E-07 0.330974160 -0.480205911 282.13 2.821E-07 0.391668721 -0.407081110 320.82 3.208E-07 0.445380354 -0.351268944 360.73 3.607E-07 0.500785659 -0.300348117 411.97 4.120E-07 0.571919906 -0.242664787 472.94 4.729E-07 0.656561887 -0.182724332 546.91 5.469E-07 0.759251198 -0.119614514 602.57 6.026E-07 0.836521538 -0.077522873 689.41 6.894E-07 0.957077706 -0.019052800 784.60 7.846E-07 1.089225813 0.037117925 

### A plot of log10 

### Vp versus reciprocal temperature for Run 3 gives the following statistical data using an unweighted least squares treatment. Slope Standard errorin slope Intercept Standard errorin intercept -3.61 X 

### 103 

### 38.6 14.1 0.153 Extrapolation using the slope and intercept above yields a vapor pressure (Pa) at 298.15 K with a commonlogarithm of 1.97.

I] I Ill

# ( Harlan**~tudy** Number: 41304287 Sponsor Reference: 130188 Report Page 17



### Figure3.4 Run 3- Graph ofLog10

### VaporPressurevs Reciprocal Temperature

0.00388 0.00392 0.00396 1/Temp(K) 0.00400 0.00404

## 0.1t------------+------------~----------~----------~

0.0

-0.1



# - :: i -0.2 :I 

# :: l!! G.


# ... 0 

### a.

## ~0 ·0.3 

### ci 0 ...J

·0.4

-0.5

·0.6

### ....._-------------~--------....1Harlan :Study Number:**4BU4l~7** SponsorReference: 130188


### Summary ofResults Report Page 18



### The values of vapor pressure at 25 

# oc extrapolated from each graph are summarized in the following table: 

### Table3.4 Summary ofVaporPressureData
Run Logto [Vp(25 °C)]

1 1.98 2 1.95 3 1.97 Mean 1.97 VaporPressure 93 

### The test item did not change in appearance under the conditions used inthe determination.


### 3.1.6 Discussion A total of 3 runs were completed for the main sequence. Equilibrium with regard to vapor pressure was assessed to have been reached prior to the main sequence therefore all 3 runs were used to calculate the definitive vapor pressure value forthetest item. During run 3 the shutter on the instrument became stuck on the final measurement. Therefore, although the run was continued to -15 °C, no data was obtained for the final point. As the correlation using the 10 points obtained was acceptable and the result from the run was in line with the results from runs 1 and 2 this is considered not to have adversely affected the result of the study.


### 3.1.7 Conclusion The vapor pressure ofthe test item has been determined to be 93 Pa at 25 

# oc, using the vapor pressure balance method.

I il! Ill( \ nanan**~moy** J'IUIDOer: 4UU4.LlS/ SponsorReference: 130188



## APPENDICES Appendix 1 Certificate ofAnalysis Keport

..

## ~J 

# wr

# n:**~r** 

# a -tt I 

# Ja fft =ft"

## ~**~ ~**  

# WI ZHEJIANG 

### XINNONG CHEMICAL CO., LTD. SANLIXI, XIANJU COUNTY, ZHEJIANG PROVINCE, CHINA. 

### CERTIFICATE OF ANALYSIS
CXIXNTll-03 NO ·20130811 

### .. PRODUCT 0,0-DIETHYL ?HOSPHOROCHLORIDOTH(DEPCT) BATCH NO. C03201308l501 TANKNO. EXFU 8104300 QUANTITY 20000.00kg SAMPLE 2000 TESTING DATE 2013.8.15 INSPECTING STANDARD Q/XNJSOI(B)·2011 DATEOF EXPIRY 2013.8.15-2014.8.14 NO SPCIFICATlON STANDARD RESULT 1 APPEARANCE COLOURLESS CLEAR LIQUID COLOURLESS CLEAR LIQUID 2 O,O-oietbylphosphorochloridothioate :?!98.5% 99.13% 3 MOISTURE (ppm)**~300** 284 4 CHROMA**~100** 70 s 0,0-dietbylphosphorochloridate**~0.2%** 0.04% 6 0-ethyl phosphorodichloridothioate**~0.300/o** 0.16% 7 0,0,0-triethylphosphorothioate**~0.2%** 0.100/o 8 O,O,S-triiethyl phosphorothioate ,;;:o.IS% 0.10% 9 0,0-dimethyl phosphorochloridothioat<**~0.1%** N/A 10 0,0,0,0-tetraethyl dithiodiphosphate**~0.05%** 0.03% Ethyl mercaptan, diethylsulfide,**~0.10%** 0.08% II diethyl Disulfide, total 12 OTHER**~0.10%** 0.10% QUALIFIED CONCLUSION AUGUST IS. 2013

•• - .., J**,~.<~---** .... REMARK "' ,. .. "' '**~** ' 

### .. •.· ....... ANALYZED**BY.·Q!Nl'.~N** CHECKED BY:HUl'll'L
...c•...........JiXAMJNE AND VERIFIED BY: QIN TJNGC.4J ' ,,., ,.., ):..•:**~ ·-~--"**  I I

.--"':"'**ft-~** t ,• •**~** • ,··: • IN CASE OF SPILL, LEAK, EXPOSURE OR**ACC!DENT,cALi;::~EMERGENC}'** I;'_JWNE:INFOTRACT AT S00-992-5994. DOW .AGROSCIENCES LLC IND!ANAfo!fs•, 1 

### IN**~62'6s',** 

### . DOW AGROSCIENCES P..P.W MATERIAL Sl?ECIFICAT::ON 21126R101. PJRCHASE ORDER NO.: 16/45545329 Page 19nanan >:>tuuy 1''1umoer: 4 UU4.<::C:S 1 SponsorReference: 130188 Keport



### Appendix2 MonitoringAuthority StatementofGLPCompliance


# J.

# nH) Department

### ~ ofHealth
THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM GOOD LABORATORY PRACTICE

STATEMENT OF COMPLIANCE IN ACCORDANCE WITH DIRECTIVE 2004/9/EC TEST FACILITY Harlan Laboratories Ltd Shardtow Business Park London Road Shard!ow Derby DE722GD

DATE OF INSPECTION

10 July2012 TEST TVPE(S) Analytical/Clinical Chemistry Environmental Toxicity Environmental Fate Mutagenicity Phys/Chem. Tests Toxicology

An inspection for compliance with the Principles of Good Laboratory Practice was carried out at the above test facility as part of the UK Good Laboratory Practice Compliance Monitoring Programme. This statement confirms that, on the date of issue, the UK Good Laboratory Practice Monitoring Authority were satisfied that the above test facility was operating in compliance with the OECD Principles of Good Laboratory Practice. This statementconstitutes a Good Laboratory Practice Instrument (as defined In the UK Good Laboratory Practice Regulations 1999).

# ~~ -:.30

## /u/tz... Dr. AndrewJ. Gray

### ~ Head, UK GLP MonitoringAuthority

### ~

1111, Ill Page 20

\I \



### DEPCT Appendix 

### 0 Stability in Water Hydrolysis Study Protocol



### 16

### \ \\\\ \\\,/- 

### \

i

# " PRELIMINARY PROTOCOL TOXICOLOGY & ENVIRONMENTAL RESEARCH AND CONSULTING THE DOW CHEMICAL COMPANY 

## PROTOCOL 1803 BUILDING, MIDLAND, MICHIGAN 48674
TITLE: PHOSPHOROCHLORIDOTHIOIC ACID, 0,0-DIETHYL ESTER (DEPCT): DETERMINATION OF HYDROLYSIS AS A FUNCTION OFpH ACCORDING TO OECD GUIDELINE 111

KEYNUMBERS FILE#: STUDYID: PTR#: CAS#: SPONSOR: DowAgroSciences LLC Indianapolis, Indiana PLANNED DATES EXPERIMENTAL START: EXPERIMENTAL TERMINATION: ESTIMATED FINAL REPORT: GLP STUDY: Yes

TESTING FACILITY: The Dow Chemical Company Toxicology & Environmental Research and Consulting 1803 Building Midland, Michigan48674 REQUIRED SIGNATURES: STUDY DIRECTOR:

LEAD SCIENTIST:
MANAGEMENT APPROVAL:
SPONSORorDESIGNEE:

MANAGEMENT ASSIGNED STUDY PERSONNEL: Technical Reviewer: ADDITIONAL STUDY CONTACTS: Business Consultants: Document Management: QualityAssurance Auditor: StudyDirector in Training: /DATE
/DATE
/DATE
/DATE1711 Ill

### A. TITLE THE DOW CHEMICAL COMPANY STUDYID: PAGE2

PHOSPHOROCHLORIDOTHIOIC ACID, 0,0-DIETHYLESTER (DEPCT): DETERMINATION OF HYDROLYSIS AS A FUNCTION OF pH ACCORDING TO OECD GUIDELINE 111 

### B. INTRODUCTION Purpose The objective ofthis studyis to investigate the hydrolysis ofthe testmaterial following the guidelines ofthe Organisation for Economic Co-Operation and Development (OECD) method 111 "Hydrolysis as a Function ofpH" [OECD, 2004]. 

### TestGuidelines OECD Organisation for Economic Co-operation and Development, OECD Guidelines for Testing ofChemicals. Guideline Number 111 Hydrolysis. 13 April2004.


### MATERIALSAND METHODS TestMaterialName phosphorochloridothiotic acid, 0,0-diethylester 

### ChemicalName 0,0-Diethylesterphosphorochloridothioic acid 

### Synonyms Diethylchlorothiophosphate, DEPCT 

### Supplier, City, State (Lot, Reference Number) The DowChemical Company, Midland, Michigan (Lot#C032013081501) 

### Purity/Characterization (Method ofAnalysis and Reference) The non-GLP certificate ofanalysis lists the purity as 99.13% 0,0-Diethyl phosphorochloridothioate (Qinyan, 2013). Further GLP characterization oftest materialpuritywillbe performedpriorto initiation ofthis study. The final protocol/ 

### Ill I illTHE DOW CHEMICAL COMPANY STUDYID: PAGE3 and studyreport will include relevant information on identity andpurity, as determined from this GLP characterization. Characteristics MolecularFormula C4H10ClOzPS MolecularWeight 188.6 Chemical Structure

CAS Number 2524-04-1 Test Solutions Stock solutions usedfor dosing ofthe testmaterial to hydrolysis reaction mixtures willbepreparedin an appropriate anhydrous and water-miscible aprotic organic solvent 

### (e.g., acetonitrile). Stabilityofthe substance inthe selected solvent willbe demonstratedpriorto, oras part of, this study. Additional stock solutions ofthe test material maybe prepared in a hydrophobic, anhydrous, water-immiscible solvent (e.g, toluene), for preparation ofanalytical calibration standards. Stabilityinthis solvent will also bedeterminedpriorto, or concurrentlywith, this study. Previous Information Hydrolysis canbe an important abiotic transformationprocess for chemicals that enterthe aqueous environment. A tieredtesting approach is recommendedbythe OECD to assess the hydrolytic fate ofa substance in water. A preliminary 1sttier studyis usually conducted at 50°C to determine the susceptibility ofthe test

### III\ Ill( THE DOW CHEMICAL COMPANY STUDYID: PAGE4 compoundto hydrolysis atpH4, 7, and 9. A hydrolysis study at pH 1.2 and 37

## oc may also be includedifa physiologicallyrelevant pHis requiredto support toxicology studies. Ifthe compound is shownto be hydrolyticallyunstable inthe preliminary study, a more extensive investigation (2nd tier) maybe carried out at different temperatures to betterdefine the hydrolysis kinetics for the compound and identifyhydrolysis products formed. A previous studybyCheminovaA/S (2010) provides a preliminaryinvestigation of test material stabilityin demineralized water (non-buffered) at ambient laboratory temperature (**~** 25 °C assumed). This studyindicatedthatthe test material has a hydrolysis half-life ofapproximately 14 minunderthese conditions, andis thus consideredto be hydrolyticallyunstable at environmentally-relevanttemperature and pH. A single hydrolysis product, which itselfappeared (andis expected) to be hydrolytically stableunderthese test conditions, was identified as phosphorothioic acid, 0,0-diethyl ester (CAS. No. 2465-65-8). The OECD Guideline 111 requires thathydrolysis ofa substance is evaluated at a concentration which does not exceed O.OlM, orone-halfthe water solubility limit concentration. Due to the hydrolytic instability ofthe testmaterial, a water solubility limit cannotbe accurately determined. Estimatedwater solubilityvalues for the test material, which are basedonthe structure-fragment contribution calculation (WATERNTvl.Ol) andon correlation oflogPowwith water solubility (WSKOW v1.42) methods ofthe U.S. EPAEPI Suite software (US EPA, 2010) are 4,296 and 1,249 mg/L 

### (i.e., 0.023 and 0.007 M), respectively. The testmaterialhas a measured vaporpressure ofapproximately0.7 mm Hg at 25 °C, andcalculated Henry's Law Constant, based onthe ratio ofthis vaporpressure and estimated water solubility, of approximately 1 x 104 atm*mole/m3 at 25 

## oc (Appendix A). This indicates the substance to be highlyvolatile from water, andthat appropriate experimental design and procedures willbenecessaryto avoid confounding ofresults from volatilization losses. Similarly, the water solubility values for the expectedhydrolysis product (CAS No. 2465-65-8) are 24,000 (WSKOWv1.42) and 198,000 (WATERNTvl.Ol)mg/L. Estimatedvaporpressure for this substance is approximately 0.04 mm Hg at 25 

## oc (US EPA, 2010; Appendix B. Therefore, the expectedhydrolysis product ofthe test material is expectedto be much more soluble in and less volatile from water.\!



### \ 

### II\\ \\\THE DOW CHEMICAL COMPANY STUDYID: PAGES Consideringthis information, further testing ofhydrolysis according to the 2ndTier ofOECD Guideline 111 is required to fulfill (inpart) informationrequirements ofthe U.S. EPA HPV Program. Although the OECD Guideline recommends testing at three temperatures falling between 10 and 70 °C, itis expectedthat complete hydrolysis attemperatures above 40 °C would occur within timeframes which would notpermitmeaningful determination ofkinetics. To betterunderstand the temperature- andpH-dependence, and associated daughter products ofDEPCT hydrolysis, testing willbe conductedunderthe following conditions:
• Approximately 10 °C at initial pHof4, 7, 9 • Approximately25 

## oc at initial pHof4, 7, 9 • Approximately40

## oc at initial pHof 1.2, 7, 9 

### PreliminaryTest A preliminarytest, as recommendedin OECD Guideline 111, will notbe included as partofthis study. Information from a previous study (Cheminova 2010), as well as information from structure-activityrelationships, have indicatedthe testmaterial to behighlyunstable in water. 

### C. EXPERIMENTALDESIGN Hydrolysis studies willbe conducted following the procedures described in the OECD Guideline 111 (OECD, 2004). Aqueousbuffer solutions**(~0.05M)** willbeprepared at pH 1.2, pH4, pH 7, andpH9, usingbuffercompositions selectedfrom those recommendedinthe OECD Guideline, andhaving components which are notexpected to react directly with the test material orinterfere with analytical determinations. Final pH adjustments willbe made with O.lN HCl or0.1NNaOH. To minimize possible oxidationreactions, test solutions will be spargedwith inert gas to remove dissolved oxygen. To reduce the potential for biodegradation ofthe test compound, the buffer solutions willbe filter-sterilized withNalgene disposable filterware (Nalge Company, Rochester, New York) containing0.22-J..lm nylon membranes. The test solutions willbe incubatedinthe darkto minimizepossible photochemical reactions. Glassware and equipment 

### (i.e., septa, pipette tips, etc.) will be sterilizedbyautoclaving as appropriate.III I Ill ITHE DOW CHEMICAL COMPANY STUDYID: PAGE6 The concentration ofDEPCT and its expectedhydrolysis product intest solutions will not exceed 0.01M or one-halfthe water solubility. Portions ofthe test solutions will be addedto serum bottles (identifiedbyappropriate code), and sealedwith Tefloncoatedrubber septa and aluminum crimp seals. The pH 1.2, 4, 7 and 9 reaction mixtures willbeplaced inthermostated waterbath orincubator set at 10, 25, or40

## oc (temperature control±0.5°C). ThepH ofselected, representative samples willbe measuredduring the experiment. Blank solutions (buffered solutionwithouttest material added) will be prepared and analyzed as appropriate to quantify any backgroundinterferences inthebuffered solutions. Forthe hydrolysis experiment, a sufficientnumber ofreplicate test solutions willbepreparedin separate serumbottles to meetthe samplingrequirements outlined in the OECD guideline. A minimum of six datapoints between 10 and 90percenthydrolysis ofthe test compound (replicate samples) will be obtainedto define the degradationkinetics for the parent compound. ThepH ofrepresentative samples willbe determined at selected samplingtime points. Hydrolysis experiments willbeterminated after 90% degradationhas occurred, orafter 30 days.



### D. STUDY SPECIFIC PARAMETERS Hydrolysis Reaction Mixtures The hydrolysis reaction mixtures willbeprepared andincubated in 10 mL glass serum bottles, which are rinsed withultrapure water and sterilized in an autoclave priorto use. The serum bottles willbe sealedusing PTFE-linedbutyl rubber septum closures, secured with aluminum crimp caps. The serumbottles willbe filled with sterilebuffer solution withno remainingheadspace, sealed, andpre-equilibrated at the target incubationtemperature. Theindividual reaction mixtures willbe spikedto the target initial test material concentrationusing a gas-tight glass syringe, anda test material stock solutionprepared in anappropriate water-miscible aprotic solvent. A sufficientnumber ofreplicate reaction mixtures willbepreparedto allow for triplicate analyses at each targetedtime interval, andfor determination ofpH and extractionblankmatrix ofun-spikedbuffer solutions.1111 IllTHE DOW CHEMICAL COMPANY STUDYID:

At selectedincubationtime intervals, triplicate reaction mixtures for each pH/temperature combination, will be sacrificed for extraction and analysis of remainingtestmaterial and associatedhydrolysis products.
Analytical Procedures PAGE7

The test material is expectedto be stable when extracted from water into an anhydrous aprotic solvent (e.g., hexane, toluene). Hydrolysis occurring inaqueous samples collected at selected sampling intervals willbe quenchedbyimmediate extraction into such an immiscible organic solvent. Aliquots ofthe organic solvent phase willbe transferred to sealed autosampler vials, and analyzedbygas chromatography. The concentration ofDEPCTin test solutions willbe determinedbygas chromatographywith appropriate detection 

### (e.g., electron capture, mass spectrometry). Instrument and analysis conditions willbe detailed inthe final report. An attempt will be made to quantifyhydrolysis productsusing chromatographic retention timematches with analytical standards ofthe expectedproducts (where available commercially), and/orbymatches ofmass spectra againstthose obtained from commercial spectral libraries. The pH oftestsolutionsprepared atnominalpH of 1.2, 4, 7, and 9 willbe measured periodicallybefore, during, and afterincubation ofthe hydrolysis reaction mixtures. Priorto pHmeasurements, the pHelectrode/meterwillbe calibrated at two ormore pHvaluesusing standardbuffer solutions. The recordedpHwillbe correctedfor, and reported at, the target temperature ofthe hydrolysis test solutions. Periodic measurements ofthe waterbathtemperature willbe made andrecorded using a NIST traceable thermocouple to confirmproperoperation. Data and Reporting Hydrolysis half-lives for DEPCTwillbereported at eachtemperature andpHtested usingthe calculations described inthe OECD Guideline 111. Ifthe hydrolysis reaction follows pseudo-first orderkinetics, a plot ofthe natural logarithm ofthe concentrationversus time will result in a straight line. The slope ofthe line will equalIT! i IllTHE DOW CHEMICAL COMPANY STUDYID: PAGES -k, the pseudo-first orderrate constant. The halflife for the hydrolysis reaction will be equal to t112 - In 2/k:. Concentrations ofhydrolysis products formed at greater than 10% yield will be reported at each samplingpoint. Computermodeling maybe usedto estimate halflives for measuredhydrolysis products where appropriate. Details for modeling will be includedinthe final report. 

### E. STATISTICS Descriptive statistics (mean, standard deviation) will be usedwhere applicable in this study. 

### F. FINAL REPORT A final report ofthe definitive study will be submittedto the study sponsor andwill include butnotbe limitedto the following: 1. Narne and address ofthe facility performingthe study and the dates onwhich the studyis initiated and completed, 2. Objectives andprocedures statedinthe approved protocol, including any changes in the original protocol, 3. Statisticalmethods employed for analyzing the data, 4. Description oftest and/or control substance (e.g., lot#, purity, physical characteristics, and method ofpreparingtest concentrations) 5. Description ofthe analytical methods used, 6. A description ofthe test concentrationused duringthe study, 7. A description ofall circumstances that mayhave affectedthe quality orintegrity ofthe study, 8. Location ofraw data, 9. List and signatures ofstudypersonnel, 10. Statement signedbythe QualityAssurance Unit. 

### G. REGULATORY COMPLIANCE GLP Standards Organisation for Economic Co-Operation and Development (OECD)

-- OECD Series onPrinciples ofGoodLaboratoryPractice and Compliance Monitoring, Number 1. OECD Principles onGood LaboratoryPractice (as revised in 1997) ENV /MC/CHEM(98)17I 11 I

. IllUS Environmental ProtectionAgency-TSCA GLP THE DOW CHEMICAL COMPANY STUDYID: PAGE9

Title 40 CFR, Part 792-Toxic Substances ControlAct (TSCA); Good Laboratory Practice Standards, Final Rule QualityAssurance The study conduct, data, protocol, protocol changes/revisions, and final report willbe inspectedbythe QualityAssurance Unit, Toxicology & Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan. Study Archives The data, protocol, protocol changes/revisions, and final report willbe archivedby the Toxicology & Environmental Research and Consulting archivist and stored atThe Dow Chemical Company, Midland, Michigan. H. SAFETY Allpersonnel involvedinthe studywill be advised ofthe safetyprecautions to follow whenhandling the test material. Chemical safetyinformation willbe made available.I II! - /ifI. REFERENCES THE DOW CHEMICAL COMPANY STUDYID:

PAGElO

Cheminova (2010). StabilityofEP1 (CASRN 298-06-6) and EP2 (CASRN 252-04- 1) in water. Unpublishedreport ofCheminovaA/S Laboratories, Harboore, Denmark. October2010. Used with permission ofCheminova. OECD (2004). OECD Guidelines for the Testing ofChemicals. Test Guideline 111: Hydrolysis as a Function ofpH. Adopted 13 April 2004. Qinyan (2013). Certificate ofAnalysis ofO,O-Diethylphosphorochloridoth (DEPCT), Lot C032013081501. ZhejiangXinnong Chemical Co., Ltd., Sanlixi, Xianju County, Zhejiang Province, China. U.S. EPA (2010). EPI Suite Software v4.1, including KOWWIN v1.68, WSKOWIN v1.42, WATERNT v1.01, HENRYWIN v3.20, andMPBPVP v1.43. United States Environmental ProtectionAgency, Office ofPollutionPrevention and Toxics, Washington, D.C.\\\\ \\\APPENDIX A THE DOW CHEMICAL COMPANY STUDYID:

PAGE 11


### Estimated Physical/Chemical Properties for DEPCT (CAS No. 2524-04-1)

SMILES 0 (P (OCC) (=S) CL) CC CHEM Phosphorochloridothioic acid, 0,0-diethyl ester MOL FOR: C4 H10 CL1 02 P1 S1 MOL WT : 188.61 EPI SUMMARY (v4.10)

-------------------- Physical Property Inputs: Log Kow (octanol-water) : Boiling Point (deg C) Melting Point (deg C) -20.00 Vapor Pressure (mm Hg) 0.7 Water Solubility (mg/L): Henry LC (atm-m3/mole) :ITil - Ill/ 

## \. THE DOW CHEMICAL COMPANY STUDYID:

PAGE12
KOWWIN Program (v1.68) Results:
Log Kow(version 1.68 estimate): 2.37 SMILES O(P(OCC) (=S)CL)CC CHEM Phosphorochloridothioic acid, 0,0-diethyl ester MOL FOR: C4 H10 CL1 02 P1 S1 MOL WT : 188.61 

## -------+-----+--------------------------------------+---------+-------- TYPE I NUM I LOGKOW FRAGMENT DESCRIPTION COEFF VALUE 

## -------+-----+--------------------------------------+---------+-------- Frag 2 -CH3 [aliphatic carbon) 0.5473 1.0946 Frag 2 -CH2- [aliphatic carbon) 0.4911 0.9822 Frag 1 -CL [chlorine, aliphatic attach) 0.3102 0.3102 Frag 1 S=P [thio=phosphorus] 1-0.6587 -0.6587 Frag 2 -0-P [aliphatic attach) 1-0.0162 -0.0324 Factor! 1 Phosphorus-halogen correction 0.4500**1 0.4500 Const I Equation Constant 0.2290 

## -------+-----+--------------------------------------+---------+-------- NOTE An estimated coefficient (**) used 

## -------+-----+--------------------------------------+---------+-------- Log Kow 2.3749\\\\ \\\THE DOW CHEMICAL COMPANY STUDYID:

MPBPVP (v1.43} Program Results:
Experimental Database Structure Match: Name 0,0-Diethyl chlorothiophosphate CAS Num 002524-04-1 Exp MP (deg C): Exp BP (deg C) : 45 @ 3 mm Hg Exp VP (mm Hg}:

SMILES O(P(OCC} (=S}CL}CC CHEM Phosphorochloridothioic acid, 0,0-diethyl ester MOL FOR: C4 H10 CL1 02 P1 S1 MOL WT : 188.61

------------------------ SUMMARY MPBVP v1.43

-------------------- Boiling Point: 198.05 deg C (Adapted Stein and Brown Method}
Melting Point: -150.31 deg C (Adapted Joback Method} Melting Point: 1.98 deg C (Gold and Ogle Method} Mean Melt Pt -74.17 deg C (Joback; Gold,Ogle Methods} Selected MP: -74.17 deg C (Mean Value}
Vapor Pressure Estimations (25 deg C): (Using BP: 198.05 deg C (estimated}} (MP not used for liquids} VP: 0.432 mm Hg (Antoine Method}: 57.6 Pa (Antoine Method} PAGE 13

VP: 0.363 mm Hg (Modified Grain Method}: 48.5 Pa (Modified Grain Method}\\\\ \\\/ I \ THE DOW CHEMICAL COMPANY STUDYID:

PAGE14
VP: 0.587 mm Hg (Mackay Method): 78.2 Pa (Mackay Method) Selected VP: 0.398 mm Hg (Mean of Antoine & Grain methods) 53 Pa (Mean of Antoine & Grain methods)
-------+-----+--------------------+----------+--------- TYPE I NUM I BOIL DESCRIPTION COEFF VALUE
-------+-----+--------------------+----------+--------- Group 2 -CH3 21.98 43.96 Group 2 -CH2- 24.22 48.44 Group 1 -Cl 34.08 34.08 Group 2 -0- (nonring) 25.16 50.32 Group 1 >P=S 100.00 100.00 

### * Equation Constant 198.18
=============+====================+==========+========= RESULT-uncorrl BOILING POINT in deg Kelvinl 474.98 RESULT- corr I BOILING POINT in deg Kelvinl 471.211

BOILING POINT in deg C 198.05

-------+-----+--------------------+----------+--------- TYPE I NUM I MELT DESCRIPTION COEFF VALUE
-------+-----+-------------------+----------+--------- Group 2 -CH3 -5.10 -10.20 Group 2 -CH2- 11.27 22.54 Group 1 -Cl 13.55 13.55 Group 2 -0- (nonring) 22.23 44.46 Group 1 >P=S -70.00 -70.00 * Equation Constant 122.50
=============+====================+==========+=========Illi ill ITHE DOW CHEMICAL COMPANY STUDYID:

PAGE15
RESULT MELTING POINT in deg Kelvin 122.85 Special-limit! MELTING POINT in deg Kelvin 122.85 MELTING POINT in deg C I -150.31

Water Sol from Kow (WSKOW vl.42) Results:

Water Sol: 1249 mg/L SMILES 0 (P (OCC) (=S) CL) CC CHEM Phosphorochloridothioic acid, 0,0-diethyl ester MOL FOR: C4 HlO CLl 02 Pl Sl MOL WT : 188.61

---------------------------------- WSKOW vl.42 Results

---------------- Log Kow (estimated) 2.37 Log Kow (experimental) : not available from database Log Kow used by Water solubility estimates: 2.37
Equation Used to Make Water Sol estimate: LogS (mol/L) = 0.693-0.96 log Kow-0.0092(Tm-25)-0.00314 MW + Correction Melting Pt (Tm) -20.00 deg C (Use Tm 25 for all liquids) Correction(s): Value

No Applicable Correction Factors
Log Water Solubility (in moles/L) -2.1791111 IllTHE DOW CHEMICAL COMPANY STUDYID:

PAGE 16
Water Solubility at 25 deg C (mg/L): 1249

WATERNT Program (vl.01) Results:

Water Sol (v1.01 est): 4296.2 mg/L SMILES O(P(OCC) (=S)CL)CC CHEM Phosphorochloridothioic acid, 0,0-diethyl ester MOL FOR: C4 H10 CL1 02 P1 S1 MOL WT : 188.61 

## -------+-----+--------------------------------------------+----------+-

TYPE I NUM IWATER SOLUBILITY FRAGMENT DESCRIPTION I COEFF VALUE 

## -------+-----+-------------------------------------+----------+-------- Frag 2 -CH3 [aliphatic carbon] 1-0.3213 -0.6425 Frag 2 -CH2- [aliphatic carbon] 1-0.5370 -1.0740 Frag 1 -CL [chlorine, aliphatic attach] 1-0.2872 -0.2872 Frag 1 S=P [thio=phosphorus] I 0.8774 0.8774 Frag 2 -0-P [aliphatic attach] 1-0.3827 -0.7653 Const Equation Constant 0.2492 

## -------+-----+--------------------------------------------+----------+- Log Water Sol (moles/L) at 25 dec C -1.6425 Water Solubility (mg/L) at 25 dec C 4296.21111 Ill

### APPENDIXB: THE DOW CHEMICAL COMPANY STUDYID:

PAGE17


### Estimated Physical/Chemical Properties for the Expected Hydrolysis Product ofDEPCT

SMILES : O=P(S) (OCC)OCC CHEM 0,0-DIETHYL PHOSPHOROTHIOATE MOL FOR: C4 H11 03 P1 S1 MOL WT : 170.16

------------------------------ EPI SUMMARY (v4.10)

-------------------- Physical Property Inputs: Log Kow (octanol-water) : Boiling Point (deg C) Melting Point (deg C) Vapor Pressure (mm Hg) Water Solubility (mg/L) : Henry LC (atm-m3/mole) KOWWIN Program (v1.68) Results: Log Kow(version 1.68 estimate): 0.48 SMILES : O=P(S) (OCC)OCC CHEM 0,0-DIETHYL PHOSPHOROTHIOATE MOL FOR: C4 H11 03 P1 S1 MOL WT : 170.16 

## -------+-----+--------------------------------------+---------+-------- TYPE I NUM I LOGKOW FRAGMENT DESCRIPTION COEFF VALUE1111 IllTHE DOW CHEMICAL COMPANY STUDYID:

PAGElS

-------+-----+--------------------------------------------+---------+--



### ------ Frag 2 -CH3 [aliphatic carbon] I 0.5473 1.094 6 Frag 2 -CH2- [aliphatic carbon] I 0.4911 0.9822 Frag 2 -0-P [aliphatic attach] 1-0.0162 -0.0324 Frag 1 -S-P [sulfur, phosphorus attach] I 0.6270 0.6270 Frag 1 O=P 1-2.4239 -2.4239 Const Equation Constant I 0.2290 -------+-----+--------------------------------------------+---------+-- ------Log Kow 0.4765

MPBPVP (v1.43) Program Results: Experimental Database Structure Match: no data SMILES : O=P(S) (OCC)OCC CHEM 0,0-DIETHYL PHOSPHOROTHIOATE MOL FOR: C4 H11 03 P1 S1 MOL WT : 170.16

------------------------ SUMMARY MPBVP v1.43

-------------------- Boiling Point: 241.03 deg C (Adapted Stein and Brown Method) Melting Point: Melting Point: Mean Melt Pt Selected MP: -23.77 deg C (Adapted Joback Method) 27.07 deg C (Gold and Ogle Method)

1.65 deg C (Joback; Gold,Ogle Methods) 1.65 deg C (Mean Value) Vapor Pressure Estimations (25 deg C) : (Using BP: 241.03 deg C (estimated)) (MP not used for liquids) VP: 0.0456 mm Hg (Antoine Method) 6.08 Pa (Antoine Method) VP: 0.0421 mm Hg (Modified Grain Method) 5.61 Pa (Modified Grain Method) VP: 0.0734 mm Hg (Mackay Method) 9.78 Pa (Mackay Method) Selected VP: 0.0439 mm Hg (Mean of Antoine & Grain methods) 5.85 Pa (Mean of Antoine & Grain methods)
-------+-----+--------------------+----------+--------- TYPE I NUM I BOIL DESCRIPTION COEFF VALUE -------+-----+--------------------+----------+--------- Group 2 -CH3 21.98 43.96 Group 2 -CH2- 24.22 48.44 Group 2 -0- (nonring) 25.16 50.32 Group 1 -SH 81.71 81.71 Group 1 O=P< 107.23 107.23 * I I Equation Constant 198.18 =============+====================+==========+========= RESULT-uncorrl BOILING POINT in deg Kelvin 529.84 RESULT- corr I BOILING POINT in deg Kelvin 514.191111 IllTHE DOW CHEMICAL COMPANY STUDYID:

PAGE19
BOILING POINT in deg C 241.03

-------+-----+--------------------+----------+--------- TYPE I NUM I MELT DESCRIPTION I COEFF I VALUE -------+-----+--------------------+----------+--------- Group I 2 I -CH3 I -5.10 I -10.20 Group I 2 I -CH2- I 11.27 I 22.54 Group I 2 I -0- (nonring) I 22.23 I 44.46 Group I 1 I -SH I 20.09 I 20.09 Group I 1 I O=P< I 50.00 I 50.00 * I I Equation Constant I I 122.50 =============+====================+==========+========= RESULT MELTING POINT in deg Kelvin 249.39 Special-limit! MELTING POINT in deg Kelvin I 249.39 MELTING POINT in deg C I -23.77

Water Sol from Kow (WSKOW vl.42) Results:
Water Sol: 2.405e+004 mg/L SMILES : O=P(S) (OCC)OCC CHEM 0,0-DIETHYL PHOSPHOROTHIOATE MOL FOR: C4 Hll 03 Pl Sl MOL WT : 170.16

---------------------------------- WSKOW vl.42 Results

---------------- Log Kow (estimated) 0.48 Log Kow (experimental) : not available from database Log Kow used by Water solubility estimates: 0.48 Equation Used to Make Water Sol estimate: LogS (mol/L) = 0.796

- 0.854 log Kow- 0.00728 MW +Correction (used when Melting Point NOT available) Correction(s): Value No Applicable Correction Factors Log Water Solubility (in moles/L) -0.850 Water Solubility at 25 deg C (mg/L): 2.405e+004

WATERNT Program (vl.Ol) Results:

### Ill I IllTHE DOW CHEMICAL COMPANY STUDYID:

PAGE20
Water Sol (vl.Ol est): 1.9809e+005 mg/L SMILES : O=P(S) (OCC)OCC CHEM 0,0-DIETHYL PHOSPHOROTHIOATE MOL FOR: C4 Hll 03 Pl Sl MOL WT : 170.16 -------+-----+--------------------------------------------+----------+- TYPE I NUM I VALUE WATER SOLUBILITY FRAGMENT DESCRIPTION COEFF
-------+-----+--------------------------------------------+----------+-



### -------- Frag I 2 -CH3 [aliphatic carbon] 1-0.3213 I 

### - 0.6425 Frag I 2 -CH2- [aliphatic carbon] 1-0.5370 I 

### - 1.0740 Frag I 2 -0-P [aliphatic attach] 1-0.3827 I 

### - 0.7653 Frag I 1 -S-P [sulfur, phosphorus attach] 1-0.9761 I 

### - 0.9761 Frag I 1 O=P 3.2748 3.2748 Const I Equation Constant 0.2492 -------+-----+--------------------------------------------+----------+- Log Water Sol (moles/L) at 25 dec C 0.0660 Water Solubility (mg/L) at 25 dec C =1.9809e+005
HENRYWIN (v3.20) Program Results:
Bond Est : 8.02E-007 atm-m3/mole (8.13E-002 Pa-m3/mole) Group Est: Incomplete SMILES : O=P(S) (OCC)OCC CHEM 0,0-DIETHYL PHOSPHOROTHIOATE MOL FOR: C4 Hll 03 Pl Sl MOL WT : 1 70
. 16

--------------------------- HENRYWIN v3.20 Results

-------------------- ----------+---------------------------------------------+---------+---- CLASS BOND CONTRIBUTION DESCRIPTION I COMMENT I VALUE ----------+---------------------------------------------+---------+----



### ------ HYDROGEN 10 Hydrogen to Carbon (aliphatic) Bonds -1.1968 HYDROGEN 1 Hydrogen to Sulfur Bonds 0.2247 FRAGMENT 2 

### c-c 0.2326 FRAGMENT 2 

### c-o 2.1709 FRAGMENT 2 0-P 0.7861 FRAGMENT 1 S-P 0.6334 FRAGMENT 1 O=P 1.6334\\\\ \\ITHE DOW CHEMICAL COMPANY STUDYID:

PAGE21 

## ----------+---------------------------------------------+---------+---- RESULT BOND ESTIMATION METHOD for LWAPC VALUE ITOTALI 4.484 

## ----------+-------------------------------------------+---------+------ HENRYs LAW CONSTANT at 25 deg C = 8.02E-007 atm-m3/mole 3.28E-005 unitless = 8.13E-002 Pa-m3/mole 

## --------+-----------------------------------------------+------------+- GROUP CONTRIBUTION DESCRIPTION COMMENT VALUE 

## --------+-----------------------------------------------+------------+-

------- 2 CH3 (X) I -1.24 2 CH2 (C) (0) I -0.26 MISSING Value for: p (S) (0) (0) (=0) MISSING Value for: SH (P) MISSING Value for: 0 (C) (P) MISSING Value for: 0 (C) (P) 

## --------+-----------------------------------------------+------------+- RESULT I GROUP ESTIMATION METHOD for LOG GAMMA VALUEIINCOMPLETEI -1.50 

## --------+-----------------------------------------------+------------+- For Henry LC Comparison Purposes: Exper Database: none available User-Entered Henry LC: not entered Henrys LC [via VP/WSol estimate using User-Entered or Estimated values]: HLC: 4.087E-007 atm-m3/mole (4.141E-002 Pa-m3/mole)1111 IllAppendix E Reverse Mutation Assay "Ames test" Study Protocol



### 17 

### DEPCT

### ITil IllJRF StudyNumber: 481-1-06-XXXX Dow StudyNumber: 130186 

## PROTOCOL Bacterial Reverse Mutation Assay Page 1 of15



### BACTERIAL REVERSE MUTATION ASSAY
GUIDELINES: OECD N° 471, OPPTS 870.5100, EEC B.13/14 andJMAFF 2-1-19-1


## SPONSOR DOWEUROPE GmbH BACHTOBELSTRASSE 3 CH-8810 HORGEN

SWITZERLAND

STUDY DIRECTOR: NIRALIN. PATEL



## TEST FACILITY JAIRESEARCHFOUNDATION DEPARTMENT OFTOXICOLOGY VALVADA- 396108 DIST. VALSAD GUJARAT INDIA

JANUARY- 2014
Study ProtocolJRF StudyNumber: 481-1-06-XXXX Dow StudyNumber: 130186 Bacterial Reverse Mutation Assay Page2 of15
TITLEOFSTUDY: Salmonella-Escherichia coli Mammalian-Microsome Reverse Mutation Assay Pre-incubation Method with a ConfirmatoryAssay 1. PURPOSE To evaluate the mutagenic potential ofthe test item by utilizing a pre-incubation method to measure the test material's ability to induce reverse mutations at selected loci ofseveral strains ofSalmonella typhimurium and at the tryptophan locus ofEscherichia coli WP2uvrA (pKMlOl) in the presence and absence ofS9 activation. 2. TESTITEM A. Source :The test item will be received from the Sponsor. B. Identification: The test item will be identified using information provided by the Sponsor, including the productname, lot number, expiration date, and storage condition. C. SampleDescription: Details listed in the attached Protocol Annexure Form. D. Characterization ofTestItem The Sponsor will be directly responsible for determination and documentation ofthe analytical purity an composition ofthe test item. E. AnalyticalVerification and StabilityofTestItem Dose Formulations Stability ofthe test item in the vehicle, as well as analytical verification ofthe test item dose formulations, will be conducted by the Chemistry Department ofJai Research Foundation using a validated analytical method, which will be furnished in an amendment to the study protocol. Sampling Upon preparation for use in the confirmatory mutation assay, the following samples will be collected in duplicate, as follows: If dose formulations are solutions, required aliquots of the vehicle and all dose formulations will be collected. Ifdosing formulations are suspensions, aliquots from the top (T), middle (M), and bottom (B) ofeach dose formulation will be collected for homogeneity and concentration verification. The vehicle control will be sampled from the middle portion only. Samples aliquots (solutions or suspensions) will be stabilized by immediately freezing or by chemical preservation in an effort to quench any degradation ofthe test material. After a minimum of4 hours, samples (solutions or suspensions) will be taken from the lowest and highest dose formulations for stability analysis and preserved as above. One aliquot of each respective concentration verification, homogeneity and/or stability sample will bl Study Protocol

1111 IllJRF Study Number: 481-1-06-XXXX Dow Study Number: 130186 Bacterial Reverse MutationAssay Page 3 of15 analyzed at Jai Research Foundation. The second aliquot will be stored at Jai Research Foundation as a backup and will be analyzed only as needed. All test item formulations stored as backup samples will be discarded upon fmalization ofthe report. Stability ofthe Test Item in the Selected Vehicle Stability ofthe test item in the selected vehicle will be established for a minimum of4 hours, at the lowest and the highest doses being tested. Samples will be analyzed for stability using the following equation: [(Dosee- Doses) IDoses] x 100 =percent change Where: Doses= analytical concentration at '0' hour (start) Dosee =analytical concentration after4 hours (end) Dose Formulation Concentration Analysis The samples will be analyzed for concentration verification and/or homogeneity. The results from samples taken from the middle portion of each dose will serve as a confirmation of concentration of the formulation. Ifhomogeneity was analyzed, the average of all replicates will serve as the concentration verification. Acceptance Criteria The acceptable specification range for dose concentration verification is 85% to 115% ofnominal. The test item is considered homogenous if the % relative standard deviation (RSD) is < I0% of a minimum n=3 aliquots for the low and high concentrations. For the test item to be considered stable in the vehicle for this test system, the stability calculation result must be :::;15%. The results will be appended to the raw data and presented in the report. In the event that a sample is outside ofthe acceptable specification range, the Study Director willjustifY the acceptability ofthe results, suggest re-analysis ofthe backup samples, orretest the affected portion of the study. F. TestItem Retention Sample Since the in-life portion ofthis study is less than four weeks in duration, Jai Research Foundation will not retain a reserve sample ofthe test item.

Study ProtocolJRF Study Number: 481-1-06-XXXX Dow Study Number: 130186
G. Disposition ofTest Item Bacterial Reverse MutationAssay Page 4 of15

Residual dosing preparations, excluding those saved for concentration or homogeneity analysis (ifany), will be disposed offollowing administration to the test system. Following issue ofthe final report, unless otherwise specified by the Sponsor, all remainingtest item will be returned to the Sponsororproperly disposed of as indicated in the attached Protocol Annexure Form. Records ofsample return or disposition will be maintained by the test facility. H. Storage The test item will be stored as per the details in the Protocol Annexure Form. I. Hazards Appropriate routine safety precautions will be exercised in the handling ofthe test item, including any special requirements indicated by the sponsor in the MSDS and/orProtocol Annexure Form. 3. TEST SYSTEM The approved test system underthe 1997 guidance ofOECD allows the use ofthe following strains ofbacteria: Histidine auxotrophic strains ofSalmonella typhimurium viz., TA98, TAlOO, TA1535 and TA1537. Tryptophan auxotrophic strain ofEscherichia coli WP2uvrA (pKMlOl). Each S. typhimurium tester strain contains, in addition to a mutation in the histidine operon, additional mutations that enhance sensitivity to some mutagens. The rfa mutation results in a cell wall deficiency that increases the permeability ofthe cell to certain classes ofchemicals such as those containing large ring systems that would otherwise be excluded. The deletion in the uvrB gene results in a deficient DNA excision-repair system. Tester strains TA98 and TAlOO also contain the pKM101 plasmid (carrying theR-factor). Ithas been suggested that the plasmid increases sensitivity to mutagens by modifYing an existing bacterial DNA repair polymerase complex involved with the mismatch-repairprocess. TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. TA100 is reverted by both frameshift and base substitution mutagens and TA1535 is reverted only by mutagens that cause base substitutions. The E. coli tester strain has an AT base pair at the critical mutation site within the trpE gene (Wilcox et a!., 1990). Tester strain WP2uvrA (pKM101) has a deletion in the uvrA gene resulting in a deficient DNA excision-repair system. Tryptophan revertants can arise due to a base change at the originally mutated site orby a base change elsewhere in the chromosome causing the original mutation to be suppressed. Thus, the specificity of the reversion mechanism is sensitive to base-pair substitution mutations (Green and Muriel, 1976).

Study Protocol

1111 Ill

### ( Bacterial Reverse Mutation Assay JRF Study Number: 481-1-06-XXXX Dow Study Number: 130186 A. Source ofthe Test System Salmonella typhimurium Molecular Toxicology Inc., I57 Industrial Park Dr. Boone, NC 28607 U.S.A. B. Storage ofthe Test System Page 5 of15

Escherichia coli Molecular Toxicology Inc., I57 Industrial Park Dr. Boone, NC 28607 U.S.A.

Stock cultures of tester strains in Oxoid nutrient broth No. 2 are stored in the test facility as frozen permanents in liquid nitrogen. Fresh cultures for the experiment will be prepared by inoculating frozen cultures into a flask containing I0 mL ofsterile nutrient brothN° 2 (Oxoid Unipath, England). C. Genotypic Characterization ofthe Test System Genotype TA98 TAIOO TAI535 TA1537 WP2uvrA (pKMIOI) Tryptophan/Histidine requirement His+ His+ His+ His+ Trp+ Not rfa mutation + + + + applicable uvrB IuvrA mutation uvrB uvrB uvrB uvrB uvrA Not Not R-factor + + applicable applicable + The growth requirements and the genetic identity of strains like histidine or tryptophan requirement, sensitivity to UV radiation, resistance ofstrains TA98, T A I00, and WP2uvrA (pKMI0I) to ampicillin and rfa mutation of Salmonella typhimurium strains will be checked along with the range of spontaneous revertants afterpreparation ofthe masterplates. 4. TEST MEDIUM, SOLUTIONS AND OTHER CHEMICALS I VB(Minimal Glucose**~gar)** agar 2 Soft agar 3 Soft agar (Top agar) containing 0.5 rnM histidine and biotin 4 Soft agar containing 5 Jlg/mL tryptophan 5 Nutrient agar 6 Oxoid NutrientbrothNo. 2 7 0.2 M sodium phosphate buffer The chemicals used in the study wiii be documented in the raw data and presented in the report along withtheir batch/lotnumbers and the name ofthe manufacturer.
Study ProtocolJRF Study Number: 481-1-06-XXXX Dow Study Number: 130186 5. POSITIVE CONTROL SUBSTANCES Bacterial Reverse Mutation Assay Page 6 of15

The batch/lot number along with the name ofthe manufacturer ofthe following positive control substances that will be used in the study will be documented in the raw data and presented in the report. Positive control CAS No. 2-nitrofluorene 607-57-8 2-aminoanthracene 613-13-8 9-Aminoacridine hydrochloride hydrate 52417-22-8 4-nitroquinoline-1-oxide 56-57-5 Sodium azide 26628-22-8 6. METHODS A. Outline ofthe Method The principle ofthe Bacterial Reverse Mutation Test is that it detects mutations that revert the mutations already present in the test strains thereby restoring the functional capability ofthe bacteria to synthesize an essential amino acid. Such revertant bacteria are detected by their ability to grow in the absence ofthe amino acid (histidine c, tryptophan) in the culture medium as required by the parent strain. In the pre-incubation assay, the test constituents are mixed with the bacteria inside a tube, incubated in an incubator shaker, mixed with overlay agar and plated immediately onto minimal medium his- for Salmonella typhimurium and trp- for Escherichia coli, respectively. After a suitable period ofincubation, the revertant colonies are counted and compared with the number of spontaneous revertants in the solvent/vehicle control plates. B. Preparation ofS9 Homogenate and Activation Mixture Aroclor 1254 induced rat liver S-9 homogenate will be used as the metabolic activation system. Aroclor 1254 induced rat liver S-9 fraction (Maron and Anies, 1983; Venitt, eta!., 1984) procured from Molecular Toxicology Inc., USA and stored at -80 ± 5 °C, will be used in the experiment. Each batch ofS9 is being characterised by the supplier for its protein content, enzyme activity and ability to metabolize the promutagens 2-aminoanthracene and benzo(a)pyrene to mutagens using Salmonella typhimurium TAIOO strain. Certificate ofanalysis will be included in the fmal report. S-9 homogenate will be thawed immediately before use and is buffered and supplemented with the essential co-factors such as**~-NADP** and Glucose-6-phosphate to form the "S9 mix". The details ofS-9 homogenate and activation mixture used in the study will be documented inthe raw data and presented in the report.

Study Protocol

1111 Ill

## (

JRF Study Number: 481-1-06-XXXX Dow Study Number: 130186 C. Preliminary Solubility Test Bacterial Reverse Mutation Assay Page 7 of15

As needed, a solubility determination will be conducted to determine the maximum soluble concentration or workable suspension as indicated below. Vehicles compatible with this test system, in order of preference, include but are not limited to water, dimethyl sulfoxide (CAS 67-68-5), ethanol (CAS 64-17-5), and acetone (CAS 67-64-1). The vehicle of choice will be the solvent, selected in order of preference, which permits preparation ofthe highest workable or soluble stock concentration, up to 50 mg/mL for aqueous vehicles and up to 500 mg/mL for organic vehicles. Upon completion ofthe solubility test, the Sponsor will be notified ofthe chosen vehicle prior to starting the study. Based on the molecular weight of the test item, the solvents to be tested and the dose to be achieved in the assay, alternate stock concentrations may be tested, as needed. The details ofsolubility test will be documented in the raw data and presented in the report. D. TesterStrain Cultures Each ofthe tester strains from the frozen stock will be grown in Oxoid Nutrient broth No.2 to a density of 1-2 x I09 cells/mL. After plating for the mutagenicity assay, the bacterial suspension ofeach tester strain will be diluted up to 10"6 in PBS and 100 J-LL ofthe diluted suspension from each tester strain will be plated onto nutrient agar plates in triplicate. The plates will be incubated at 37 ± 1 

## oc for 24 to 48 hours. After incubation, the number ofcolonies in each plate will be counted and expressed as number ofcolony forming units per mL ofthe bacterial suspension.
E. Initial Toxicity-MutationAssay Selection of dose levels for the confirmatory mutagenicity assay will be based upon the toxicity and precipitation profile ofthe test item assessed in an initial toxicity-mutation assay. The test item will be tested at a minimum ofeight dose levels along with appropriate vehicle and positive controls with tester strains TA98, TAlOO, TA1535, TA1537, and WP2uvrA (pKMlOl) without and with S9 activation. All dose levels oftest item, vehicle controls, and positive controls will be plated in duplicate. Unless indicated otherwise by the Sponsor, the highest dose will be the highest workable concentration in the vehicle of choice but not to exceed 5 mg/plate. Solubility or workability permitting, the dose levels will be 5000, 1500, 500, 150, 50, 15, 5.0 and 1.5 J-Lg per plate. In selecting dose levels for the confirmatory mutagenicity assay the following guidelines will be employed. Doses will be selected such that precipitate does not interfere with manual and or colony counter scoring. Whenever possible, the highest dose for the confirmatory mutagenicity assay will be selected to give some indication of toxicity without exceeding 5 mg/plate. For freely soluble, nontoxic test items, the highest dose level will be 5 mg/plate. For precipitating, nontoxic test items, the highest dose level may be selected in an attempt to yield precipitate at only the top one or two dose levels. The Sponsor will be consulted regarding dose selection if (1) the maximum dose level is selected based on precipitation and this dose level is less than 5 mg/plate or (2) the maximum achievable test item dose level is less than 5mg/plate and this dose level is nontoxic. The doses selected for the confirmatory mutagenicity assay will be documented in the raw data and presented in the report.
Study ProtocolJRF Study Number: 481-1-06-XXXX Dow Study Number: 130186 F. ConfirmatoryMutation Assay Bacterial ReverseMutationAssay Page 8 of15

The test item will be tested at a minimum offive dose levels along with appropriate vehicle and positive controls with tester strains TA98, TA100, TA1535, TA1537, and WP2uvrA (pK.Ml01) without and with S9 activation. All dose levels oftest item, vehicle controls, and positive controls will be plated in triplicate. G. Controls Vehicle Control Appropriate vehicle controls will be plated for each tester strain without and with S9 activation. The vehicle control will be the vehicle alone, unless there is no historical basis for use ofthe selected vehicle. In the latter case, both untreated and vehicle controls will be used. Positive Control No analyses will be performed on the positive control substances orthe positive control dose formulations. The neat positive control substances and the vehicles used to prepare the test item and positive control formulations will be characterized by the Certificates ofAnalysis provided by the Supplier(s). Copies ofthe Certificates ofAnalysis will be kept on file at Jai ResearchFoundation. The positive controls that will be plated concurrently with the assay are listed below. Results obtained from these substances will be used to assure responsiveness ofthe test system but not to provide a standard for comparison with the test item.

Positive Control Vehicle Bacterial Strains Concentration Metabolic Activation Sodium azide TA1535 0.5 11g/plate (CAS No 26628-22-8) Sterile distilled TA100 5 11g/plate 4-nitroquinoline-1-oxide water WP2uvrA (CAS No 56-57-5) (pKM101) 0.5 11g/plate Absence ofS9 9-Aminoacridine mix hydrochloride hydrate TA1537 75 11g/plate (CAS No 52417-22-8]_ 2-Nitrofluorene TA98 7.5 11g/plate (CAS No. 607-57-8) Dimethyl sulfoxide TA1537, TA1535 10 11g/plate 2-Aminoanthracene TA98, TAIOO 5 11g/plate Presence ofS9 (CAS W 613-13-8) mix WP2uvrA 10 11g/plate i2_K.Ml01)
Study Protocol

1111 IllJRF Study Number: 481-1-06-XXXX Dow Study Number: 130186 Sterility Controls Bacterial Reverse MutationAssay Page 9 of15

The most concentrated test item dilution, the vehicle control, S9 mix, 0.2 M phosphate buffer, Oxoid nutrient broth solution and top agarwill be checked for sterility.
H. Performance oftheAssay Test Item Preparation Unless specified otherwise, test item stock and dilutions will be prepared immediately prior to use. All test item dosing will be at room temperature underyellow light. Labeling The petri-dishes will be labeled to indicate the study number, strain number, treatment group, assay phase and activation. NumberofReplicates There will be two replicates for the initial toxicity-mutation assay and three replicates for the confirmatory mutation assay. Plating Procedure The initial toxicity-mutation, as well as the confirmatory mutation assay will be conducted using the preincubation assay method. A. Presence ofmetabolic activation a) 50 or 100 f..lL test dose/vehicle/appropriate positive control b) 100 f..lL bacterial culture c) 500 f..lL S-9 mix B. Absence ofmetabolic activation a) 50 or 100 f..lL test dose/vehicle/appropriate positive control b) 100 f..lL bacterial culture c) 500 f..lL of0.2 M phosphate buffer These test constituents will be transferred into sterile test tubes and will be kept in an incubator shaker for approximately 20 ± 2 minutes at 37± 1°C. After this period, 2 mL ofsoft agar containing histidine-biotin I tryptophan will be added to each ofthe tubes. These constituents will be overlaid onto VB agar plates. Afterthe soft agar sets, theplates will be incubated at 37 ± 1

## oc for 48 to 72 hours.

Study ProtocolBacterial Reverse Mutation Assay JRF Study Number: 481-1-06-XXXX Dow Study Number: 130186 Page 10 of15
7. SCORING The revertant colonies will be counted manually or using colony counter and the plates wiii be examined for bacterial background lawn. The condition ofthe bacterial background lawn will be evaluated for evidence of test item toxicity and precipitate. Evidence oftoxicity will be scored relative to the vehicle control plate and recorded along with the revertant count for that plate. Toxicity will be evaluated as a decrease in the number of revertant colonies per plate and/or a thinning or disappearance ofthe bacterial background lawn. Precipitation will be evaluated after the incubation period by visual examination without magnification. 8. CRITERIAFORACCEPTABILITYOFTHEASSAY The Salmonella typhimurium and Escherichia coli reverse mutation test is considered acceptable ifit meets the following criteria:

• Tester strain integrity All Salmonella typhimurium tester strains must exhibit sensitivity to crystal violet and to ultraviolet light to demonstrate the presence ofrfa mutation and uvr B mutation, respectively. The Escherichia coli tester strain must exhibit sensitivity to ultraviolet light to demonstrate th presence ofuvrA mutation. Salmonella typhimurium strains TA98 and TAIOO and Escherichia coli strain WP2uvrA (pKMIOl) must exhibitresistance to ampicillin to demonstrate the presence ofthe plasmid R-factor.

• The spontaneous reversion rates in the solvent/vehicle control**m~st** be in the range of in-house historical data

• All tester strain culture titers must be in the range of 1-2 x 109 cells/mL to ensure that appropriate numbers ofbacteria are used for plating.
• Each mean positive control value must exhibit at least a 3.0-fold increase over the respective mean vehicle control value for each tester strain
. 

## • • No bacterial contamination is observed in the sterility control cultures.

• Toxicity: A minimum ofthree non-toxic dose levels will be required to evaluate assay data. A dose level is considered toxic if it causes a >50% reduction in the mean number ofrevertants per plate relative to the mean vehicle control value (this reduction must be accompanied by an abrupt dose-dependent drop in the revertant count) or a reduction in the background lawn. In the event that less than three non-toxic dose levels are achieved, the affected portion ofthe assay will be repeated with an appropriate change in dose levels.

Study Protocol

111 Ill

# (

( \ Bacterial Reverse Mutation Assay JRF Study Number: 481-1-06-XXXX Dow Study Number: 130186 Page 11 of15
9. EVALUATIONANDINTERPRETATIONOFRESULTS The conditions necessary for determining a positive result are, there should be a dose related increase in the mean revertants per plate ofat least one tester strain over a minimum oftwo increasing doses ofthe test item either in the absence orpresence ofthe metabolic activation system. Strains TA98, TA1535, and TA1537 Data sets will bejudged positive ifthe increase in mean revertants at the peak ofthe dose response is equal to orgreater than 3.0-times the mean vehicle control value. Strain TAtOO and WP2uvrA (pKMlOl) Data sets will bejudged positive ifthe increase in mean revertants at the peak ofthe dose response is equal to orgreater than 2.0-times the mean vehicle control value. A response that does not meet all three of the above criteria (magnitude, concentration-responsiveness, reproducibility) will notbe evaluated as positive. 10. OBSERVATIONS

11. Effect on Bacterial Background Lawn The condition ofthe bacterialbackground lawn will be evaluated for evidence ofthe test item toxicity. Precipitation The precipitation ofthe test item will be evaluated after the incubation periodby visual examination without magnification. Number ofRevertants Revertantcolonies for all given strains and groups will be counted manually or using colony counter. Viable Counts Viable counts for the different bacterial strains on nutrient agar plates will be counted manually or using colony counter. DATA COMPILATION All findings observed (e.g., precipitation, signs of toxicity) will be recorded and presented in the report. Individual revertant colonies for a given strain will be presented as appendices and the summarized results will be presented as tables. Viable counts ofthe overnight culture will be expressed as colony forming units (CFU) permL ofbacterial suspension. Study ProtocolJRF Study Number: 481-1-06-XXXX Dow Study Number: 130186 12. FINALREPORT Bacterial Reverse Mutation Assay Page 12 of15

A signed study report will be provided to the Sponsor. This report will include, but not be limited to, the following specific information:
• Information on the item tested and vehicle(s) used
• Test system information (e.g., strain, strain characteristics,number ofcells used)
• Test conditions (amount of test item per plate, media used, type and composition of metabolic activation system, treatmentprocedures)
• Results (signs oftoxicity, precipitation, individual plate counts, mean number ofrevertant colonies perplate and standard deviation, dose-response relationship, if·applicable, concurrent vehicle and positive control data, historical vehicle and positive control data)
• Treatment, evaluation and interpretation ofthe results and the conclusions drawn by the Study Director
• A compliance statement signed by the Study Director that states that the report accurately reflects the raw data obtained duringthe performance ofthe study andthat all applicable GLP regulations were followed in the conduct ofthe study. Any exceptions to GLP compliance wiii be included in the compliance statement • A Quality Assurance Unit statement, including the dates ofQAactivities and the dates reported to study director and management • Copy ofstudy protocol, protocol amendments and protocol deviations as annexure/appendixto the report 13. STUDYCONDUCT A. Laboratory This test will be conducted at Jai Research Foundation, Gujarat, India. B. Protocol Annexure Form A Protocol Annexure Form accompanies and represents an extension ofthis protocol. When completed by the Sponsor, it will contain information that is unique to the individual study (i.e., Study number, test item identification, characterization, expiration date, description, precautions, GLP compliance directive, etc.), as well as otherinformation related to the specific study. C. GLP Compliance This study will be conducted in compliance with Good Laboratory Practice (GLP) regulations that are specifically mentioned in the Protocol Annexure Form. Exceptionsto GLP compliance will be listed inthe final report.

Study Protocol



### Ill IllJRF Study Number: 481-1-06-XXXX Dow Study Number: 130186 D. Test Procedure Guidelines Bacterial Reverse Mutation Assay Page 13 of15

Theprocedures as described inthis protocol are basedonthe following selectedtesting guidelines: 

### rgJ OECD Guideline 471 (Genetic Toxicology: Bacterial Reverse Mutation Test), Ninth Addendum to the OECD Guidelines for the Testing ofChemicals, published by OECD, 1997 

### rgJ U.S. EPA: Health Effects Test Guidelines, OPPTS 870.5100, Bacterial Reverse Mutation Test, August 1998 

## D EC: B.l3!14, Mutagenicity

- Reverse Mutation Test Using Bacteria. Official Journal of the European Communities, L142, 31/05/2008 

### D METI: Ministry ofEconomy, Trade and Industry

-- Reverse Mutagenicity Test on Bacteria, Methods of Testing New Chemical Substances (Section 5.1-1 to 5.1-11): Japanese Act on the Evaluation ofChemical Substances and Regulation of Their Manufacture, Act No. 117, Ministerial Ordinance No.1-3. Enforced April2004 

### D The Japanese Ministry ofAgriculture, Forestry and Fisheries. Testing Guidelines for Toxicology Studies, 2000. Notification No. 12-Nousan-8147. November24, 2000. Reverse Mutation Studies 2-1-19-1. 14. QUALITY ASSURANCE This study plan has been verified by JRF Quality Assurance Unit (QAU) and documented (Number XXXXX). The QAU ofJRF will inspect the critical phase(s) ofthe study by study based inspection and/or process based inspection. The raw data, draft and final reports will be audited to ensure that the fmal report accurately reflect the raw data. The audit/inspection reports will be provided to the Study Director and the Test Facility Management. The date of audits/inspections and reporting of fmdings to the Study Director and the Test Facility Managementwill be incorporated in the study report.. 15. ARCIDVING The original raw data, protocol and final report ofthis study, following signing ofthe fmal report, will be sent to the Sponsor for retention at their location: The Dow Chemical Company, Toxicology and Environmental Research and Consulting, Midland, MI 48674. Exact photocopies ofeach document (stamped on first/cover page as "Exact True Copy ofthe Original: Page 1to XXX", signed and dated) will be retained at the archives oftest facility for two years for the purpose ofany auditby Monitoring/Regulatory Authorities. No test item shall be archived at the test facility as the study is not more than 4 weeks of experimental duration. At the end ofretention period, the documents retained at the test facility will be disposed after documented Quality Assurance inspection and related records maintained, however, no intimation ofsuch disposal will be informed to the sponsor. In the event ofany inspection by Monitoring/Regulatory Authorities seeking original raw data, protocol or fmal report for verification, the sponsor would be responsible to send the originals to eitherthe test facility or the Monitoring/Regulatory Authorities.

Study ProtocolJRF Study Number: 481-1-06-XXXX Dow Study Number: 130186 16. PROTOCOLAMENDMENTSANDDEVIATIONS Bacterial Reverse MutationAssay Page 14 of15

Modification of the protocol may be accomplished during the course of this investigation. However, no changes will be made in the study design without the verbal or written permission ofthe Sponsor. In the event that the Sponsor verbally requests or approves a change in the protocol, such changes will be made by appropriate documentation in the form ofa protocol amendment. All alterations ofthe protocol and reasons for the modification(s) shall be documented, signed and dated by the Study Director, QAU and test facility management and maintained with the protocol. Any deviation from this protocol will be recorded in the raw data and documented in the fmal report. 17. REFERENCES 1. EC, 2008. B.l3/14, Mutagenicity

- Reverse Mutation Test Using Bacteria. Official Journal ofthe European Communities. 2. Green, M.H.L. and W.J. Muriel (1976). Mutagen testing using trp+ reversion in Escherichia coli. Mutation Research 38:3-32. 3. Maron, D. M. and Ames, B. N. 1983. Revised methods for Salmonella mutagenicity test. Mut. Res., 113: 173-215. 4. Mortelmans, K. and Zeiger, E., 2000: "The Ames Salmonella Microsome Mutagenicity Assay'· Mutation Research, 455, pp. 29-60. 5. METI, 2004. Ministry of Economy, Trade and Industry

-- Reverse Mutagenicity Test on Bacteria, Methods of Testing New Chemical Substances (Section 5.1-1 to 5.1-11): Japanese Act on the Evaluation of Chemical Substances and Regulation of Their Manufacture, Act No. 117, Ministerial Ordinance No. 1-3. Enforced April2004. 6. OECD, 1997. Guideline for testing of chemicals 471. Adopted: 21 July 1997. Bacterial Reverse Mutation Test, PageNo.1

- 11. 7. Sword, I. P. and Thomson, R. 1980. Standard Operating Procedures. In vitro Toxicology, MTP Press Ltd., Lancaster, England. pp. 268. 8. U.S. EPA, 1998. Health Effects Test Guidelines, OPPTS 870.5100, Bacterial Reverse Mutation Test. 9. Vogel, H. J. and Bonner, D. M. 1956. Acetylomithinase of E. coli: Partial purification and some properties, J. Bioi. Chern., 218: 97-106. 10. Wilcox, P., Naidoo, A., Wedd, D. J. and Gatehouse, D. G. 1990. Comparison of Salmonella typhimurium TA!02 with Escherichia coli WP2 tester strains. Mutagenesis, 5:285-291. 11. Venitt, S., Crofton-Sleigh, C. and Forster, R. 1984: "Bacterial Mutation Assays using Reverse Mutation", In: Mutagenicity Testing

- A Practical Approach, Venitt, S. and Parry, J.M. [Eds], pp. 45-98.

Study Protocol



### Ill Ill /JRF Study Number: 481-1-06-XXXX Dow Study Number: 130186 18. PROTOCOLAPPROVAL- SPONSOR Address ofSponsor: DowEurope GmbH Bachtobelstrasse 3 CH-8810 Horgen Switzerland
Responsibility Name SponsorStudy Monitor DonnaAnderson
19. PROTOCOL APPROVAL- TEST FACILITY Address ofTest Facility, StudyDirector and Quality Assurance: Jai ResearchFoundation PostBoxNo. 30, GJ.D.C., Vapi- 396 195 Dist. Valsad, Gujarat India Responsibility Name Study Director NiraliN. Patel Quality Assurance Unit Dr. L. U. Sanghani TestFacility Management Dr. T. G. Marvania
20. SfUDYSCHEDULE Proposed Study Initiation Date Proposed Experimental StartingDate February
.., 2014 March
.., 2014 Signature

Signature

Proposed Experimental Completion Date Latestby March 25, 2014 Proposed Draft Report Date Latest by March 3I,2014 Proposed Study Completion Date Latestby April20, 2014 Study Protocol Bacterial Reverse MutationAssay Page 15 of15

Date

DateDow Study No:_I30Jg6._ JRF study No:

------- JRF Protocol No:
_____ _



### -------·--- PROTOCOL ANNEXURE FORM 

## *

*import:mf*

## * Pleaseprm'ide thefollowing**it~formation** EXACTLYas the sponsor wouldlike it to appear in thefinal report(s). l. STUDY SPONSOR I. Company: Dow Europe GmbH 2. Address: Bachtobdstrasse :i CH-8810 Hnrgen Switzerland II. TEST ITEM L Test item name: phosphorochloridothiotic acid, 0.0-dicthvl ester 2. Lot#: C03201 308150I If"lot" does not apply, please write the appropriate wording (e.g. Batc!t, Code, Fonnula) in the .1pace provided.
3. Chemical name: 0,0-Diethyl ester phosphorochloridothioic acid Synonym(s): diethylchlorothiophosphatc; DEPCT CAS No.: 2524-04-1 Molecular Weight:**-~18"'8""'.~6-----------------** 4. Is the documentation ofthe methods ofsynthesis/fabrication ofthe test item located atthe sponsor's facility'! 

### [2JYcs 0No (lfno, a location must be specified):-------------- 4. Physical Description (color+ physical state): colorless to light yellow liquid 5. Composition; for reasons ofconfidentiality, it may be appropriate to include percentages only for the active ingrcdient(s). All remaining [inert] components can be reported together as "other ingredients": Purity (as per 

## r8J Cert.ificale ofAnalysis 0MSDS 0Sponsor): The non-GLP certificate ofanalvsis lists the puritv as 99. I3% 0,0-Diethyl phosnhorochloridothioate (()invan, 2013). Specil!c gravity I density (include unit and temperature): (21To be performed by JRF QSponsor provided:
____ _ Certificate ofAnalysis: (21Availablc 

## 0 Not availabil' Cbaracterit.ation oftest item: 

## 0 GLP Onon-GLP* 

### 0 In Progress *To be rellectcu as exception to GLP compliance 6. pH:
____ [g)Not Available. please conductpH test.

Protocol Annexure h)rm Page 1 of3

·111 Ill \IDow Study No:_130J86_ JRF study No:
______ _ JRF Protocol No:
____**~--·-**

-=--·-------------- 0Water 0DMSO 0Iv!t:thanol 0Ethanol 0Acetont: 7. Solubility (check appropriate): 0Corn Oil 0Mineral Oil OOther (spccily):
____ _ [;8JNot Availablc, please conduct a solubility test (results to he provided to the Sponsor prior to treatment date(s). 8. Handling Precautions (Please supply MSDS ifavailable): [;8JMSDS Availahle**0Bm~ic** 00thcr (specify): 9. Storage Conditions: 12$1Ambient [;8JOthcr (specify): Nitrogen pad 0Refrigcrated 0Frozcn 0No MSDS

JO. Is the product expected to be stable under standard conditions for the duration ofthe study? [gjYes 0No I l. Recertification/Expiration Date (MM/DD/YY):

------- *To be reflected as exception to GLP compliance
III. ANALYTICAL CHEMISTRY (IF APPLICABLE) (g!Not Available*

I. Conductdose formulation stability, homogeneity, and concentration analysis: IZJYcs, atJRF

## 0 No* *To be reflected as exception to GLP compliance 2. Analytical Method Information: IZJAvailahle, provided by Sponsor 0Not applicable 

## 0 Not available from Sponsor, JRF to develop a feasible analytical method Analytical Monitoring Scientist Name:**""GC.!.Cn:!!ln.!!:t-=.M~ar~tyl--------------­** Title: Analytical Study Monitor Address: The Dow Chemical Company. 1803 Bldg.- MIOPS, Midland. MI 48674 USA

IV. GLPCOMPLIANCE: OEPA-FIFRA 0JMAFF (Check the appropriate agency(ies)) IZJEPA-TSCA IZJOECD 0METI OOther (specify):----------
V. INFORlVIATION FOR SAMPLE RETURN ORDISPOSAL: Disposition ofremaining sampk (not the retention sampks in archives) after testing is complete: 0Rcturn 0Disposc/Non-Ha:atrdous IZ!Disposc/Hazardous 0Return Charge: Acwal
Hazardous Shipping Name:
_______ _ DOT Class oftest itern (e.g. flammable, poison, corrosive, ell:.):-----·

Prowco! Annexure Form Pagt.: 2 ol. 3Dow Study No:_i30lH6_ JRF study No: JRF Protocol No:
________ _**---~-·-~------·** 

### -·-------·------------- Disposition ofthe retention samples in archives (after the rett•ntion period): 0Return [8JDispost: Ifthe sample is to he returned, shipping info will be provided by email. VI. STODY RESULTS: Would tlw sponsor !.ike to receive a summary oftesting results upon experiment completion? [8JYcs 0No
VII. REPORTS: I. The sponsor must review a quality assurance audited draft report prior to the final report being issued: t8JYes Method ofTransmission: t8JE-mail (No Charge) 2. The final report and raw data will be shipped to the Sponsor upon study completion. VIU. APPROVAL: l. Sponsor's Study Monitor By typing Monitoring Scientist's name on the line belowandtransmitting thh,· information to the Test l''acility ver(f.v that the information contained in this document is, to the best ofhidherknowledge,factual and can he usedby the Test Facility for theptaposes of conducting the approvedstudies undpreparing a jinal report. Monitoring Scientist Name: D"'-'-'o_,__,n_,_,n,_a__._A"''-'-'1c"-'!e'"'r""s(,_,JJ"-l
____________________ _ Title: Study Monitor 11 /'}**.,,~.r** /'./ ' ;",

..(' (..!i •. ' ,,. ' (,.·,"**·~** ,:l...,.,,f"'lf
... fd'J.,A

.••**-~1...** 1 I Signature:**---""'-"-·-------~.l~/ ''**  :"''.(""1(='- • .(i'""':o...:t_·'_!

.. _J:...f_>_·::·_·c_.,_·''

__ L .i'' "'"·-- Date: 13 January 2014

Protocol Annexure rorm Page 3 of3

Ill Ill

# (

Appendix F Mammalian Erythrocyte Micronucleus Proposed Protocol

(

. '-, /' 

### 18 DEPCT\\ \ \\\PRELIMINARY PROTOCOL TOXICOLOGY AND ENVIRONMENTAL RESEARCH AND CONSULTING THE DOW CHEMICAL COMPANY 

# PROTOCOL
1803 BUILDING, MIDLAND, MICHIGAN 48674
TITLE: EVALUATION OF PHOSPHOROCHLORIDOTHIOTIC ACID, 0,0-DIETHYL ESTER (DEPCT) IN THE MOUSE PERIPHERAL BLOOD MICRONUCLEUS TEST

KEY NUMBERS FILE#: DR-0006-I347-00I STUDY ID: I3II28 PTR#: IOOOI843-29I-I CAS #: 2524-04-I PLANNED DATES EXPERIMENTAL START: 04 March 20I4 EXPERIMENTAL TERMINATION: I5 July 20I4 ESTIMATED FINAL REPORT: II August 20I4 GLP STUDY: Yes SPONSOR: DowAgroSciences LLC 9330 Zionsville Road Indianapolis, Indiana 46268 TESTING FACILITY: The Dow Chemical Company Toxicology and Environmental Research and Consulting I803 Building Midland, Michigan 48674 REQUIRED SIGNATURES: STUDY DIRECTOR/: LEAD SCIENTIST M. R. Schisler, B.S. MANAGEMENT APPROVAL: M. R. Woolhiser, Ph.D. SPONSORor DESIGNEE: D. R. Juberg, Ph.D. MANAGEMENT ASSIGNED STUDY PERSONNEL: Technical Reviewer: M. J. LeBaron, Ph.D. Toxicology Technologist: L. K. Sosinski, B.S. ADDITIONAL STUDY CONTACTS: Analytical Chemist: L. A. Douglass, A.S. Document Management: K. Collier Quality Assurance Auditor: H. M. Mahan, B.S., M.T. (A.S.C.P.) /DATE

/DATE
/DATE

### A. TITLE THE DOWCHEMICAL COMPANY STUDY ID: 131128 PAGE2



### EVALUATION OF PHOSPHOROCHLORIDOTHIOTIC ACID, 0,0-DIETHYL ESTER (DECPT) IN THE MOUSE PERIPHERAL BLOOD MICRONUCLEUS TEST B. 

### INTRODUCTION Purpose The purpose ofthe studywill be to determine the cytogenetic effects of phosphorochloridothiotic acid, 0,0-diethyl ester (DEPCT) in the mouse by examining the incidence ofmicronuclei inperipheralblood reticulocytes. Test Guidelines The assay will be conducted to meet the following guidelines. OECD

USEPA
EC Guidelines for Testing of Chemicals, 

### #474: Genetic ToxicologyMicronucleus Test, OECD Publication Service, 2 rue AndrePascal, 75775 Paris Cedex 16, France (1997). Office ofPrevention, Pesticides and Toxic Substances, OCSPP 870.5395. MammalianErythrocyte Micronucleus Test (1998). B.12, Mutagenicity- In Vivo 

### MammalianErythrocyte Micronucleus Test. Official Journal ofthe European Communities, L142, 31/05/2008. C. TEST MATERIAL INFORMATION Test Material Name phosphorochloridothiotic acid, 0,0-diethyl ester ChemicalNarne 0,0-Diethyl esterphosphorochloridothioic acid Synonyms Diethylchlorothiophosphate, DEPCT Supplier, City, State (Lot, Reference Number) The Dow Chemical Company, Midland, Michigan (Lot# C032013081501)

·111 Ill 

### )\. THE DOW CHEMICAL COMPANY STUDY ID: 131128 PAGE3 

### Purity/Characterization (Method ofAnalysis and Reference) The non-GLP certificate ofanalysis lists the purity as 99.13% 0,0-Diethyl phosphorochloridothioate (Qinyan, 2013). 

### Characteristics Molecular Formula

Molecular Weight 188.6 Chemical Structure

CAS Number 2524-04-1 

### D. POSITIVE CONTROL CHEMICAL ChemicalNarne Cyclophosphamide monohydrate (CP) 

### CAS Number 6055-19-2 

### Source Sigma, St. Louis, Missouri 

### E. DOSE, ROUTE, AND JUSTIFICATION Animal Dosing The test material, the vehicle (negative control), and CP (positive control) will be administered byoral gavage. Oral gavage is one ofthe generally accepted routes ofTHE DOW CHEMICAL COiviPANY STUDY ID: 131128 PAGE4 

### administering the test material in this assay system. CP, when administered by oral gavage, has been shown to induce micronuclei in polychromatic erythrocytes ofCD-I mice (De Boeck 

### et al., 2005; Gollapudi 

### et al., I986). Dosing solutions will be administered in aliquots ofI0-20 ml/kg bodyweight. Dose Levels 

### Test Material The dose levels for the micronucleus test (MNT) will be selected based upon the range finding test. The maximum dose for the micronucleus study will be the estimated maximumtolerated dose (MTD). MTD is defined as the dose that produces clear signs oftoxicity without having a major effect onthermal regulation orthe survival ofthe animals. For changes in thermal regulation, a body temperature increase ofat least I

# oc or a decrease ofat least 3

# oc for five or more hours will be declared as having exceeded an MTD. Body temperature changes outside this range have been previouslyreportedto cause an increase in micronucleus formation in absence ofchemical treatment (Asanami and Shimono, I997; Asanami 

### etal., I998). With some test materials, death ofthe treated animals may be the only sign oftoxicity at doses above the MTD and no other clinical signs oftoxicity may be apparent at the MTD. In such cases, the MTD will be a dose in betweenthe doses that induced some mortalities and none. These are only general guidelines in determining the MTD, and the Study Director will take into consideration other factors such as the dose-mortality curve, any other available toxicological data, etc. Relatively non-toxic compounds will be tested up to 2 g/kg bodyweight/day. The middle- and lowdoses will be approximately I/2 and 114 ofthe maximum dose, respectively. The test material will be administered ontwo consecutive days, approximately 24 hours (± 1hour) apart. The dose level for CP (40-60 mg/kg) was based upon our experience, which indicated a pronounced micronuclei induction in CD-I mice at this dose level. CP will be administered only once, approximately 48 hours(± I hour) before sacrifice
.

.Til. Ill

## ( THE DOW CHEMICAL COMPANY STUDY ID: 131128 PAGES 

### Rationale for Selection ofSampling Intervals for MNT It

### takes approximately 48 hours for the treated erythroblasts to undergo one posttreatment division and mature into reticulocytes and reach a peak incidence in the peripheral blood (CSGMT, 1992). However, ifthe treatment induces cell cycle delay, orthere is a delayed absorption and metabolism ofthe test material, a longer sampling (such as 72 hours) may be needed to recover the damage induced in the erythroblasts. The sampling time used in the current protocol will provide opportunityto recover any delayed effect (i.e., effect induced by the first treatment) as well as a more immediate effect 

### (i.e., effect induced bythe second treatment) in the same treated animals. Preparation ofDosing Solutions 

### The test material will be mixed in a suitable matrix for dosing. The details ofthe preparation ofthe dosing matrix will be incorporated in the study file. CP will be mixed in distilled water. Only freshly prepared solutions ofthe test material will be used for dosing unless there are data indicating the stability ofthe test material inthe vehicle. Either a freshly prepared solution or a frozen stock solution ofthe positive control will be used for dosing. Analytical Verification ofDosing Solutions 

### For the MNT, uponmixing, the selected concentrations ofthe test material inthe dosing matrix, used for the first day ofdosing, will be checked by the Analytical Chemistry Laboratory, Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan. Samples will be analyzed by gas chromatographyusing electrochemical detection (GC/ECD) and external standard quantitation. Homogeneity analysis will be performed in cases where the test material is administered as a suspension. Dose Range Finding Test (RFT) 

### The RFT may be composed ofone or more phases. Initially, two dose groups for each sex will be evaluated in the RFT. The number ofanimals included in each dose group will be based upon the use ofthe dose selection criteria (Appendix A). Two extra groups ofthree animals will be retained and used iffurther dose levels ofthe test article need to be evaluated due to excessive toxicity in the first two dose levels.THE DOW CHEMICAL COMPANY STUDY ID: 131128 PAGE6 

### The mice will be observed daily for at least 72 hours or until day 4 ofthe study (day 1 is defined as the frrst day ofdosing). Signs oftoxicity and deaths occurring during the observationperiod will be recorded. All mortalities and/or moribund animals will be necropsied to ascertain whether the condition is related to a dosing mishap. F. TEST SYSTEM 

### Principle ofthe Test System The MNT is a short-term in vivo cytogenetic assay for detecting agents that induce chromosomal breakage and spindle malfunction (Mavournin 

### etal., 1990). Micronuclei arise when acentric chromosomal fragments and/or whole chromosomes are excluded from the main nucleus at the telophase stage ofthe cell division. The MNT has several advantages over a cytogenetic assay involving direct examination ofmetaphase chromosomes. The MNT provides information on chromosome loss due to spindle impairment whereas such information cannot be readily obtained with metaphase chromosome analysis. The end point scored in the MNT is less subjective than in metaphase analysis. The significance ofmicronuclei is comparatively clear versus some parameters measured in metaphase analysis, e.g., gaps. 

### Species and Sex Male and female mice will be used for the RFT. Only males will be used for the MNT ifthe results ofthe RFT showno apparent difference inthe toxicity between the sexes. Otherwise, both sexes will be used for the MNT as well. 

### Strain Outbred Crl:CD1(ICR) mice 

### Criteria for Selectingthe Strain

1. Availability ofhistorical negative control data. 2. Suitability for utilization in the 

### MNT: 3. General suitability for toxicity testing. 

### Supplier Charles River (Kingston, New York)

·11 I Ill

### Age at Study Start 

### Approximately 8-12 weeks Physical and Acclimation THE DOW CHEMICAL COMPANY STUDY ID: 131128 PAGE7



### During the acclimationperiod each animal will be evaluated by a laboratory veterinarian, or a trained animal/toxicologytechnician under the direct supervision of a laboratory veterinarian, to determine the general health status and acceptability for study purposes. The Toxicology and Environmental Research and Consulting Laboratory is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). The animals will be housed one per cage in stainless steel cages, in rooms designed to maintain adequate conditions (temperature, humidity, and photocycle), and acclimated to the laboratory for at least one week priorto the start ofthe study. Housing 

### After assignment, animals will be housed one per cage in stainless steel cages. Cages have wire mesh floors and are suspended above absorbent paper. Non-woven gauze will be placed inthe cages to provide a cushion from the flooring for the rodents' feet. The gauze will also provide environmental enrichment. Cages will contain a hanging feeder and a pressure activated lixit valve-type watering system. The following environmental conditions will be maintained in the animal room. Temperature: Humidity: Air Changes: Photoperiod: 22°C with a tolerance of± 1 

### °C (and a maximum permissible excursion of± 3°C) 40-70% 12-15 times/hour (average) 12-hour light/dark (on at 6:00a.m. and offat 6:00p.m.) Randomization and Identification 

### Before administration oftestmaterial begins, animals will be stratified by body weight and thenrandomly assigned to treatment groups using a computerprogram designed to increase the probability ofuniformgroup meanweights and standard deviations at the start ofthe study. Animals that are placed on studywillbe uniquely identified via subcutaneously implanted transponders (BioMedic Data Systems,THE DOW CHEMICAL COMPANY STUDY ID: 131128 PAGES 

### Seaford, Delaware) that are correlated to unique alphanumeric identification numbers. Ifa transponder stops functioning or is lost, it will be replaced with a new transponder that will be correlated with the unique animal number. Feed and Water Feed and municipal water will be provided 

### adlibitum. Animals will be provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in pelleted form. Analyses ofthe feed will be performed by PMI Nutrition International to confirmthe diet provides adequate nutrition and to quantify the levels ofselected contaminants. Drinking water obtained from the municipal water source is periodically analyzed for chemicalparameters and biological contaminants bythe municipal water department. In addition, specific analyses for chemical contaminants are conducted at periodic intervals by an independent testing facility. Copies ofthese analyses are maintained in the study file. Animal Welfare In accordance with the U.S. Department ofAgriculture Animal Welfare Regulations, 9 CPR, Subchapter A, Parts 1-4, the animal care and use activities required for conduct ofthis study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). The IACUC has determined that the proposed activities are in full accordance with these Final Rules. The IACUC-approved Animal Care and Use Activities to be used for this study are Genetic Tox 01, Blood Collection 01, Humane Endpoints 01, and Animal ID 01. G. STUDY DESIGN Micronucleus Test Groups ofmice (six/dose) will be administered dose levels ofthe test material ontwo consecutive days based onthe results ofthe RFT. Extra animals (up to six mice) may be included at the highest dose level to serve as possible replacements for study animals in the case ofexcessive toxicity or death. The decisionto dose the extra animals will be made bythe StudyDirector based uponthe results ofthe RFT. The mice will be observed daily, and signs oftoxicity or deaths occurring during the observationperiod will be recorded. All mortalities and/or moribund animals will be necropsied to ascertainwhether the condition is related to a dosing mishap.

11 I Ill

# ( THE DOW CHEMICAL COMPANY STUDY ID: 131128 PAGE9 

### Approximately 48 hours following the last dosing, a peripheral blood sample will be collected from the orbital sinus ofall surviving animals following anesthesia with isoflurane. Animal Observations and BodyWeight Determination 

### The mice will be observed for positive signs oftoxicity at least once onthe day of dosing and subsequent days. The mice will be weighed prior to dosing and onthe day oftheir scheduled sacrifice in orderto assess the extent ofstress experienced by the animals following treatment (Gollapudi 

### et al., 1986). Body Temperature Data Collection 

### The relative bodytemperatures ofthe treated animals will be monitored during the RFT and provisionally during the MNT using programmable transponders (BioMedic Data Systems, Seaford, Delaware). The temperatures will generally be collected by scanning the microchip prior to each dosing, approximately 2, 5, and 24 hours after each dosing as well as prior to sacrifice. Peripheral Blood 

### Micronucleus formation in peripheral blood reticulocytes (RETs) will be determined by flow cytometrywithtraditional blood smears prepared as a backup. Samples will be prepared and analyzed as per instructions inthe mouse MicroFlow Micronucleus Analysis Kit Manual (Litron Laboratories, Rochester, New York). Briefly, blood samples from all animals in all experimental groups will be obtained viaretro-orbital sampling into anticoagulant solution and fixed in ultracold (-70 to -80°C) methanol within five hours ofcollection. All fixed blood samples will be stored at -80°C. Fixed blood samples will be washed with a cold, buffered salt solution and isolated by centrifugation. The resulting cell pellets will be stored at 4°C until staining. Blood samples will ultimately be incubated with RNAse to degrade the high levels of RNA present inthe RET and a fluorescently labeled antibody to the transferrin receptor (anti-CD71-FITC) to stain the RETs. A propidium iodide solutionwill be added to each sample immediately before flow cytometry (FCM) analysis to stain the DNA, including that ofmicronuclei. Blood samples will be analyzed by high-speed FCM. Inthis system, the sample is moved at a highvelocitypast a laser set to provide 488 nmexcitation. The fluorescent wavelengths emitted by each cell will be collected by photomultiplier tubes. Using the previously described stainingTHE DOW CHEMICAL COMPANY STUDY ID: 131128 PAGE10 

### procedure, the propidium iodide-stained DNA ofthe micronuclei emits a red fluorescence, and the anti-CD71-FITC antibody emits a high green fluorescent signal permitting differentiation between cells with and without micronuclei. In addition to obtaining fluorescent profiles, FCM simultaneouslyprovides cell size information by determining the light scatter properties ofeach cell or combination ofcells. 

### Cells Examined Whenever possible, approximately 5,000 RETs will be analyzed per blood sample. The number ofnormochromatic erythrocytes (NCE), MN-NCE, RET, and MN-RET will be recorded for each sample, and the frequency ofMN-RET calculated to provide an indication ofgenotoxic potential. The frequency ofRETsrelative to total erythrocytes will be determined to provide an indication ofperturbations in hematopoietic activity indicative ofcell toxicity. For each ofthe treatment groups, a mean and standard deviation will be calculated to describe the frequency ofRET and MN-RET observed. Analyses will be conducted utilizing a Beckman Coulter Gallios flow cytometer. 

### Details ofSlide Preparation Duplicate cell smears will be prepared and storedto serve as backups in the event that flow cytometric analyses are not possible. Bloodwill be collected into a microtainer tube coated with EDTA (Becton Dickinson, Franklin Lakes, New Jersey). Wedge smears will be prepared, fixed in methanol, and stored at roomtemperature. If needed, the slides will be stained with Acridine Orange and manually scored. 

### H. STATISTICS AND CALCULATIONS MN-RET and percent RET will be tested for equality ofvariance using Bartlett's test (alpha= 0.01; Winer, 1971). Iftheresults from Bartlett's test are significant, then the data for the parameter may be subjected to a transformation to obtain equality ofthe variances. The transformations that will be examined are the common log, the inverse, and the square root in that order. The datawill be reviewed and an appropriate form ofthe data selected and subjected to the following analysis. Based onthe results ofthe sex effect in the RFT, the MN-RET data and the data on percent RET may be analyzed by a two-way analysis ofvariance (when both sexes are used in the study) or a one-way analysis ofvariance (when only males are used)

11 i Ill( \"- THE DOW CHEMICAL COMPANY STUDY ID: 131128 PAGE 11 

### (Winer, 1971). The sex by dose interaction in the two-way analysis will be reviewed and ifsignificant, a one-way analysis ofvariance will be performed for each sex. If different dose levels ofthe test material are used for each sex, the data will be separated by sexprior to statistical analysis. Pairwise comparisons oftreated vs. control groups will be done, ifthe dose effect is significant, by Dunnett's t-test, onesided (upper) for MN-RET and two-sided for the percent RET (Winer 1971). Linear dose-related trend tests will be performed ifany ofthe pairwise comparisons yield significant differences. The alpha level at which all tests will be conducted is 0.05. The MN-NCE will not be analyzed statistically and will only be used as an adjunct end pointto evaluate the biological significance ofthe MN-RET results. The final interpretation ofbiological significance ofthe responses is based on both statistical outcome and scientificjudgment. A test is considered valid ifall ofthe following conditions are met:
• The range ofMN-RETvalues in the negative controls are within reasonable limits ofthe recent laboratory background range. • There is a significant increase inthe incidence ofMN-RET in the positive control treatment as compared to the concurrent negative controls.
• The mean for percent RET value in one ormore ofthe test material treated groups should be 2:: 

### 20% ofthe control value indicating no undue effect on erythropoiesis (toxicity). A test material is considered positive inthis assay ifthe following criterion is met:
• Statistically significant increase in MN-RET frequency at one ormore dose levels accompanied by a dose response. A test material is considered negative in this assay ifthe following criterion is met:
• No statistically significant dose-related increase in MN-RET when compared to the negative control. A test result not meeting the criteria for either the positive orthe negative response will be considered to be equivocal.

### I. FINAL REPORT THE DOW CHEMICAL COMPANY STUDY ID: 131128 PAGE12



### A final report ofthe definitive study will be submittedto the study sponsor and will include but not be limited to the following:

1. Name and address ofthe facility performing the study and the dates onwhich the study is initiated and completed, 2. Objectives and procedures stated in the approved protocol, including any changes inthe originalprotocol, 3. Statistical methods employed for analyzing the data, 4. Description oftest and/or control substance (e.g., 

### lot#, purity, physical characteristics, and method ofpreparing test concentrations), 5. Stability ofthe test and control articles under the conditions ofadministration and description ofthe analytical methods used, 6. A description ofthe test systemused including, when applicable, the number of animals used, sex, body weight range, source ofsupply, species, strain, and substrain, age, and procedure used for identification, 7. A description ofthe dosage, dosage regimen, route ofadministration, and duration, 8. A description ofall circumstances that may have affected the quality orintegrity ofthe study, 9. The results ofthe study, discussion ofresults, and conclusions, 10. Location ofraw data and specimens, 11. List and signatures ofstudypersonnel, Statement signed by the Quality Assurance Unit. 

### J. REGULATORY COMPLIANCE GLP Standards Organisation for Economic Co-Operation and Development (OECD)

-- OECD Series onPrinciples ofGood LaboratoryPractice and Compliance Monitoring, Number 1. OECD Principles on Good Laboratory Practice (as revised in 1997) ENV/MC/CHEM(98)17 US Environmental ProtectionAgency

-- TSCA GLPs Title 40 CFR, Part 792

- Toxic Substances Control Act TSCA); Good Laboratory Practice Standards, Final Rule 

### Quality Assurance The study conduct, data, protocol, protocol deviation(s)/amendment(s) ifapplicable, and final report will be inspected bythe Quality Assurance Unit, Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan.

11 I IllI \ 

### Study Archives THE DOW CHEMICAL COMPANY STUDY ID: 131128 PAGE13

The data, protocol, protocol deviation(s)/amendment(s) ifapplicable, and final report will be archived bythe Toxicology and Environmental Research and Consulting archivist and stored at The Dow Chemical Company, Midland, Michigan. 

### K. SAFETY Allpersonnel involved in the studywill be advised ofthe safetyprecautions to follow when handling the test and positive control materials. Chemical safety information will be made available. 

### L. REFERENCES Asanami, S. and Shimono, K. (1997). Highbodytemperature induces micronuclei in mouse bone marrow. 

### Mutat. Res. 390, 79-83. Asanami, S., Shimono, K., and Kaneda, S. (1998). Transient hypothermia induces micronuclei in mice. 

### Mutat. Res. 413, 7-14. CSGMT (The Collaborative Study Group for the Micronucleus Test) (1992). Micronucleus test withmouse peripheral blood erythrocytes by acridine orange supravital staining: the summaryreport ofthe 5thcollaborative studyby CSGMT/JEMS.MMS. The Collaborative Study Group for the Micronucleus Test. De Boeck, M., Vander Leede, B., Van Goethem, F., De Smedt, A., Steemans, M., Lampo, A., and Vanparys, P. (2005). Flow cytometric analysis of micronucleated reticulocytes: Time- and dose-dependent response ofknow mutagens inmice using multiple blood sampling. 

### Environmental andMolecular Mutagenesis 46, 30-42. Gollapudi, B. B., Bruce, R. J., Linscombe, V. A., Watanabe, P. G., and Sinha, A. K. (1986). Effect offood and water deprivation onthe parameters ofthe mouse bone marrowmicronucleus test. 

### Mutat. Res. 173, 121-125. Mavournin, K. H., Blakey, D. H., Cimino, M. C., Salamone, M. F., and Heddle, J. A. (1990). The 

### in vivo micronucleus assay in mammalianbone marrow and peripheral blood. A report ofthe U.S. Environmental ProtectionAgency GeneTox Program. 

### Mutat. Res. 239, 29-80. Qinyan (2013). Certificate ofAnalysis ofO,O-Diethylphosphorochloridoth (DEPCT), Lot C032013081501. Zhejiang Xinnong Chemical Co., Ltd., Sanlixi, Xianju County, Zhejiang Province, China.THE DOW CHEMICAL COMPANY STUDY ID: 131128 PAGE 14 

### Winer, B. J. 

### (1971). Statistical Principles in ExperimentalDesign, 2nd ed., McGrawHill, 

### New York.

11 I Ill

## ( THE DOW CHEMICAL COMPANY STUDY ID: 131128 PAGElS EVALUATION OF 0,0-DIETHYL PHOSPHOROCHLORIDOTHIOATE IN THE MOUSE PERIPHERAL BLOOD MICRONUCLEUS TEST 

### Appendix A. 

### Dose Selection Criteria 1. Acute oral toxicity datais available inthe rat ormouse. a. The existing dataindicates no toxicity and the calculated oral LD50 

### is > 2000 mg/kg

- The initial dose levels evaluated in the RFT will be 2000 and 1000 mg/kg/day b. The existing data indicates the calculated oral LD50 

### is < 

### 2000 mg/kg

- The initial dose levels evaluated inthe RFT will be LD50 

### and 

### Yz LD50 

### 2. Acute oral toxicity datais not available.
a. Dose 1 animal/sex at 2000 and 1000 mg/kg and observe animals for 24 hrs after the second dosing -Ifall animals survive, dose an additional2 animals/sex at dose levels of2000

and 1000 mg/kg.

- Ifthe animal dosed with 2000 mg/kg dies and clinical signs are present in the

animal receiving 1000 mg/kg, dose an additional 2 animals/sex at 1000 and 3 animals/sex at 500 mg/kg.

- Ifanimal dosed with 2000 mg/kg dies and NO clinical signs are present at 1000

mg/kg, discuss with study director/lead scientist regarding the addition ofa dose level between 2000 and 1000 mg/kg.111 IllAppendix G Stability in Rodent Feed and Corn Oil: Preliminary Study Protocol



### 19 

### DEPCT! I

111 Ill )\ )FINAL PROTOCOL TOXICOLOGY AND ENVIRONMENTAL RESEARCH AND CONSULTING THE DOW CHEMICAL COMPANY 

# PROTOCOL
1803 BUILDING, MIDLAND, MICHIGAN 48674
TITLE: DIETHYLCHLOROTHIOPHOSPHATE (DEPCT): METHOD VALIDATION AND STABILITY IN RODENT FEED AND CORN OIL

KEY NUMBERS FILE#: K-006538-013 STUDY ID: 141009 PTR#: 1 0001843-324-1 CAS #: 2524-04-1 PLANNED DATES EXPERIMENTAL START: 01/27/2014 EXPERIMENTAL TERMINATION: 04/02/2014 ESTIMATED FINAL REPORT: 7/26/2014 GLP STUDY: YES SPONSOR: Dow Agrosciences LLC 9330 Zionsville Road Indianapolis, IN 46268 TESTING FACILITY: The Dow Chemical Company Toxicology and Environmental Research and Consulting 1803 Building Midland, Michigan 48674 REQUIRED SIGNATURES: LEAD SCIENTIST/ STUDY DIRECTOR:
MANAGEMENT APPROVAL:
SPONSOR orDESIGNEE: A. W. Perala, B.S.

# /lt4£WJLM. R. Woolhiser, Ph.D. D. R. Juberg, Ph.D.
MANAGEMENT ASSIGNED STUDY PERSONNEL: Technical Reviewer: K. A. Brzak, B.S. Chemist: J. R. McFadden, B.S. Pharmacy Contact: M. K. Scherzer, B.S. Tox Contact C. L. Zablotny, B.S. Tox Tech J. J. Fisher ADDITIONAL STUDY CONTACTS: Business Consultant: R. J. Rasoulpour, Ph.D. Document Management: K. Collier Quality Assurance Auditor: T. H. DeLisle, B.S. Study Director in Training: J. R. McFadden, B.S. 1 !J!>ATE

# ~**1/.2'(/J~** /DATE

### A. TITLE THE DOWCHEMICAL COMPANY STUDY ID: 141009 PAGE2



### DIETHYLCHLOROTHIOPHOSPHATE (DEPCT): METHOD VALIDATION AND STABILITY IN RODENT FEED AND CORN OIL B. 

### INTRODUCTION Purpose The purpose ofthis study is to validate the analytical method for determination of diethylchlorothiophosphate (DEPCT) in rodent feed and com oil and to evaluate the stability ofdiethylchlorothiophosphate (DEPCT) in rodent feed for a period of approximately 65 days and in com oil and toluene for a period ofapproximately 24 days. Work is to be performed in accordance with Standard Operating Procedure ACL-GEN-017.03. 

### Test Guidelines C. TESTMATERIAL INFORMATION There are no test guidelines for this study. 

### TestMaterial Name phosphorochloridothiotic acid, 0,0-diethyl ester 

### Chemical Name 0,0-Diethylester phosphorochloridothioic acid 

### Synonyms Diethylchlorothiophosphate, DEPCT 

### Supplier, City, State (Lot, Reference Number) The Dow Chemical Company, Midland, Michigan (Lot#C032013081501) 

### Purity/Characterization (Method ofAnalysis and Reference) The non-GLP certificate ofanalysis lists the purity as 99.13% 0,0-Diethyl phosphorochloridothioate (Qinyan, 2013). 

### Characteristics Molecular Formula

111 Ill )I \ 

### MolecularWeight 188.6 Chemical Structure

CAS Number 2524-04-1
Previous Stability Information None D. EXPERIMENTAL DESIGN THE DOW CHEMICAL COMPANY STUDY ID: 141009 PAGE3



### Stabilitytime points for Diethylchlorothiophosphate (DEPCT) in rodent feed will be done on days 0, 2, 4, and 8. Ifday 8 recovery is greaterthan 85% at any ofthe test concentrations, analyses on days 12, 24, 36, and 65 will be performed as well. Stabilitytime points for Diethylchlorothiophosphate (DEPCT) in corn oil and toluene will be done on days 0, 2, 4, and 8. Previous stabilities will be taken into consideration before continuing with subsequent analyses. All time points maybe adjusted at the discretion ofthe study director based upon scheduling and data obtained from earliertime points. Analytical method validation will beperformedbyfortification ofrodent feed and corn oil with Diethylchlorothiophosphate (DEPCT) at the concentration range of interest, as perTERC SOP ACL-GEN-013. This will include at least 6-9 fortified (spiked) samples (e.g., 

### duplicate or triplicate analyses at three concentration levels). Samples ofcontrol matrix will be analyzed as well. Appropriately sized aliquots of the samples will be analyzed to obtain an accurate determination oftest chemical concentration (e.g., 

### 2-5 grams for animal feed, 0.01

- 5 mL for solutions, 0.5- 5 grams for suspensions).THE DOW CHEMICAL COMPANY STUDY ID: 141009 PAGE4


### E. STUDY SPECIFIC PARAMETERSRoute and Justification This study will be performed to determine stability ofthe test material in the test system in support oftoxicological studies where this test system will be used. The method ofadministration is by direct addition ofneat material to the vehicle. This is the acceptable method for preparation ofthe test article for use in toxicological studies. 

### Data and Report Concentration oftest material in the test matrix, relative to the initial analysis results (day 0 or 1), will be reported for each test day analysis. Accuracy and precision for recovery oftest material from samples will be determined byanalyzing freshly prepared matrix fortified samples with each analysis set. These recoveries will be applied to the determined sample concentrations in an appropriate manner as defined in the raw data. A method validation report will include a representative standard curve and table, spike recoveries, and example chromatograms ofa standard solution, fortified sample, and matrix blank. 

### Vehicle Lab Diet Certified Rodent Diet #5002M, Lot#TBD PMINutrition International St. Louis, Missouri Corn Oil, Lot# MKBL8756V Sigma-Aldrich, St. Louis, Missouri Solvent- Toluene, Lot#lll565 Fisher Scientific, FairLawn, New Jersey



### Identification Samples are identified by a unique sample identifier (i.e. 

### concentration level) that will be used throughout the study for identification and tracking.

Ill Ill

## ( Matrix THE DOWCHEMICAL COMPANY STUDY ID: 141009 PAGES 

## [K] Feed (rodenUcanine) 

## [K] Solvent 

# I X

# ISolution/suspension 

### Stability Timepoints for Rodent Feed (Days) 

## [K] 

# o 

# D1 

## [K] 2 

## []l 4 

## []l 8 

# 00**12~** 24 

## []l 36[]] 65 

### StabilityTimepoints for Corn Oil (Days) 

## [K] o 

# D1 

## [K] 2

### ~ 4

### ~ 8 

# [R]**12~** 24 

# D 36 

# D 65 

### Stability Timepointsfor Solvent (Days) 

## [K] o 

# D1 

## [K] 2

### ~ 4 

# CKJ 8 

# CKJ 12 []] 24 

# D 36 

# D 65 

### Stability Concentrations 

# [K] 10%/0.5%/0.05%/0.005%/0.0005% (w/w), (Rodent Feed) 

## [K] 250/25/2.5/0.25 (mg/mL), (Corn Oil) 

## [K] 10,000 ([!g/mL), (Toluene) Note: time points and concentrations may change at discretion ofstudy director actual time points and dates will be recorded in the raw data file. 

### Analysis Method 

## [Kj GC 

# D HPLC 

# D GC/MS 

# D LC/MS Reference: give previous report reference ifavailable

Standard concentrations will bracket the sample concentrations, after preparation for analysis. The target LLQ will be 11100 ofthe lowest sample concentration. Note: LLQ may be changed at discretion ofstudy director, based on method sensitivity Specific details ofthe analytical methods will be included in the study file.THE DOW CHEMICAL COMPANY STUDY ID: 141009 PAGE6 

### Storage Conditions - Rodent Feed, Corn Oil, and Toluene 

# I X

# IAmbient (- 25°C) 

# 0 Refrigerator (- 5°C) 

# I X

# IExposed to light 

# 0 Amber glass 

### F. STATISTICS AND CALCULATIONS 

# 0 Freezer (--l5°C) 

# 0 Light proofcontainer or storage location
Descriptive statistics will be used, 

### i.e., mean± standard deviation, or relative standard deviation (standard deviation/mean). All calculations will be conducted using Microsoft Excel and Applied Biosystems/MDS SCIEX Analyst spreadsheets in full precision mode (15 digits ofaccuracy). 

### G. FINAL REPORT A final report ofthe definitive study will be submitted to the study sponsor and will include but not be limited to the following: 1. Name and address ofthe facility performing the study and the dates on which the study is initiated and completed, 2. Objectives and procedures stated in the approved protocol, including any changes in the original protocol, 3. Statistical methods employed for analyzing the data, 4. Description oftest and/or control substance (e.g., lot#, 

### purity, physical characteristics, and method ofpreparing test concentrations), 5. Stability ofthe test and control articles under the conditions ofadministration and description ofthe analytical methods used, 6. A description ofthe concentrations, 7. A description ofall circumstances that may have affected the quality orintegrity ofthe study, 8. Location ofraw data, 9. List and signatures ofstudypersonnel, 10. Statement signedbythe Quality Assurance Unit. 

### GLPStandards Organization for Economic Co-Operation and Development (OECD)

-- OECD Series on Principies ofGood LaboratoryPractice and Compliance Monitoring, Number 1. OECD Principles on Good Laboratory Practice (as revised in 1997) ENV/MC/CHEM(98)17 US Environmental Protection Agency

-- TSCA GLPs Title 40 CFR, Part 792

- Toxic Substances Control Act TSCA); Good Laboratory Practice Standards, Final Rule US Environmental Protection Agency

-- FIFRA GLPs Title 40 CFR, Part 160

- Federal Insecticide, Fungicide and Rodenticide Act (FIFRA); Good Laboratory Practice Standards, Final Rule

'111 Ill \**~-** 

### ( THE DOW CHEMICAL COMPANY STUDY ID: 141009 PAGE7 

### The Japanese Ministry ofAgriculture, Forestry and Fisheries (JMAFF) --Good Laboratory Practice Standards, 11 NohSan, Notification No. 6283- 1October 1999 revised by 12 NohSan, Notification No. 8628- 6 December, 2000 Quality Assurance The study conduct, data, protocol, protocol deviation(s)/amendment(s) ifapplicable, and final report will be inspectedbythe Quality Assurance Unit, Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan. StudyArchives The data, protocol, protocol deviation(s)/amendment(s) ifapplicable, and final report will be archived bythe Toxicology and Environmental Research and Consulting archivist and stored at The Dow Chemical Company, Midland, Michigan. 

### H. SAFETY All personnel involved in the study will be advised ofthe safetyprecautions to follow when handling the test material prior to studyinitiation. Chemical safetyinformation will be 

### made available. 

### I. REFERENCES TERC Standard Operating Procedures ACL-GEN-006, ACL-GEN-013, and ACLGEN-0117. http://ehs.intranet.dow.com/teamsandgroups/terc/sop/sopindex/ACL.htm Qiilyan (2013). Certificate ofAnalysis of0,0-Diethylphosphorochloridoth (DEPCT), Lot C032013081501. ZhejiangXinnong Chemical Co., Ltd., Sanlixi, Xianju County, Zhejiang Province, China.ill 

### IllAppendix H Oral Gavage Range Finding Protocol for Mammalian ReproductivityToxicity Study

20 

### DEPCT•II I IllPRELIMINARY PROTOCOL
TOXICOLOGY & ENVIRONMENTAL RESEARCH AND CONSULTING THE DOW CHEMICAL COMPANY 

# PROTOCOL 1803 BUILDING, MIDLAND, MICHIGAN 48674
TITLE: DIETHYLCHLOROTHIOPHOSPHATE (DEPCT): 14 DAY ORAL GAVAGE RANGEFINDING STUDY IN Crl:CD(SD) RATS

KEY NUMBERS PLANNED DATES EXPERIMENTAL START: TBD EXPERIMENTAL TERMINATION: TBD ESTIMATED FINAL REPORT: TBD GLP STUDY: No FILE#: K-006538-014 STUDY ID: 140001 PTR#: 10001843 207 1 CAS #: 2524-04-1 SPONSOR: DowAgroSciences LLC 9330 Zionsville Road Indianapolis, Indiana 46268 TESTING FACILITY: The DowChemical Company Toxicology & Environmental Research and Consulting 1803 Building Midland, Michigan 48674 REQUIRED SIGNATURES: STUDY DIRECTOR: C. L. Zablotny, B.S.

LEAD SCIENTIST: A. K. Andrus, M.S. MANAGEMENTAPPROVAL: M. R. Woolhiser, Ph.D. SPONSOR or DESIGNEE: D. R. Juberg, Ph.D. MANAGEMENT ASSIGNED STUDY PERSONNEL: Pathologist: R. Sura, B.V.Sc. & A.H., Ph.D. Animal Technician: J. N, Fischer, B.S., LVT, LAT Technical Reviewer: M. S. Marty, Ph.D. Toxicology Technologist(s): J. K. Passage, B.S., LVT, E Collins, B.S. ADDITIONAL STUDY CONTACTS: Lead Scientist in Training: B. R. Hannas. Ph.D.

Document Management: D.P. Curell /DATE
/DATE
/DATE
/DATE

### A. TITLE THE DOWCHEMICAL COMPANY STUDY ID: 140001 PAGE2



### DIETHYLCHLOROTHIOPHOSPHATE (DEPCT): 14 DAY ORAL GAVAGE RANGE-FINDING STUDY IN Crl:CD(SD) RATS B. INTRODUCTION Purpose The purpose ofthis studyis to evaluate the potential toxicity of diethylchlorothiophosphate (DEPCT), inrats following oral gavage administration for 14 days. Red blood cell and brain cholinesterase activities will also be determined. The results ofthis study will be used to select dose levels for a subsequent oral gavage reproductive/developmental screening study. Test Guidelines This studyis not designed to fulfill any specific guideline requirements. C. TEST MATERIAL INFORMATION Test Material Name phosphorochloridothiotic acid, 0,0-diethyl ester Chemical Name 0,0-Diethyl ester phosphorochloridothioic acid 

### Synonyms Diethylchlorothiophosphate, DEPCT Supplier, City, State (Lot, Reference Number) The Dow Chemical Company, Midland, Michigan (Lot# C032013081501) Purity/Characterization (Method ofAnalysis and Reference) The non-GLP certificate ofanalysis lists the purity as 99.13% 0,0-Diethyl phosphorochloridothioate (Qinyan, 2013). Characteristics Molecular Formula



### Ill Ill )( \._ - 

### Molecular Weight 188.6 Chemical Structure

CAS Number 2524-04-1
Cornoil (Sigma Aldrich, St. Louis, Missouri) THE DOW CHEMICAL COMPANY STUDY ID: 140001 PAGE3



### Corn oil was the chosen vehicle because the test material was unstable in water. Previous Toxicity Information 

### DEPCT is an intermediate used in the manufacturing process ofchlorpyrifos. DEPCT and chlorpyrifos are structurally similar molecules and are bothknownto inhibit cholinesterase. Relevant oral toxicity data onDEPCT are limited and consist primarily oftwo single dose oral gavage studies in rats. Therefore, oral toxicity data onchlorpyrifos will also be used to aid in dose level selection for this range-finding study. DEPCT A single dose study was performed with Sherman rats to evaluate cholinesterase inhibition ofDEPCT (Litchfield and Norris, 1969). Test animals were administered 0, 63, 126, or252 mg/kg DEPCT in a 5% cornoil mixture by oral gavage. Cholinesterase (ChE) levels were measured in the blood 24 hours post-dosing. Plasma and red blood cell (RBC) ChE inhibitionoccurred at all dose levels and ranged from 25-46% inthe plasma and 21-59% inthe RBCs (Text Table 1). An acute oral toxicity study determined the oral LD50 to be 1.87 g/kg for bothmale and female rats (Keeler, 1975). Additionaltest animals were administered 0, 63, 126, 252, or 500 mg/kg DEPCT in a 10% cornoil mixture by oral gavage. ChE levels wereTHE DOW CHEMICAL COMPANY STUDY ID: 140001 PAGE4


### measured in the blood 24 hours post-dosing. Plasma and RBC ChE inhibition occurred at all dose levels and ranged from 10-59% in the plasma and 23-53% in the RBCs (Text Table 1 ). Text Table 1. 24 Hour ChE Inhibition Following Single Oral Dose DEPCT in Corn Oil Litchfield and Norris, 1969 Keeler, 1975 Dose (mg/kg) Plasma RBC Plasma RBC 63 29.5% 21.2% 9.8% 22.6% 126 25.0% 36.5% 13.7% 38.4% 252 45.5% 59.4% 33.3% 39.5% 500 NA NA 58.8% 53.1%
Chlorpyrifos A probe studywas conducted to obtainpreliminary data onthe dermal absorption of chlorpyrifos in the rat (Nolan 

### et al., 1981). One component ofthe study included groups of2 or 3 male Fischer 344 rats that were administered a single oral gavage dose of0 or 5 mg chlorpyrifos/kg in corn oil, respectively. Rats were euthanized twenty-four hours after dosing. Blood was obtained bycardiac puncture and plasma and RBC ChE activities were determined. ChE was inhibited 30% inthe plasma and 59% in the RBCs following a single dose of5 mg/kg chlorpyrifos. A dose response study was conducted focusing oninhibition ofChE and neurotoxic esterase in female Fischer 344 rats receiving single oral doses ofchlorpyrifos (Dittenber, 1997). Six rats/dose level were administered a single dose ofO, 0.5, 1, 5, 10, 50 or 100 mg chlorpyrifos/kg by oral gavage in corn oil. Assays ofbrain NTE and plasma, RBC, heart muscle and brain ChE were conducted 24 hours post-gavage. There were no significant differences in NTE activity values in brain homogenates compared with the control group at any dose level. Treatment-related inhibition of plasma, RBC, and heart ChE was observed inrats dosed with 5, 10, 50 or 100 mg/kg ofchlorpyrifos. Percent ChE inhibition in the plasmaranged from 45-91% and increased in a dose-dependent fashion. Percent inhibition in the RBCs ranged from



### Ill IllI \ THE DOW CHEMICAL COMPANY STUDY ID: I4000I PAGE5 

### 17-52%, and also increased dose dependently, but had a similar level ofinhibition at 50 and 100 mg/kg chlorpyrifos (52 and 51%, respectively). Mattsson 

### et al. (1998) evaluated ChE inhibition in Sprague-Dawleyrats following daily exposure ofdams on gestation day (GD) 6 through lactation day (LD) 10 to doses ofO, 0.3, 1or 5 mg/kg/day chlorpyrifos via oral gavage in corn oil. ChE activity was measured in dams approximately 4 hours after dosing on GD 20 and LDs 1and 5
.. ChE inhibition in the plasma and RBCs ofdams is shown in Text Table 2. Inhibition ofbothplasma and RBC ChE occurred at all dose levels, in a dose dependent manner, at all sampling time points. In addition, dams exposed to 5 mg/kg/day had approximately 80% inhibition ofbrain ChE on LD 5. Text Table 2. ChE Inhibition in Dams Following Oral Dosingwith Chlorpyrifos in Corn Oil GD 6- LD 10 Sample Day Dose (mg/kg/day) Plasma(%) RBC (%) GD20 5 88.0 95.I LD I 5 93.9 98.8 LD5 5 92.2 97.3 GD20 I 61.5 82.4 LD I I 77.I 87.3 LD5 I 69.3 84.0 LD20 0.3 32.8 26.3 LD I 0.3 52.2 39.2 LD5 0.3 31.7 Il.l


### D. DOSE, ROUTE AND JUSTIFICATIONS Route and Justification, Method ofAdministration, Frequency and Duration A possible route ofhuman exposure to the test materialwould be via accidental exposure during the manufacturing process. The purpose ofthis study is to set dose levels for a subsequent reproductive/developmental screening studywhere the preferred route ofadministration is oral. Dietary administration ofthe test material is not feasible due to instability in rodent feed. Thus, oral administration ofthe test material to rats via gavage represents an appropriate means ofexposure. Animals will be administered DEPCT once daily, seven days/week for 14 days. Dosing RegimenTHE DOW CHEMICAL COMPANY STUDY ID: 140001 PAGE6


### Female rats (6 per group) will be given 0, 5, 25, 50 or 100 mg/kg/day DEPCT by oral gavage in corn oil from test day (TD) 1-14. Dose Levels and Justification 

### Very little data is available onprevious toxicity ofDEPCT. Based onthe data cited above, ChE inhibition appears to be less sensitive to DEPCTthan chlorpyrifos administrationwith acute exposure. Using the available data, DEPCT is approximately 10-50 times less potent for inhibition in the RBCs following a single oral gavage dose. Additionally, an approximate two-fold increase in RBC ChE inhibition can be expected following repeated compared to single dose chlorpyrifos via oral gavage in corn oil. Therefore, it can be predicted based onthe available data that a high-dose level of 100 mg/kg/day DEPCT mayresult in RBC ChE inhibition in excess of75%. Dose levels for this range-finding study (Text Table 3) will include a high-dose level of 100 mg/kg/daywhich is expected to produce significant RBC ChE inhibition, and may result in other systemic effects. The intermediate dose levels of50 and 25 mg/kg/day are expected to provide a dose-response relationship for any treatment-related effects, and the low dose of5 mg/kg/day is expected to be a NOEL for all parameters with the potential exception ofRBC ChE inhibition. Additional dose levels may be included ifdeemed necessary. Text Table 3: Dose Levels 

### Dose Level (mg/kg/day)* No. ofRats/Dose Level 0 6 5 

### 6 25 6 50 6 100 6



## ------ Total= 30 

### *ConcentratiOns will notbe adjusted for purity. 

### Dose Preparation 

### All dosing solutions will be prepared by mixing the test material in corn oil at concentrations ofO, 1.7, 8.3, 16.7, or 33.3 mg/ml and administered at a dose volume of 3 ml/kg bodyweight to achieve the targeted dose levels. Dose volumes will be

111 IllTHE DOW CHEMICAL COMPANY STUDY ID: 140001 PAGE? 

### adjusted daily based onindividual body weights. Dose solutions will not be corrected for purity ofthe test material. 

### Analysis Dose Confirmation, Homogeneity, and Stability Dose confirmation and homogeneity analyses will notbe conducted due to the probe nature ofthis study. DEPCT was found to be stable in corn oil at concentrations ranging from 0.25 to 250 mg/ml for at least 16 days (Perala, in progress). Dose solutions will be mixed and used within the stability limits. Retainer Samples A sample ofthe bulk test material will be retained. No samples ofdose solutions will be retained. 

### E. TEST SYSTEM Species and Sex Rat, female (nulliparous and nonpregnant) 

### Strain and Justification Crl:CD(SD) rats were selected because oftheir general acceptance and suitability for toxicity testing; the subsequent study will be conducted using this strain ofrat, and the reliability ofthe commercial supplier. 

### Supplierand Location Charles River (Portage, Michigan) 

### Age at Study Start Approximately 7 weeks 

### Physical andAcclimation During the acclimationperiod, each animal will be evaluated by a laboratory veterinarian, or a trained animal/toxicology technicianunder the direct supervision of a laboratory veterinarian, to determine the general health status and acceptability for studypurposes (fully accredited bythe Association for Assessment and Accreditation ofLaboratory Animal Care International- AAALAC International). The animals will be housed two-three per cage in stainless steel cages, in rooms designed to maintainTHE DOW CHEMICAL COMPANY STUDY ID: 140001 PAGES 

### adequate conditions (temperature, humidity, and photocycle), and acclimated to the laboratory for approximately one week prior to the start ofthe study. Housing After assignment, animals will be housed one per cage in stainless steel cages. Cages will have solid floors with corncob bedding and shredded aspen for enrichment. Cages will contain a feed crock and a pressure activated lixit valve-type watering system. The following environmental conditions will be maintained in the animal room.: Temperature: Humidity: Air Changes: Photoperiod:
Enrichment 22°C with a range of20°C-26°C 50% with a range of30-70% 12-15 times/hour (average) 12-hour light/dark (on at 6:00a.m. and offat 6:00p.m.)

Enrichment for rats includes the use ofground corn cob and shredded Aspenbedding (Harlan Laboratories, Inc., Indianapolis, Indiana) and open areas onthe cage sides for visualization ofother rats. Randomization and Identification Before administration ofthe test material begins, animals will be stratified by body weight and thenrandomly assigned to treatment groups using a computer program designed to increase the probability ofuniform group mean weights and standard deviations at the start ofthe study. Animals placed on studywill be uniquely identified via subcutaneously implanted transponders (BioMedic Data Systems, Seaford, Delaware) that are correlated to unique alphanumeric identification numbers. Ifa transponder stops functioning oris lost, it will be replaced with a new transponder that will be correlated with the unique animal number. Feed and Water Animals will be provided LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form. Feed and municipal water will be provided 

### adlibitum. Analyses ofthe feed will be performed by PMI Nutrition International to confirmthe diet provides adequate nutrition and to quantify the levels

111 IllTHE DOW CHEMICAL COMPANY STUDY ID: 140001 PAGE9


### ofselected contaminants. Drinking water obtained from the municipal water source is periodically analyzed for chemicalparameters and biological contaminants by the municipal water department. In addition, specific analyses for chemical contaminants are conducted at periodic intervals by an independent testing facility. Copies ofthese analyses will be maintained in the study file. 

### AnimalWelfare In accordance withthe U.S. Department ofAgriculture animal welfare regulations, 9 CFR, Subchapter A, Parts 1-4, the animal care and use activities required for conduct ofthis studywere reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). The IACUC has determined that the proposed Activities are in full accordance withthese Final Rules. The IACUC-approved Animal Care and Use Activities to be used for this study are Subchronic/Chronic Tox 01, Bloodcollection 01, Humane Endpoints 01, and Animal ID 01. STUDY DESIGN 

### Summary Groups ofsix female Crl:CD(SD) rats will be administered DEPCT in corn oil via oral gavage at dose levels ofO, 5, 25, 50 or 100 mg/kg/day from TD 1-14. The parameters summarized in Text Table 4 will be measured. Specific details ofcollected parameters are outlined below in the appropriate sections.THE DOWCHEMICAL COMPANY STUDY ID: 140001 PAGE 10


### Text Table 4. A Summary ofStudy Parameters

Study Parameters BodyWeights Feed Consumption Cage-Side and Clinical Observations RBC and Brain ChE Activity Necropsy Organ Weights (Liver and Kidney) 

### F. STUDY SPECIFIC PARAMETERS Daily Observations No. ofAnimals/Dose 6 6 6 6 6 6



### A cage-side examination will be conducted twice daily, preferably at the same times each day on all animals. This examination is typicallyperformed with the animals in their cages and is designed to detect significant clinical abnormalities that are clearly visible upon a limited examination, and to monitor the general health ofthe animals. The animals are not hand-held for these observations unless deemed necessary. Significant abnormalities that could be observed include, but are not limited to: decreased/increased activity, repetitive behavior, vocalization, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor, and twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in fecal consistency, and fecal/urinary quantity. Moribund animals not expected to survive until the next observationperiod will be humanely euthanized that day. In addition, all animals will be observed for morbidity, mortality, and the availability offeed and water at least twice daily. Clinical Observations 

### Clinical observations will be conducted onall animals at least once daily approximately one hour following dosing. Clinical observations include a careful, hand-held examination ofthe animal with an evaluation ofabnormalities in the eyes, urine, feces, gastrointestinal tract, extremities, movement, posture, reproductive system, respiration, skin/hair-coat and mucous membranes, as well as an assessment of general behavior, injuries or palpable mass/swellings.

11 I Ill \,

)( \ 

### Body Weights/Body Weight Gains THE DOW CHEMICAL COMPANY STUDY ID: 140001 PAGE 11



### All rats will be weighed pre-exposure and daily during the dosing period. Body weight gains will be calculated relative to day 1. Feed Consumption 

### The amount offeed consumed will be determined daily throughout the treatment period for all animals. Feed consumption is determined by weighing feed containers at the start and end ofa measurement cycle. Feed consumption will be calculated using the following equation: Feed consumption (g/day) =(initial weight offeed container- final weight offeed container) ( # ofdays in measurement cycle) ( # ofanimals per cage) 

### Anatomic Pathology 

### Animals submitted alive for necropsy will be anesthetized by the inhalation of isoflurane/02 

### vapors and blood will be collected from the inferior vena cava once deep anesthesia is achieved. During blood collection, care will be takento minimize clotting and limit red blood cell lysis. Following exsanguination, the animals will be decapitated, the cranial cavity will be opened, and the brainwill be removed. Blood samples for ChE measurements will be kept onice until centrifuged to separate plasma and RBCs. RBCs will be stored at -80°C until shipped for ChE analysis. A complete gross necropsywill be conducted onall animals by a veterinary pathologist assisted by a team oftrained individuals. The necropsy will include an examination ofthe external tissues and all orifices. The head will be removed from all animals, the cranial cavity opened and the brainwill be removed, rinsed with saline, and blotted dry. The brain willthen be dissected into the right and left hemispheres. The right hemisphere will be weighed. Both sections will be individually quick frozen in liquid nitrogen and will be used for assessment ofbrain ChE activity (assay described below). The skull containing the pituitary will be placed in neutral, phosphate-buffered 10% formalin. The eyes will be examined 

### in situ by application ofa moistened microscope slide to each cornea. The skin will be reflected from the carcass, the thoracic and abdominal cavities will be opened and the viscera examined. The heart and a section ofthe diaphragm will be removed from all animals, rinsed and/or flushed with saline, blotted dry and individually quick frozen in liquid nitrogen for future potential assessment ofChE.. The liver and kidneys will be weighed and preserved. The ratios ofliver or kidney weight to terminal bodyweight will be calculated. Any obvious gross pathologic alterations and relevantTHE DOW CHEMICAL COMPANY STUDY ID: 140001 PAGE12


### gross lesions will be recorded and tissues preserved. Microscopic examination oftissues will not be conducted unless deemed necessary. Moribund animals that are not expected to survive until the next observationperiod will be humanely euthanized. These animals, as well as any animals found dead, will be necropsied onthat day during the normal work week. However, animals euthanized or found dead after routine working hours oronweekends, and holidays will be refrigerated until the next scheduled workday, at whichtime they will be necropsied. Similar necropsyprocedures will be followed with the exceptionthat organs will not be weighed and tissues will notbe saved for ChE activity. 

### G. SPECIMEN COLLECTION Summary 

### Levels ofChE activity in RBCs and brain will be measured following DEPCT exposure. RBC 

### The samples will be placed on ice, and then centrifuged packed RBCs will be harvested. RBCs will be diluted 1:20 in 1% TritonX-100. The samples will be frozen at -80°C. Samples will be shipped to a contractresearch laboratory for ChE analyses. 

### Brain The head will be removed, the cranial cavity opened and the brainwill be removed, rinsed with saline, and blotted dry. The brain will then be dissected into right and left hemispheres. The right hemisphere will be weighed. Both sections will be individually quick frozen in liquid nitrogen with the right halfofthe brain specifically identified. The weighed right halfofthe brain will be shipped to a contractresearch laboratory for ChE analyses. 

### ChE Activity Samples will be shipped on dry ice to WIL Research Laboratories; LLC, Ashland, OH for assessment ofthe total ChE activity ofRBC and brain. ChE activities will be determined using an assay based on a modification ofthe Ellman reaction (Ellman et al., 1961; Hunter et al., 1997). The principal investigator, Dr. Jonathan Toot, will be responsible for oversight ofall ChE analyses conducted at WIL Research Laboratories. Details ofthephase plan.



### Ill Ill \,

### H. STATISTICS THE DOW CHEMICAL COMPANY STUDY ID: 140001 PAGE 13



### Descriptive statistics 

### (i.e., mean± standard deviation) will be calculated for all measured parameters. Statistical outliers (alpha= 0.02) will be identified by the sequential method ofGrubbs (1969) and will only be excluded from analysis for documented, scientifically sound reasons. Feed consumption values will be excluded from analysis ifthe feed is spilled or scratched. Determination oftreatment-related effects will be based on sound scientific principles such as biologicalplausibility, comparison to concurrent and historical control data, and dose response relationships. Equivocal findings will be noted appropriately and addressed inthe subsequent oral gavage reproductive/developmental screening study. 

### I. FINAL REPORT A final report ofthe definitive studywill be submitted to the study sponsor and will include but not be limited to the following: 1. Name and address ofthe facility performing the study and the dates onwhich the study is initiated and completed, 2. Objectives and procedures stated in the approved protocol, including any changes in the original protocol, 3. Statistical methods employed for analyzing the data, 4. Description oftest and/or control substance (e.g., lot#, purity, physical characteristics, and method ofpreparing test concentrations), 5. A description ofthe test systemused including, when applicable, the number of animals used, sex, bodyweight range, source ofsupply, species, strain, and substrain, age, and procedure used for identification, 6. A description ofthe dosage, dosage regimen, route ofadministration, and duration, 7. A description ofall circumstances that may have affected the quality or integrity ofthe study, 8. Location ofraw data and specimens, 9. List and signatures ofstudypersonnel. 

### StudyArchives The data, protocol, protocol deviation(s)/amendment(s) ifapplicable, and final report will be archived bythe Toxicology and Environmental Research and Consulting archivist and stored at The Dow Chemical Company, Midland, Michigan.

### J. SAFETY THE DOWCHEMICAL COMPANY STUDY ID: 140001 PAGE 14

All personnel involved in the studywill be advised ofthe safetyprecautions to follow when handling the test material and treated animals priorto study initiation. Chemical safety informationwill be made available. 

### K. REFERENCES Dittenber, D. A. (1997). Chlorpyrifos: Evaluation ofSingle Oral Doses on Chloinesterase and Neurotoxic Esterase Inhibition in F344 Rats, Report ofHealth and Environmental Research Laboratories, The ToxicologyResearch Laboratory, The Dow Chemical Company. Ellman, G. L., Courntey, 

### K. D., Andres, Jr. V., and Featherstone, R.M. (1961). A new and rapid colorimetric determination ofacetylcholinesterase activity. 

### Biochemical Pharmacology 7, 88-95. Grubbs, F. E. (1969). Procedures for detecting outlying observations in samples. 

### Technometrics 11:1-21. Hunter, P. L., Marshal, R. S., and Padilla, S. (1997). Automated instrument analysis ofCholinesterase Activity in Tissues from Carbamate-treatedAnimals: A CautionaryNote. 

### Toxicology Methods, 7, 43-53. Keeler, P. A. (1975). Toxicological Properties and Industrial Handling of: 0, 0- Diethyl Phosphorchloridothioate (DEPCT), Report ofToxicology Research Laboratory, The Dow Chemical Company. Litchfield, N., Norris, 

### J. M. (1969). Cholinesterase Inhibition Study Conducted on 0, 0-Diethyl Phosphorchloridothioate, Report SummaryofBiochemical Research Laboratory, The Dow Chemical Company. Mattsson, 

### J. L., Maurissen, 

### J. P., Spencer, M. S., Brzak, 

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