# ~cr,am~or, ~Tedtnol ies" VIA FEDEX December 20, 2012 

# Mg-e, W 7t/(~ RECEIVED OPPT CBIC 

## 2012 DEC 26 PM12

: 03

Document Control Office (7407M) EPA East Bldg ;, RM

E6428 Office of Pollution Prevention and Toxics (OPPT) US Environmental Protection Agency 1200 Pennsylvania Ave, NW Washington DC 20004 Attn : 40 CFR 799.5089 Docket ID No: EPA-HQ-OPPT-2009-0112 Champion Technologies, Inc. (Champion) has previously committed to meet the testing requirements for of Ethane, 1,1'-oxybis[2-chloro- (CAS #111-44-4) . Champion has entered into an agreement with Buckman Laboratories, Inc. to share in the development costs for these studies. Attached please find a copy of the study plans for the following tests: " Toxicity to plants (Algae) (Test Symbol C6) " Combined Repeated Dose Toxicity Study with Reproduction / Developmental Toxicity Screening (Test Symbol F1) We also request an extension for the submission of the data to June 2013. This is due to the unavailability of laboratory space and will allowsufficient time for review of the draft reports. If you have any questions or require any additional information, please feel free to contact me at +1-713-423-7875 or via email @kevin.drake(aD-champ-tech.comor Dr . Carl Watson of Buckman Laboratories at +1-901-272-6228 or via email @ cfwatson(a)buckman.com . 

## Sincerely, Kevin D Drake Director, Global Product Stewardship IIIIII IIIII IIIII1IIIII7IIIII IIIII IIIII IIIII IIIII IIIIIIIIIIIIII IIII 

# CC : Carl Watson CONTAINS NOCBI

3200 Southwest Freeway, Suite 2700 ~ Houston, TX 77027 PO Box 27727 ~ Houston, TX 77227-7727 ~ P (713) 627-3303 ~ F (713) 627-9353 ~ CHAMP-TECH.COM

## STUDY SPECIFIC SUPPLEMENT To General Study Plan: 908-24 CAS# 111-44-4: Algal Growth Inhibition Test 

# harlan® ti

Data Requirements / Test Guidelines: " OECDGuidelines for Testing ofChemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" 

### " Method C

.3 of Commission Regulation (EC) No. 761/2009 " US EPA Draft Ecological Effects Test Guidelines OPPTS 850.5400 "Algal Toxicity, Tiers I and II" Study Director: HVryenhoef BSc Sponsor : Champion Technologies, Inc 3200 Southwest Freeway Suite 2700 Houston Texas USA 77027 UNITED STATES OF AMERICA Harlan Study Number: 41201182 Proposed Schedule: Experimental Starting Date: September 2012 Experimental Completion Date: November 2012 Draft Report Date : December 2012

Page 1 of 2 L~J N n r\) -o f`J O W 

### CD M -`',7i 7 c-a < u~ Mc-Jc

# CONTAINS NOCBIHarlan Study Number : 41201182 Study Specific Supplement



### Test Item Information as provided by the Sponsor . Identification: CAS# 111-44-4 

### Special Conditions Study specific parameters Test System

: Pseudokirchneriella subcapitata Test Duration: 96 hours 

## SIGNATURES Study Director : H Vryenhoef BSc

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Date:



### For the Sponsor: p. 3 Page 2

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Date:

## GENERAL STUDY PLAN : 908-24 hh

# rlan® Algal Growth Inhibition Test

Data Requirements/Test Guidelines: 

### " OECD Guidelines for Testing ofChemicals (2006) No 201 "FreshwaterAlga and Cyanobacteria, Growth Inhibition Test" " Method C

.3 of Commission Regulation (EC) No. 761/2009 " US EPADraft Ecological Effects Test Guidelines OPPTS 850 .5400 "Algal Toxicity, Tiers I and IP " JMAFF Test Guidelines 2-7-7 Test Facility: Tiarlan Laboratories Ltd. Shardiow

Business Park Shardlow Derbyshire DE72 2GD 

### UK Signatures : Author: HVryenhoef BSc



# .. ..~~s./ .~.K.~ Date. AUG Test Facility Management: C Mea 

## 011 ............

. . . . . . .7. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 

## Date: - 2 AUG 2011 QualityAssurance: J GRiley BSc (Hons) MRQA

.. . .. . . .. . . .. . .. . . .. . .. . . . .. . . .. . . .. . . . .. . . .. . . .. . 

## Date: 0,2 AUG 2011 Page 1 of20General Study Plan: 908-24



## CONTENTS p. 5 Page 2



### CONTENTS. .. . . .. . . .. . .. . . . .. .. . . . .. . . .. . . .. . . .. . . .. . .. . .. . . .. .. . . . .. . .. . . .. . .. . . . .. . .. . . . .. . . .. .. .. . . .. . . .. . .. . . .. . . .. . . .. . .. . . .. . .. .. . . . . .. .2

. ., ., ., . . . . . . . . . . . . . . . . . . . . . . . . . ., . . ., ., . .. .. . . .. . . .. . . .. . .. . . . .. . .. . . . .. . . .. .. .. . . .. . . .. . .. . . .. . . .. . . .. .. . . . .. . .. .. . . . . .. . ., 2 GENERAL INFORMATION . . . . .. .. . . .. . . .. .. . . .. . . .. . .. . . .. . . .. . . .. . . . .. . . .. .. .. . . .. . . .. . .. . . .. . . .. . . .. . .. . . .. . .. . . . . . . .. .4 Amendments and Deviations to Study Plan . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4 Quality Assurance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4 Reporting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4 Archiving . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4 

### 3 INTRODUCTIONANDPURPOSE. .. .. .. . .. . . . .. . .. . . .. . . .. . . .. . . . .. . . .. . . .. . . .. . . .. . .. . . .. . .. : . . .. .. . . .. . . .. . . . . . .. . .5 3.1 Guidelines / Regulations .. . .. . .. .. .. . .. . . . .. . .. . . .. . . .. . . .. . . . .. . . .. . . .. . . .. . . .. . .. . . .. . .. . . .. . . .. . . .. . .. . . . . . .. . .5 4 TEST AND REFERENCE ITEMS .. . .. .. .. . .. . . . .. . .. . . .. . . .. . . .. . . . .. . . .. . . .. . .. . . .. . . .. . . .. . .. . . .. . .. . . .. . . .. . . . . . .. . .6 4 .1 TestItem

. .. . . .. . . .. . . . .. . .. .. . . . .. . .. . .. . . .. . .. . . . .. . .. . . .. . . .. . . .. . . . .. . . .. . . .. . .. . . .. . .. . . . .. . .. . . .. . .. . . .. . . .. . . . . . .. . .6 4 .2 Reference Item

.. . . . .. . .. .. . . . .. .. . . .. . . .. . .. . . .. . . .. . . .. . . .. . . .. . . . .. . . .. . . .. . .. . . .. . . .. . . .. . .. . . .. . .. . . .. . . .. . . . . . .. . .6 5 MATERIALS ANDMETHODS . .. .. . . .. . . .. . .. . . .. . . .. . .. . . .. . . . .. . . . .. . . .. . . .. . .. . . .. . .. . . . .. . .. . .. . . .. . . .. . . .. . . . . . .. . .7 5.1 Definitions and Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 5.2 Test System

. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 5.3 Test Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8 5.4 Dosage . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 5.5 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8 5.6 Experimental Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9 5.7 Study Design. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9 5.8 Re-growth Experiment. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 5.9 Colored Test Items. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . l l 5.10 Evaluations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11 5 .10.1 Test OrganismObservations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11 5 .10.2 Water Quality Criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12 5 .10.3 Analysis ofTest ItemConcentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .12 

### 5.11 Data Analysis. . . .. . . .. . . . .. . .. . . .. . .. . .. . . .. . .. . . . .. .. . . . . .. . .. . . .. . . . . .. . . .. . .. . . . .. . .. .. .. . .. . .. . . . .. . .. . . . .. . . . .. . .14 5.11 .1 Plotting Growth Curves. . .. . . .. . .. . . . . .. .. . . . .. . . . . .. . . .. . .. . .. . . . .. .. .. . .. . .. . . . .. . .. . . . .. . . . .. . .14 5.11 .2 Calculation of Growth Rates . .. . . . .. . .. . . . .. . . . . .. . . .. . .. . .. . . . .. . . .. . .. . .. . . . .. . .. . . . .. . . . .. . .14 5.11 .3 Yield . . .. . . .. . . .. . .. . . .. . . .. .. . . . .. . .. . . .. . .. . . . .. . .. . . . .. . . . . .. . .. . . .. . . . . . . .. .. .. . .. . .. . . . .. . .. . . . .. . . . .. . .15 5.11 .4 Calculation of Biomass Integral . .. . .. . . . .. . . . .. . . .. . . .. . .. . . .. . .. . . . .. . .. . . . .. . .. . . . .. . . . .. . .15 5.11 .5 Plotting Concentration-Response Curves .. . . .. . . .. . .. . . .. . .. .. . .. . .. . . . .. . .. . . . .. . . . .. . . 16 5.11 .6 Statistical Analysis . .. . . .. . . .. . . .. . .. . . . .. . .. . . . .. . . . . .. . .. . . .. . .. . . . .. .. .. . .. . .. . . . .. . .. . . . .. . .. .. . .16 5.12 Validity Criteria . . .. . . .. . .. . . . .. . . .. .. . . .. . . .. . . .. . .. . . . .. . .. . . . .. . . . . .. .. . . . .. . .. . . .. . .. . . . .. . .. . . . .. . .. . . . .. . .. . . . .17 6 REFERENCES .. . . .. . . .. .. . .. . . .. . . .. . .. . . . .. . . .. .. . . .. . . .. . . .. . .. . . . .. . .. . . . .. . . . .. . . .. . . .. . .. . . . .. .. . . . .. . .. . . .. . . .. . . . .. . .. . . . .18 7 APPENDICES .. . . . .. . . .. . . . .. . . .. . . .. . .. . . . .. . . .. .. . . .. . . .. . . .. . .. . . . .. . .. . . . .. . . . .. . . .. . . .. . .. . . . .. .. . . . .. . .. . . . .. . .. . . . .. . .. . . . .19 Appendix 1 Culture Medium . . .. . . .. . .. . . . .. .. .. .. . . .. . . .. . . .. . .. . . . .. . .. . . . .. . . . .. . .. . . . .. . .. . . .. . .. . . .. . . .. . . . .. . .. . . . .. . .. .. . .19 Appendix 2 Testing ofVolatile Test Items . . .. . . .. . .. . . .. . . . .. . .. . . . .. . . . .. . .. . . . .. . .. . . .. . .. . . .. . . .. . . .. . . .. . . . .. . .. . . . .20p. 6 General Study Plan: 908-24 Page 3 

## 1 GOOD LABORATORY PRACTICE Testing will be performed in compliance with UK GLPstandards (Schedule 1, Good Laboratory Practice Regulations 1999 (ST 1999/3106 as amended by S12004/0994)) . These regulations are in accordance with GLP standards published as OECD Principles on Good Laboratory Practice (revised 1997, ENV/MC/CHEM(98)17) ; and are in accordance with, and implement, the requirements ofDirectives 2004/9/EC and 2004/10/EC .General Study Plan: 908-24



## 2 GENERAL INFORMATION Amendments and Deviations to Study Plan p. 7 Page 4

Amendments (planned changes) to the study plan (general study plan and study specific supplement) will be made only by completion of an amendment to study plan approved by the Study Director. Amendments will be distributed to all recipients ofthe study plan. 

### Deviations (unplanned changes) to the study plan (general study plan and study specific supplement) will be documented andacknowledged by the Study Director. 

### Quality Assurance Test facility quality assurance will audit and inspect study-related procedures and will report any audit and inspection results in writing to the study director andtest facility management . 

### Reporting A full report containing a description ofthe test itemand detailed description ofthe experimental procedures will be presented. The report will contain observations, discussion and interpretation ofthe resultswhere relevant. Adraft report will be sent to the Sponsor for reviewand comments before issue ofthe Final Report. 

### Archiving Unless instructed otherwise by the Sponsor, all orginal data and the final report will be retained in the Harlan Laboratories Ltd, Shardlow, UK archives for five years after which instructions will be sought as to further retention or disposal . Further retention or return ofthe data will be chargeable to the Sponsor.p. 8 General StudyPlan: 908-24 Page 5 

## 3 INTRODUCTIONAND PURPOSE The purpose ofthis study is to assess theacute toxicity of the test itemto afreshwater alga. The test endpoint is inhibition of growth, expressed as the logarithmic increase in algal biomass (average growth rate) during the exposure period. The toxicity will be expressed as the concentration (or loading rate) bringing about aspecified x% inhibition of growth (e.g. 50%) and expressed as the ErCx (or EL,) . The Lowest Observed Effect Concentration (LOEC) or Lowest Observed Effect Loading rate (LOEL), and the No Observed Effect Concentration (NOEC) or No Observed Effect Loading rate (NOEL) will also be determined. An additional response variable recommended by the Test Guidelines is yield, which may be required to fulfil specific regulatory requirements in some countries. Results may therefore also be expressed in terms of inhibition ofyield (EyCX or EyLx) . 

### The biomass integral (area under the growth curve) may also be used as an additional endpoint. If required the results may therefore also be expressed in terms of inhibition of biomass integral (EyC, or EyL,~) . The study will use one of the following species of freshwater alga which are representative ofa wide variety of natural habitats, and can therefore be considered as an important non-target organismin freshwater ecosystems : " Pseudokirchneriella subcapitata " Desmodesmus subspicatus (formerly knownas Scenedesmus subspicatus) " Anabaenaflos-aguae " Navicula pelliculosa The dispersal of the test item in the surrounding mediumis considered to represent the most probable route ofexposure in theenvironment. 

### 3 .1 Guidelines / Regulations The test methods described are designed to be compatible with the procedures indicated by the following internationally accepted guidelines and recommendations: " OECDGuidelines forTesting of Chemicals (2006) No 201 "FreshwaterAlga and Cyanobacteria, Growth Inhibition Test" " Method C

.3 of Commission Regulation (EC) No . 761/2009 " US EPADraft Ecological Effects Test Guidelines OPPTS 850.5400 "Algal Toxicity, Tiers I and II" 

### " JMAFF Test Guidelines 2-7-7General Study Plan : 908-24 

## 4 TEST AND REFERENCE ITEMS 4.1 Test Item p. 9 Page 6



### Thetest item

will be supplied by or on behalfof the Sponsor including thefollowing information if known ; description, batch, purity, expiry 1 retest date, storage conditions and safety precautions to be taken

. 

### 4 .2 Reference Item For evaluation of the quality of the algal culture and the experimental conditions, potassium dichromate (zinc chloride for OPPTS guidelines) will be tested as a positive control twice a year to demonstrate satisfactory conditions ofthe test . The reference itemwill be supplied by a commercial supplier and will be stored as specified by the supplier.General StudyPlan: 908-24 Page 7



## 5 MATERIALSAND METHODS 5.1 Definitions and Abbreviations Biomass: Measurement variable (fluorescence or algal cell density) as surrogate measure for algal biomass. EC,/E4 : The calculated concentration or loading rate of test itemthat results in an x%reduction ofthe respective growth parameter relative to the control (or solvent control, if applicable) within a stated exposure period. Growth: The increase in biomass over the test period. 

### Growth Rate (Average Specific Growth Rate) : The logarithmic increase in biomass during the exposure period. 

### Yield: The value of the measurement variable at the end of the exposure period minus the measurement variable's value at the start of the exposure period, calculated to express biomass increase during the test . LOEC (Lowest Observed Effect Concentration) or LOEL (Lowest Observed Effect Loadingrate) : NOW (No Observed Effect Concentration) or NOEL (-No Observed _Effect Loading rate) : Treatment: Section-by-Section Growth Rate: AUC (Area Under the Growth Curve) : 

### The lowest test concentration or loading rate at which a statistically significant inhibition of growth is determined relative to thecontrol (or solvent control, if applicable) . The highest test concentration or loading rate at which no statistically significant inhibition of growth is determined relative to the control (or solvent control, ifapplicable) . Comprises test itemtreatments, control (test medium only) and solvent control (ifapplicable) . Specific growth rate for each dayduring the course of the test (days 0-1, 1-2, 2-3 and, 3-4whereappropriate) . Biomass integral calculated as area under the growth curve .General Study Plan: 908-24 Page 8



### 5 .2 Test System The study will be performed using either Pseudokirchneriella subcapitata, Desmodesmus subspicatus (formerly known as Scenedesmus subspicatus), Anabaena flos-aguae or Navicula pelliculosa. These freshwater alga species are recommended by the relevant test guideline. The test species used will be identified in the study-specific supplement ofthe corresponding study. 

### An inoculumculture of algae will be set up three to four days before the start o£the test . The algae will be cultivated under the test conditions and will be kept in the exponential growth phase until inoculation ofthe test solutions. 

### 5.3 Test Water Culture medium

as defined in Aronendix 1 . 

### 5 .4 Dosage If necessary, pre-experiments will be performed to determine the solubility and stability of the test item in the test water. According to the findings of these pre-experiments, appropriate procedures will be chosen to prepare thetestmedia. Thetest itemwill be dissolved or dispersed in thetest water once at thestart ofthe test. Methods such as prolonged mixing, ultrasonication, high shear force stirring or the production of Water Accommodated Fractions (WAFs) or saturated solutions may be used depending on the nature of the test item

. If auxiliary solvents or surfactants are used to aid in the dispersion, they will be used at a maximumconcentration of 100 pL/L (final volume) . If the test itemfalls into the category of a "difficult substance" as defined by the OECD Guidance Document (OECD . 2002) , the method of preparation ofthe test media will be based on the recommendations ofthis document. 

### If the test itemis photodegradable, the preparation ofthe test media will be performed as far as possible in thedark. Details ofthe method ofpreparation will be documented in the data. 

### 5.5 Materials Glass test vessels containing at least 15 mL test mediumwill be used. The test vessels will be covered/plugged to reduce evaporation and to avoid entry of dust into the solutions. Volatile chemicals will be tested in completely filled and stoppered test vessels (i .e. test performed in a closed system) . The size o£the test vessels and the volume oftest solution will be documented in the data.General Study Plan: 908 "24 Page 9 

### The test vessels will be labeled with the study number and all necessary information to ensure unique identification. 

### 5 .6 Experimental Conditions Theenvironmental parameters will be as used to maintain the stock organisms. Thetest flasks will be incubated under one ofthefollowing conditions: 1NFORS Multitron®Version 2incubator under constant illumination (between 4440 and 8880 lux) provided by warm white lighting (380 - 730nn) at 24 :1: 1°Cand agitated by orbital shaker (approximately 150 rpm) . Temperature, light intensity and oscillation rate ofthe incubation chamber will be recorded daily throughout thetest. Temperature-controlled water bath under constant illumination (between 4440 and 88801ux) provided by fluorescent tubes installed above the test flasks. The light intensity will notvary by more than ±15% from the average light intensity over the incubation area. The temperature will be maintained at between 21 and 24°C and maintained at t 2°C . Thetest solutions will be continuously stirred by magnetic stirrers. 

### 5.7 Study Design The test design will include three replicates per test concentration, six replicates of the control and, if appropriate, six replicate solvent control flasks. If the presence of particles in the test media could interfere with the determination of the algal biomass, eg ifusing a Coulter® Particle Counter, one additional replicate per test concentration containing thetest media without algaewill be prepared in the definitive test as particle controls. These additional replicates will be incubated under the same conditions as the test media with algae and will be used for the correction of the algal biomass determination. In cases where preliminary work indicates no counts due to dispersed test item, the blankwill consist of culture medium alone. Depending on the specific requirements of the relevant test guidelines, the following conditions will be met: OECD/EC OPPTS JMAFF Initial cell density (cells/mL) 2-5 x 103 1 x 104 5x 103 - 1 x 104 Test duration (hours) 72 96 72 Astatic test design will be applied . At least five concentrations of the test itemarranged in a geometric series will be tested. The spacing factor between the test concentrations will preferably not exceed 2.2 (2.0 for OPPTS guidelines) . The actual test concentrations are assigned according to preliminary range-finding tests or available toxicity data and will be documented in the data prior to the start of theGeneral Study Plan: 908-24 Page 10 

### definitive test . If in the range-finding test a flat concentration-effect relationship is determined and, thus, a large concentration range will have to be tested, a spacing factor exceeding 2.2 (e.g. 3.2) will be applied

. The lowest test concentration should have no significant effect on the growth of algae. The concentration series should preferably cover the range causing 5 to 75% inhibition of algal growth rate. In accordance with the test guidelines PA 1996; EC . 20021 , concentrations ofthe test itemhigher than 100 mg/L (1000 mg/L for industrial chemicals tested according to OPPTS guidelines or agrochemical formulations tested according to JIv1AFF guidelines), or above the limit of solubility of the test itemin the test water (whichever is the lower) will usually not be tested. 

### If no effect is observed in the preliminary range-finding test at the highest concentration recommended in the relevant test guideline, or the limit o£ solubility of the test itemin the test water (whichever is the lower), a Limit Test may be conducted using only this concentration and the corresponding control(s) . In this case, the number of test concentration replicates will be doubledto six

. The control group will be exposed to the test water alone. Data fromthe control group may be shared with similar concurrent studies. 

### Where auxiliary solvents/surfactants are used, a second control will be employed. The test organisms will be exposed to a concentration of solvent equal to that employed in the test series, this solvent concentration will not exceed 100 pL/L. Data fromthe solvent control group may be shared with similar concurrent studies. The test will be carried out without adjustment of pH . If the pHfalls outside the range 6-9, the Sponsor will be contacted for advice on whether a second test should be performed with adjustment of pHto that of the test water. However, pHadjustment will not be carried out ifthe test solutions are changed to any significant extent and if a chemical reaction or precipitation is caused. 

### 5.8 Re-growth Experiment For OPPTS requirements, at the end of the definitive test a re-growth experiment will be performed to determine whether the toxicity observed is due to the inhibitory (algistatic) or toxic (algicidal) nature ofthetest item . At the end ofthe definitive test, samples will be removed from each replicate of the control (or solvent control) cultures and each test group. The replicate samples will be pooled and used to inoculate fresh culture medium at a ratio of 1 .5 mL/100 ml, for each test concentration. Thesamples will be incubated for afurther 9days (maximum) under test conditions with daily observations on the cultures to provide a visual assessment of when/if re-growth occurs. For JMAFF requirements, a re-growth experiment will be conducted as above using the control (or solvent control) cultures and a single test group that has shown significant inhibition ofGeneral Study Plan: 908-24 Page 11



### growth. The samples will be incubated for a further 10 days (maximum) under test conditions with samples of the cultures being taken at 0 hours and periodically thereafter for the measurement of algal biomass to determinewhen/ifre-growth occurs. The re-growth experiment will not be carried out ifa "limit test" has been conducted. 

### 5.9 Colored Test Items Colored test items may absorb biologically relevant wavelengths and thereby may impair algal growth due to a reduction in the light available for photosynthetic growth. Using the standard algal test method it is not possible to differentiate between reduced algal growth by light absorption ofthe colored test solutions or an inherent toxic effect of the test item . The absorption spectra of chlorophyll a, the principal photoreceptor in the chloroplasts of green plants, shows a double peak (Bidwell . 1979) with maximum absorption occurring at approximately 460run and 665 min. As such, if colored solutions formed by the test itemshow significant absorbance at either ofthese wavelengths it would reduce the light intensity available for photosynthetic growth to occur. In cases wherethe test solutions are shownto have distinct absorbance at 460run or 665 nn, the range-finding test and the definitive test will therefore be carried out according to the modification to the standard methodology currently recommended by various competent authorities. This includes reducing the test volume in the range of 5- 25 mL in order to shorten the light path through the test medium and increasing the light intensity to approximately 88801ux (t15%) in order to reduce as much as possible the light absorbance effect ofthe colored test solutions. If, after conducting a range-finding test using the modified method, a reduction in algal growth is observed, current regulatory advice is to change the test method to a Lemna Growth Inhibition Test (OECD

. 2006 ) as Lemna are a floating plant species and hence colouration of the test solutions does not affect photosynthetic growth. 

### 5 .10 Evaluations 5 .10 .1 Test OrganismObservations A small volume of the algal suspension will be withdrawn fromeach test flask daily for the measurement ofalgal biomass, andwill not be replaced. The algal concentration of each sample is determined by direct counting using a Coulter® Particle Counter, haemocytometer and light microscope or by fluorescence measurement . Three determinations are made for each sample. Observations will be made on thetest itempreparations daily during the exposure period.General Study Plan: 908-24 Page 12

To determine a potential effect ofthe test itemon the appearance of algal cells, a sample will be taken fromthe test and control cultures at the end ofthe test. The shape and size ofthe algal cells will be inspected microscopically and any abnormalities recorded. 5 .10.2 Water Quality Criteria 

### The pHof the test media will be measured in all treatments at the start and end of the test. The pHof the controls should not normally deviate by more than 1 .5 pH units during the test. An explanation will be provided in the report if pHdeviations in excess of 1 .5 units are observed. The water temperature will be measured daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks. For OPPTS Guideline tests, the temperature will be monitored at least every hour. The appearance ofthe test mediawill be recorded daily . 5 .10 .3 Analysis ofTest ItemConcentrations 

### 5.10 .3.1 Sampling Samples of the test media will be taken at initiation and termination of both the range-finding and definitive tests for analysis. Where possible duplicate samples will be taken at each occasion, these samples may also be analyzed should the need arise. Analysis of samples taken fromthe range-finding test is performed in order to determine the stability of the test itemunder test conditions over the exposure period. The replicate test vessels at each concentration will be pooled to give one sample. The test concentrations provided for analysis will be documented by the Study Director in the data. 

### For the purposes ofthe definitive test, the replicate test and control (or solvent control) vessels will be pooled to give one sample. Should the analysis of the range-finding test samples show a decline in measured test concentration over the test duration then additional test samples may be prepared at test initiation with the omission of algal cells and incubated alongside the test from which samples will be takenfor analysis at test termination. Ifrequired, in order to investigate possible losses of test itemdue to volatility when the test item vessels are opened on a daily basis, a fourth replicate vessel may be prepared at 0 hours and incubated alongside the other exposure vessels. This additional replicate will remain unopened until termination ofthetest when it will be analyzed for test itemconcentration. If the test mediumvolume fromthe actual test is too small to performthe required analyses, requisite volumes of the freshly prepared test media will be incubated separately, but under identical conditions as in the actual test. Final details ofthe samples to be taken andthe sample size, etc, will be documented in thedata.General Study Plan: 908-24 Page 13



### 5.10 .3.2 Storage and sample preparation All samples will be analyzed immediately after sampling or stored at approximately -20°Cuntil analysis. If the test itemhas been shown to be unstable under the storage conditions, the samples will be analyzed immediately after sampling without prior storage or measures will be taken (e.g. pH adjustment or addition oforganic solvents) in order to maintain the stability of the test itemuntil analysis. It may be necessary for some chemicals which form emulsions or dispersions to filter or centrifuge some samples as part of the pre-test validation process to determine the proportion of test itemin the dissolved form . 5.10 .3.3 Analysis of samples The test item formulations will be analyzed for concentration and stability by Harlan Laboratories Ltd, Shardlow, UKAnalytical Services. The test item concentrations will be measured in at least one of the duplicate test medium samples. However, the samples from test concentrations belowthe 72-Hour NOEC will not normally be analyzed. Theremaining samples may be analyzed, if necessary. Stock solutions may also be analyzed, if necessary, for example if the test concentrations are below the limit ofquantitation ofthe analytical method or for reasons of stability. If the test itemis a formulation, test itemconcentrations will be determined by the measured concentrations of the active ingredient(s) . WARwill be characterized by analysis of the main test itemcomponent(s) . The analytical method (eg HPLC, LC-MS, GC, AAS or photospectrometry) will either be provided by the Sponsor and adapted, if necessary, to the analytical conditions at Harlan Laboratories or developed by Harlan Laboratories. Adaptation or development o£the analytical method will not be performed under GLP . Details about the analytical method used will be given in an Appendix of the final report. The accuracy of the analytical method will be verified by recoveries spanning the concentration range to be analyzed within this study. Analytical pre-experiments may be performed within a different Harlan Laboratories study.General Study Plan: 908-24 Page 14



### 5 .11 Data Analysis 5 .11 .1 Plotting Growth Curves The cell density in the control and test cultures together with the test item concentrations and times of measurement, recorded to the nearest whole hour, are tabulated and growth curves are constructed by plotting cell density (logarithmic axis) against time (arithmetic axis) . All data points are critically examined and if it is obvious that procedural mistakes can be identified and/or considered highly likely, the specific data point is marked as an outlier and not included in subsequent statistical analyses. Reasons for rejection of a data point as an outlier will be clearly stated in the final report. Acceptable reasons are only procedural mistakes and notjust bad precision. Outliers (marked as such) will be retained amongst the data points shown in the final report . 

### 5.11.2 Calculation of Growth Rates The average specific growth rate for a specified period is calculated as the logarithmic increase in biomass from

the equation: R= 

### In N . -In N, tn -ti 

### where : N Ni N tl t 

### average specific growth rate from time ti to t . cell concentration at ti . cell concentration at t . time offirst measurement . time ofnth measurement . 

### The average specific growth rate for each replicate control and test itemvessel is calculated for the periods 0- 24, 0 - 48, 0 - 72 and 0 - 96 hours (if applicable) using the nominally inoculated cell concentration as the starting value rather than the measured starting value in order to increase the precision ofthe calculation. In addition the section-by-section growth rate (0 - 24, 24 - 48, 48 - 72 and 72 - 96 hours (if applicable)) is calculated for the control cultures and the results examined in order to determine whether the growth rate remainsconstant. Percentage inhibition of growth rate for each replicate test itemvessel is calculated using the following equation:General StudyPlan: 908-24 Page 15



### 1, = p'' -p` x 100 wo 

### where: h Nc 

### Nt percentage inhibition ofaverage specific growth rate.

mean average specific growth rate for the control cultures. average specific growth rate for the test culture . Where auxiliary solvents/surfactants are used, percentage inhibition in the test cultures is calculated by comparison to thesolvent control cultures. 5.11 .3 Yield Yield is calculated as the increase in biomass over a specified period using the following equation: Y=N -N, where: Y N, N 

### For each test concentration and control, the mean value for yield, along with the standard deviation is calculated at 24, 48, 72 and 96 hours (if applicable) . Percentage inhibition of yield is calculated using the following equation: Ty = (Y`,Y Y') x 100

Yield. cell concentration at the beginning ofthespecified period, cell concentration at the end oftheend of the specified period.

Where ly YC Ye 

### percentage inhibition ofyield

meanvalue for yield in the control group meanvalue for yieldfor thetreatment group 5.11.4 Calculation ofBiomass Integral The biomass integral (area under the growth curve) may be used as an additional endpoint . The biomass integral at 24, 48, 72 and 96 hours (if applicable) is calculated using the following equation:General Study Plan: 908-24 Page 16



### A= N, 2No x tt+ Ni+N~-2Ne x(t2-tI)+ N"_,+ 2 -2Ne x(ta-t

-:) where: A No NI N, tt ta 

### area.

nominal cell concentrationat start oftest. measured cell concentration at ti. measured cell concentration at t . time of first measurement after beginning of test. time ofnth measurement after beginning of test . 

### Percentage inhibition of the biomass integral for each replicate test item vessel is calculated using the following equation: I = A, A A` x 100 where : I Ae A, 

### percentage inhibition of the biomass integral .

mean biomass integral for the control cultures. biomass integral for the test culture. 5 .11 .5 Plotting Concentration-Response Curves 

### For each individual test vessel, percentage inhibition (arithmetic axis) is plotted against test concentration (logarithmic axis) and a line fitted by computerised interpolation e,g. Xlfit or Toxltat . Any data points identified as outliers in the first phase of data evaluation are disregarded when fitting theconcentration-response curve. The ECIo, EC2o, ECso (or ELIo, ELzo, ELso) values are read fromthegraph. Where appropriate 95%confidence limits for the ECso (or ELse) will be calculated, using either the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949) or by Fieller's Theorem(1944) . All ECso (or ELse) values and 95%confidence limits will be rounded to two significant figures. 

### 5 .11 .6 Statistical Analysis The Lowest Observed Effect Concentration (LOEC) or Lowest Observed Effect Loading rate (LOEL) is the lowest concentration (or loading rate) tested at which significant (P < 0 .05) inhibition of growth relative to the control is observed within a given exposure time. However, all test concentrations above the LOEC (or LOEL) must have an effect equal to or greater than those observed at the LOEC . When these two conditions cannot be satisfied, a full explanation of how the LOEC has been selected will be given in thetest report.General Study Plan: 908-24 Page 17

The LOEC (or LOEL) for all requested growth variables will be determined using analysis of variance (ANOVA) incorporating a statistical test for homogeneity of variance and an adequate post hoc test (e.g. Dunnett test) . The No Observed Effect Concentration (NOEC) or No Observed Effect Loading rate (NOEL) is the test concentration (or loading rate) immediately belowthe LOEC (or LOEL) which, when compared to the control, has no statistically significant effect (P >- 0 .05), within a given exposure time. LOEC (or LOEL) and NOEC (or NOEL) values at 72 and 96 hours (if applicable) will be determined. 

### 5.12 Validity Criteria For the test to be valid, the following performance criteria should be met: The cell concentration of the control cultures must increase by a factor of at least 16 after 72 hours (OECD/EC/JIv1AFF Guidelines) and by a factor of at least 100after 96 hours (OPP"1'S Guidelines) . The mean of the coefficients of variation of the daily section-by-section specific growth rate in the control cultures after 72 hours (0 - 24, 24 - 48 and 48 - 72 hours) must not exceed 35% (OECD/JMAFF Guidelines). The coefficient variation of the average specific growth rate in replicate control cultures after 72hours must not exceed 7%(OECD/JMAFF Guidelines) . The coefficient of variation o£ the cell density values in replicate control cultures must not exceed 20%

after 72 and 96 hours (OPPTS Guidelines) .General Study Plan: 908-24 Page 18



## 6 REFERENCES BIDWELL, R

. G . S. (1979) PlantPhysiology. 2nd ed. London: Collier Macmillan . FIELLER, E. C

. (1944) A fundamental formula in the statistics of biological assay, and some applications. Quarterly Journal ofPharmacy andPharmacology, 17, pp . 117-123. 

### HERMAN, D

. C ., INNISS, W

. E. and MAXFIELD, C . 1. (1990) Impact of volatile aromatic hydrocarbons, alone and in combination, on growth of the freshwater alga Selenastrum capricornuturn. Aquatic Toxicology, 18, pp . 87 -100 . INTERNATIONAL STANDARDS ORGANISATION (ISO) (1999) Standard 14442 : Water quality - Guidelines for algal growth inhibition tests with poorly soluble materials, volatile compounds, metals andwaste water. Geneva: International Standards Institute. LITChIFIELD, J . T

. and WILCOXON, F. (1949) A Simplified Method of Evaluating DoseEffect Experiments. J. Pharmacol Exp . Ther., 96, pp

. 99-113. OECD (2002) Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures. OECDSeries onTesting and Assessment, No. 23. OECD Publishing. OECD (2006) Test No

. 221 : Lemna sp

. Growth Inhibition Test. OECD Guidelines for the Testing of Chemicals, Section 2 : Effects on Biotic Systems. OECD Publishing. EUROPEAN COMMISSION (2002) Sanco/3268/2001 rev . 4 (final) Guidance Document on Aquatic Ecotoxicology in the context ofDirective 91/414/EEC . ToxRat Professional, ToxRat®Solutions GmbH US EPA (1996) Ecological Effects Test Guidelines OPPTS 850 .1000:Special Considerations for ConductingAquatic Laboratory Studies. 

### Xlfit, ID Business Solutions Ltd.General Study Plan: 908-24 Page 19



## 7 APPENDICES Appendix 1 Culture Medium Reconstituted test water (AAP/ASTM medium) prepared according to OECD Test Guideline No 201 will be used for algal cultivation and testing. Analytical grade salts will be dissolved in purified water to obtain the following final concentrations :

Ingredients Concentration Macro-nutrients NaHC03 15.0 m

K2HP04 1 .044nt /L 

### M S04.7H20 14.6 m M CI2.6H20 12.16 m 

### CaCI2.2H20 4 .41 m NaN03 25.5 m Trace elements H3EO3 186.0 L



### MnC12 .4Hz0 415.0 ZnC12 3.27 CoC1Z.6H20 1 .43 L CuC12.21420 0.012 Na2Mo04.2H20 7 .26 L PeC13.6H20 160 .0 L Na2EDTA.2H20 300 .0 The pH will be adjusted to 7.5. The water hardness (calculated) of the test water will be 0 .15 mmol/L (=15 mg/L as CaCO3) . In tests conducted using Navicula pelliculosa the culture mediumwill be supplemented with Na2Si03.9HZ0at afinal concentration of 1 .4 mg Si/LGeneral Study Plan: 908-24 Page 20



### Appendix 2 Testing of Volatile Test Items In cases where the test itemis known (or suspected) to be volatile, the following amendments to the study design may be made in order to minimize possible losses of test itemover the test period: " The test vessels used will be glass conical flasks completely filled with test preparation and sealed with ground glass stoppers. " Additional sodium bicarbonate (up to 500 mg/L final concentration) may be added to the prepared culture medium as defined by Herman et al (1990) to counter act the change in pH due to algal growth. The pH ofthe culture medium will be adjusted to 7.5 f 0 .1 with 0 . IN NaOHor HCl prior to use. 

### " HEPES buffer (1430 mg/L) may be added to the prepared culture mediumto counter act the change in pHdueto algal growth (ISO . 1999) " In order to investigate possible losses of test itemdue to volatility when the test vessels are opened on a daily basis a fourth replicate at each concentration will be prepared at 0 hours and incubated alongside the other exposure vessels. This fourth replicate will remain unopened until termination of the test when it will be analyzed for test item concentration. 

### Details oftheseamendments to thestudy design will be documented in the data.p. 24 

# harlan®

STUDY PLAN Test Item Study Type

Harlan Laboratories Project Number Proposed Experimental Starting Date Proposed Experimental Completion Date Proposed Draft Report Date 

### Sponsor : CAS* 111-44-4

. Oral (Gavage) Combined Repeat Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422 with Recovery Groups) : 41201180 : 26 September 2012 : 21 November 2012

. March 2013 Champion Technologies, Inc 3200 Southwest Freeway Suite 2700 Houston Texas USA 77027 UNITED STATES OF AMERICA

N O N n CTI N

FOR SPONSOR -o t'v 0 DATE: . . . . . . . . . . . . . . . . . . . . . . . . . . . . STUDY DIRECTOR: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DATE: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SM Fulcher BA (Hons) FIAT This study plan is issued without signature by the Study Director to enable changes to be made if necessary prior to authorisation. Sponsors should sign and return the document to indicate approval and GLPauthorisation will be

confirmed by the Study Director's signature prior to the start of the study. Z7 rrI Tl (") 

### -+m c-) L 

### c5o



# CONTAINS NOCS[1 . INTRODUCTION AND OBJECTIVES The purpose of this study is to establish the effects of repeated oral administration of the test item to rats over a period of up to eight weeks. The results of the study are believed to be of value in predicting the toxicity of the test itemto man and can identify the organs and tissues which may be injured by exposure, can enable detection of possible cumulative toxicity and the estimation of the "No Observed Effect Level" (NOEL) . The study is also designed to screen for potential adverse effects on reproduction and offspring development . The test method described is designed to be compatible with the following internationally accepted guidelines and recommendations: i) OECD Guidelines for Testing of Chemicals No 422 "Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test" (adopted 22.03.96) . Where testing is conducted in accordance with specific guidelines this will be documented and reflected in the final report. With the exception of the histopathology examination phase, testing will be 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 2004/10/EC . The histopathology examination phase of the study will be undertaken at the Test Site in accordance with the Swiss Ordinance relating to Good Laboratory Practice, adopted May 18 2005 [SR 813.112.1] . These principles are compatible with Good Laboratory Practice regulations specified by regulatory authorities throughout The European Community, The United States (EPA and FDA), and Japan (MHLW, MAFF and METI) . The Principal Investigator will be responsible for compliance with their national GLP regulations of work performed at the Test Site and data provided to the Study Director for inclusion in the final report. Project No

: 41201180 Page 2 of 212. RESPONSIBILITIES Study Director: SM Fulcher Test Facility: Harlan Laboratories Ltd. ShardlowBusiness Park Shardlow Derbyshire DE72 2GD UK Lead Quality Assurance: Harlan Laboratories Ltd. Quality Assurance, Shardlow, UK Study Phases: Histology Processing Principal Investigator : N Fower Propath UK Limited Test Site: WillowCourt Netherwood Road Hereford HR2 6JU 

### UK Test Site Quality Assurance: Study Phase: Principal Investigator : AF International Consultancy, Cambridgeshire, UK 

### Histopathology Examination To be confirmed Test Site: AnaPath GmbH Buchsweg 56 4625 Oberbuchsiten Switzerland Test Site Quality Assurance: AnaPath GmbHQuality Assurance, Oberbuchsiten, Switzerland

Project No

: 41201180 Page 3 of 213. ANIMALS Specification Species: Strain: Sub-strain: Source of supply: Number: Body weight range: Sex: Age: Other :

Justification Rat Wistar HanT^" RccHanT^^ :WIST Harlan Laboratories U.K. Ltd., Oxon, UK 100 Approximately 190 to 350g at start of treatment Males and females Approximately 12 weeks at start of treatment Females must be nulliparous and non-pregnant Weight variation must not exceed t 20%of the mean weight for either sex at start of treatment

Preferred species of choice as historically used for safety evaluation studies and specified by appropriate regulatory authorities. 4 . ANIMAL HUSBANDRY Environment Environmental conditions will be maintained within the following target parameters: Target temperature: 22 ± 3°C Target humidity: 30 to 70% Lighting : Twelve hours of continuous artificial light in each twenty-four hour period Ventilation: At least fifteen air changes per hour Animals will be housed in accordance with the 'Code of Practice for the Housing and Care of Animals Used in Scientific Procedures' and in alignment with the United Kingdom Home Office recommendations and requirements. Project No: 41201180 Page 4 of 21Housing During the pre-mating phase: Animals will be housed in groups of five by sex in polypropylene cages with stainless steel mesh lids and furnished with softwood flake bedding (Datesand Ltd ., Cheshire, UK) . Recovery aniamls will be housed under these conditions throughout the study. Certificates of analysis for the bedding will be provided by the supplier . 

### The bedding is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. During the mating phase: Non-recovery animals will be paired on a one male:one female basis within each dose group in polypropylene cages with stainless steel mesh lids and grid flooring suspended over polypropylene tray liners with absorbent paper . Following mating: Non-recovery males will be re-housed in their original pre-mating cages. Non-recovery females will be individually housed in solid floor polypropylene cages with stainless steel mesh lids and furnished with softwood flake bedding (Datesand Ltd., Cheshire, UK) for the gestation, birth and lactation phases of the study. Diet and Water The animals will be provided with Rodent 2018C Teklad Global Certified Diet (Harlan Laboratories U.K. Ltd ., Oxon, UK) and tap water ad libitum . Drinking water is routinely analysed (at least annually) and certificates of analysis for diet will be provided by the supplier. The diet and water are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. Project No: 41201180 Page 5 of 216. ANIMAL WELFARE Environmental Enrichment The animals will be provided with environmental enrichment items: wooden chewblocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) or suitable alternatives. 

### Certificates of analysis for the environmental enrichment items will be provided by the supplier. These enrichment items are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. Environmental enrichment will not be provided to animals during the mating phase or for females during the gestation and lactation phases of the study. Study Conduct The study was designed and will be conducted to cause the minimumsuffering or distress to the animals consistent with the scientific objectives and in accordance with the Harlan Laboratories Ltd., Shardlow, UK policy on animal welfare and the requirements of the United Kingdom's Animals (Scientific Procedure) Act 1986 . This study plan is subject to reviewand the conduct of the study may be retrospectively reviewed, as part of the Harlan Laboratories Ltd., Shardlow, UK Ethical ReviewProcess. 6 . PRE-TEST PROCEDURES Acclimatisation Period At least five days prior to the start of treatment . Allocation Animals will be allocated to dose groups using a randomisation procedure based on body weight.

Project No: 41201180 Page 6 of 21Identification Each animal, selected at random, will be uniquely identified within the study by ear-punch. A colour-coded cage card will be prepared with details of test item, project number, dose level, sex, numbers of animals, route of administration and Study Director responsible for the study. 7. TEST ITEM

AND EXPERIMENTAL PREPARATION 

### Supply The test itemwill be supplied by the Sponsor with details of purity and hazardous properties if known. The integrity of data supplied relating to the identification, purity and stability of the test itemwill be the responsibility of the Sponsor . Storage Room temperature, in the dark, unless otherwise specified by Sponsor. 

### Preparation The test itemwill be dissolved or suspended in a suitable vehicle (subject to confirmation of stability) . Wherever possible, an aqueous formulation will be used, followed by consideration of formulation in oil (eg Arachis oil BP), then other specified vehicles. The method of preparation will be documented in the study records. The frequency of test itemformulation will be dependent on known stability. Analysis Details of identification of the test item will be supplied by the Sponsor. The test item formulations will be analysed for concentration by Harlan Laboratories Ltd ., Shardlow, UK Analytical Services. Stability and homogeneity of the formulations will be determined if this information has not been provided froma previous study.

Project No: 41201180 Page 7 of 218. STUDY DESIGN Definitions Day 1 of the study is defined as the day the test itemis first administered to the animals. Day 0 of gestation is defined as the day a vaginal/cage plug is found (uncertain mating) or sperm is present in a vaginal smear (confirmed mating) . Day 0 post partum is defined as the day of birth; that is, when parturition is recorded as complete. Administration Once daily, by gavage, throughout the treatment period (with the exception of females during parturition, if applicable), using a suitable dosing cannula attached to a graduated syringe. Dosing will be performed at a similar time each day wherever possible. Dose Groups Animals will be allocated to dose groups as follows: Necropsy Day Dose Groups Group Size &Sex Non-recovery animals Males Females Recovery animals 1 . Control 15 males & 15 females 438 Day 5post partume 57° 2 . Low 10 males & 10 females 43 Day 5post partum - 3 . Intermediate 10 males & 10females 43 Day 5post partum - 4 . High 15 males & 15 females 43s Day 5post partume 57° ` Thefirst ten males and ten females fromthis treatment groupare allocated as the non-recovery groups andwill be paired for mating on Day 15 " The remaining five males and five females from this treatment group are allocated as recovery group animals and will not be paired for mating Dose levels will be based on available toxicity data, up to a maximum dose of 1000 mg/kg bw/day. The dose levels to be used in the study will be documented as an amendment to study plan, together with the treatment volume. Control animals will be treated with the vehicle alone Project No: 41201180 Page 8 of 21and administered at the same dose volume as that of test animals. The recovery groups will be used for evaluation of reversibility of toxic findings but will not be used for evaluation of reproductive effects. The volume of test and control item administered to each animal will be based on the most recent body weight and adjusted accordingly. Chronological Sequence of Study Non-recovery Groups i) Groups of ten male and ten female rats will be dosed according to dose group for fourteen days prior to pairing. ii) Once before the first dose, and once weekly thereafter, detailed clinical observations will be made for all non-recovery animals in an open arena to observe functional/behavioural changes. iii) On Day 15, non-recovery animals will be paired on a one male:one female basis within each dose group for a maximumof fourteen days and smearing will commence. Dosing will continue throughout the mating phase. iv) At the end of the mating phase, males will be returned to their original cages and females will be transferred to individual cages with suitable nesting material . Dosing will continue for both sexes during the subsequent female gestation and lactation phases. v) Once during the study, in the final week of treatment, the first five males randomly allocated to each non-recovery dose group will be evaluated for sensory reactivity to stimuli, grip strength and motor activity assessment . The same males will be used for the remaining extended investigations. vi) Pregnant females are allowed to give birth and maintain their offspring until Day 5 post partum . Evaluation of each litter will be performed during this period.

Project No: 41201180 Page 9 of 21vii) On Day 4 post partum, five females per dose group will be selected for sensory reactivity to stimuli, grip strength and motor activity assessments and blood sampling for blood chemistry and haematology assessments . viii) On Day 5 post partum, following completion of functional assessments all females and offspring will be killed and examined macroscopically. Any females considered to be nonpregnant will be killed on or after Day 25 post coitum . ix) Urinalysis will be performed on five randomly selected males from each dose group during the final week of dosing . x) Blood sampling will be performed on males on the five selected males per group on Day 42 for blood chemistry and haematology assessments. xi) On Day 43 of the study, subject to confirmation of successful mating, males will be killed and examined macroscopically. 

### Recovery Groups i) Two groups of five male and five female rats will be dosed with either the vehicle or the high dose daily, up to the point of sacrifice of non-recovery males at which time treatment will be discontinued . ii) The males and females will be maintained without treatment for fourteen days. iii) Urinalysis will be performed for all males during the final week of the recovery period. iv) Dependent upon previous results, blood sampling and behavioural investigations may be performed on Day 56. v) After fourteen days without treatment, males and females will be killed and examined macroscopically.

Project No: 41201180 Page 10 of 219. OBSERVATIONS Morbidity/Mortality Inspection Twice daily, early and late during the working period . Clinical Observations Individual clinical observations will be performed immediately before dosing, up to 30 minutes post-dosing and one hour after dosing. An additional observation will be made five hours after dosing during the normal working week (not at weekends or on public holidays) . Recovery groups will be observed once daily during the treatment-free period. All observations will be recorded. Functional Observations Detailed clinical observations will be performed on all non-recovery test and control group animals before the first exposure to the test itemand once weekly thereafter. Observations will also be performed on females with litters on Day 4 postpartum . These observations will be performed outside the home cage, in a standard arena, at least two hours after dosing (where applicable) to ensure that any effects of treatment are identified. All observations will be recorded . In addition, sensory reactivity to different stimuli (eg auditory, visual and proprioceptive), grip strength (fore and hind limb) and motor activity (using a 44 photobeamunit) will be measured, in five randomly selected non-recovery males per group, once during the final week of treatment, at least two hours after dosing. Five selected non-recovery females per group will be similarly evaluated at Day 4 postpartum . Investigations may be extended in to the final week of the treatment-free period for recovery animals at the discretion of the Study Director . Body Weights Individual body weights will be recorded for non-recovery males on Day 1 and then weekly until termination. Individual body weights will also be recorded at terminal kill . Project No: 41201180 Page 11 of 21For non-recovery females, individual body weights will be recorded on Day 1 and then weekly until pairing. During the mating phase, females will be weighed daily until mating is confirmed (this data may be presented in the final report only if required) . Mated females will be weighed on Day 0, 7, 14 and 20 post coitum and body weights for females which give birth will be recorded on Days 1 and 4 postpartum . Body weights will also be recorded at terminal kill . Body weights for recovery group animals will be recorded on, Day 1, then weekly throughout the treatment and recovery period. Food Consumption Non-recovery male dietary intake will be recorded weekly prior to and after pairing . Nonrecovery female dietary intake will be recorded weekly prior to pairing. Weekly food efficiency (body weight gain/food intake) will be calculated for non-recovery males, (except during the mating phase) and for non-recovery females prior to pairing . Food consumptions will not be performed during the mating phase. Dietary intake for mated females will be recorded on Days 0-7, 7-14 and 14-20 post coitum . Food consumptions for females with live litters will be recorded between Days 1-4 post partum . Dietary intake for recovery group animals will be recorded weekly throughout the treatment and recovery period. Weekly food efficiency will be calculated retrospectively for the treatment and treatment-free, periods of the study. Water Consumption Monitored daily by visual inspection of water bottles. Measurement will be initiated if a treatmentrelated effect is suspected, at the discretion of the Study Director. 

### Mating Animals will be paired on a one male:one female basis within each non-recovery dose group for up to fourteen days.

Project No: 41201180 Page 12 of 21The stage of oestrus will be recorded during this period for all non-recovery females via a vaginal smear. Smearing of individual females will be discontinued when spermare found. On confirmation of mating, males will be re-housed in their original cages and females will be individually housed in solid floor cages. Uncertain mated females will continue to be housed with the male until mating is confirmed by the presence of spermin a vaginal smear, or the fourteenday mating period is complete . 

### Pregnancy and Parturition For each pregnant female the following will be recorded : i) Date of pairing 

### ii) Date of mating iii) Date of parturition Litter Data Offspring will be individually identified within each litter by tattoo. For each litter the following will be recorded : i) Number of offspring born ii) Number of offspring alive recorded daily iii) Sex of offspring on Day 1 and 4 post partum iv) Results of the surface righting assessment on Day 1 post partum v) Clinical condition of offspring frombirth to Day 5 post partum vi) Individual offspring weights on Day 1 and 4 post partum(litter weights will be calculated retrospectively fromoffspring weights) Laboratory Investigations Haematological and blood chemical investigations will be performed on five males and five females, selected fromeach non-recovery dose group on Day 42 (males) and Day 4 post partum (females) . If a treatment-related effect is suspected, these investigations may be undertaken on Project No: 41201180 Page 13 of 21all recovery group animals at the end of the fourteen day treatment-free period. Blood samples will be withdrawn fromthe lateral tail vein. Repeat blood samples will be withdrawn by cardiac puncture at termination if required . Urinalytical investigations will be performed on five males selected fromeach non-recovery dose group during the final week of dosing and on all recovery group males during the final week of the fourteen day treatment-free period . Urine samples will be collected by housing the animals overnight in metabolismcages under normal hydration but without access to food . Haematology Haemoglobin Prothrombin time Haematocrit Activated partial thromboplastin time Erythrocyte count Platelet count Total leucocyte count Reticulocyte count* Differential leucocyte count Erythrocyte indices : mean cell haemoglobin : mean cell volume : mean cell haemoglobin concentration * Blood film will be prepared but only examined at Study Director's discretion Blood Chemistry Blood Urea Total Protein Albumin Albumin/Globulin ratio (by calculation) Sodium Potassium Chloride Calcium Inorganic phosphorus Creatinine Alkaline phosphatise Alanine aminotransferase Aspartate aminotransferase Glucose Total cholesterol Total bilirubin Bile acids Urinalysis Volume pH Glucose Bilirubin Project No: 41201180 Page 14 of 21Specific gravity Urobilinogen Protein Blood Ketones Post MortemStudies Post mortemstudies will be performed on animals found dead or killed in extremis during the study and on all adult animals killed by intravenous overdose of sodiumpentobarbitone followed by exsanguination at termination. Offspring will be killed by intracardiac overdose of sodium pentobarbitone. Gross Examination Full external and internal examination of all animals including offspring . The corpora lutea of all ovaries fromall non-recovery females will be counted at necropsy. The uterine implantation sites will be counted. The procedure may be enhanced using the technique proposed by Salewski (1964) . Organ Weights The following organ weights will be recorded for all surviving animals at termination: Adrenals Prostate Brain Seminal vesicles Epididymides Spleen Heart Testes Kidneys Thyroid/Parathyroid (post-fixation) Liver Thymus Ovaries Uterus and Cervix 

### Pituitary (post-fixation) Histopathology Samples of the following tissues from all animals will be preserved in buffered 10% formalin, except where stated, and despatched to the histology processing Test Site: Project No: 41201180 Page 15 of 21Adrenals Aorta (thoracic) Bone and bone marrow(femur including stifle joint) Bone and bone marrow(sternum) Brain (including cerebrum, cerebellumand pons) Caecum Coagulating gland Colon Duodenum Epididymides* Eyes* Gross lesions Heart Ileum (including payer's patches) Jejunum Kidneys Liver Lungs (with bronchi)# Lymph nodes (mandibular and mesenteric) Mammary gland

+ preserved in Bouins fluid then transferred to 70% Industrial Methylated Spirits (IMS) approximately 48 hours later * Eyes fixed in Davidson's fluid # Lungs inflated to approximately normal inspiratory volume with 10%formalin before immersion in fixative Muscle (skeletal) Oesophagus Ovaries Pancreas Pituitary Prostate Rectum Salivary glands (submaxillary) Sciatic nerve Seminal vesicles Skin (hind limb) 

### Spinal cord (cervical, mid-thoracic and lumbar) Spleen Stomach Testes* Thymus Thyroid/Parathyroid Trachea Urinary bladder Uterus and Cervix Vagina

Project No: 41201180 Page 16 of 21Initially all tissues from : a) all animals that die or are killed in extremis during the study b) five males and females fromthe non-recovery control and high dose groups and reproductive organs (tissues shown in bold) from all non-recovery control and high dose animals, any animal failing to produce a pregnancy and any gross lesions will be routinely processed to paraffin wax, sectioned and stained with Haematoxylin and Eosin . In addition, sections of testes and epididymides will also be stained with Periodic Acid-Schiff (PAS) stain. Detailed qualitative examination of the testes will be made, taking into account the tubular stages of the spermatogenic cycle. The examination will be conducted in order to identify treatmentrelated effects such as missing germcell layers or types, retained spermatids, multinucleated or apoptotic germcells and sloughing of spermatogenic cells into the lumen . Any cell-or stagespecificity of testicular findings will be noted . Prepared slides will be sent to the histopathology examination Test Site for histopathology examinations by the Pathologist acting as the Principal Investigator for this study, whereupon special staining techniques may be used, where appropriate. A peer reviewof the histopathology examinations will also be included in this phase of the study. The Study Pathologist will inform the Study Director of any treatment-related changes. Where treatment-related lesions are seen in the high dose group, histology processing and histopathological examinations of the affected tissue(s) will be extended to five animals of either sex from the remaining dose groups. In the case of reproductive organs, examination will be extended to all animals fromthe remaining dose groups. On completion, a histopathological examination phase report will be sent to the Study Director. 10 . EVALUATION OF DATA All data will be summarised in tabular form and analysed statistically, where appropriate, to assess the significance of intergroup differences.

Project No: 41201180 Page 17 of 21Quantitative recovery group data, non-recovery male data, non-recovery female data prior to pairing, functional performance test data, blood chemical and haematology data will be analysed by the ProvantisT"" Tables and Statistics Module. For each variable, the most suitable transformation of the data will be found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA on ANCOVA and Bartlett's test . The transformed data will be analysed to find the lowest treatment level that shows a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data . If no dose response is found, but the data shows non-homogeneity of means, the data will be analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant difference fromthe control group. Finally, if required, pair-wise tests are performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric) . Non-recovery female gestation and lactation data and offspring data will be assessed, where appropriate, by linear regression analysis (for dose response relationships) followed by one way analysis of variance (ANOVA) incorporating a test for homogeneity of variance. Where variances are shown to be homogenous, pairwise comparisons will be conducted using Dunnetts's test . In the case of recovery group data, the analysis performed will be a two-tailed t-test incorporating Levene's test for homogeneity of variance . Where Levene's test shows unequal variances among either non-recovery or recovery group data, the affected parameters will be analysed using non-parametric methods: Krukal-Wallis ANOVA and Mann-Whitney "U" test. Reproductive and viability indices will be statistically analysed by Fisher's exact test or chisquared probability test may be used, where applicable (Appendix 1) . Histopathological data for the reproductive tissues may be statistically analysed using Armitage/Cochran analysis and/or Fisher's exact test if an effect of treatment is suspected. Further analyses may be performed at the discretion of the Study Pathologist if required. 11 . QUALITY ASSURANCE The study plan will be reviewed for GLP compliance and the final report will be audited by Harlan Laboratories Ltd., Shardlow, UK Quality Assurance Unit . Study phases will be inspected as determined by Harlan Laboratories Ltd., Shardlow, UK Quality Assurance Unit . The Test Site(s) quality assurance should inspect study-related work according to their own SOPs, reporting any inspection results promptly in writing to the Principal Investigator, Test Site management, Study Director, Test Facility management and lead quality assurance. Project No: 41201180 Page 18 of 2112. STUDY PLANAMENDMENTS AND DEVIATIONS Amendments to the Study Plan will be made only by completion of an amendment to study plan approved by the Study Director . The Principal Investigator will document deviations to the study plan, acknowledge and report themto the Study Director. Deviations to the study plan will be documented and acknowledged by the Study Director . 13. REPORT Any phase report or data provided by a Principal Investigator should include a signed statement of GLP compliance and a quality assurance statement signed by a representative of Test Site Quality Assurance. A full report containing a description of the test itemand detailed description of the experimental procedures will be presented . The report will contain observations, discussion and interpretation of the results where relevant . A draft report will be sent to the Sponsor for reviewand comments before issue of the final report . 14. ARCHIVE The archive section has been amended to the following: Unless instructed otherwise by the Sponsor, and with the exception of unprocessed wet tissue samples, all original data including tissue blocks and tissue slides generated at the histology processing Test Site and all data generated at the Test Facility, including the final report and a copy of the histopathology examination phase report will be retained in the Harlan Laboratories Ltd., Shardlow, UK archives for five years, after which instructions will be sought as to further retention or disposal . Unprocessed wet tissue samples will be retained in these archives for two years prior to disposal . AnaPath GmbH, 4625 Oberbuchsiten, Switzerland will retain a copy of the rawdata generated for the histopathology examination phase, together with an electronic copy of the phase report and any duplicate signature report pages, for at least ten years. Project No: 41201180 Page 19 of 2115. REFERENCE Salewski E (1964) . F9rbemethode zummakroskopischen Nachweis von Implantationsstellen am Uterus der Ratte. Naunyn - Schmiedebergs Arch Exp Path Pharmacol 247 367.

Project No: 41201180 Page 20 of 21Appendix 1 Reproductive Indices Number of animals mated Mating Index = X100

Number of animals paired Pregnancy Index - Number of pregnant females x 100

Number of animals mated Parturition Index = Number of females delivering live offspring x 100 Number of pregnant females Live Birth Index - Number of offspring alive on Day 1 X100

Number of offspring born Viability Index = Number of offspring alive on Day 4 X100

Number of offspring alive on Day 1 Pre - implantation Loss = Number of corpora Iutea - number of implantati ons X100 Number of corpora lutea Post-implantation Loss= Number of implantations -total number of offspring born X100 Number of implantations %Male offspring (Sex Ratio) = Number of male offspring X100 Total number of offspring

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