~ Dow AgroSciences 

Dow AgroSciences LLC 

9330 Zionsville Road Indianapolis, IN 46268 USA 

Bldg 308/2E 

25 February, 2014 

Document Control Office (DCO) (7407M) 

Office of Pollution Prevention and Toxice (OPPT) 

U. S. Environmental Protection Agency 

1200 Pennsylvania Ave., NW., 

Washington, DC 20460-0001 

l\Ecc::..;c.o 

R t S '2.\)\~ 

Attention: 40 CFR 799.5089; Docket ID Number EPA-HQ-OPPT-2007-0531 

CBIC Control Number 

II 

358722 

www.dowagro.com 

& --r/L{ ']:ck I ' 

Scanned & Processed in 

CIS by: SHARON 

Attached is the study plan for the testing of phosphorochloridothiotic acid, 0,0-diethyl ester (CASRN 2524-04-1) (aka 

diethylchlorothiophosphate or DEPCT) complying with EPA ruling under section 4 of the Toxic Substance Control Act 

(TSCA) on January 7, 2011 (76 FR 1067). In summary, Dow Chemical's testiniiifans for this substance are listed below: 

• 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 will be conducted to account for the very short half-life of this material in water. The protocol 

is attached and this study will iilform the protocols for the water solubility determination, ready biodegradation 

study, the aquatic tox studies. The hydrolysis study results and protocols for each of these subsequent studies will 

be submitted to EPA by 30 September 2014 

• Protocol is attached for the Bacterial Reverse Mutation study (Ames test) and it will be submitted by 30 

September 2014 

• Mammalian Erythrocyte Micronucleus Test protocol is attached and Dow intends to conduct the study and 

submit the report 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 then submit the study reports for both studies by June 30, 2015. Some preliminary 

testing regarding stability and dose range finding is required prior to initiating these two studies. Protocols for , ~? 

these preliminary tests are attached. 

If you have questions regarding this study plan, please contact Darin Lickfeldt at 317-337-3774 or dwlickfeldt@dow .com. 

We look forward to your review and feedback of the attached study plan and study reports. 

Respectfully, 

~- /~_,//~ ·-~~~~-~~~, 

Darin W. Lickfeldt, Ph.D 

Regulatory Manager 

Dow AgroSciences LLC 

®TM Trademark of The Dow Chemical Company ("Dow") or an affiliated company of Dow 

··.~ 

(_ 

( 

ORPHAN HPV TESTING PROGRAM 

Study Plan 

for 

phosphorochloridothiotic acid, 0,0-diethyl ester 

(aka diethylchlorothiophosphate or DEPCT) 

CASRN 2524-04-1 

PREPARED BY: 

Dow AgroSciences LLC 

Indianapolis, IN 

25 February 2014 
Manufacturing 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 nut trees, and vegetable crops. 

(i) Identity of the test rule. 

This study plan includes the detailed information and proposed plans for fulfilling requirements for the 

EPA ruling under section 4 of the 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 of that rule to be covered by the study plan 

Table 1 

Test Study Study Testing Lab 

Test Type Monitors 

A1 Melting Point: ASTM E 324-99 Joyce Battjes 

Harlan, Study 

(capillary tube) report attached in 

Appendix B 

A2 Boiling Point: ASTM E 1719-05 Joyce Battjes Harlan,Study report 

(ebulliometry) attached in 

Appendix B 

A3 Vapor Pressure: ASTM E 1782-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 

E 729-96(2002); protocols for these 

3. Toxicity To Plants (Algae): ASTM three studies 

E 1218-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 Screening Test: 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 for this 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 of the testing facilities and the names, addresses, and telephone 

numbers of the 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 new future testing will be conducted at one of the 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 of Toxicology 

TERC Office 236 Shard low 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) Brief summaries of the training and experience of each professional involved in the study, 

including study director, lead scientist and technical reviewer are attached in Appendix A with 

CVs of key personnel conducting testing at Dow Chemical Co. or Harlan Laboratories Ltd. 

(iv) Identity and data on the chemical substance(s) being tested, including physical constants, 

spectral data, chemical analysis, and stability under test and storage conditions, as 

appropriate. 

phosphorochloridothiotic acid, 0,0-diethyl ester 

CASRN 2524-04-1 

(aka diethylchlorothiophosphate or DEPCT) 

DEPCT 

5 
Table 3- Physical Constants for DEPCT: 

Property Value Method Reference 

Freeze Point* < -2o ·c OECD Guideline 102: Harlan Laboratories 

Freezing Temperature Report of Study No. 

Method (GLP Compliant) 41304286 (2014), See 

Appendix B 

Boiling Point* 202 ·c OECD Guideline 103: Harlan Laboratories 

@ 98.3 kPa Differential Scanning Report of Study No. 

Calorimetry Method (GLP 41304286 (2014), See 

Compliant) Appendix B 

Vapor 93 Pascal OECD Guideline 104: Harlan Laboratories 

Pressure* @ 25 Vapor Pressure Balance Report of Study No. 

·c Method (GLP Compliant) 41304287{2014),See 

Appendix C 

Water Undefined, due to rapid hydrolysis. Further testing of water 

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 

Paw with water solubility (WSKOW v1.42) methods of the 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 of substance with water 

Pow) and 1-octanol. A 

calculated value is 

provided, according to 

structure fragment 

constant method of 

KOWWIN v1.68 

*properties determined for test substance of 99.13% purity 

6 

DEPCT 

Ill 

I 
Spectral data for DEPCF: •I •, •; t 

:·~·-'/·~··:) :.~·= ./: ... :·. :-r.i : ...... : ··_(~:: )·i· ,·; .·: •. ··., •• ·:,.·\·:- ':::·:···-~: \ .·.: • ~ ,_.; 

An infrared sp~;!ctrum was obtained for the substan1=e (99.13% purity), An aliquot of the .. _l.::··~·\::f;·~·:.~·:·.·.).:·:.:·.t<·:·t!~- .. --:!~:':~;-·:.:·~ ,·.·::· ·1 ... 1 . .' · -~ '.:.' ·, ,·. •· .. , -~-.~·:=.i··':.-~.-= ,·. •i:V·-' ~ ,_·;. .. ::···· . .. ·1: 

. . . ~1.1bstar,ce wc,~.s sc.anned .·directly ysipg .AtteniJatedJQtCI! Refl~ctan~e (ATR) over the range ·, ,•·,-','.-;;.'},,·.,; :.,,·'~:!•,\.,,.,' :. ,;; •,l;'j., ~·,·, .,·o o' 't(.~\''o~''•,,\ ', ')ol •,: I:':: .·.~·: ,. C • 1,·,,,-1,; ~-.' 

of 4000 to 600 cm-1. The we~v.e numbers and assignments of the most structurally ·., ,·,.,;: ·::. ;.~ · ;n·;· \l"' .~". ··-~·- .:,1· '\-1\.',: · ~:;<··f ;·.-~:· -.:,:. ': ,.:.' · .:. ::v .. !- •· •.• :-: ... ,_ .·::•. • • ;' ·:1 ·• 

significant bands were' recorded, a'nd are consisteritwith the.expectedabsorbance ,·,;_•:--.~;~!·'t:·::· -~-· ' ::··.~ .• ·,;· 

bands associated with the structural features of this 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 DEPCT Test Material: 

Dow anticipates the test material for new testing will be > 99% pure DEPCT, but this will be 

, validated with a GLP characterization studies. The GLP characterization study will likely include 

NMRand MS spectral information. .. · · . , · 

.' \ 

Stability u·nder testconditions:. '!.· 

_.,· -'. -~ 

The test material (a diethyl ester of o-thiophosphoric acid chlorid.e or DEPCT) has reactivity 

similar to that of carboxylic acid chlorides (e.g., acetyl chloride). As such, the substance will 

readily react with water, proteins, alcohols, a mines, etc. (i.e., any nucleophilic substance). This 

reactivity has significant influence on how toxicological and ecological hazard testing is 

conducted on the substance, and makes certain physical and hazard property determinations ·. : ' :"".~ .,··-~~------~:.-....... · .. ~. ,.,,._{·,· ·t' -~-· (_~'- ... · ·· ..• ·:·. •·.:. ': ,,. >:.: .: '·:··· ·_:,·~ · .... -·''· -~~ ,·) ·'~ . ~. ',•;;' '-:'. ,\·.;' 

. qifficult or irrelevant. These aspeq$ of substance 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 stored under: appropriate" -

conditions. For determinations of freeze point, boiling point, and ;vapor pressure,.the 

7 
substance evaluated was determined to be of suitable purity (99;13 %) prior: to or 

concurrently with performing these measurements. The properties of water 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= . • ·:. •.• :· '>,:· ... ;. ·• 

water solubility ahd' log Kow are provided (Table 3), as determined according to • '<·;..·:, :·;,~!: -.",;·• .. ·.·,: ·1 ;··>: (i ):',\: "T···.~··.' ·.•.• .: ·:_'; ,· :·; . _'· ·J' :;. 

· accepted QSAR relationships. ) .. •' 

Environmental Fate and Pathways: 

For studies which determine the fate of DEPCT in the environment, stability of the pure 

applied test substance is of importance/relevance; however, it is the purpose of such 

studies to understand stability of this pure substance subsequent to its addition to test 

systems containing water, wastewater, soil, etc. The study of hydrolysis (protocol in 

Appendix D) would demonstrate the instability of that pure substance in water, also 

indicates relative stability of the primary hydrolysis product (0,0-diethyl hydrogen 

phosphorothioate; CASRN 2465-65-8). 

Aquatic Toxicity: 

In accordance with OECD guidance on aquatic toxicity testing of difficult substances, the 

parent DEPCT substance would be allowed to hydrolyze to the primary (and stable) 

hydrolysis product (0,0-diethyl hydrogen phosphorothioate) priorto exposure of the 

test organisms. In each of these studies, the concentration and stability of this 

hydrolysis product would be determined over the course ofthe aquatic exposures, with 

these measured concentrations as basis for the reported E/LC50 and NOEC values. 

Mammalian Toxicitv-Genotoxicity ,, 

The stability of DEPCT in various vehicles/media is not known and before protocols for 

: the Reproduction I Developmental· Toxicity Screen (OECD 421) can be written (with 

known vehicles to which DEPCT would be added)j there is a need to 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 most stable for each respective test. Because of this need, a protocol ' . . ' . . . . . . .· . -~ ' ·, . : . : 

that evaluates stability in food and corn oil, conducted under GLP, is attached in this 

study plan as Appendix G-this work is essential to determining' stability of the test 

material in the 421 screen. ·, . : ~ . ' \ ·,,, ' . ~! ' 

··:.' 

~ . , . l ~ i . . 

. (vl study prototol, .ihc:ludillg 1:'11e ~ational'e 'tor any 'combination of te~t protbcols; the rationale 

. for' species/stra'in' selection; dose selectiotl (and supportil1g data'); ~o~te(s) tir method(~) of 

exposure; description of diet to be used and its source; inc;:ludi.ng m•~r!ert~s and: contaminants 

and their concentrations; for in vitro test systems, a description of culture medium and its 

source; and a summary of expected spontaneous chronic diseases (including tumors), 

genealogy, and life span; •· ·· · · · ' 

8 

DEPCT 

Ill 
I 

\ 

Each study to be conducted is addressed in the following appendices {Table 4) with either a 

protocol, a reference to previously conducted study, or justification for preliminary studies that 

will be required in order to conduct the EPA requested testing. 

Table 4 

Study Appendix Study Study Plan 

Monitor 

Melting Point: Recently conducted study of 

Harlan Laboratories, attached 

ASTM E 324-99{capillary tube) 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 E 1782-Q3 thermal in Appendix C 

analysis 

n-Octanoi/Water Partition Testing is not feasible. 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 not feasible. 

Estimated values of 4,296 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 prior to writing 

biodegradation protocol. Dow 

Pathways-Ready will conduct the hydrolysis 

Biodegradation: Preferred Laura Laughlin study and submit 

method not yet known biodegradation protocol by 

September 30, 2014 with study 

report submitted by 30 June 

2015 

9 

DEPCT 
The 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 E 729-96{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 E 729-96{2002); 

2015 

Analytical monitoring of the 

3. Toxicity To Plants (Algae): concentrations of DEPCT and its 

ASTM E 1218-04e1; primary acid degradation 

product shall be conducted 

during the dosing phase of the 

toxicity test. 

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 

preliminary testing in Appendix 

G & H. Following the 

preliminary testing, 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 quantify the 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 prior to creating protocols for the Ready 

Biodegradation and the three Aquatic Toxicity studies. The hydrolysis study along with 

protocols for these 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 preliminary testing regarding stability and dose range finding is required 

prior to initiating these two studies. Protocols for these preliminary tests are attached. 

11 

DEPCT 
Appendices 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 

I 
Appendix A 

Curriculum Vita for Study Directors, Lead Scientists, and Technical Reviewers 

13 

DEPCT 
Ill 

I 
HARLAN LABRATORIES L TO 

Department of Physico-Chemical Properties 

CURRICULUM VITAE 

EMPLOYEE NAME DUNCAN TARRAN 

POSITION STUDY DIRECTOR 

SITE ADDRESS SHARDLOW BUSINESS PARK 

CURRENTHARLANEMPLOYMENT 

~--·-·--. -· ------- ------·-------......._.., ................................... """"' "''"''"""''"''"''"'' ........ . 

I DATE FROM I POSITION & BRIEF DESCRIPTION 

!o-ct~b~; 2o1_1 __ · --~ As study Director in ttie Ptiysico~Chemical Properties testing laboratories if is 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 out as ancj 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 of the 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 2 
EDUCATION/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 of Science in 

Applied Experimental Psychology 

1998: Central Michigan University. Mount Pleasant, MI, Bachelors of Science 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,v and Teratolo[!,y 25:543-553. 

2) Andrus, A, K., Marable, B. R., Dunbar, G. L., Reilly, M.P., Maurissen, J.P. J. 

2007. Effects of intensity and type of prepulse stimulus on prepulse inhibition in 

scopolamine treated rats. Pharmacol, Biochem, and Behavior 87:481-488. 

3) Dryzga MD, Yano BL, Andrus AK, Martsson JL. 2007. Evaluation of the safety 

and nutritional equivalence of a 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 of indole-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 of the Auditory Startle Response: Effects of 

Prepulse Stimulus Duration, Frequency and Intensity. Poster presented at the 29th 

Annual Meeting of the 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. Poster presented at the SOT 44th 

Annual Meeting, March 6-1 0, 2005 

3) Marable, B.R., A.K. Andrus and J.P. Maurissen. 2001. Assessment of the 

Validity of an Automated Auditory Stm1le Response System with or without 

Chemicals. Presented at the Annual Meeting of the Society. of Toxicology, 

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 our children's future? 

Presented at the Dow Global Scientists' Organization meeting, Galveston, TX, 

Oclober 23-25, 2000. 

5) A. K. Andrus 2002. Resistance of different materials and cables to rodent attacks. 

6) 

Presented at the TERC Science Discussion Group, December 2, 2002. 

Andrus, A. K. Scopolamine and Prepulse Inhibition of the Auditmy Startle 

Response: Effects of Prepu1se Stimulus Duration, Frequency and intensity. 

Seminar presented on 2115/05, TERC Science Seminar 

DOW RESTRICTED 

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) 

DOW RESTRICTED 
2 

.PUBLICATIONS 

I) Breslin, W. J., Ciezlak, F. S., Zablotny, C. L., Corley, R. A., Yano, B. L. and 

Verschuuren, H. G. ( 1990). Developmental toxicity of inhaled 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 with picloram triisopropanolamine salt and 

picloram 2-ethylhexyl ester in 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. Abstract No. 580. 

4) Zablotny, C. L., Breslin, W. J. and Kociba, R. J. (1992). Developmental Toxicity 

of Orthophcnylphenol (OPP) in New Zealand 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 of indole-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 of isobutyl heptyl ketone (IBHK) by oral gavage in an OECD 

422 rat reproduction/developmental screening study with systemic and 

neurological endpoints. 44th Annual Society of Toxicology 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. 

DOW RESTRICTED 

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( 

CURRICULUM VITAE 

NAME: Melissa R. Schisler 

.JOB TITLE: Study Director 

EDUCATION: 

1996: Ferris State University, Big Rapids, Michigan, Bachelor of Science 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-jj 4 /;_!:)_ _____ _ (Date) 

DOW RESTRICTED 
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 lymphoma cells, 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 of gene 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 of EDST AC 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 of Mutagenic 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 of DNA adduct ofthymidylyl(3'-5')thymidine methyl 

phosphotriester by liquid chromatography/negative electrospray tandem 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 B Analyt 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 of lower levels of DNA adduct ofThymidyly (3'-5') 

thymidine methyl phosphotriester by liquid chromatography/negative atmospheric 

pressure chemical ionization tandem mass spectrometry. Rapid Commun Mass 

Spectrom, 2 I: I 043- I 048. 

8) Zhang, F., Stott, W.T., Clark, A.J., Schisler, M.R.. Grundy, J.M., GoiJapudi, B.B., 

Bartels, M.J. (2007). Quantitation of 8-hydroxydeoxyguanosine in DNA by liquid 

chromatography/positive atmospheric pressure photoionization 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 of glutathione by liquid 

chromatography/positive electrospray ionization tandem mass spectrometry. 

Rapid Comrnun Mass Spectrom, 22(22):3608-3614. 

DOW RESTRICTED 

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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 of methylated 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): 138-4 7. 

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 of oxidative 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 of deoxyinosine adduct: Can it he a 

reliable tool to assess oxidative or nitrosative 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 of counting methodology on erythrocyte ratios in the mouse 

micronucleus test. Environ Mol Mutagen 54(3):222-228. 

DOW RESTRICJl:l) 
PRESENTATIONS 

1) Dow MRDTG Poster Session (2000), Short-Term Tests for Identifying Chemical 

Mutagens and Carcinogens. 

2) Dow MRDTG Poster Session (2004), Mode of Action for the In Vitro 

Mutagenicity of Bioban 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 of Environmental 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 at 47th 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 

Phosphotriester by 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 of DNA adducts and peripheral blood micronucleus in 

rats treated with MMS. Presented at 38th Annual Meeting of the 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 II b of mouse lymphoma cells treated 

with taxol. Presented at 38111 Annual Meeting of the 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 of methylated 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 of 8-hydroxydeoxyguanosine in 

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

DOW RESTRICTED 

Ill 
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Conference on Mass Spectrometry and Allied Topics, Indianapolis, Indiana, June, 

( 2007. 

I 0) 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., Schisler M.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 of genetic 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 of the U. S. National Toxicology 

Program (NTP) using International Workshop on Genotoxicity Tests (IWGT) 

criteria. Presented at 41st Annual Meeting ofthe Environmental-MutagenSociety 2010. 

I 5) 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 of the Environmental-Mutagen-Society 20 I 0. 

16) LeBaron, MJ, Schisler, MR, Kan, HL, Thomas, J, Eisenbrandt, DL and Gollapudi, 

BB (20 11 ). Analysis of Molecular, Cellular, and Biochemical Changes in the 

Liver of Male B6C3F1 Mice Treated With Nitrapyrin- 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 

of Propylene Oxide (PO)-Induced Mutagenicity in a p53-Competent, HumanDerived Cell Line, TK6. Presented at 42nd Annual Meeting of the 

Environmental-Mutagen-Society October 15-19, 2011. 

DOW RESTRICTED 
18) Schisler, M.R., Gollapudi, B.B., McDaniel, L.P., and Moore, M.M. (2012) 

Reevaluation of the mouse lymphoma forward mutation assay (MLA) results on 

342 chemicals from the U.S. National Toxicology Programs (NIP) Database. 

Presented at 42nd Annual Meeting of the 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 of the EnvironmentalMutagen-Society September 8-12, 2012. 

20) Gollapudi, B.B., Schisler, M.R., McDaniel, L.P., and Moore, M.M. (2012). 

6 

Reevaluation of the 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 of molecular, cellular, and biochemical changes 

in the liver of male CD-I mice treated with the herbicide Pronamide. Presented at 

SOT Annual Meeting & Tox ExpoTM. 20 12. 

DOW RESTRICTED 

Ill ~ i 
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 

Master of Veterinary Veterinary 

Science (M.V.Sc.) Microbiology 

Bachelor of Veterinary All veterinary science 

Science & Animal Subjects 

Husbandry 

(B.V.Sc. & A. H.) 

(Equivalent to DVM) 

12th Standard Physics, Chemistry, 

Higher Secondary Biology, English 

Certificate Examination Crop Science 

(H.S.C.) 

lOth Standard English, Hindi, 

Secondary School Marathi, Social 

Certificate Examination Science, Science 

(S.S.C.) 

Name and Address of Institution 

College: Bombay Veterinary College, 

Mumbai, Pin- 400012, India 

University: 

Maharashtra Animal & Fishery Sciences 

University, Seminary Hills, Nagpur, Pin -

440006, Maharashtra, India 

College: Bombay Veterinary College, 

Mumbai, Pin -400 012, Maharashtra, India 

University: Maharashtra Animal & 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 1996 
Job 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 of Toxicology 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 of study results. 

4. Conducted Toxin 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. To provide technical support for genetic toxicity studies in terms of regulatory submission to the sponsors. 

9. To monitor the genetic toxicity studies of different clients conducted at JRF. 

I 0. To review the genetic toxicity study protocols and reports of in-house validations conducted at Section Mutagenicity 

(Jai Research Foundation). 

TRAINING, WORKSHOP AND SEMINAR ATTENDED: 

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 Molecular Biology Conference at New Delhi. 

4. Participated in National Seminar organized by Jai Research Foundation on" REACH" in 2006. 

5. Under gone training on Molecular techniques for 15 days at Indian Institute of Science (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 ECV AM/ESAC SkinEthic Skin 

irritation Validated protocols" at SkinEthic Laboratories (Lyon, France), during September 10- 13, 2012. 

14. Attended 52"ct, Society of Toxicology's Annual Meeting, at the Henry Gonzalez Convention Center San Antonio, Texas 

during March 10-14, 2013. 

LIFE MEMBERSHIP OF PROFESSIONAL SOCIETIES: 

1. Life member of Indian Journal of Environment 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 Study Director for 

Residue and Persistence studies in Soil, Water 

and Crop from 1993 to 1996. 

~ Worked as QA officer from 1996 to 2000 

~ Heading QA of JRF since 2001 

~ Head of Global QA of Jai Research Foundation 

since 2011 and co-coordinating & auditing of 

different sites of JRF in US and UK 

Training/Competency ~ Trained Study Director for Residue field trials 

and GLP studies 

~ Trained as GLP inspector by National GLP 

Compliance Monitoring Authority, Government 

oflndia 

~ Trained Laboratory Assessor of National 

Accreditation Board for Laboratories for auditing 

the laboratory operated as per IS0-17025 

~ Development and implementation ofGLP quality 

system as per OECD, US-EPA and FDA GLP 

requirements 

~ Attended 8SQA Annual meetings, 2 Global QA 

conferences, 5 RQA conference and various 

quality assurance trainings since 2006. 

~ Development of SOPs, Validation plans and 

policies under GLP. Auditing of Computerized 

system 

~ Qualified trainer for training on various aspects 

ofGLP 

Total Experience 21 years 

Academic Society ~ Member of Society of Quality Assurance, USA 

Membership ~ Member of Research Quality Association, UK 

~ Member and founder Director of Indian Chapter 

of Society of Quality Assurance (ICSQA) 

Publication ~ Published 3 technical research papers and 7 

_l)_osters on GLP quality system 
) 
RESUME 

Name Ms. Kirti E. Tendulkar 

Home Address 2/45, M.H.B. Colony, Mithagar Road, Mulund East, Mumbai- 400081. 

Business Address Jai Research Foundation, Post Box No 30, GIDC, Vapi- 396 195, Gujarat, India 

Phone Number: Home (+91) 9687483386 Office (+91) 260-6540-242 

E-Mail Address: kirti.tendulkar@jrfonline.com 

tendulkar.kirti@gmail.com 

Degree Received Major 

Master of Applied Genetics Applied Genetics 

(M.Sc.) 

Bachelor of Biotechnology 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 of Institution 

College: Acharya Institute of Life Sciences 

University: Bangalore University, 

Karnataka, India 

College: K.J Somaiya Intitute of Science and 

Commerce 

University: Mumbai University, Mumbai, 

Maharashtra, India 

V.P.M Junior College, Mulund East, 

Mumbai, Maharashtra, India 

V.P.M English High School, Mulund East, 

Mumbai, Maharashtra, India 

Year of 

Passing 

March 2009 

July 2007 

June 2004 

June 2002 
Job 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 in Mice 

~ 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 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 of study results. 

TRAINING ATTENDED: 

1. Completed GLP training organized at Jai Research Foundation. 

2. Attended 32"d Annual Conference of Society of Toxicology (STOX) and International Symposium on ''New frontiers in 

Toxicology" held at Lucknow, in December 2012. 

Ill 
CV- DR. T.G. MARVANIA 

Name 

Date of Birth 

Qualification 

Present Designation 

Responsibility 

Tulsidas Gordhan Marvania 

1 ih June, 1965 

Ph.D., M.Sc. (Agri.) 

Deputy Director, JRF 

Project Coordination and Monitoring, JRF 

Total Job Experience 

Study Director for Field studies (Efficacy, Compatibility and 

Phytotoxicity) and Ecotox studies 

24 years 

1 Since 1993 till Date: Jai Research Foundation, Joined as a Research Officer and present position is 

Deputy Director 

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 of the 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 of resources for Study Group and monitoring of the 

ongoing projects with respect to contractual terms 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 of new formulations 

of insecticides including house hold insecticides. 

2 1989-91 Worked with United Phosphorus Limited, Mumbai for one year as Sales Representative 

The job profile includes the establishment of Product distribution chain (appointment of Distributors 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 Officer for Gujarat Land Development 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 of2 
Page 2 of2 

I~-- Ill 
TOXICOLOGY AND ENVIRONMENTAL RESEARCH AND CONSULTING 

CURRICULUM VITAE 

NAME: Chad A. 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: Delta College, University Center, MI, Associates of Science 

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 Dow Chemical 

Company, Midland, MI, Laboratory Technologist 

1995-1997: The Dow Chemical Company, Midland, MI, Larkin 

Lab 

N/A 

N/A 

See Attached 

Employee Signature: -----'/Vt"'-__:..:.~.....:.....!.lQ/t__!........:~=---Date. __ 2}'--l_f("--t-y __ _ 
Ill 
PRESENTATIONS 

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 of two Propionic Esters to Freshwater Fish. 27th 

Annual Meeting of the 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 

of a Novel Flow-Through Exposure System in Evaluating the 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: 

20 I 0-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 on Assignment at Roche Diagnostics, Indianapolis In, 

V aJ idation Engineer 

2002-2007: Pfizer Inc. Ann Arbor, Ml, Associate Scientist, Analytical Research and 

Development 

PUBLICATIONS: See Attached 

PRESENT ATlONS: N/ A 

Reviewed/Revised: ....... ~?:---~~ ::2 --------- Signature) 
) 

I I Ill 
l'lJBUCA TIONS 

An evaluation ofJ(mr commercial HPLC chiral detectors: A comparison of three 

polarimeters and a circular dichroism detector, Laila Kott*, W. Brian .H.olzheuer, Megan 

M. Wong and Gregory K. Webster Journal ofPhammceutical and Biomedical Analysis 

(2005) 

Application of a Low Cost Amperornetric Sensor f'iJr Detection of Alcohols in Process 

Related Headspace Applications;with Gregory K. Webster, Tracy l-lobson and W. Brian 

Holzheucr, submitted J. of Proc Anal Chem (2007) 

2 

A Comparison of IJPLC and SFC Chiral Method Development Screening Approaches for 

Compounds of Pharmaceutical Interest, Megan M Wong, William B Holzheuer, 

and Gregory K Webster Curr Phann Anal 4(2): 1 0 1-l 05 (2008) 

Reversed Phase Chiral Method Development Screening f(>r Compounds of 

Phannaceutical Interest, William B Holzheucr, Megan M Wong, and Gregory K Webster, 

Curr Pharm Anal 5(4):346-357 (2009) 

CURRICULUM VITAE 

NAME: Robert J. West 

JOB TITLE: Group Leader 

EDUCATION: 

1992: Central Michigan University, Bachelor of Science, Chemistry 

1989: St. Clair County Community College, Associate of Science Degree 

1987: Croswell-Lexington High School 

EMPLOYMENT: 

2005-Present: The Dow Chemical Company, Midland, MI, Senior Research Specialist, 

Environmental Chemistry Research Laboratory 

2000-2005: The Dow Chemical Company, Midland, MI, Research Specialist, 

Environmental Chemistry Research Laboratory 

1997-2000: The Dow Chemical Company, Midland, MI, Senior Research Chemist, 

Environmental Chemistry Research Laboratory 

1994-1997: The Dow Chemical Company, Midland, Ml, Research Chemist, 

Environmental Chemistry Research Laboratory 

1992-1994: The Dow Chemical Company, Midland, MI, Chemist, Environmental 

Toxicology & Chemistry Research 

1990-1992: The Dow Chemical Company, Midland, MI, Student Technologist, 

Environmental Toxicology & Chemistry Research Laboratory 

1987-1989: Carpenter-Self employed, Croswell, MI 
I! ill 
2 

MEMBERSHIPS: 

Society of Environmental Toxicology and Chemistry 

American Chemical Society-Environmental Chemistry Division 

PUBLICATIONS: See Attached 

PRESENTATIONS: See Attached 

Reviewed/Revised: 

(Signature) (Date) 

PUBLICATIONS 

1) Schupp, T. and West, R. J. (2007) Primary aromatic amines: The contribution 

of sediment organisms to human exposure. EXCLI Journal 6: 187-196. 

2) West, R. J., Davis, J. W., Pottenger, L. H., Banton, M. I., and Graham, C. (2007) 

Biodegradability relationships among propylene glycol substances in the 

Organization for Economic Cooperation and Development ready- and seawater 

biodegradability tests. Environ. Toxicol. Chern. 26:862-871. 

3) Klecka, G. M., Gonsior, S. J., West, R. 1., and Markham, D. A. (2002) 

Biodegradation of bisphenol A in aquatic environments- River die-away. 

Environ. Toxicol. Chern. 20:2725-2735. 

4) West, R. J., Goodwin, P.A., and Klecka, G. M. (2001) Assessment of the ready 

biodegradability of bisphenol 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., and Decarolis, J. G. (2002) Natural attenuation of 

chlorinated solvents at Area 6, Dover Air Force 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) The natural attenuation of chlorinated ethenes 

at Dover Air Force Base, Delaware, USA. Proceedings of the 6th International 

FZKITNO Conference on 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 of triethanolamine. 

Environ. Toxicol. Chern. 15:472-480. 

lO)Gonsior, S. J. and West, R. J. (1995) Biodegradation of glycol ethers in soil. 

Environ. Toxicol. Chern. 14:1273-1279. 

3 
I rl Ill 
PRESENTATIONS 

I) Lowe, E. R., Rick, D. L., West, R. J., and Bartels, M. J. The Evaluation of 

Quantitative Structure Property Relationship Predictions of Tissue: Blood 

Partition Coefficients of Highly Lipophilic Chemicals. 14th North 

American ISSX Meeting, International Society for the Study of Xenobiotics 

(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 of a Novel Flow-Through Exposure System in Evaluating the Chronic 

Toxicity of Chloroform to Sediment-Dwelling Organisms. 7th Annual Meeting of 

the Society for Environmental Toxicology and Chemistry, North America, held in 

Montreal, Quebec, November 5-9, 2006. 

3) West, R. J. and Mihaich, E. (2005). Overview of the Persistence, 

Bioaccumulation Potential, and Inherent Ecotoxicity of Diisocyanate Substances. 

Presented to Existing Substances Assessment Division, Environment Canada, 

Montreal, Quebec. Jan. 17, 2005. 

4) West, R. J; Henry, K. S; and Davis, J. W. (2004). Examination of feeding modes 

and sediment equilibration time used in evaluating chronic toxicity of 

toluenediamine (TDA) to sediment-dwelling organisms. Presented at Fourth 

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 intrinsic biodegradation of carbon 

tetrachloride in a fractured bassalt aquifer. In Leeson, A. (Ed.) Proceedings of the 

6th International In-Situ and On-Site Bioremediation Symposium, San Diego, CA 

June 4-7, 2:89-96 
I fl Ill 
( 

1, 

Appendix B 

Melting Point, Freezing Point, Boiling Point Study Report 

14 

DEPCT 
Ill Ill -
( 

I 

\ 

REPORT 

PHOSPHOROCHLORIDOTHIOTIC ACID, 

0,0-DIETHYL ESTER (DEPCT): 

Determination of Melting/Freezing Temperature and Boiling 

Temperature 

Study Director: 

Test Facility: 

Sponsor: 

Harlan Study Number: 

Sponsor References: 

Study Completion Date: 

DA Tarran 

Harlan Laboratories Ltd. 

Shardlow Business Park 

Shard! ow 

Derbyshire 

DE722GD 

UK 

The Dow Chemical Company 

Building 1803 

Midland 

Michigan 48674 

UNITED STATES OF AMERICA 

41304286 

130187 & 130189 

07 February 2014 

Pal!e 1 of 19 
Harlan Study Number: 41304286 

Sponsor References: 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 INTRODUCTION AND 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 of the Test. .................................................................................. 9 

3.1.3 Performance ofthe Test. ............................................................................ 9 

3 .1.4 Data Handling ............................................................................................ 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 ofthe Test. ................................................................................ 14 

3.2.3 Performance ofthe Test. .......................................................................... 14 

3.2.4 Data Handling .......................................................................................... 15 

3.2.5 Results ..................................................................................................... 16 

3.2.6 Conclusion ............................................................................................... 17 

APPENDICES ............................................................................................................................... 18 

Appendix 1 

Appendix 2 

Certificate of Analysis ........................................................................................... 18 

Monitoring Authority Statement of GLP Compliance .......................................... 19 

Ill, Ill 
Harlan Study Number: 41304286 

Sponsor References: 130187 & 130189 

Report Page3 

~ STUDY DIRECTOR STATEMENT OF GLP COMPLIANCE \. 

Harlan Laboratories Ltd., Shardlow Business Park, Shardlow, Derbyshire, DE72 2GD, UK 

Harlan Study Number: 

Study Title: 

41304286 

PHOSPHOROCHLORIDOTHIOTIC ACID, 

0,0-DIETHYL ESTER (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 of Directives 2004/9/EC and 2004/1 0/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 (MHL W, MAFF and METI). 

This report fully and accurately reflects the procedures used and data generated. There were no 

circumstances considered to have affected the integrity of the study or the validity of the data. 

Study Director: D A Tarran 

Date: 0 7 FEB 20H 
Harlan Study Number: 41304286 

Sponsor References: 130187 & 130189 

Report Page4 

QUALITY ASSURANCE STATEMENT 

Harlan Study Number: 

Study Title: 

41304286 

PHOSPHOROCHLORIDOTHIOTIC ACID, 

0,0-DIETHYL ESTER (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-related procedures conducted at the test facility were audited and inspected. The details of 

these audits and inspections are given below. 

Reported to the relevant 

Dates and Types of QA Inspections Study Director and Test 

Facility Management 

Date of Inspection Type of Inspection Phase Inspected Report Date 

05 December 2013 Study Plan Verification N/A 05 December 2013 

22 January 2014 Process - based Melting/Freezing 22 January 2014 

Temperature 

14 January 2014 Study- based Boiling Temperature 14 January 2014 

06 February 2014 Report Audit N/A 06 February 2014 

Verification of study plan amendments for GLP compliance were performed during the course of 

the study. 

This statement confirms that this final report reflects the raw data and the procedures followed. 

Quality Assurance: 

.L .. 

0 7 FEB 2014 

Ill Ill 
( 

\ 

Harlan Study Number: 41304286 

Sponsor References: 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 

summarized below: 

Melting/Freezing Temperature. Less than -20 °C (253 K) using a method designed to be 

compatible with Method Al Melting/Freezing Temperature of Commission Regulation (EC) No 

440/2008 of 30 May 2008 and Method 102 of the OECD Guidelines for Testing of Chemicals, 

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 of Chemicals, 27 July 1995. 
Harlan Study Number: 41304286 

Sponsor References: 130187 & 130189 

Report 

GENERAL INFORMATION 

Schedule 

Experimental Starting Date: 

Experimental Completion Date: 

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 of all original data and the final report will be retained in the Harlan 

Laboratories Ltd, Shardlow, UK archives for a minimum of one monitoring authority inspection 

cycle, in accordance with current GLP requirements. 

Ill· Ill 
( 

\. 

Harlan Study Number: 41304286 

Sponsor References: 130187 & 130189 

Report 

1 INTRODUCTION AND PURPOSE 

Page 7 

The purpose of this study is to evaluate the melting/freezing temperature and boiling temperature 

of the 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 and recommendations: 

• Commission Regulation (EC) No 440/2008 (as amended) of30 May 2008. 

• OECD Guidelines for Testing of Chemicals. 
Harlan :Study Number: 413042!!6 

Sponsor References: 130187 & 130189 

2 TESTITEM 

Report Page 8 

Information as provided by the Sponsor. The Certificate of Analysis provided by the Sponsor is 

presented as Appendix 1. 

Identification: 

Description: 

Batch: 

Purity: 

Expiry Date: 

Storage Conditions: 

Ill Ill 

PHOSPHOROCHLORIDOTHIOTIC ACID, 

0,0-DIETHYL ESTER (DEPCT) 

pale yellow liquid 

C032013081501 

99.13% 

14 August 2014 

room temperature in the dark under nitrogen 
Harlan Study Number: 41304286 

Sponsor References: 130187 & 130189 

3 TESTS 

Report 

3.1 Melting/Freezing temperature 

3.1.1 Test System 

Page9 

The determination was 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 

and Method 102 ofthe OECD Guidelines for Testing of Chemicals, 27 July 1995. 

3.1.2 Principle of the Test 

An aliquot of the 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 Performance of the Test 

An aliquot (~15 mL) of test 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 test tube was surrounded by a glass jacket and then placed into 

a large beaker containing a dry ice/acetone mixture. The temperature and observations of the 

test item were recorded every 30 seconds. 

The determination was carried out in duplicate. 

3.1.4 Data Handling 

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 

Sponsor References: 130187 & 130189 

3.1.5 Results 

Keport 

Cooling data for Determinations A and B are shown in the 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 in appearance during cooling. 

Result: Freezing temperature <-20 °C (253 K). 

I 11 Ill 

Page lU 

Temperature (K) 

264 

263 

262 

261 

261 

260 

259 

259 

258 

257 

256 

256 

256 

255 

255 

254 

254 

253 

253 

252 
Harlan Study Number: 41304286 

Sponsor References: 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 Study Number: 41304286 

Sponsor References: 130187 & 130189 

Report 

Figure 3.1 Determination A 

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) 

Figure 3.2 Determination B 

Freezing Temperature Determination 2 

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 12 
Harlan Study Number: 41304286 

Sponsor References: 130187 & 130189 

Report 

The results are summarized in the following table: 

Table3.3 Freezing Points 

Freezing Point 

[OC] 

1st defmitive test <-20 

2nd definitive test < -20 

Mean value <-20 

Both samples did not freeze under the conditions of the test. 

3.1.6 Conclusion 

Page 13 

[K] 

<253 

<253 

<253 

The freezing temperature of the test item has been determined to be less than -20 °C (253 K). 
Harlan Study Number: 41304286 

Sponsor References: 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 of the 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 with the 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 in heat flow between the sample crucible and the reference crucible, is registered. 

3.2.3 Performance of the 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 within the 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 after the test, the appearance of the samples was determined. 

• Value quoted by supplier 

Ill: Ill 
Harlan Study Number: 41304286 

Sponsor References: 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 of a 

liquid is the same as the standard pressure (101.325 kPa). During thermal analysis the peak width 

of an endothermic reaction also depends on sample amount and heating rate. Therefore, the onset 

point of the relevant endothermic peak is usually stated as the boiling temperature. Where the 

endothermic peaks/effects are poorly defined, a range will be stated in the result section. 

Measured temperatures were converted from °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 

with regard to the ambient pressure during measurement. 
Harlan ~tudy Number: 413042.86 

Sponsor References: 130187 & 130189 

3.2.5 Results 

Report Page 16 

Thermo grams and thermographic data for determinations 1 and 2 are shown in Figure 3.3 and 

Figure 3.4 and in the following tables. 

Figure 3.3 Thermogram - Determination 1 

.-------------------------------------·-----------------------------

20 

mW 

Method: PIJT640 

eft 1.00 s 

100.o-300.00C 20.00°C/mln 

Synchronization enabled 

TI178946, 17.01.2014 11:05:04 

TI178946, 4.5000 mg 

Residue: No visible residue 

Table 3.5 

Thermal Event 

Endotherm 

Ill: Ill 

Integral -619.00 mJ 

Onset 201.59 oc 

Thermographic Data - Determination 1 

Interpretation 

Temperature 

oc K 

Boiling temperature 201.59 475 

Atmospheric pressure: 98.3 kPa 
( 

\ 

Harlan Study Number: 41304286 

Sponsor References: 130187 & 130189 

Figure 3.4 

Report Page 17 

Thermogram - Determination 2 

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 ' 

Table 3.6 Thermographic Data - Determination 2 

Temperature 

Thermal Event Interpretation oc K 

Endotherm Boiling temperature 201.90 475 

Atmospheric pressure: 98.3 kPa 

Overall boiling temperature: 202 ± 0.5 °C (475 ± 0.5K) 

3.2.6 Conclusion 

The boiling temperature of the test item has been determined to be 202 ± 0.5 oc (475 ± 0.5K) at 

98.3 kPa. 
Harlan Study Number: 41304286 

Sponsor References: 130187 & 130189 

APPENDICES 

Appendix 1 Certificate of Analysis 

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 SPClFICA TION STANDARD RESULT 

1 APPEARANCE COLOURLESS CLEAR LIQUID COLOURLESS CLEAR UQUID 

2 0,0-Diethyl phospborochloridothioate ;:.?>98.5% 99.!3% 

3 MOISTURE (ppm) ~300 284 

4 CHROMA ~100 70 

5 0,0-diethyl phosphorochloridate ~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 CHECKED BY:HUJIN:..... .. . ........ -iXAMINE AND VERIFIED 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 Study Number: 41304286 

Sponsor References: 130187 & 130189 

Report 

Appendix 2 Monitoring Authority Statement of GLP 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 July 2012 

TEST TYPE(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 statement constitutes a Good Laboratory Practice Instrument (as defined in the 

UK Good Laboratory Practice Regulations 1999). 

~~~ '30 /o (tz_ 

Head, 

Dr. Andrew 

UK GLP 

J. Gray 

Monitoring Authority 

~ 

~ 

Page 19 
Ill; Ill 
Appendix C 

Vapor Pressure Study Report 

15 

DEPCT 
Ill: Ill 

\ 

I 

J 
( 

REPORT 

PHOSPHOROCHLORIDOTHIOTIC ACID, 

0,0-DIETHYL ESTER (DEPCT): 

Determination of Vapor Pressure 

Study Director: 

Test Facility: 

Sponsor: 

Harlan Study Number: 

Sponsor Reference: 

Study Completion Date: 

DA Tarran 

Harlan Laboratories Ltd. 

Shardlow Business Park 

Shardlow 

Derbyshire 

DE722GD 

UK 

The Dow Chemical Company 

Building 1803 

Midland 

Michigan 48674 

UNITED STATES OF AMERICA 

41304287 

130188 

18 February 2014 

Pag:e 1 of20 
Harlan Study Number: 41304287 

Sponsor Reference: 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 Vapor Pressure ......................................................................................................... 9 

3.1.1 Test System ............................................................................................... 9 

3.1.2 Principle ofthe Test ................................................................................... 9 

3.1.3 Performance of the 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 of Analysis ........................................................................................... 19 

Monitoring Authority Statement of GLP Compliance .......................................... 20 

Ill Ill 

\ 

j 
( 

' 

Harlan :Study Number: 41JU42H7 

Sponsor Reference: 130188 

Report 

STUDY DIRECTOR STATEMENT OF GLP COMPLIANCE 

Harlan Laboratories Ltd., Shardlow Business Park, Shardlow, Derbyshire, DE72 2GD, UK 

Harlan Study Number: 

Study Title: 

41304287 

PHOSPHOROCHLORIDOTHIOTIC ACID, 

0,0-DIETHYL ESTER (DEPCT): 

Determination of Vapor 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 of Directives 2004/9/EC and 2004/1 0/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 (MHL W, MAFF and METI). 

This report fully and accurately reflects the procedures used and data generated. There were no 

circumstances considered to have affected the integrity of the study or the validity of the data. 

Study Director: D A Tarran 

Date: 1 8 FEB 2014 
Harlan Study Number: 41304287 

Sponsor Reference: 130188 

Report Page4 

QUALITY ASSURANCE STATEMENT 

Harlan Study Number: 

Study Title: 

41304287 

PHOSPHOROCHLORIDOTHIOTIC ACID, 

0,0-DIETHYL ESTER (DEPCT): 

Determination of Vapor Pressure 

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-related procedures conducted at the test facility were audited and inspected. The details of 

these audits and inspections are given below. 

Reported to the relevant 

Dates and Types of QA Inspections Study Director and Test 

Facility Management 

Date of Inspection Type of Inspection Phase Inspected Report Date 

05 December 2013 Study Plan Verification N/A 05 December 2013 

13 January 2014 Process - based Vapor Pressure 13 January 2014 

11 February 2014 Report Audit N/A 11 February 2014 

Verification of study plan amendments for GLP compliance were performed during the course of 

the study. 

This statement confirms that this final report reflects the raw data and the procedures followed. 

Quality Assurance: 

1 7 FEB 2014 

Ill i Ill 
( 

Harlan Study Number: 41304287 

Sponsor Reference: 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 Vapour Pressure of Commission Regulation (EC) No 

440/2008 of 30 May 2008 and Method 104 of the OECD Guidelines for Testing of Chemicals, 

23 March 2006. 
Harlan Study Number: 41304287 

Sponsor Reference: 130188 

Report 

GENERAL INFORMATION 

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 of archiving at The Dow 

Chemical Company. A copy of all original data and the final report will be retained in the Harlan 

Laboratories Ltd, Shardlow, UK archives for a minimum of one monitoring authority inspection 

cycle, in accordance with current GLP requirements. 

Ill• Ill 
( 

Harlan :Study Number: 4 13042!!7 

Sponsor Reference: 130188 

Report 

1 INTRODUCTION AND PURPOSE 

The purpose ofthis study is to evaluate the vapor pressure of the 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) of30 May 2008. 

• OECD Guidelines for Testing of Chemicals. 
Harlan Study Number: 41304287 

Sponsor Reference: 130188 

2 TEST ITEM 

Report Page 8 

Information as provided by the Sponsor. The Certificate of Analysis 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 August 2014 

room temperature in the dark under nitrogen 
' I 

Harlan Study Number: 41304287 

Sponsor Reference: 130188 

3 TESTING 

3.1 Vapor Pressure 

3.1.1 Test System 

Report Page9 

The determination was carried out using a procedure designed to be compatible with Method A4 

Vapour Pressure of Commission Regulation (EC) No 440/2008 of 30 May 2008 and Method 104 

of the OECD Guidelines for Testing of Chemicals, 23 March 2006. 

Instrument: Vapor pressure balance. 

3.1.2 Principle of the Test 

The vapor pressure is determined using a vapor pressure 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 of the Test 

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 of the cross-section of the vapor pressure balance is represented in Figure 3 .1. After 

evacuating the system, opening the shutter above the sample oven causes the escaping vapor jet 

to be directed at the scale pan. The difference in mass readings with the orifice covered and 

uncovered is proportional to the vapor pressure at the given oven temperature. 

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 of three runs was started after a sample of test 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 between runs. 
Hanan ~tuay Numoer: 4UU4:LIS'/ 

Sponsor Reference: 130188 

Report 

Figure 3.1 Schematic Diagram of the Apparatus Used 

3 

~ 

2 

4 5 

1 Microbalance 

2 Oven 

3 Glass viewing panel 

4 Balance pan 

5 Shutter with orifice 

6 Test sample 

Ill Ill 

Page 10 
Hartan ::stuay Number: 4UU42~/ 

Sponsor Reference: 130188 

Keport 

3.1.4 Data Handling 

The vapor pressure is related to the observed mass difference by Equation 3 .1. 

where: 

Vp 

om 

g 

A 

= 

= 

V om.g p=--A 

vapor pressure (Pa) 

mass difference (kg) 

acceleration due to gravity (9.813 m s"2

) 

area of the orifice (7.06858x10-6 m2

) 

Vapor pressure is related to temperature by Equation 3 .2. 

where: 

Vp 

T 

= 

= 

. slope . Log10 [Vp] = --+ mtercept T 

vapor pressure (Pa) 

temperature (K) 

Page 11 

Equation 3.1 

Equation 3.2 

Therefore a plot of log10 Vp versus reciprocal temperature gives a straight line graph. 

The vapor pressure of the sample was measured over a range of temperatures to enable 

extrapolation to 298.15 K. 
Harlan Study Number: 41304287 

Sponsor Reference: 130188 

3.1.5 Results 

Report Page 12 

Recorded temperatures, mass differences and the resulting calculated values of vapor pressure 

are shown in the following tables and figures: 

Runl 

Table 3.1 Vapor Pressure Data 

Temperature Temperature Reciprocal Mass Mass Vapor Temperature Difference Difference Logw Vp CO C) (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 0.00399760 I 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 of log10 Vp versus reciprocal temperature for Run 1 gives the following statistical data 

using an unweighted least squares treatment. 

Slope 

Standard error in slope 

Intercept 

Standard error in intercept 

-3.64 X 103 

40.1 

14.2 

0.158 

Extrapolation using the slope and intercept above yields a vapor pressure (Pa) at 298.15 K with a 

common logarithm of 1.98. 

Ill i Ill 
( 

rtanan ~may Numoer: 4UU4L~1 

Sponsor Reference: 130188 

Report Page l3 

Figure 3.2 Run 1 - Graph of Log10 Vapor Pressure vs 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 Study Number: 41304287 

Sponsor Reference: 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 of log10 Vp versus reciprocal temperature for Run 2 gives the following statistical data 

using an unweighted least squares treatment. 

Slope 

Standard error in slope 

Intercept 

Standard error in intercept 

-3.58x 103 

30.9 

14.0 

0.122 

Extrapolation using the slope and intercept above yields a vapor pressure (Pa) at 298.15 K with a 

common logarithm of 1.95. 

I Tl I Ill 
( 

\ 

.ttartan ::stuay Number: 4UU42lr/ 

Sponsor Reference: 130188 

Report Page 15 

Figure 3.3 Run 2- Graph ofLogto Vapor Pressure vs 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 

Sponsor Reference: 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 

Vapor Pressure 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 error in slope 

Intercept 

Standard error in 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 

common logarithm of 1.97. 

I] I Ill 
( 

Harlan ~tudy Number: 41304287 

Sponsor Reference: 130188 

Report Page 17 

Figure 3.4 Run 3- Graph ofLog10 Vapor Pressure vs 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 ....._-------------~--------....1 
Harlan :Study Number: 4BU4l~7 

Sponsor Reference: 130188 

Summary of Results 

Report Page 18 

The values of vapor pressure at 25 oc extrapolated from each graph are summarized in the 

following table: 

Table 3.4 Summary of Vapor Pressure Data 

Run Logto [Vp(25 °C)] 

1 1.98 

2 1.95 

3 1.97 

Mean 1.97 

Vapor Pressure 93 

The test item did not change in appearance under the conditions used in the 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 for the test 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 of the 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 of Analysis 

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 TANK NO. EXFU 8104300 

QUANTITY 20000.00kg SAMPLE 2000 

TESTING DATE 2013.8.15 INSPECTING STANDARD Q/XNJSOI(B)·2011 

DATE OF EXPIRY 2013.8.15-2014.8.14 

NO SPCIFICATlON STANDARD RESULT 

1 APPEARANCE COLOURLESS CLEAR LIQUID COLOURLESS CLEAR LIQUID 

2 O,O-oietbyl phosphorochloridothioate :?!98.5% 99.13% 

3 MOISTURE (ppm) ~300 284 

4 CHROMA ~100 70 

s 0,0-dietbyl phosphorochloridate ~0.2% 0.04% 

6 0-ethyl phosphorodichloridothioate ~0.300/o 0.16% 

7 0,0,0-triethyl phosphorothioate ~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•, 1IN ~62'6s', . DOW AGROSCIENCES P..P.W MATERIAL Sl?ECIFICAT::ON 21126R101. 

PJRCHASE ORDER NO.: 16/45545329 

Page 19 
nanan >:>tuuy 1''1 umoer: 4 UU4.<::C:S 1 

Sponsor Reference: 130188 

Keport 

Appendix 2 Monitoring Authority Statement of GLP Compliance 

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 July 2012 

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 statement constitutes a Good Laboratory Practice Instrument (as defined In the 

UK Good Laboratory Practice Regulations 1999). 

~~ 

-:.30 /u/tz... 

Head, 

Dr. Andrew 

UK GLP 

J. Gray 

Monitoring Authority 

~ 

~ 

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Page 20 

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DEPCT 

Appendix 0 

Stability in Water Hydrolysis Study Protocol 

16 
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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 OF pH ACCORDING TO OECD 

GUIDELINE 111 

KEY NUMBERS 

FILE#: 

STUDYID: 

PTR#: 

CAS#: 

SPONSOR: Dow AgroSciences 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, Michigan 48674 

REQUIRED SIGNATURES: 

STUDY DIRECTOR: 

LEAD SCIENTIST: 

MANAGEMENT APPROVAL: 

SPONSOR or DESIGNEE: 

MANAGEMENT ASSIGNED STUDY PERSONNEL: 

Technical Reviewer: 

ADDITIONAL STUDY CONTACTS: 

Business Consultants: 

Document Management: 

Quality Assurance Auditor: 

Study Director in Training: 

/DATE 

/DATE 

/DATE 

/DATE 
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A. TITLE 

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STUDYID: 

PAGE2 

PHOSPHOROCHLORIDOTHIOIC ACID, 0,0-DIETHYL ESTER (DEPCT): 

DETERMINATION OF HYDROLYSIS AS A FUNCTION OF pH ACCORDING 

TO OECD GUIDELINE 111 

B. INTRODUCTION 

Purpose 

The objective of this study is to investigate the hydrolysis of the test material 

following the guidelines of the Organisation for Economic Co-Operation and 

Development (OECD) method 111 "Hydrolysis as a Function of pH" [OECD, 2004]. 

Test Guidelines 

OECD Organisation for Economic Co-operation and Development, OECD 

Guidelines for Testing of Chemicals. Guideline Number 111 Hydrolysis. 

13 April2004. 

MATERIALS AND METHODS 

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 of Analysis and Reference) 

The non-GLP certificate of analysis lists the purity as 99.13% 0,0-Diethyl 

phosphorochloridothioate (Qinyan, 2013). Further GLP characterization oftest 

material purity will be performed prior to initiation of this study. The final protocol 
/ 

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and study report will include relevant information on identity and purity, as 

determined from this GLP characterization. 

Characteristics 

Molecular Formula 

C4H10ClOzPS 

Molecular Weight 

188.6 

Chemical Structure 

CAS Number 

2524-04-1 

Test Solutions 

Stock solutions used for dosing of the test material to hydrolysis reaction mixtures 

will be prepared in an appropriate anhydrous and water-miscible aprotic organic 

solvent (e.g., acetonitrile). Stability of the substance in the selected solvent will be 

demonstrated prior to, or as part of, this study. 

Additional stock solutions of the test material may be prepared in a hydrophobic, 

anhydrous, water-immiscible solvent (e.g, toluene), for preparation of analytical 

calibration standards. Stability in this solvent will also be determined prior to, or 

concurrently with, this study. 

Previous Information 

Hydrolysis can be an important abiotic transformation process for chemicals that 

enter the aqueous environment. A tiered testing approach is recommended by the 

OECD to assess the hydrolytic fate of a substance in water. A preliminary 1st tier 

study is usually conducted at 50°C to determine the susceptibility of the test 
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PAGE4 

compound to hydrolysis at pH 4, 7, and 9. A hydrolysis study at pH 1.2 and 37 oc 

may also be included if a physiologically relevant pH is required to support 

toxicology studies. If the compound is shown to be hydrolytically unstable in the 

preliminary study, a more extensive investigation (2nd tier) may be carried out at 

different temperatures to better define the hydrolysis kinetics for the compound and 

identify hydrolysis products formed. 

A previous study by Cheminova A/S (2010) provides a preliminary investigation of 

test material stability in demineralized water (non-buffered) at ambient laboratory 

temperature ( ~ 25 °C assumed). This study indicated that the test material has a 

hydrolysis half-life of approximately 14 min under these conditions, and is thus 

considered to be hydrolytically unstable at environmentally-relevant temperature and 

pH. A single hydrolysis product, which itself appeared (and is expected) to be 

hydrolytically stable under these test conditions, was identified as phosphorothioic 

acid, 0,0-diethyl ester (CAS. No. 2465-65-8). 

The OECD Guideline 111 requires that hydrolysis of a substance is evaluated at a 

concentration which does not exceed O.OlM, or one-half the water solubility limit 

concentration. Due to the hydrolytic instability of the test material, a water solubility 

limit cannot be accurately determined. Estimated water solubility values for the test 

material, which are based on the structure-fragment contribution calculation 

(WATERNT vl.Ol) and on correlation oflog Pow with water solubility (WSKOW 

v1.42) methods of the U.S. EPA EPI Suite software (US EPA, 2010) are 4,296 and 

1,249 mg/L (i.e., 0.023 and 0.007 M), respectively. The test material has a measured 

vapor pressure of approximately 0.7 mm Hg at 25 °C, and calculated Henry's Law 

Constant, based on the ratio of this vapor pressure and estimated water solubility, of 

approximately 1 x 104 atm*mole/m3 at 25 oc (Appendix A). This indicates the 

substance to be highly volatile from water, and that appropriate experimental design 

and procedures will be necessary to avoid confounding of results from volatilization 

losses. 

Similarly, the water solubility values for the expected hydrolysis product (CAS No. 

2465-65-8) are 24,000 (WSKOW v1.42) and 198,000 (WATERNT vl.Ol) mg/L. 

Estimated vapor pressure for this substance is approximately 0.04 mm Hg at 25 oc 

(US EPA, 201 0; Appendix B. Therefore, the expected hydrolysis product of the test 

material is expected to be much more soluble in and less volatile from water. 
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Considering this information, further testing of hydrolysis according to the 2nd Tier 

of OECD Guideline 111 is required to fulfill (in part) information requirements of the 

U.S. EPA HPV Program. Although the OECD Guideline recommends testing at 

three temperatures falling between 10 and 70 °C, it is expected that complete 

hydrolysis at temperatures above 40 °C would occur within timeframes which would 

not permit meaningful determination of kinetics. 

To better understand the temperature- and pH-dependence, and associated daughter 

products of DEPCT hydrolysis, testing will be conducted under the following 

conditions: 

• Approximately 10 °C at initial pH of 4, 7, 9 

• Approximately 25 oc at initial pH of 4, 7, 9 

• Approximately 40 oc at initial pH of 1.2, 7, 9 

Preliminary Test 

A preliminary test, as recommended in OECD Guideline 111, will not be included as 

part of this study. Information from a previous study (Cheminova 2010), as well as 

information from structure-activity relationships, have indicated the test material to 

be highly unstable in water. 

C. EXPERIMENTAL DESIGN 

Hydrolysis studies will be conducted following the procedures described in the OECD 

Guideline 111 (OECD, 2004). Aqueous buffer solutions (~0.05M) will be prepared at 

pH 1.2, pH 4, pH 7, and pH 9, using buffer compositions selected from those 

recommended in the OECD Guideline, and having components which are not expected 

to react directly with the test material or interfere with analytical determinations. Final 

pH adjustments will be made with O.lN HCl or 0.1N NaOH. To minimize possible 

oxidation reactions, test solutions will be sparged with inert gas to remove dissolved 

oxygen. To reduce the potential for biodegradation of the test compound, the buffer 

solutions will be filter-sterilized with Nalgene disposable filterware (Nalge Company, 

Rochester, New York) containing 0.22-J..lm nylon membranes. The test solutions will be 

incubated in the dark to minimize possible photochemical reactions. Glassware and 

equipment (i.e., septa, pipette tips, etc.) will be sterilized by autoclaving as appropriate. 
I II I Ill 

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The concentration of DEPCT and its expected hydrolysis product in test solutions will 

not exceed 0.01M or one-half the water solubility. Portions of the test solutions will 

be added to serum bottles (identified by appropriate code), and sealed with Tefloncoated rubber septa and aluminum crimp seals. The pH 1.2, 4, 7 and 9 reaction 

mixtures will be placed in thermostated water bath or incubator set at 10, 25, or 40 oc 

(temperature control± 0.5°C). The pH of selected, representative samples will be 

measured during the experiment. Blank solutions (buffered solution without test 

material added) will be prepared and analyzed as appropriate to quantify any 

background interferences in the buffered solutions. For the hydrolysis experiment, a 

sufficient number of replicate test solutions will be prepared in separate serum bottles 

to meet the sampling requirements outlined in the OECD guideline. A minimum of 

six data points between 10 and 90 percent hydrolysis of the test compound (replicate 

samples) will be obtained to define the degradation kinetics for the parent compound. 

The pH of representative samples will be determined at selected sampling time 

points. Hydrolysis experiments will be terminated after 90% degradation has 

occurred, or after 30 days. 

D. STUDY SPECIFIC PARAMETERS 

Hydrolysis Reaction Mixtures 

The hydrolysis reaction mixtures will be prepared and incubated in 10 mL glass 

serum bottles, which are rinsed with ultrapure water and sterilized in an autoclave 

prior to use. The serum bottles will be sealed using PTFE-lined butyl rubber septum 

closures, secured with aluminum crimp caps. The serum bottles will be filled with 

sterile buffer solution with no remaining headspace, sealed, and pre-equilibrated at 

the target incubation temperature. The individual reaction mixtures will be spiked to 

the target initial test material concentration using a gas-tight glass syringe, and a test 

material stock solution prepared in an appropriate water-miscible aprotic solvent. A 

sufficient number of replicate reaction mixtures will be prepared to allow for 

triplicate analyses at each targeted time interval, and for determination of pH and 

extraction blank matrix ofun-spiked buffer solutions. 
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THE DOW CHEMICAL COMPANY 

STUDYID: 

At selected incubation time intervals, triplicate reaction mixtures for each 

pH/temperature combination, will be sacrificed for extraction and analysis of 

remaining test material and associated hydrolysis products. 

Analytical Procedures 

PAGE7 

The test material is expected to be stable when extracted from water into an 

anhydrous aprotic solvent (e.g., hexane, toluene). Hydrolysis occurring in aqueous 

samples collected at selected sampling intervals will be quenched by immediate 

extraction into such an immiscible organic solvent. Aliquots of the organic solvent 

phase will be transferred to sealed autosampler vials, and analyzed by gas 

chromatography. 

The concentration ofDEPCT in test solutions will be determined by gas 

chromatography with appropriate detection (e.g., electron capture, mass 

spectrometry). Instrument and analysis conditions will be detailed in the final report. 

An attempt will be made to quantify hydrolysis products using chromatographic 

retention time matches with analytical standards of the expected products (where 

available commercially), and/or by matches of mass spectra against those obtained 

from commercial spectral libraries. 

The pH oftest solutions prepared at nominal pH of 1.2, 4, 7, and 9 will be measured 

periodically before, during, and after incubation of the hydrolysis reaction mixtures. 

Prior to pH measurements, the pH electrode/meter will be calibrated at two or more 

pH values using standard buffer solutions. The recorded pH will be corrected for, and 

reported at, the target temperature of the hydrolysis test solutions. 

Periodic measurements of the water bath temperature will be made and recorded 

using a NIST traceable thermocouple to confirm proper operation. 

Data and Reporting 

Hydrolysis half-lives for DEPCT will be reported at each temperature and pH tested 

using the calculations described in the OECD Guideline 111. If the hydrolysis 

reaction follows pseudo-first order kinetics, a plot of the natural logarithm of the 

concentration versus time will result in a straight line. The slope of the line will equal 
IT! i Ill 
THE DOW CHEMICAL COMPANY 

STUDYID: 

PAGES 

-k, the pseudo-first order rate constant. The halflife for the hydrolysis reaction will 

be equal to t112 - In 2/k:. 

Concentrations of hydrolysis products formed at greater than 10% yield will be 

reported at each sampling point. Computer modeling may be used to estimate halflives for measured hydrolysis products where appropriate. Details for modeling will 

be included in the final report. 

E. STATISTICS 

Descriptive statistics (mean, standard deviation) will be used where applicable in this 

study. 

F. FINAL REPORT 

A final report of the definitive study will be submitted to the study sponsor and will 

include but not be limited to the following: 

1. N arne and address of the 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 of test and/or control substance (e.g., lot#, purity, physical 

characteristics, and method of preparing test concentrations) 

5. Description of the analytical methods used, 

6. A description of the test concentration used during the study, 

7. A description of all circumstances that may have affected the quality or integrity 

of the study, 

8. Location of raw data, 

9. List and signatures of study personnel, 

10. Statement signed by the Quality Assurance Unit. 

G. REGULATORY COMPLIANCE 

GLP Standards 

Organisation for Economic Co-Operation and Development (OECD) -- OECD Series 

on Principles of Good Laboratory Practice and Compliance Monitoring, Number 1. 

OECD Principles on Good Laboratory Practice (as revised in 1997) 

ENV /MC/CHEM(98) 17 
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US Environmental Protection Agency-TSCA GLP 

THE DOW CHEMICAL COMPANY 

STUDYID: 

PAGE9 

Title 40 CFR, Part 792-Toxic Substances Control Act (TSCA); Good Laboratory 

Practice Standards, Final Rule 

Quality Assurance 

The study conduct, data, protocol, protocol changes/revisions, and final report will be 

inspected by the Quality Assurance Unit, Toxicology & Environmental Research and 

Consulting, The Dow Chemical Company, Midland, Michigan. 

Study Archives 

The data, protocol, protocol changes/revisions, and final report will be archived by 

the Toxicology & 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 of the safety precautions to follow 

when handling the test material. Chemical safety information will be made available. 
I II! - /if 
I. REFERENCES 

THE DOW CHEMICAL COMPANY 

STUDYID: 

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Cheminova (2010). Stability ofEP1 (CASRN 298-06-6) and EP2 (CASRN 252-04-

1) in water. Unpublished report ofCheminova A/S Laboratories, Harboore, 

Denmark. October 2010. Used with permission ofCheminova. 

OECD (2004). OECD Guidelines for the Testing of Chemicals. Test Guideline 111: 

Hydrolysis as a Function of pH. Adopted 13 April 2004. 

Qinyan (2013). Certificate of Analysis ofO,O-Diethyl phosphorochloridoth 

(DEPCT), Lot C032013081501. Zhejiang Xinnong 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, and MPBPVP v1.43. United 

States Environmental Protection Agency, Office of Pollution Prevention and 

Toxics, Washington, D.C. 

APPENDIX A 

THE DOW CHEMICAL COMPANY 

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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) : 
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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} 

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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 

=============+====================+==========+========= 
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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) -2.179 
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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 4296.2 
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Estimated Physical/Chemical Properties for the Expected Hydrolysis Product of DEPCT 

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 VALUE 
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-------+-----+--------------------------------------------+---------+--

------

Frag 2 -CH3 [aliphatic carbon] I 0.5473 1. 0 94 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 514.19 
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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 Ill 
THE 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 7 0 . 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 

THE 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 Ill 
Appendix E 

Reverse Mutation Assay "Ames test" Study Protocol 

17 

DEPCT 
I Til Ill 
JRF Study Number: 481-1-06-XXXX 

Dow Study Number: 130186 

PROTOCOL 

Bacterial Reverse Mutation Assay 

Page 1 of15 

BACTERIAL REVERSE MUTATION ASSAY 

GUIDELINES: OECD N° 471, OPPTS 870.5100, EEC B.13/14 and JMAFF 2-1-19-1 

SPONSOR 

DOW EUROPE GmbH 

BACHTOBELSTRASSE 3 

CH-8810 HORGEN 

SWITZERLAND 

STUDY DIRECTOR: NIRALI N. PATEL 

TEST FACILITY 

JAI RESEARCH FOUNDATION 

DEPARTMENT OF TOXICOLOGY 

VALVADA- 396108 

DIST. VALSAD 

GUJARAT 

INDIA 

JANUARY- 2014 

Study Protocol 
JRF Study Number: 481-1-06-XXXX 

Dow Study Number: 130186 

Bacterial Reverse Mutation Assay 

Page 2 of15 

TITLE OF STUDY: Salmonella-Escherichia coli Mammalian-Microsome Reverse Mutation Assay 

Pre-incubation Method with a Confirmatory Assay 

1. PURPOSE 

To evaluate the mutagenic potential of the test item by utilizing a pre-incubation method to measure the test 

material's ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and 

at the tryptophan locus of Escherichia 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 

product name, lot number, expiration date, and storage condition. 

C. Sample Description: Details listed in the attached Protocol Annexure Form. 

D. Characterization of Test Item 

The Sponsor will be directly responsible for determination and documentation of the analytical purity an 

composition of the test item. 

E. Analytical Verification and Stability of Test Item Dose Formulations 

Stability of the test item in the vehicle, as well as analytical verification of the test item dose formulations, 

will be conducted by the Chemistry Department of Jai 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. 

If dosing formulations are suspensions, aliquots from the top (T), middle (M), and bottom (B) of each 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 of the test material. 

After a minimum of 4 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 Ill 
JRF Study Number: 481-1-06-XXXX 

Dow Study Number: 130186 

Bacterial Reverse Mutation Assay 

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 of the report. 

Stability of the Test Item in the Selected Vehicle 

Stability of the test item in the selected vehicle will be established for a minimum of 4 hours, at the lowest 

and the highest doses being tested. 

Samples will be analyzed for stability using the following equation: 

[(Dosee- Doses) I Doses] x 100 =percent change 

Where: 

Doses= analytical concentration at '0' hour (start) 

Dosee =analytical concentration after 4 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. If homogeneity 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% of nominal. 

The test item is considered homogenous if the % relative standard deviation (RSD) is < I 0% 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 of the acceptable specification range, the Study Director will justifY 

the acceptability of the results, suggest re-analysis of the backup samples, or retest the affected portion of 

the study. 

F. Test Item Retention Sample 

Since the in-life portion of this study is less than four weeks in duration, Jai Research Foundation will not 

retain a reserve sample of the test item. 

Study Protocol 
JRF Study Number: 481-1-06-XXXX 

Dow Study Number: 130186 

G. Disposition of Test Item 

Bacterial Reverse Mutation Assay 

Page 4 of15 

Residual dosing preparations, excluding those saved for concentration or homogeneity analysis (if any), 

will be disposed of following administration to the test system. Following issue of the final report, unless 

otherwise specified by the Sponsor, all remaining test item will be returned to the Sponsor or properly disposed of 

as indicated in the attached Protocol Annexure Form. Records of sample 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 of the test item, including any 

special requirements indicated by the sponsor in the MSDS and/or Protocol Annexure Form. 

3. TEST SYSTEM 

The approved test system under the 1997 guidance ofOECD allows the use of the following strains of bacteria: 

Histidine auxotrophic strains of Salmonella typhimurium viz., TA98, TAlOO, TA1535 and TA1537. 

Tryptophan auxotrophic strain of Escherichia 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 of the cell to certain classes of chemicals 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). It has been 

suggested that the plasmid increases sensitivity to mutagens by modifYing an existing bacterial DNA repair 

polymerase complex involved with the mismatch-repair process. 

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 or by 

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 of the Test System 

Salmonella typhimurium 

Molecular Toxicology Inc., 

I 57 Industrial Park Dr. Boone, 

NC 28607 

U.S.A. 

B. Storage ofthe Test System 

Page 5 of15 

Escherichia coli 

Molecular Toxicology Inc., 

I 57 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 I 0 mL of sterile nutrient broth N° 2 (Oxoid Unipath, England). 

C. Genotypic Characterization of the Test System 

Genotype TA98 TAIOO TAI535 TA1537 WP2uvrA 

(pKMIOI) 

Tryptophan/ Histidine 

requirement His+ His+ His+ His+ Trp+ 

Not 

rfa mutation + + + + applicable 

uvrB I uvrA 

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 of strains T A98, T A I 00, and WP2uvr A (pKM I 0 I) to ampicillin and 

rfa mutation of Salmonella typhimurium strains will be checked along with the range of spontaneous 

revertants after preparation of the master plates. 

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 Nutrient broth No. 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 with their 

batch/lot numbers and the name of the manufacturer. 

Study Protocol 
JRF 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 of the manufacturer of the 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 of the Method 

The principle of the Bacterial Reverse Mutation Test is that it detects mutations that revert the mutations 

already present in the test strains thereby restoring the functional capability of the bacteria to synthesize an 

essential amino acid. 

Such revertant bacteria are detected by their ability to grow in the absence of the 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 of incubation, the revertant colonies are counted and compared with the number of 

spontaneous revertants in the solvent/vehicle control plates. 

B. Preparation of S9 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 of S9 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 of analysis 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 in the 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 of the 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 of the solubility test, the 

Sponsor will be notified of the 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 of solubility test will be documented in the raw data 

and presented in the report. 

D. Tester Strain Cultures 

Each of the tester strains from the frozen stock will be grown in Oxoid Nutrient broth No.2 to a density of 

1-2 x I 09 cells/mL. 

After plating for the mutagenicity assay, the bacterial suspension of each tester strain will be diluted up to 

10"6 in PBS and 100 J-LL of the 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 of colonies in each plate will be counted and expressed as number of colony forming units per mL 

of the bacterial suspension. 

E. Initial Toxicity-Mutation Assay 

Selection of dose levels for the confirmatory mutagenicity assay will be based upon the toxicity and 

precipitation profile of the test item assessed in an initial toxicity-mutation assay. The test item will be 

tested at a minimum of eight 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 of test 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 5 mg/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 Protocol 
JRF Study Number: 481-1-06-XXXX 

Dow Study Number: 130186 

F. Confirmatory Mutation Assay 

Bacterial Reverse Mutation Assay 

Page 8 of15 

The test item will be tested at a minimum of five 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 of test 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 of the 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 or the 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 of Analysis provided by the Supplier(s). Copies of the 

Certificates of Analysis will be kept on file at Jai Research Foundation. 

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 of the 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 of S9 

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 of S9 

(CAS W 613-13-8) mix 

WP2uvrA 10 11g/plate i2_K.Ml01) 

Study Protocol 

1111 Ill 
JRF Study Number: 481-1-06-XXXX 

Dow Study Number: 130186 

Sterility Controls 

Bacterial Reverse Mutation Assay 

Page 9 of 15 

The most concentrated test item dilution, the vehicle control, S9 mix, 0.2 M phosphate buffer, Oxoid 

nutrient broth solution and top agar will be checked for sterility. 

H. Performance of the Assay 

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 under yellow light. 

Labeling 

The petri-dishes will be labeled to indicate the study number, strain number, treatment group, assay phase 

and activation. 

Number of Replicates 

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 of metabolic 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 of metabolic 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 of soft agar containing histidine-biotin I 

tryptophan will be added to each of the tubes. These constituents will be overlaid onto VB agar plates. 

After the soft agar sets, the plates will be incubated at 37 ± 1 oc for 48 to 72 hours. 

Study Protocol 
Bacterial 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 of the bacterial background lawn will be evaluated for evidence of 

test item toxicity and precipitate. Evidence of toxicity 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 of the bacterial background lawn. Precipitation 

will be evaluated after the incubation period by visual examination without magnification. 

8. CRITERIA FOR ACCEPTABILITY OF THE ASSAY 

The Salmonella typhimurium and Escherichia coli reverse mutation test is considered acceptable if it 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 of rfa mutation and uvr B mutation, respectively. 

The Escherichia coli tester strain must exhibit sensitivity to ultraviolet light to demonstrate th 

presence of uvrA mutation. 

Salmonella typhimurium strains TA98 and TAIOO and Escherichia coli strain WP2uvrA (pKMIOl) 

must exhibit resistance to ampicillin to demonstrate the presence of the 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 of bacteria 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 of three 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 of revertants 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 of the 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 of at least one tester strain over a minimum of two increasing doses of the test item 

either in the absence or presence of the metabolic activation system. 

Strains TA98, TA1535, and TA1537 

Data sets will be judged positive if the increase in mean revertants at the peak of the dose response is equal to 

or greater than 3.0-times the mean vehicle control value. 

Strain TAtOO and WP2uvrA (pKMlOl) 

Data sets will be judged positive if the increase in mean revertants at the peak of the dose response is equal to 

or greater 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 not be evaluated as positive. 

10. OBSERVATIONS 

11. 

Effect on Bacterial Background Lawn 

The condition of the bacterial background lawn will be evaluated for evidence of the test item toxicity. 

Precipitation 

The precipitation of the test item will be evaluated after the incubation period by visual examination 

without magnification. 

Number of Revertants 

Revertant colonies 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 of the overnight culture will be expressed as colony forming units (CFU) 

per mL of bacterial suspension. 

Study Protocol 
JRF 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 of cells used) 

• Test conditions (amount of test item per plate, media used, type and composition of metabolic activation 

system, treatment procedures) 

• Results (signs of toxicity, precipitation, individual plate counts, mean number of revertant colonies per plate and 

standard deviation, dose-response relationship, if· applicable, concurrent vehicle and positive control data, 

historical vehicle and positive control data) 

• Treatment, evaluation and interpretation of the 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 during the performance of the study and that all applicable GLP regulations were followed in the conduct 

of the study. Any exceptions to GLP compliance wiii be included in the compliance statement 

• A Quality Assurance Unit statement, including the dates ofQA activities and the dates reported to study director 

and management 

• Copy of study protocol, protocol amendments and protocol deviations as annexure/appendix to the report 

13. STUDY CONDUCT 

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 of this 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 other information 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. 

Exceptions to GLP compliance will be listed in the final report. 

Study Protocol 

Ill Ill 
JRF Study Number: 481-1-06-XXXX 

Dow Study Number: 130186 

D. Test Procedure Guidelines 

Bacterial Reverse Mutation Assay 

Page 13 of15 

The procedures as described in this protocol are based on the following selected testing guidelines: 

rgJ OECD Guideline 471 (Genetic Toxicology: Bacterial Reverse Mutation Test), Ninth Addendum to the 

OECD Guidelines for the Testing of Chemicals, 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, L 142, 31/05/2008 

D METI: 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 

D The Japanese Ministry of Agriculture, Forestry and Fisheries. Testing Guidelines for Toxicology Studies, 

2000. Notification No. 12-Nousan-8147. November 24, 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 of JRF will inspect the critical phase(s) of the 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 Management will be incorporated in the study report .. 

15. ARCIDVING 

The original raw data, protocol and final report of this study, following signing of the 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 of each document (stamped on first/cover 

page as "Exact True Copy of the Original: Page 1 to XXX", signed and dated) will be retained at the archives 

of test facility for two years for the purpose of any audit by 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 of retention period, the documents retained at the test facility will be disposed after documented 

Quality Assurance inspection and related records maintained, however, no intimation of such disposal will be 

informed to the sponsor. In the event of any 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 

either the test facility or the Monitoring/Regulatory Authorities. 

Study Protocol 
JRF Study Number: 481-1-06-XXXX 

Dow Study Number: 130186 

16. PROTOCOLAMENDMENTSANDDEVIATIONS 

Bacterial Reverse Mutation Assay 

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 of the 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 of a protocol amendment. All alterations of the 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 of the 

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, Page No.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: 

Dow Europe GmbH 

Bachtobelstrasse 3 

CH-881 0 Horgen 

Switzerland 

Responsibility Name 

Sponsor Study Monitor Donna Anderson 

19. PROTOCOL APPROVAL- TEST FACILITY 

Address of Test Facility, Study Director and Quality Assurance: 

Jai Research Foundation 

Post Box No. 30, GJ.D.C., Vapi- 396 195 

Dist. Valsad, Gujarat 

India 

Responsibility Name 

Study Director Nirali N. Patel 

Quality Assurance 

Unit Dr. L. U. Sanghani 

Test Facility 

Management Dr. T. G. Marvania 

20. SfUDY SCHEDULE 

Proposed Study Initiation Date 

Proposed Experimental Starting Date 

February .. , 2014 

March .. , 2014 

Signature 

Signature 

Proposed Experimental Completion Date Latest by March 25, 2014 

Proposed Draft Report Date Latest by March 3 I, 2014 

Proposed Study Completion Date Latest by April20, 2014 

Study Protocol 

Bacterial Reverse Mutation Assay 

Page 15 of15 

Date 

Date 
Dow Study No:_I30Jg6._ 

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

-------· ---

PROTOCOL ANNEXURE FORM 

* *import:mf* * Please prm'ide the following it~formation EXACTLY as the sponsor would like it to appear in 

the final report(s). 

l. STUDY SPONSOR 

I. Company: Dow Europe GmbH 

2. Address: Bachtobdstrasse :i 

CH-881 0 Hnrgen 

Switzerland 

II. TEST ITEM 

L Test item name: phosphorochloridothiotic acid, 0.0-dicthvl ester 

2. Lot#: C0320 1 308150 I 

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 of the methods of synthesis/fabrication of the test item located at the sponsor's 

facility'! 

[2JY cs 0No (lf no, a location must be specified):--------------

4. Physical Description (color+ physical state): colorless to light yellow liquid 

5. Composition; for reasons of confidentiality, 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 of Analysis 0MSDS 0Sponsor): The non-GLP certificate of analvsis 

lists the puritv as 99. I 3% 0,0-Diethyl phosnhorochloridothioate (()invan, 2013). 

Specil!c gravity I density (include unit and temperature): 

(21To be performed by JRF QSponsor provided: ____ _ 

Certificate of Analysis: (21Availablc 0 Not availabil' 

Cbaracterit.ation of test item: 0 GLP Onon-GLP* 0 In Progress 

*To be rellectcu as exception to GLP compliance 

6. pH: ____ [g)Not Available. please conduct pH test. 

Protocol Annexure h)rm Page 1 of 3 

111 Ill 

\ 

I 
Dow Study No:_130 J 86_ 

JRF study No: ______ _ 

JRF Protocol No: ____ ~--·-

-=--·-------------- 7. Solubility (check appropriate): 0Water 0DMSO 0Iv!t:thanol 0Ethanol 0Acetont: 

0Corn Oil 0Mineral Oil OOther (spccily): ____ _ 

[;8JNot A vailablc, please conduct a solubility test (results to he provided to the Sponsor prior 

to treatment date(s). 

8. Handling Precautions (Please supply MSDS if available): [;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 of the 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. Conduct dose formulation stability, homogeneity, and concentration analysis: 

IZJY cs, at JRF 0 No* 

*To be reflected as exception to GLP compliance 

2. Analytical Method Information: 

IZJA vailahle, 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. GLP COMPLIANCE: 

OEPA-FIFRA 

0JMAFF 

(Check the appropriate agency(ies)) 

IZJEPA-TSCA IZJOECD 

0METI OOther (specify):----------

V. INFORlVIA TION FOR SAMPLE RETURN OR DISPOSAL: 

Disposition of remaining 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 of test itern (e.g. flammable, poison, corrosive, ell:.):-----· 

Prowco! Annexure Form Pagt.: 2 ol. 3 
Dow Study No:_i30lH6_ 

JRF study No: 

JRF Protocol No: ________ _ 

---~-·-~------· -·-------·------------- Disposition of the retention samples in archives (after the rett•ntion period): 

0Return [8JDispost: 

If the sample is to he returned, shipping info will be provided by email. 

VI. STODY RESULTS: 

Would tlw sponsor !.ike to receive a summary of testing 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 of Transmission: 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 below and transmitting thh,· information to the Test 

l''acility ver(f.v that the information contained in this document is, to the best ofhidher knowledge, factual 

and can he used by the Test Facility for the ptaposes of conducting the approved studies und preparing 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 of 3 

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: Dow AgroSciences 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. 

SPONSOR or 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 DOW CHEMICAL 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 of the study will be to determine the cytogenetic effects of 

phosphorochloridothiotic acid, 0,0-diethyl ester (DEPCT) in the mouse by 

examining the incidence of micronuclei in peripheral blood 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. Mammalian Erythrocyte Micronucleus Test (1998). 

B.12, Mutagenicity- In Vivo Mammalian Erythrocyte 

Micronucleus Test. Official Journal ofthe European 

Communities, L142, 31/05/2008. 

C. TEST MATERIAL INFORMATION 

Test Material Name 

phosphorochloridothiotic acid, 0,0-diethyl ester 

Chemical N arne 

0,0-Diethyl ester phosphorochloridothioic 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 of Analysis and Reference) 

The non-GLP certificate of analysis 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 

Chemical N arne 

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 by oral gavage. Oral gavage is one of the generally accepted routes of 
THE DOW CHEMICAL COiviP ANY 

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 of CD-I 

mice (De Boeck et al., 2005; Gollapudi et al., I986). Dosing solutions will be 

administered in aliquots of I 0-20 ml/kg body weight. 

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 maximum tolerated dose (MTD). MTD is defined as the dose that 

produces clear signs of toxicity without having a major effect on thermal 

regulation or the survival of the animals. For changes in thermal regulation, a 

body temperature increase of at least I oc or a decrease of at least 3 oc for five or 

more hours will be declared as having exceeded an MTD. Body temperature 

changes outside this range have been previously reported to cause an increase in 

micronucleus formation in absence of chemical treatment (Asanami and Shimono, 

I997; Asanami et al., I998). With some test materials, death of the treated 

animals may be the only sign of toxicity at doses above the MTD and no other 

clinical signs of toxicity may be apparent at the MTD. In such cases, the MTD 

will be a dose in between the 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 body weight/day. The middle- and lowdoses will be approximately I/2 and 114 of the maximum dose, respectively. The 

test material will be administered on two consecutive days, approximately 

24 hours (± 1 hour) 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 
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THE DOW CHEMICAL COMPANY 

STUDY ID: 131128 

PAGES 

Rationale for Selection of Sampling 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, 

or there is a delayed absorption and metabolism of the 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 

opportunity to recover any delayed effect (i.e., effect induced by the first treatment) 

as well as a more immediate effect (i.e., effect induced by the second treatment) in the 

same treated animals. 

Preparation of Dosing Solutions 

The test material will be mixed in a suitable matrix for dosing. The details of the 

preparation of the dosing matrix will be incorporated in the study file. CP will be 

mixed in distilled water. Only freshly prepared solutions of the test material will be 

used for dosing unless there are data indicating the stability of the test material in the 

vehicle. Either a freshly prepared solution or a frozen stock solution of the positive 

control will be used for dosing. 

Analytical Verification of Dosing Solutions 

For the MNT, upon mixing, the selected concentrations of the test material in the 

dosing matrix, used for the first day of dosing, 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 

chromatography using 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 of one or more phases. Initially, two dose groups for 

each sex will be evaluated in the RFT. The number of animals included in each dose 

group will be based upon the use of the dose selection criteria (Appendix A). Two 

extra groups ofthree animals will be retained and used if further 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 of the study (day 1 

is defined as the frrst day of dosing). Signs of toxicity and deaths occurring during 

the observation period 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 of the Test System 

The MNT is a short-term in vivo cytogenetic assay for detecting agents that induce 

chromosomal breakage and spindle malfunction (Mavournin et al., 1990). 

Micronuclei arise when acentric chromosomal fragments and/or whole chromosomes 

are excluded from the main nucleus at the telophase stage of the cell division. 

The MNT has several advantages over a cytogenetic assay involving direct 

examination of metaphase 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 of micronuclei 

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 if the results of the RFT show no apparent difference in the toxicity between 

the sexes. Otherwise, both sexes will be used for the MNT as well. 

Strain 

Outbred Crl:CD1(ICR) mice 

Criteria for Selecting the 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 acclimation period each animal will be evaluated by a laboratory 

veterinarian, or a trained animal/toxicology technician 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 prior to the start of the 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 in the 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 off at 6:00p.m.) 

Randomization and Identification 

Before administration oftest material begins, animals will be stratified by body 

weight and then randomly assigned to treatment groups using a computer program 

designed to increase the probability ofuniform group mean weights and standard 

deviations at the start of the study. Animals that are placed on study will be 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. 

If a 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 ad libitum. Animals will be provided 

LabDiet Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, 

Missouri) in pelleted form. Analyses of the feed will be performed by PMI Nutrition 

International to confirm the diet provides adequate nutrition and to quantify the levels 

of selected contaminants. Drinking water obtained from the municipal water source 

is periodically analyzed for chemical parameters 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 of these 

analyses are maintained in the study file. 

Animal Welfare 

In accordance with the U.S. Department of Agriculture Animal Welfare Regulations, 

9 CPR, Subchapter A, Parts 1-4, the animal care and use activities required for 

conduct of this 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 of mice (six/dose) will be administered dose levels ofthe test material on two 

consecutive days based on the results of the 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 of excessive toxicity or death. The decision to dose the extra 

animals will be made by the Study Director based upon the results of the RFT. The 

mice will be observed daily, and signs of toxicity or deaths occurring during the 

observation period will be recorded. All mortalities and/or moribund animals will be 

necropsied to ascertain whether the condition is related to a dosing mishap. 

11 I Ill 
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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 of all surviving animals following anesthesia with 

isoflurane. 

Animal Observations and Body Weight Determination 

The mice will be observed for positive signs of toxicity at least once on the day of 

dosing and subsequent days. The mice will be weighed prior to dosing and on the 

day of their scheduled sacrifice in order to assess the extent of stress experienced by 

the animals following treatment (Gollapudi et al., 1986). 

Body Temperature Data Collection 

The relative body temperatures of the 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 cytometry with traditional blood smears prepared as a backup. Samples will 

be prepared and analyzed as per instructions in the mouse MicroFlow Micronucleus 

Analysis Kit Manual (Litron Laboratories, Rochester, New York). Briefly, blood 

samples from all animals in all experimental groups will be obtained via retro-orbital 

sampling into anticoagulant solution and fixed in ultracold ( -70 to -80°C) methanol 

within five hours of collection. 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 in the RET and a fluorescently labeled antibody to the transferrin 

receptor (anti-CD71-FITC) to stain the RETs. A propidium iodide solution will be 

added to each sample immediately before flow cytometry (FCM) analysis to stain the 

DNA, including that of micronuclei. Blood samples will be analyzed by high-speed 

FCM. In this system, the sample is moved at a high velocity past a laser set to 

provide 488 nm excitation. The fluorescent wavelengths emitted by each cell will be 

collected by photomultiplier tubes. Using the previously described staining 
THE DOW CHEMICAL COMPANY 

STUDY ID: 131128 

PAGE10 

procedure, the propidium iodide-stained DNA of the 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 simultaneously provides cell size information by 

determining the light scatter properties of each cell or combination of cells. 

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 of genotoxic potential. The frequency ofRETs relative to total 

erythrocytes will be determined to provide an indication of perturbations in 

hematopoietic activity indicative of cell toxicity. For each of the treatment groups, a 

mean and standard deviation will be calculated to describe the frequency of RET and 

MN-RET observed. Analyses will be conducted utilizing a Beckman Coulter Gallios 

flow cytometer. 

Details of Slide Preparation 

Duplicate cell smears will be prepared and stored to serve as backups in the event that 

flow cytometric analyses are not possible. Blood will 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 room temperature. 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 of variance using Bartlett's test 

(alpha= 0.01; Winer, 1971). Ifthe results from Bartlett's test are significant, then the 

data for the parameter may be subjected to a transformation to obtain equality of the 

variances. The transformations that will be examined are the common log, the 

inverse, and the square root in that order. The data will be reviewed and an 

appropriate form of the data selected and subjected to the following analysis. 

Based on the results of the sex effect in the RFT, the MN-RET data and the data on 

percent RET may be analyzed by a two-way analysis of variance (when both sexes 

are used in the study) or a one-way analysis of variance (when only males are used) 

11 i Ill 
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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 if significant, a one-way analysis of variance will be performed for each sex. If 

different dose levels of the test material are used for each sex, the data will be 

separated by sex prior to statistical analysis. Pairwise comparisons of treated vs. 

control groups will be done, if the 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 if any of the 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 point to evaluate the biological significance of the MN-RET results. 

The final interpretation ofbiological significance of the responses is based on both 

statistical outcome and scientific judgment. 

A test is considered valid if all of the following conditions are met: 

• The range ofMN-RET values in the negative controls are within reasonable 

limits of the recent laboratory background range. 

• There is a significant increase in the incidence ofMN-RET in the positive 

control treatment as compared to the concurrent negative controls. 

• The mean for percent RET value in one or more of the test material treated 

groups should be 2:: 20% of the control value indicating no undue effect on 

erythropoiesis (toxicity). 

A test material is considered positive in this assay if the following criterion is met: 

• Statistically significant increase in MN-RET frequency at one or more dose 

levels accompanied by a dose response. 

A test material is considered negative in this assay if the 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 or the negative response 

will be considered to be equivocal. 
I. FINAL REPORT 

THE DOW CHEMICAL COMPANY 

STUDY ID: 131128 

PAGE12 

A final report of the definitive study will be submitted to the study sponsor and will 

include but not be limited to the following: 

1. Name and address of the 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 of test and/or control substance (e.g., lot#, purity, physical 

characteristics, and method of preparing test concentrations), 

5. Stability of the test and control articles under the conditions of administration 

and description of the analytical methods used, 

6. A description of the test system used including, when applicable, the number of 

animals used, sex, body weight range, source of supply, species, strain, and 

substrain, age, and procedure used for identification, 

7. A description of the dosage, dosage regimen, route of administration, and 

duration, 

8. A description of all circumstances that may have affected the quality or integrity 

ofthe study, 

9. The results ofthe study, discussion of results, and conclusions, 

1 0. Location of raw data and specimens, 

11. List and signatures of study personnel, 

Statement signed by the Quality Assurance Unit. 

J. REGULATORY COMPLIANCE 

GLP Standards 

Organisation for Economic Co-Operation and Development (OECD) -- OECD Series 

on Principles of Good Laboratory Practice 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 

Quality Assurance 

The study conduct, data, protocol, protocol deviation( s )/amendment( s) if applicable, 

and final report will be inspected by the Quality Assurance Unit, Toxicology and 

Environmental Research and Consulting, The Dow Chemical Company, Midland, 

Michigan. 

11 I Ill 
I 

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Study Archives 

THE DOW CHEMICAL COMPANY 

STUDY ID: 131128 

PAGE13 

The data, protocol, protocol deviation(s)/amendment(s) if applicable, and final report 

will be archived by the Toxicology and Environmental Research and Consulting 

archivist and stored at The Dow Chemical Company, Midland, Michigan. 

K. SAFETY 

All personnel involved in the study will be advised of the safety precautions 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). High body temperature 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 with mouse peripheral blood erythrocytes by acridine orange 

supravital staining: the summary report of the 5th collaborative study by 

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 in mice using multiple blood sampling. Environmental and Molecular 

Mutagenesis 46, 30-42. 

Gollapudi, B. B., Bruce, R. J., Linscombe, V. A., Watanabe, P. G., and Sinha, A. K. 

(1986). Effect of food and water deprivation on the parameters ofthe mouse 

bone marrow micronucleus 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 mammalian bone marrow and 

peripheral blood. A report of the U.S. Environmental Protection Agency GeneTox Program. Mutat. Res. 239, 29-80. 

Qinyan (2013). Certificate of Analysis ofO,O-Diethyl phosphorochloridoth 

(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 Experimental Design, 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 data is available in the rat or mouse. 

a. The existing data indicates 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 in the RFT will be LD50 and Yz LD50 

2. Acute oral toxicity data is not available. 

a. Dose 1 animal/sex at 2000 and 1000 mg/kg and observe animals for 24 hrs after 

the second dosing 

-If all animals survive, dose an additional2 animals/sex at dose levels of2000 

and 1000 mg/kg. 

- If the 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 1 000 and 3 

animals/sex at 500 mg/kg. 

- If animal 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 of a dose 

level between 2000 and 1 000 mg/kg. 
111 Ill 
Appendix 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-006538-0 13 

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 or DESIGNEE: 

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 DOW CHEMICAL COMPANY 

STUDY ID: 141009 

PAGE2 

DIETHYLCHLOROTHIOPHOSPHATE (DEPCT): METHOD VALIDATION 

AND STABILITY IN RODENT FEED AND CORN OIL 

B. INTRODUCTION 

Purpose 

The purpose of this study is to validate the analytical method for determination of 

diethylchlorothiophosphate (DEPCT) in rodent feed and com oil and to evaluate the 

stability of diethylchlorothiophosphate (DEPCT) in rodent feed for a period of 

approximately 65 days and in com oil and toluene for a period of approximately 24 

days. Work is to be performed in accordance with Standard Operating Procedure 

ACL-GEN-017.03. 

Test Guidelines 

C. TEST MATERIAL INFORMATION 

There are no test guidelines for this study. 

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 of Analysis and Reference) 

The non-GLP certificate of analysis lists the purity as 99.13% 0,0-Diethyl 

phosphorochloridothioate (Qinyan, 2013). 

Characteristics 

Molecular Formula 

111 Ill 

) 
I 

\ 

Molecular Weight 

188.6 

Chemical Structure 

CAS Number 

2524-04-1 

Previous Stability Information 

None 

D. EXPERIMENTAL DESIGN 

THE DOW CHEMICAL COMPANY 

STUDY ID: 141009 

PAGE3 

Stability time points for Diethylchlorothiophosphate (DEPCT) in rodent feed will be 

done on days 0, 2, 4, and 8. If day 8 recovery is greater than 85% at any of the test 

concentrations, analyses on days 12, 24, 36, and 65 will be performed as well. 

Stability time 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 may be adjusted at the discretion of the study director based 

upon scheduling and data obtained from earlier time points. 

Analytical method validation will be performed by fortification of rodent feed and 

corn oil with Diethylchlorothiophosphate (DEPCT) at the concentration range of 

interest, as per TERC 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 of control matrix will be analyzed as well. Appropriately sized aliquots of 

the samples will be analyzed to obtain an accurate determination of test 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 of the test material in the test 

system in support of toxicological studies where this test system will be used. The 

method of administration is by direct addition of neat material to the vehicle. This is 

the acceptable method for preparation of the test article for use in toxicological 

studies. 

Data and Report 

Concentration of test 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 of test material from samples will be determined by analyzing 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 of a standard solution, fortified 

sample, and matrix blank. 

Vehicle 

Lab Diet Certified Rodent Diet #5002M, Lot# TBD 

PMI Nutrition International St. Louis, Missouri 

Corn Oil, Lot# MKBL8756V 

Sigma-Aldrich, St. Louis, Missouri 

Solvent- Toluene, Lot#lll565 

Fisher Scientific, Fair Lawn, 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 DOW CHEMICAL COMPANY 

STUDY ID: 141009 

PAGES 

[K] Feed (rodenUcanine) 

[K] Solvent 

I X I Solution/suspension 

Stability Timepoints for Rodent Feed (Days) 

[K] o D 1 [K] 2 []l 4 []l 8 00 12~ 24 []l 36[]] 65 

Stability Time points for Corn Oil (Days) 

[K] o D 1 [K] 2 ~ 4 ~ 8 [R] 12~ 24 D 36 D 65 

Stability Timepoints for Solvent (Days) 

[K] o D 1 [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 of study 

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 if available 

Standard concentrations will bracket the sample concentrations, after preparation for 

analysis. The target LLQ will be 11100 of the lowest sample concentration. 

Note: LLQ may be changed at discretion of study director, based on method 

sensitivity 

Specific details of the 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 I Ambient (- 25°C) 0 Refrigerator (- 5°C) 

I X I Exposed to light 0 Amber glass 

F. STATISTICS AND CALCULATIONS 

0 Freezer (--l5°C) 

0 Light proof container 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 of accuracy). 

G. FINAL REPORT 

A final report of the definitive study will be submitted to the study sponsor and will 

include but not be limited to the following: 

1. Name and address of the 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 of test and/or control substance (e.g., lot #, purity, physical 

characteristics, and method of preparing test concentrations), 

5. Stability of the test and control articles under the conditions of administration 

and description of the analytical methods used, 

6. A description of the concentrations, 

7. A description of all circumstances that may have affected the quality or integrity 

of the study, 

8. Location of raw data, 

9. List and signatures of study personnel, 

10. Statement signed by the Quality Assurance Unit. 

GLP Standards 

Organization for Economic Co-Operation and Development (OECD) -- OECD Series 

on Princi pies of Good Laboratory Practice 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 

\ 
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( 

THE DOW CHEMICAL COMPANY 

STUDY ID: 141009 

PAGE7 

The Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF) --Good 

Laboratory Practice Standards, 11 NohSan, Notification No. 6283- 1 October 1999 

revised by 12 NohSan, Notification No. 8628- 6 December, 2000 

Quality Assurance 

The study conduct, data, protocol, protocol deviation(s)/amendment(s) if applicable, 

and final report will be inspected by the Quality Assurance Unit, Toxicology and 

Environmental Research and Consulting, The Dow Chemical Company, Midland, 

Michigan. 

Study Archives 

The data, protocol, protocol deviation(s)/amendment(s) if applicable, and final report 

will be archived by the 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 of the safety precautions to follow 

when handling the test material prior to study initiation. Chemical safety information 

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 of Analysis of 0,0-Diethyl phosphorochloridoth 

(DEPCT), Lot C032013081501. Zhejiang Xinnong Chemical Co., Ltd., Sanlixi, 

Xianju County, Zhejiang Province, China. 
ill Ill 
Appendix H 

Oral Gavage Range Finding Protocol for Mammalian Reproductivity Toxicity Study 

20 

DEPCT 
II I Ill 
PRELIMINARY 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: Dow AgroSciences LLC 

9330 Zionsville Road 

Indianapolis, Indiana 46268 

TESTING FACILITY: The Dow Chemical 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. 

MANAGEMENT APPROVAL: 

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 DOW CHEMICAL COMPANY 

STUDY ID: 140001 

PAGE2 

DIETHYLCHLOROTHIOPHOSPHATE (DEPCT): 14 DAY ORAL GAVAGE 

RANGE-FINDING STUDY IN Crl:CD(SD) RATS 

B. INTRODUCTION 

Purpose 

The purpose of this study is to evaluate the potential toxicity of 

diethylchlorothiophosphate (DEPCT), in rats following oral gavage administration for 

14 days. Red blood cell and brain cholinesterase activities will also be determined. 

The results of this study will be used to select dose levels for a subsequent oral gavage 

reproductive/developmental screening study. 

Test Guidelines 

This study is 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 of Analysis and Reference) 

The non-GLP certificate of analysis 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 

Corn oil (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 of chlorpyrifos. DEPCT 

and chlorpyrifos are structurally similar molecules and are both known to inhibit 

cholinesterase. Relevant oral toxicity data on DEPCT are limited and consist 

primarily of two single dose oral gavage studies in rats. Therefore, oral toxicity data 

on chlorpyrifos 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, or 252 mg/kg DEPCT in a 5% corn oil mixture by oral gavage. 

Cholinesterase (ChE) levels were measured in the blood 24 hours post-dosing. Plasma 

and red blood cell (RBC) ChE inhibition occurred at all dose levels and ranged from 

25-46% in the plasma and 21-59% in the RBCs (Text Table 1). 

An acute oral toxicity study determined the oral LD50 to be 1.87 g/kg for both male 

and female rats (Keeler, 1975). Additional test animals were administered 0, 63, 126, 

252, or 500 mg/kg DEPCT in a 10% corn oil mixture by oral gavage. ChE levels were 
THE 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 study was conducted to obtain preliminary data on the dermal absorption of 

chlorpyrifos in the rat (Nolan et al., 1981 ). One component of the study included 

groups of 2 or 3 male Fischer 344 rats that were administered a single oral gavage 

dose of 0 or 5 mg chlorpyrifos/kg in corn oil, respectively. Rats were euthanized 

twenty-four hours after dosing. Blood was obtained by cardiac puncture and plasma 

and RBC ChE activities were determined. ChE was inhibited 30% in the plasma and 

59% in the RBCs following a single dose of 5 mg/kg chlorpyrifos. 

A dose response study was conducted focusing on inhibition of ChE and neurotoxic 

esterase in female Fischer 344 rats receiving single oral doses of chlorpyrifos 

(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 of brain 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 in rats dosed with 5, 10, 50 or 100 mg/kg 

of chlorpyrifos. Percent ChE inhibition in the plasma ranged from 45-91% and 

increased in a dose-dependent fashion. Percent inhibition in the RBCs ranged from 

Ill Ill 
I 

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THE DOW CHEMICAL COMPANY 

STUDY ID: I4000I 

PAGE5 

17-52%, and also increased dose dependently, but had a similar level of inhibition at 

50 and 100 mg/kg chlorpyrifos (52 and 51%, respectively). 

Mattsson et al. (1998) evaluated ChE inhibition in Sprague-Dawley rats following 

daily exposure of dams on gestation day (GD) 6 through lactation day (LD) 10 to 

doses ofO, 0.3, 1 or 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 1 and 5 .. 

ChE inhibition in the plasma and RBCs of dams is shown in Text Table 2. Inhibition 

of both plasma 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 Dosing with 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 of Administration, Frequency and Duration 

A possible route of human exposure to the test material would be via accidental 

exposure during the manufacturing process. The purpose of this study is to set dose 

levels for a subsequent reproductive/developmental screening study where the 

preferred route of administration is oral. Dietary administration of the test material is 

not feasible due to instability in rodent feed. Thus, oral administration of the test 

material to rats via gavage represents an appropriate means of exposure. Animals will 

be administered DEPCT once daily, seven days/week for 14 days. 

Dosing Regimen 
THE 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 on previous toxicity of DEPCT. Based on the data cited 

above, ChE inhibition appears to be less sensitive to DEPCT than chlorpyrifos 

administration with 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 on the available data that 

a high-dose level of 100 mg/kg/day DEPCT may result 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/day which is expected to produce significant RBC ChE 

inhibition, and may result in other systemic effects. The intermediate dose levels of 50 

and 25 mg/kg/day are expected to provide a dose-response relationship for any 

treatment-related effects, and the low dose of 5 mg/kg/day is expected to be a NOEL 

for all parameters with the potential exception of RBC ChE inhibition. Additional 

dose levels may be included if deemed 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 body weight to achieve the targeted dose levels. Dose volumes will be 

111 Ill 
THE DOW CHEMICAL COMPANY 

STUDY ID: 140001 

PAGE? 

adjusted daily based on individual body weights. Dose solutions will not be corrected 

for purity of the test material. 

Analysis 

Dose Confirmation, Homogeneity, and Stability 

Dose confirmation and homogeneity analyses will not be conducted due to the 

probe nature of this 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 of the bulk test material will be retained. No samples of dose 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 of their general acceptance and suitability for 

toxicity testing; the subsequent study will be conducted using this strain of rat, and the 

reliability of the commercial supplier. 

Supplier and Location 

Charles River (Portage, Michigan) 

Age at Study Start 

Approximately 7 weeks 

Physical and Acclimation 

During the acclimation period, each animal will be evaluated by a laboratory 

veterinarian, or a trained animal/toxicology technician under the direct supervision of 

a laboratory veterinarian, to determine the general health status and acceptability for 

study purposes (fully accredited by the 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 maintain 
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adequate conditions (temperature, humidity, and photocycle), and acclimated to the 

laboratory for approximately one week prior to the start of the 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 off at 6:00p.m.) 

Enrichment for rats includes the use of ground corn cob and shredded Aspen bedding 

(Harlan Laboratories, Inc., Indianapolis, Indiana) and open areas on the cage sides for 

visualization of other rats. 

Randomization and Identification 

Before administration of the test material begins, animals will be stratified by body 

weight and then randomly assigned to treatment groups using a computer program 

designed to increase the probability of uniform group mean weights and standard 

deviations at the start of the study. Animals placed on study will be uniquely 

identified via subcutaneously implanted transponders (BioMedic Data Systems, 

Seaford, Delaware) that are correlated to unique alphanumeric identification numbers. 

If a 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 

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 ad libitum. Analyses of the feed will be performed by PMI Nutrition 

International to confirm the diet provides adequate nutrition and to quantify the levels 

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of selected contaminants. Drinking water obtained from the municipal water source is 

periodically analyzed for chemical parameters 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 of these 

analyses will be maintained in the study file. 

Animal Welfare 

In accordance with the U.S. Department of Agriculture animal welfare regulations, 9 

CFR, Subchapter A, Parts 1-4, the animal care and use activities required for conduct 

of this 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 Subchronic/Chronic Tox 01, Blood collection 

01, Humane Endpoints 01, and Animal ID 01. 

STUDY DESIGN 

Summary 

Groups of six 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 of collected parameters 

are outlined below in the appropriate sections. 
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Text Table 4. A Summary of Study Parameters 

Study Parameters 

Body Weights 

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. of Animals/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 typically performed 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 of the 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 observation period will be humanely euthanized that 

day. In addition, all animals will be observed for morbidity, mortality, and the 

availability of feed and water at least twice daily. 

Clinical Observations 

Clinical observations will be conducted on all animals at least once daily 

approximately one hour following dosing. Clinical observations include a careful, 

hand-held examination of the animal with an evaluation of abnormalities 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. 

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Body Weights/Body Weight Gains 

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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 of feed 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 of a measurement cycle. Feed consumption will be calculated 

using the following equation: 

Feed consumption (g/day) =(initial weight of feed container- final weight of feed container) 

( # of days in measurement cycle) ( # of animals 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 taken to minimize clotting 

and limit red blood cell lysis. Following exsanguination, the animals will be 

decapitated, the cranial cavity will be opened, and the brain will be removed. Blood 

samples for ChE measurements will be kept on ice until centrifuged to separate plasma 

and RBCs. RBCs will be stored at -80°C until shipped for ChE analysis. A complete 

gross necropsy will be conducted on all animals by a veterinary pathologist assisted by a 

team of trained individuals. The necropsy will include an examination of the external 

tissues and all orifices. The head will be removed from all animals, the cranial cavity 

opened and the brain will be removed, rinsed with saline, and blotted dry. The brain 

will then 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 of brain 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 of a 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 of the 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 of ChE.. The liver and 

kidneys will be weighed and preserved. The ratios of liver or kidney weight to terminal 

body weight will be calculated. Any obvious gross pathologic alterations and relevant 
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gross lesions will be recorded and tissues preserved. Microscopic examination of tissues 

will not be conducted unless deemed necessary. 

Moribund animals that are not expected to survive until the next observation period will 

be humanely euthanized. These animals, as well as any animals found dead, will be 

necropsied on that day during the normal work week. However, animals euthanized or 

found dead after routine working hours or on weekends, and holidays will be 

refrigerated until the next scheduled workday, at which time they will be necropsied. 

Similar necropsy procedures will be followed with the exception that organs will not be 

weighed and tissues will not be 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 contract research laboratory for ChE analyses. 

Brain 

The head will be removed, the cranial cavity opened and the brain will 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 half of the brain specifically 

identified. The weighed right half of the brain will be shipped to a contract research 

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 of the Ellman reaction (Ellman et 

al., 1961; Hunter et al., 1997). The principal investigator, Dr. Jonathan Toot, will be 

responsible for oversight of all ChE analyses conducted at WIL Research 

Laboratories. Details ofthe phase plan. 

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H. STATISTICS 

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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 of Grubbs (1969) and will only be excluded from analysis for 

documented, scientifically sound reasons. Feed consumption values will be excluded 

from analysis if the feed is spilled or scratched. Determination of treatment-related 

effects will be based on sound scientific principles such as biological plausibility, 

comparison to concurrent and historical control data, and dose response relationships. 

Equivocal findings will be noted appropriately and addressed in the subsequent oral 

gavage reproductive/developmental screening study. 

I. FINAL REPORT 

A final report of the definitive study will be submitted to the study sponsor and will 

include but not be limited to the following: 

1. Name and address of the 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 of test and/or control substance (e.g., lot#, purity, physical 

characteristics, and method of preparing test concentrations), 

5. A description of the test system used including, when applicable, the number of 

animals used, sex, body weight range, source of supply, species, strain, and 

substrain, age, and procedure used for identification, 

6. A description of the dosage, dosage regimen, route of administration, and duration, 

7. A description of all circumstances that may have affected the quality or integrity 

ofthe study, 

8. Location of raw data and specimens, 

9. List and signatures of study personnel. 

Study Archives 

The data, protocol, protocol deviation(s)/amendment(s) if applicable, and final report 

will be archived by the Toxicology and Environmental Research and Consulting 

archivist and stored at The Dow Chemical Company, Midland, Michigan. 
J. SAFETY 

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All personnel involved in the study will be advised of the safety precautions to follow 

when handling the test material and treated animals prior to study initiation. Chemical 

safety information will be made available. 

K. REFERENCES 

Dittenber, D. A. (1997). Chlorpyrifos: Evaluation of Single Oral Doses on 

Chloinesterase and Neurotoxic Esterase Inhibition in F344 Rats, Report of Health 

and Environmental Research Laboratories, The Toxicology Research 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 of acetylcholinesterase 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 

of Cholinesterase Activity in Tissues from Carbamate-treated Animals: A 

Cautionary Note. Toxicology Methods, 7, 43-53. 

Keeler, P. A. (1975). Toxicological Properties and Industrial Handling of: 0, 0-

Diethyl Phosphorchloridothioate (DEPCT), Report of Toxicology Research 

Laboratory, The Dow Chemical Company. 

Litchfield, N., Norris, J. M. (1969). Cholinesterase Inhibition Study Conducted on 0, 

0-Diethyl Phosphorchloridothioate, Report Summary of Biochemical Research 

Laboratory, The Dow Chemical Company. 

Mattsson, J. L., Maurissen, J. P., Spencer, M. S., Brzak, K. A., Zablotny, C. L. 

(1998). Effects of Chlorpyrifos Administered via Gavage to CD Rats during 

Gestation and Lactation on Plasma, Erythrocyte, Heart and Brain Cholinesterase, 

and Analytical Determination of Chlorpyrifos and Metabolites, Report of 

Toxicology & Environmental Research and Consulting, The Dow Chemical 

Company, Midland, Michigan. 

Nolan, R. J., Freshour, N. L., Rick, D. L., Dittenber, D. A. (1981). Chlorpyrifos: 

Probe Study to Determine Plasma and Erythrocyte Cholinesterase Depression and 

Blood Chlorpyrifos Levels in Male Fischer 344 Rats Following a Single Oral or 

Dermal Dose, Report ofToxicology Research Laboratory, The Dow Chemical 

Company. 

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Perala, A. W. (in progress). Diethylchlorothiophosphate (DEPCT): Method 

Validation and Stability in Rodent Feed and Corn Oil.. Report of Toxicology & 

Environmental Research and Consulting, The Dow Chemical Company, Midland, 

MI. 

Qinyan (2013). Certificate of Analysis ofO,O-Diethyl phosphorochloridoth 

(DEPCT), Lot C032013081501. Zhejiang Xinnong Chemical Co., Ltd., Sanlixi, 

Xianju County, Zhejiang Province, China. 
. 

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