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## STUDY PLAN

Sodium Glucoheptonate: Acute Toxicity to Rainbow Trout

Study Director: S Harris

Test Facility: Harlan Laboratories Ltd.

Shardlow Business Park

Shardlow Derbyshire

DE72 2GD

UK

Sponsor: Harcros Chemicals Inc.
3452 Corporate Drive

Dalton GA 30720

UNITED STATES OF AMERICA

Harlan Study Number: 41402585Harlan Study Number: 41402585 Study Plan Page 2



### CONTENTS

CONTENTS ...................................................................................................................... .............. 2

SIGNATURES .................................................................................................................... ............ 3

GOOD LABORATORY PRACTICE ............................................................................................. 4

GENERAL INFORMATION ......................................................................................................... 5 Proposed Schedule ............................................................................................................. .... 5

Animal Welfare ................................................................................................................ ...... 5

Distribution List ............................................................................................................. ........ 5 Study Plan Amendments and Deviations ............................................................................... 5 Quality Assurance ............................................................................................................. ..... 6 Reporting ..................................................................................................................... .......... 6 Archiving ..................................................................................................................... .......... 6

1 INTRODUCTION AND PURPOSE ..................................................................................... 7

1.1 Guidelines / Regulations .......................................................................................... 7

2 TEST AND REFERENCE ITEMS ....................................................................................... 8

2.1 Test Item .................................................................................................................. 8

2.2 Reference Item ......................................................................................................... 8

3 MATERIALS AND METHODS ........................................................................................... 8

3.1 Definitions and Abbreviations ................................................................................. 8

3.2 Test System .............................................................................................................. 8

3.3 Test Water ................................................................................................................ 9

3.4 Dosage ..................................................................................................................... 9

3.5 Materials .................................................................................................................. 9

3.6 Experimental Conditions ......................................................................................... 9

3.7 Study Design .......................................................................................................... 10

3.8 Evaluations ............................................................................................................ 11

3.8.1 Test organism observations ..................................................................... 11

3.8.2 Water quality criteria ............................................................................... 11

3.8.3 Analysis of Test Item Concentrations ..................................................... 12

3.9 Statistical Analysis ................................................................................................ 13

3.10 Validity Criteria ..................................................................................................... 13

4 REFERENCES .................................................................................................................... 14

APPENDICES .................................................................................................................... ........... 15Harlan Study Number: 41402585 Study Plan Page 3



## SIGNATURES

Study Director: S Harris

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

Test Facility Management: C Mead

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

For the Sponsor: Harcros Chemicals Inc.

................................................. Date:Harlan Study Number: 41402585 Study Plan Page 4



## GOOD LABORATORY PRACTICE

The associated study will be performed in co mpliance with UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (S I 1999/3106 as amended by SI 2004/0994)). These regulations are in accordance with GLP st andards published as OECD Principles on Good Laboratory Practice as revised 1997 (ENV/MC/C HEM(98)17); and are in accordance with, and implement, the requirements of Directives 2004/9/EC and 2004/10/EC.

These principles are compatible with Good Laboratory Practice regul ations specified by regulatory authorities throughout the European Community, the United States (EPA and FDA), and Japan (MHLW, MAFF and METI).Harlan Study Number: 41402585 Study Plan Page 5



## GENERAL INFORMATION

Proposed Schedule

Experimental Starting Date: 31 August 2014

Experimental Completion Date: 30 September 2014

Draft Report Date: 31 October 2014



### Animal Welfare

The study is 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 a nd the requirements of the United Kingdom’s Animals (Scientific Procedure) Act 1986 Amendm ent Regulations 2012. This study plan is subject to review and the conduct of the study may be included as part of the Harlan Laboratories Ltd, Shardlow, UK Ethical Review Process.

The study will also be conducted in accordance with the UK Home Office Guidance document on Regulatory Toxicology and Safe ty Evaluation Studies and the OECD guidance document on recognition, assessment and use of clinical signs as humane endpoints for experimental animals used in safety evaluation.



### Distribution List

The original Study plan will be retained in the st udy file. Copies of the study plan along with any

amendments will be distributed as follows:

Sponsor: Harcros Chemicals Inc.

Quality Assurance: Harlan Laboratories Ltd, Shardlow, UK



### Study Plan Amendments and Deviations

Any intended change to the study pl an will result in an amendment to study plan approved by the Study Director and also signed by test facility management and the Sponsor. Amendments will

be distributed to all reci pients of the study plan.

Deviations (unplanned changes) from the study plan will be documente d and acknowledged by the Study Director.Harlan Study Number: 41402585 Study Plan Page 6



### Quality Assurance

Quality Assurance will audit and inspect study-rela ted procedures and will report any audit and inspection results in writing to the Study Director and Test Facility Management.



### Reporting

A GLP compliant report will be presented. A draft re port will be sent to the Sponsor for review and comments before issue of the final report.



### Archiving

Unless instructed otherwise by the Sponsor, the st udy plan, all raw data (p aper and electronic) 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 wi ll be chargeable to the Sponsor.

No data will be discarded without contacting the Sponsor to obtain their written consent.Harlan Study Number: 41402585 Study Plan Page 7



## 1 INTRODUCTION AND PURPOSE

The purpose of this study is to assess the acute toxi city of the test item to freshwater fish. The toxicity will be expressed as th e median lethal concentration (LC 50 ).

The No Observed Effect Concentration (NOEC) and the Lowest Observed Effect Concentration (LOEC) will also be determined if applicable.

The species used will be Rainbow Trout ( Oncorhynchus mykiss ) a freshwater species representative of a wide variet y of natural habitats, and can th erefore be considered as an important non-target organism in freshwater ecosystems.

The dispersal of the test item in the surroundi ng medium is considered to represent the most probable route of exposure in the environment.



### 1.1 Guidelines / Regulations

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

 The OECD Guidelines for Testing of Chem icals (1992) No 203 "Fis h, Acute Toxicity

Test"

 Method C.1 of Commission Re gulation (EC) No. 440/2008

 EPA Ecological Effects Te st Guidelines OPPTS 850.1075 “Fish Acute Toxicity Test,

Freshwater and Marine”Harlan Study Number: 41402585 Study Plan Page 8



## 2 TEST AND REFERENCE ITEMS

2.1 Test Item

The test item will be supplied by or on behalf of the Sponsor including the following information if known; description, batch, purity, expiry / retest date, storage conditions and safety precautions to be taken.



### 2.2 Reference Item

In the interest of animal welfare, a positive cont rol using a reference item will not be performed.



## 3 MATERIALS AND METHODS

3.1 Definitions and Abbreviations

LC 50 : The calculated concentration of test item which results in 50% mortality within the exposure period.

LOEC (Lowest Observed Effect Concentration): The lowest test concentration at which a significant toxic effect on the test organisms is observed within the exposure period.

NOEC (No Observed Effect Concentration) : The highest test concentrati on at which no significant toxic effect on the test organisms is observed within the exposure period.

Treatment: Comprises test item treatm ents and control (test water only).



### 3.2 Test System

The study will be performed using Rainbow Trout ( Oncorhynchus mykiss ) supplied by an accredited live fish supplier, details of which will be documented in the data.

Juvenile fish < 3.0 g will be used and the longest should not be more than twice the length of the shortest. The fish will be maintained at a temperature of 12 ± 2.0 o C.

Following a 48-Hour settling in period, mortalitie s are recorded and the following rejection criteria applied.

> 10% in 7 days: reje ction of entire batch 5-10% in 7 days: continue acclima tisation for an additional 7 daysHarlan Study Number: 41402585 Study Plan Page 9

< 5% in 7 days: acceptance of batch

The fish will be acclimatised for a minimum pe riod of 12 days at Harlan Laboratories Ltd, with at least 7 days acclimatisation prior to test start in water of the quality and temperature to be used

in the test.

The fish will be fed commercial fish food, details of which will be documented in the data, regularly on demand. Feeding wi ll be discontinued 48 hours prior to testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Animals will be maintained in glass or fibreglass tanks at a loading rate not exceeding 0.8 g body weight per liter in static systems. This loading rate may be exceeded in flow-through systems. Aeration will be supplied to provide  80% Air Saturation Value (ASV).



### 3.3 Test Water

The animals will be maintained in dechlori nated laboratory tap water (see Appendix 1).



### 3.4 Dosage

The test item will be dissolved in the test water on a daily basis (semi-static test).

Details of the method of preparati on will be documented in the data.



### 3.5 Materials

Glass test vessels containing an appropriate amount of test medium . The test vessels will be covered to reduce evaporation and to avoid entry of dust into the solu tions. The size of the test

vessels and the volume of test solution will be documented in the data.

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



### 3.6 Experimental Conditions

The environmental parameters will be as used to maintain the stock animals. Auxiliary aeration will not normally be supplied to the test vessels , however for ethical r easons if the dissolved oxygen levels are measured to be less than 60% ASV then gentle aera tion may be provided.Harlan Study Number: 41402585 Study Plan Page 10

The test will be performed under temper ature-controlled conditions at 12 ± 2.0 o C with a maximum deviation of ± 1 °C during the test.

A 16-hour light to 8-hour dark cycle will be ap plied with a 20 – 30 minute transition period.



### 3.7 Study Design

Seven fish will be used at each test concentration and in the control.

The loading rate will not be greater than 0.8 g bodyweight per liter of test medium.

Animals will be selected at random from the to tal number collected. The animals will not be individually identified. The anim als will not be fed during the test.

The test will be conducted with daily medium re newal (semi-static test). The actual method of

test media renewal will be documented in the data.

The test will be started by addition of the test organisms to the test vessels within 30 minutes of the test media being added to the test vessels.

At least five concentrations of the test item ar ranged in a geometric seri es will be tested. The spacing factor between the test concentrations will preferably not exceed 2.2. The actual test concentrations are assigned according to prelim inary range-finding tests or available toxicity data and will be documented in the data prior to the start of the definitive test. If in the rangefinding test a flat concentration- effect relationship is determined and, thus, a large concentration range will have to be teste d, a spacing factor exceeding 2.2 (e.g., 3.2) will be applied.

The chosen test concentrations should permit the determination of the 96-Hour LC 50 and, as far as possible, span the range of 0 to 100% mortalit y. Concentrations of the test item higher than 1000 mg/L will usually not be tested.

If no effect is observed in the preliminary ra nge-finding test at 1000 mg/L, a Limit Test may be conducted using a single test vessel at this c oncentration together w ith a control vessel.

The control group will be exposed to the test water al one. In the interests of animal welfare, data from the control group may be shared with similar concurrent studies.

The pH of the test preparat ions will not be adjusted.Harlan Study Number: 41402585 Study Plan Page 11



### 3.8 Evaluations

3.8.1 Test organism observations

The fish are inspected after 3, 6, 24, 48, 72 a nd 96 hours and at intermediate times when appropriate. Mortalities are recorded and any abnormalities in appearance or behaviour shown by surviving fish are summarised. The criteria of death are absenc e of respiratory movement and the lack of response to phys ical stimulation. Dead fish are removed when observed.

During the test, the principles mentioned in the OECD Humane Endpoint s Guidance Document No. 19 (OECD, 2002) will be taken into consider ation. Test fish, which are found in moribund condition, will be humanely killed before test end due to reasons of animal welfare. For the

calculation of the LC 50 value the sacrificed fish will be taken into account as dead fish.

The length and weight of a representative batch of fish will be determin ed e.g. fish from the stock tank prior to the start of the test or the control fish at the end of the test

3.8.2 Water quality criteria

Measurements of pH, dissolved oxygen and temper ature in each surviving test vessel (prior to and post test media renewal) will be recorded dail y. In addition the temperature will be recorded hourly in the control vess el using a data logger.

The appearance of the test media will be recorded daily.Harlan Study Number: 41402585 Study Plan Page 12

3.8.3 Analysis of Test Item Concentrations

3.8.3.1 Sampling

Samples of the test media will be taken at 0 hours (fresh media), 24, 48 and 72 hours (fresh and old media) and 96 hours (old media) of the definitive test for anal ysis. In the ra nge-finding test, samples of the test media will be taken at 0 hours and after 24 or 96 hours. Where possible duplicate samples will be taken at each occasion, these samples may also be analyzed should the

need arise.

Samples will be taken from the control and each surviving test group or at the observation point

when all fish are dead in a certain test concentration.

Analysis of samples taken from the range-finding test is performed in order to determine the stability of the test item under test conditions ov er the exposure period. The test concentrations provided for analysis will be documen ted by the Study Director in the data.

Final details of the samples to be taken and the sa mple size, etc, will be documented in the data.

3.8.3.2 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 unstabl e under the storage conditions, the samples will be analyzed immediately after sampling without prio r storage or measures will be taken (e.g. pH adjustment or addition of organic solvents) in order to maintain th e stability of the test item until analysis.

3.8.3.3 Analysis of samples

The test item formulations will be analyzed for concentr ation 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 co ncentrations below the 96-Hour NOEC will not normally be analyzed. The remaining samples may be analyzed, if necessary.

The analytical method (eg HPLC, LC-MS, GC, AAS or photospectrometry) will either be provided by the Sponsor and adapted, if necessa ry, to the analytical conditions at Harlan Laboratories Ltd or developed by Harlan Laboratories Ltd. Adaptation or development of the analytical method will not be performed under GL P. 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.Harlan Study Number: 41402585 Study Plan Page 13



### 3.9 Statistical Analysis

The LC 50 values and associated 95% confidence limits for all appropriate observation times will be calculated using an appropriate statistical method which will be documented in the data. All

LC 50 values will be rounded to two significant figures.

The No Observed Effect Concentration (NOEC) is the highest test concentration at which no significant toxic effect on the test organism s is observed within the exposure period.

The Lowest Observed Effect Concentration (LOEC) is the lowest test concentration at which a significant toxic effect on the test organisms is observed within the exposure period. If the test

has been conducted as a limit test then it is not possible to determine a LOEC.



### 3.10 Validity Criteria

For the test to be valid, the followi ng criteria should be fulfilled:

In the control, not more than one of the fish should die or show sign s of stress during the

96 hours.

The dissolved oxygen concentration at the end of the test should be ≥ 60% of Air Saturation Values (ASV) in the control and test vessels.Harlan Study Number: 41402585 Study Plan Page 14



## 4 REFERENCES

OECD (2002) Guidance Document on the Recognition, Assessment and Use of Clinical Signs as Humane Endpoints for Experiment al Animals Used in Safety Evaluation. OECD Series on Testing and Assessment, No. 19. OECD Publishing.Harlan Study Number: 41402585 Study Plan Page 15



## APPENDICES

Appendix 1 Test Water

Laboratory tap water will be dechlorinated by pa ssage through an activated carbon filter (Purite Series 500) and a proportion of the incoming wate r softened (Elga Nimbus 1248D Duplex water softener) and then remixed with the main suppl y to give a water hardness of approximately 140 mg/L as CaCO 3 and pH 6.0 - 8.5. After dechlorination and softening the water will then be passed through a series of computer controlled heat exchangers to achieve the required temperature. When a "single pass" system is not possible, the water will be recirculated via a suitable purification filter.

Chemical analysis of the water used in testing will include the following elements and

limitations on maximum concentrations:

Substance Maximum Concentration Particulate matter 20.0 mg/L Chemical Oxygen Demand (COD) 5.0 mg/L Total Organic Carbon (TOC) 2.0 mg/L Boron and fluoride <100.0 mg/L Residual chlorine 0.003 mg/L Un-ionized ammonia 0.020 mg/L Aluminum, arsenic, chromium, cobalt, copper, iron, lead, nickel, and zinc 0.001 mg/L

Cadmium, mercury, and silver <0.100 mg/L Total organophosphorus pesticides 0.050 mg/L Total organochlorine pestic ides + PCBs or organic

chlorine 0.050 mg/L

Specific conductivity <1.0 mohms

Water hardness will be measured at the beginning of the test, daily chlorine analysis will be performed.