~cr,am~or, 

~ Tedtnol ies" 

VIA FEDEX 

December 20, 2012 

Mg-e, W 7t/(~ 

RECEIVED 

OPPT CBIC 

2012 DEC 26 PM 12: 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 allow sufficient 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.com or 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 

IIIII IIIII I III IIII 

CC: Carl Watson CONTAINS NO CBI 

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: 

" OECD Guidelines for Testing of Chemicals (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: H Vryenhoef 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~ < M c-JcCONTAINS NO CBI 
Harlan 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 of Chemicals (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 IP 

" JMAFF Test Guidelines 2-7-7 

Test Facility: Tiarlan Laboratories Ltd. 

Shardiow Business Park 

Shardlow 

Derbyshire 

DE72 2GD 

UK 

Signatures : 

Author: H Vryenhoef BSc 

. . . .~~s ./.~.K.~ 

Date. AUG 

Test Facility Management: C Mea 

011

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

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

Date: - 2 AUG 2011 

Quality Assurance: J G Riley BSc (Hons) MRQA 

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

Date: 0,2 AUG 2011 

Page 1 of 20 
General 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 INTRODUCTION AND PURPOSE. . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . : . . . . . . . . . . . . . . . . . . . . . . .5 

3 .1 Guidelines / Regulations . . . . . . .. . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5 

4 TEST AND REFERENCE ITEMS .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6 

4.1 TestItem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6 

4.2 Reference Item . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6 

5 MATERIALS AND METHODS. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .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 Organism Observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .11 

5 .10.2 Water Quality Criteria . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .12 

5 .10.3 Analysis of Test Item Concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .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 of Volatile Test Items . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20 
p. 6 

General Study Plan : 908-24 Page 3 

1 GOOD LABORATORY PRACTICE 

Testing will be performed in compliance with UK GLP standards (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 of Directives 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 of the study plan. 

Deviations (unplanned changes) to the study plan (general study plan and study specific 

supplement) will be documented and acknowledged 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 and test facility management. 

Reporting 

A full report containing a description of the test item and 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 review and comments 

before issue of the 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 of the data will be 

chargeable to the Sponsor. 
p. 8 

General Study Plan : 908-24 Page 5 

3 INTRODUCTION AND PURPOSE 

The purpose of this study is to assess the acute toxicity of the test item to a freshwater 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 a specified 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 of yield (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 of a 

wide variety of natural habitats, and can therefore be considered as an important non-target 

organism in freshwater ecosystems : 

" Pseudokirchneriella subcapitata 

" Desmodesmus subspicatus (formerly known as Scenedesmus subspicatus) 

" Anabaenaflos-aguae 

" Navicula pelliculosa 

The dispersal of the test item in the surrounding medium is considered to represent the most 

probable route of exposure in the environment. 

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: 

" OECD Guidelines for Testing of Chemicals (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" 

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

4 TEST AND REFERENCE ITEMS 

4.1 Test Item 

p. 9 

Page 6 

The test item will be supplied by or on behalf of the Sponsor including the following 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 of the test . 

The reference item will be supplied by a commercial supplier and will be stored as specified by 

the supplier. 
General Study Plan: 908-24 Page 7 

5 MATERIALS AND 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 item that 

results in an x% reduction of the 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 

Loading rate): 

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 the control (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, if applicable). 

Comprises test item treatments, control (test medium only) and 

solvent control (if applicable). 

Specific growth rate for each day during the course of the test 

(days 0-1, 1-2, 2-3 and, 3-4 where appropriate). 

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 of the corresponding study. 

An inoculum culture 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 of the 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 the test media. 

The test item will be dissolved or dispersed in the test water once at the start of the 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 maximum concentration of 100 pL/L (final volume). If the test item falls into 

the category of a "difficult substance" as defined by the OECD Guidance Document (OECD. 

2002), the method of preparation of the test media will be based on the recommendations of this 

document. 

If the test item is photodegradable, the preparation of the test media will be performed as far as 

possible in the dark. Details of the method of preparation will be documented in the data . 

5.5 Materials 

Glass test vessels containing at least 15 mL test medium will 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 of test 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 

The environmental parameters will be as used to maintain the stock organisms. 

The test flasks will be incubated under one of the following conditions : 

1NFORS Multitron® Version 2 incubator under constant illumination (between 4440 and 

8880 lux) provided by warm white lighting (380 - 730 nn) at 24 :1: 1°C and agitated by 

orbital shaker (approximately 150 rpm). Temperature, light intensity and oscillation rate 

of the incubation chamber will be recorded daily throughout the test. 

Temperature-controlled water bath under constant illumination (between 4440 and 

88801ux) provided by fluorescent tubes installed above the test flasks . The light intensity 

will not vary 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. 

The test 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 if using a Coulter® Particle Counter, one additional replicate per test concentration 

containing the test media without algae will 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 blank will 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 5 x 103 - 1 x 104 

Test duration (hours) 72 96 72 

A static test design will be applied. 

At least five concentrations of the test item arranged 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 the 
General 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 of the 

test item higher 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 item in 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 item in 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 

doubled to six. 

The control group will be exposed to the test water alone. Data from the 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 from the solvent control group may 

be shared with similar concurrent studies. 

The test will be carried out without adjustment of pH. If the pH falls outside the range 6-9, the 

Sponsor will be contacted for advice on whether a second test should be performed with 

adjustment of pH to that of the test water. However, pH adjustment will not be carried out if the 

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 of the test item. At the end of the 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. The samples will be incubated for a further 9 days (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 of 
General 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 determine when/if re-growth occurs. 

The re-growth experiment will not be carried out if a "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 of the 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 460 run and 665 min. As such, if colored solutions formed by the test item show 

significant absorbance at either of these wavelengths it would reduce the light intensity available 

for photosynthetic growth to occur. 

In cases where the test solutions are shown to have distinct absorbance at 460 run 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 of the 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 Organism Observations 

A small volume of the algal suspension will be withdrawn from each test flask daily for the 

measurement of algal biomass, and will 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 the test item preparations daily during the exposure period . 
General Study Plan: 908-24 Page 12 

To determine a potential effect of the test item on the appearance of algal cells, a sample will be 

taken from the test and control cultures at the end of the test. The shape and size of the algal cells 

will be inspected microscopically and any abnormalities recorded. 

5.10 .2 Water Quality Criteria 

The pH of the test media will be measured in all treatments at the start and end of the test. The 

pH of 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 pH deviations 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 of the test media will be recorded daily. 

5.10.3 Analysis of Test Item Concentrations 

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 from the range-finding test is performed in order to determine the 

stability of the test item under 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 of the 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 taken for analysis at test termination. 

If required, in order to investigate possible losses of test item due 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 of the test when it will be analyzed for test item concentration. 

If the test medium volume from the actual test is too small to perform the 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 of the samples to be taken and the sample size, etc, will be documented in the data. 
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°C until 

analysis . 

If the test item has 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 of organic solvents) in order to maintain the stability of the test item until 

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 item in 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, UK Analytical Services. 

The test item concentrations will be measured in at least one of the duplicate test medium 

samples. However, the samples from test concentrations below the 72-Hour NOEC will not 

normally be analyzed. The remaining samples may be analyzed, if necessary. 

Stock solutions may also be analyzed, if necessary, for example if the test concentrations are 

below the limit of quantitation of the analytical method or for reasons of stability. 

If the test item is a formulation, test item concentrations will be determined by the measured 

concentrations of the active ingredient(s) . WAR will be characterized by analysis of the main 

test item component(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 

not just 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 of first measurement. 

time of nth measurement. 

The average specific growth rate for each replicate control and test item vessel 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 of the 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 remains constant. 

Percentage inhibition of growth rate for each replicate test item vessel is calculated using the 

following equation: 
General Study Plan: 908-24 Page 15 

1, = -p` p'' x 100 

wo 

where: 

h 

Nc 

Nt 

percentage inhibition of average 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 the solvent 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 of the specified period, 

cell concentration at the end of the end of the specified period. 

Where 

ly 

YC 

Ye 

percentage inhibition of yield 

mean value for yield in the control group 

mean value for yield for the treatment group 

5.11.4 Calculation of Biomass 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 concentration at start of test. 

measured cell concentration at ti . 

measured cell concentration at t . 

time of first measurement after beginning of test. 

time of nth 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 the concentration-response curve. The ECIo, EC2o, ECso (or ELIo, ELzo, 

ELso) values are read from the graph. 

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 the test 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 below the 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 100 after 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 

72 hours 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) Plant Physiology. 2nd ed. London: Collier Macmillan. 

FIELLER, E. C. (1944) A fundamental formula in the statistics of biological assay, and some 

applications . Quarterly Journal of Pharmacy and Pharmacology, 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 and waste 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. OECD Series on Testing 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 of Directive 91/414/EEC. 

ToxRat Professional, ToxRat® Solutions GmbH 

US EPA (1996) Ecological Effects Test Guidelines OPPTS 850.1000: Special Considerations for 

Conducting Aquatic 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 .044 nt /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 medium will be supplemented with 

Na2Si03 .9HZ0 at a final concentration of 1 .4 mg Si/L 
General Study Plan : 908-24 Page 20 

Appendix 2 Testing of Volatile Test Items 

In cases where the test item is known (or suspected) to be volatile, the following amendments to 

the study design may be made in order to minimize possible losses of test item over 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 of the culture medium will be adjusted to 7.5 f 0.1 with 

0. IN NaOH or HCl prior to use. 

" HEPES buffer (1430 mg/L) may be added to the prepared culture medium to counter act 

the change in pH due to algal growth (ISO 1999) . 

" In order to investigate possible losses of test item due 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 of these amendments to the study 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 GLP authorisation will be 

confirmed by the Study Director's signature prior to the start of the study. 

Z7 rrI 

Tl (") 

-+m 

c-) L 

c5o 

CONTAINS NO CS[ 
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 item to 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 21 
2. RESPONSIBILITIES 

Study Director: SM Fulcher 

Test Facility: Harlan Laboratories Ltd . 

Shardlow Business 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: Willow Court 

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 GmbH Quality Assurance, Oberbuchsiten, 

Switzerland 

Project No: 41201180 Page 3 of 21 
3. 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 21 
Housing 

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 21 
6. ANIMAL WELFARE 

Environmental Enrichment 

The animals will be provided with environmental enrichment items: wooden chew blocks 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 minimum suffering 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 review and the conduct of 

the study may be retrospectively reviewed, as part of the Harlan Laboratories Ltd., Shardlow, UK 

Ethical Review Process. 

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 21 
Identification 

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 item will 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 

item will be the responsibility of the Sponsor . 

Storage 

Room temperature, in the dark, unless otherwise specified by Sponsor. 

Preparation 

The test item will 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 item formulation 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 from a previous study. 

Project No: 41201180 Page 7 of 21 
8. STUDY DESIGN 

Definitions 

Day 1 of the study is defined as the day the test item is 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 5 post partume 57° 

2. Low 10 males & 10 females 43 Day 5 post partum - 

3. Intermediate 10 males & 10 females 43 Day 5 post partum - 

4. High 15 males & 15 females 43s Day 5 post partume 57° 

` The first ten males and ten females from this treatment group are allocated as the non-recovery groups and will 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 21 
and 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 maximum of 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 21 
vii) 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 21 
9. 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 item and 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 photobeam unit) 

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 21 
For 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 21 
The 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 sperm are 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 sperm in 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 from birth to Day 5 post partum 

vi) Individual offspring weights on Day 1 and 4 post partum (litter weights will be calculated 

retrospectively from offspring weights) 

Laboratory Investigations 

Haematological and blood chemical investigations will be performed on five males and five 

females, selected from each 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 21 
all recovery group animals at the end of the fourteen day treatment-free period . Blood samples 

will be withdrawn from the 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 from each 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 metabolism cages 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 21 
Specific gravity Urobilinogen 

Protein Blood 

Ketones 

Post Mortem Studies 

Post mortem studies will be performed on animals found dead or killed in extremis during the 

study and on all adult animals killed by intravenous overdose of sodium pentobarbitone 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 from all 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 21 
Adrenals 

Aorta (thoracic) 

Bone and bone marrow (femur including 

stifle joint) 

Bone and bone marrow (sternum) 

Brain (including cerebrum, cerebellum and 

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 21 
Initially all tissues from: 

a) all animals that die or are killed in extremis during the study 

b) five males and females from the 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 germ cell layers or types, retained spermatids, multinucleated or 

apoptotic germ cells 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 review of 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 from the 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 21 
Quantitative 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 from the 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 21 
12. STUDY PLAN AMENDMENTS 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 

them to 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 item and 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 review and 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 raw data 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 21 
15. REFERENCE 

Salewski E (1964) . F9rbemethode zum makroskopischen Nachweis von Implantationsstellen am 

Uterus der Ratte. Naunyn - Schmiedebergs Arch Exp Path Pharmacol 247 367. 

Project No: 41201180 Page 20 of 21 
Appendix 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 

Project No: 41201180 Page 21 of 21 
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