REPORT 

Sodium Glucoheptonate CAS No. 31138-65-5: 

Daphnia sp., 48-Hour Acute Immobilization Test 

Study Director: 

Test Facility: 

Sponsor: 

Harlan Study Number: 

Study Completion Date: 

S Harris 

Harlan Laboratories Ltd. 

Shardlow Business Park 

Shardlow 

Derbyshire 

DE722GD 

UK 

Harcros Chemicals Inc. 

3452 Corporate Drive 

Dalton GA 30720 

UNITED STATES OF AMERICA 

41402586 

27May201S 

Page 1 of41 
Harlan Study Number: 41402586 Report Page2 

CONTENTS 

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

STUDY DIRECTOR STATEMENT OF GLP COMPLIANCE ................................................... .4 

QUALITY ASSURANCESTATEMENT ...................................................................................... 5 

SIGNATURE OF CONTRIBUTING SCffiNTISTS ...................................................................... 6 

SUMMARY .................................................................................................................................... 7 

Introduction ............................................................................................................................ 7 

Methods ............................................................................................................................... 7 

Results .................................................................................................................................. 7 

GENERAL INFORMATION ......................................................................................................... 8 

Schedule ................................................................................................................................. 8 

Deviations from Study Plan ................................................................................................... 8 

Archiving ............................................................................................................................... 8 

1 INTRODUCTION AND PURPOSE ..................................................................................... 9 

1.1 Guidelines I Regulations .......................................................................................... 9 

2 TEST ITEM ......................................................................................................................... 10 

3 MATERIALS AND METHODS ......................................................................................... 11 

3.1 Definitions and Abbreviations ............................................................................... 11 

3.2 Test System ............................................................................................................ 11 

3.3 Test Water .............................................................................................................. 11 

3.4 Procedure ............................................................................................................... 12 

3.4.1 Range-finding Test .................................................................................. 12 

3.4.2 Definitive Test ......................................................................................... 12 

3.4.3 Evaluations .............................................................................................. 13 

3.4.4 Statistical Analysis .................................................................................. 14 

3.5 Validation Criteria ................................................................................................. 14 

4 RESULTS ............................................................................................................................ 15 

4.1 Range-finding Test ................................................................................................ 15 

4.2 Definitive Test ....................................................................................................... 15 

4.2. 1 Verification ofTest Concentrations ........................................................ 15 

4.2.2 Immobilization Data ................................................................................ l5 

4.2.3 Validation Criteria ................................................................................... 16 

4.2.4 Water Quality Criteria ............................................................................. 16 

4.2.5 Observations on Test Item Solubility ...................................................... 16 

5 CONCLUSION .................................................................................................................... 17 

6 REFERENCES .................................................................................................................... 18 

TABLES ........................................................................................................................................ 19 

FIGURES ...................................................................................................................................... 22 

APPENDICES ............................................................................................................................... 23 
Harlan Study Number: 41402586 Report Page 3 

LIST OF TABLES 

Table I 

Table 2 

Table 3 

Cumulative Immobilization Data in the Range-finding Test.. .................................... 19 

Cumulative Immobilization Data in the Definitive Test.. ........................................... 20 

Water Quality Measurements ..................................................................................... 21 

LIST OF FIGURES 

Figure 1 Testo Temperature Logger Data ................................................................................. 22 

LIST OF APPENDICES 

Appendix 1 

Appendix 2 

Appendix 3 

Appendix 4 

Appendix 5 

Appendix 6 

Certificate of Analysis ........................................................................................... 23 

Reconstituted Water - Elendt M7 Medium ........................................................... 24 

Positive Control ..................................................................................................... 25 

Reconstituted Water- ISO Medium ..................................................................... 26 

Analytical Investigations ....................................................................................... 27 

Monitoring Authority Statement of GLP Compliance ......................................... .41 
Harlan Study Number: 4 1402586 Report Page4 

STUDY DIRECTOR STATEMENT OF GLP COMPLIANCE 

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

Harlan Study Number: 41402586 

Study Title: Sodium Glucoheptonate CAS No. 31138-65-5: 

Daphnia sp., 48-Hour Acute Immobilization Test 

This study was performed in compliance with UK GLP standards (Schedule 1, Good Laboratory 

Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)). These Regulations are 

in accordance with GLP standards published as OECD Principles on Good Laboratory Practice 

(revised 1997, ENV/MC/CHEM(98)17); and are in accordance with, and implement, the 

requirements of Directives 2004/9/EC and 2004/10/EC. 

These principles are compatible with Good Laboratory Practice regulations specified by 

regulatory authorities throughout the European Community, the United States (EPA and FDA), 

and Japan (MHLW, MAFF and METI). 

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

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

Study Dir~ctor : S Harris 

... Go ...... . 

Date: 1l o5-l5 
Harlan Study Number: 4 1402586 Report Page 5 

QUALITY ASSURANCE STATEMENT 

Harlan Study Number: 41402586 

Study Title: Sodium Glucoheptonate CAS No. 31138-65-5: 

Daphnia sp., 48-Hour Acute Immobilization Test 

The general facilities and activities are inspected at least once a year and the results are reported 

to the relevant responsible person and management. 

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

these audits and inspections are given below. 

Reported to the 

Dates and Types of QA Inspections 

relevant Study 

Director and Test 

Facility Management 

Date of Inspection Type of Inspection Phase Inspected Report Date 

22 July 2014 Study Plan NIA 22 July 2014 

Verification 

13 January 2015 Process - based Test Item Preparation 13 January 2015 

13 January 2015 Process - based Test System 13 January 2015 

Preparation and 

Exposure 

15 January 2015 Process - based Assessment of 15 January 2015 

Response 

30 January 2015 Process - Based Chemical Analysis 30 January 2015 

11 May 2015 Report Audit NIA 11 May 2015 

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

Quality Assurance: U KAQ.AS 

1\ 

o;~~o; ··········· ····· · 
Harlan Study Number: 41402586 Report Page 6 

SIGNATURE OF CONTRIBUTING SCIENTISTS 

Analytical Chemistry: D M Mullee 

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

•

Date: •••• •••••••• 

2 8 M~~· Z015 
Harlan Study N urn ber: 414025 86 Report Page 7 

SUMMARY 

Introduction 

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The 

method followed was designed to be compatible with the OECD Guidelines for Testing of 

Chemicals (April2004) No 202, "Daphnia sp., Acute Immobilisation Test" referenced as Method 

C.2 of Commission Regulation (EC) No. 440/2008 and the US EPA Draft Ecological Effects 

Test Guidelines OPPTS 850.1010 (April 1996). 

Methods 

Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were 

exposed to an aqueous solution of the test item at a concentration of 100 mg active 

ingredient (ai)/L for 48 hours at a temperature of 21 oc under static test conditions. 

Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours. 

Results 

Analysis of the 100 mg ai/L test preparations at 0 and 48 hours showed measured test 

concentrations to range from 86% to 80% of nominal value and so the results are based on 

nominal test concentrations only. 

Exposure of Daphnia magna to the test item gave ECso values of greater than 1 00 mg ai/L. The 

No Observed Effect Concentration was 100 mg ai/L. 

It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg ai/L. 
Harlan Study Number: 41402586 Report 

GENERAL INFORMATION 

Schedule 

Experimental Starting Date: 

Experimental Completion Date: 

Deviations from Study Plan 

02 March 2015 

25 March 2015 

Page 8 

Deviation No.1 The study plan states that the alkalinity of the diluents must be 

0.8 mmol/L, however, the measured alkalinity was below this level. Although this was a 

deviation to the study plan, given that no adverse effects were observed in the test, this deviation 

was considered not to have had an effect on the outcome of the test. 

Deviation No.2 The study plan states that the parental daphnids are cultivated in water of 

the same quality to the test water in respect to pH, main ions and hardness. However, in order to 

comply with EPA guidelines, the hardness of the test water was reduced to 150 mg CaC03/L. 

Given that no adverse effects were observed in the control group, this was considered not to have 

has an impact on the outcome of the test. 

Archiving 

Unless instructed otherwise by the Sponsor, the study plan, all raw data (paper and electronic) 

and the fmal 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. 

No data will be discarded without contacting the Sponsor to obtain their written consent. 
Harlan Study Number: 41402586 Report Page9 

1 INTRODUCTION AND PURPOSE 

This report contains a description of the methods used and results obtained during a study to 

investigate the acute toxicity of the test item to Daphnia magna. 

Daphnia magna is a freshwater invertebrate representative of a wide variety of natural habitats, 

and can therefore be considered as an important non-target organism in freshwater ecosystems. 

1.1 Guidelines I Regulations 

This study was designed to be compatible with the procedures indicated by the following 

internationally accepted guidelines and recommendations: 

• OECD Guidelines for Testing of Chemicals (April2004) No 202 "Daphnia sp., Acute 

lrnmobilisation Test" 

• Method C.2 of Commission Regulation (EC) No. 440/2008 

• US EPA Draft Ecological Effects Test Guideline OPPTS 850.1010 "Aquatic 

Invertebrate Acute Toxicity Test, Freshwater Daphnids" 
Harlan Study Number: 41402586 Report Page 10 

2 TESTITEM 

Information as provided by the Sponsor. A Certificate of Analysis supplied by the Sponsor is 

given in Appendix 1. 

Identification: 

Physical state/ Appearance: 

Batch: 

Purity: 

Expiry Date: 

Storage Conditions: 

Intended use/ Application: 

Sodium Glucoheptonate CAS No. 31138-65-5 

Amber colored liquid 

T-461 

39.94% 

Not supplied 

Room temperature in the dark 

Not supplied 
Harlan Study Number: 41402586 Report Page II 

3 MATERIALS AND METHODS 

3.1 Definitions and Abbreviations 

Immobilization: 

ECso: 

NOEC (No Observed Effect 

Concentration): 

Treatment: 

3.2 Test System 

Those organisms which are not able to swim within 15 seconds 

after gentle agitation of the test container are considered to be 

immobile. 

The calculated concentration of test item wruch results in 50% 

immobilization within the exposure period. 

The highest test concentration at which no significant toxic 

effect on the test organisms is observed within the exposure 

period. 

Comprises test item treatments and control (test water only). 

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory 

cultures. 

Adult daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium (see 

Appendix 2) in a temperature controlled room at approximately 20 °C. The lighting cycle was 

controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk 

transition periods. Each culture was fed daily with a mixture of algal suspension (Desmodesmus 

subspicatus) and Tetramin® flake food suspension. Culture conditions ensured that reproduction 

was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such 

that the young daphnids produced overnight were less than 24 hours old. These young were 

removed from the cultures and used for testing. The diet and diluent water are considered not to 

contain any contaminant that would affect the integrity or outcome of the study. 

A positive control (Harlan Study Number 41500252) used potassium dichromate as the reference 

item. Details of the positive control are given in Appendix 3. The positive control was 

conducted between 23 February 2015 and 25 February 2015. 

3.3 Test Water 

Reconstituted water (ISO medium) used for both the range-finding and definitive tests is defined 

in Appendix 4. In order to comply with the water characteristics of the OPPTS guideline, the 

reconstituted water was diluted 50:50 with deionized reverse osmosis water prior to use to 

reduce the total water hardness. At the start of the definitive test the diluent had total water 

hardness of 146 mg/L as CaC03 and an alkalinity of 27.6 mg/L as CaC03. The water hardness 

and alkalinity were determined using the methods described in Field and On-site Test Methods 

for the Analysis of Waters (British Standards Institution, 1993). 
Harlan Study Number: 41402586 Report Page 12 

The study plan states that the alkalinity of the diluents must be 0.8 mmol/L, however, the 

measured alkalinity was below this level. Although this was a deviation to the study plan, given 

that no adverse effects were observed in the test, this deviation was considered not to have had 

an effect on the outcome of the test. 

3.4 Procedure 

3.4.1 Range-finding Test 

The test concentration to be used in the definitive test was determined by a preliminary rangefmding test. 

In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations 

of 0.1 0, 1.0, 10 and I 00 mg ai!L. At the request of the Sponsor, all test concentrations were 

corrected for a test item purity of39.94%. 

A nominal amount of test item (250.4 mg) was dissolved in test water and the volume adjusted to 

l liter to give the l 00 mg ai/L test concentration from which a series of dilutions was made to 

give further test concentrations of 0.10, 1.0 and 10 mg ai/L. 

Each prepared concentration was inverted several times to ensure adequate mixing and 

homogeneity. 

In the range-finding test l 0 daphnids were placed in each test and control vessel and maintained 

in a temperature controlled room at 21 oc with a photoperiod of 16 hours light and 8 hours 

darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Each 

250 mL test and control vessel contained 200 mL of test media and was covered to reduce 

evaporation. After 24 and 48 hours the number of immobilized daphnids were recorded. 

The control group was maintained under identical conditions but not exposed to the test item. 

A sample of each test concentration was taken for chemical analysis at 0 and 48 hours in order to 

determine the stability of the test item under test conditions. All samples were stored frozen 

prior to analysis. Only concentrations within the range to be used for the definitive test were 

analyzed. 

3.4.2 Definitive Test 

Based on the results of the range-finding test a "Limit test" was conducted at a concentration of 

100 mg ai/L to confinn that no immobilization or adverse reactions to exposure were observed. 
Harlan Study Number: 41402586 Report Page 13 

3.4.2.1 Experimental Preparation 

A nominal amount of test item (250.4 mg) was dissolved in test water and the volume adjusted to 

1 liter to give the 1 00 mg ai/L test concentration. 

The prepared concentration was inverted several times to ensure adequate mixing and 

homogeneity. 

The concentration and stability of the test item in the test preparations were verified by chemical 

analysis at 0 and 48 hours (see Appendix 5). 

3.4.2.2 Exposure Conditions 

As in the range-finding test 250 mL glass jars containing approximately 200 mL of test 

preparation were used. At the start of the test 5 daphnids were placed in each test and control 

vessel at random, in the test preparations. Four replicate test and control vessels were prepared. 

The test vessels were then covered to reduce evaporation and maintained in a temperature 

controlled room at 21 oc with a photoperiod of 16 hours light (565 to 626 lux) and 8 hours 

darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually 

identified, received no food during exposure and the test vessels were not aerated. 

The control group was maintained under identical conditions but not exposed to the test item. 

The test preparations were not renewed during the exposure period. 

3.4.3 Evaluations 

3.4.3.1 Test Organism Observations 

Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the 

start of exposure. The criterion of effect used was that daphnia were considered to be 

immobilized if they were unable to swim for approximately 15 seconds after gentle agitation. 

3.4.3.2 Water Quality Criteria 

Water temperature was recorded daily throughout the test. Dissolved oxygen concentrations and 

pH were recorded at the start and termination of the test. The pH and dissolved oxygen 

concentration were measured using a Hach HQ30d Flexi handheld meter whilst the temperature 

was measured using a Hanna Instruments HI 935 10 digital thermometer. 

The temperature was also measured every hour in one replicate of the control using a Testo 

temperature logger. 
Harlan Study Number: 4 1402586 Report Page 14 

3.4.3.3 Verification of Test Concentrations 

Samples were taken fTom the control and the 100 mg ai/L test group from the bulk test 

preparation at 0 hours and from the pooled replicates at 48 hours for quantitative analysis. All 

samples were stored frozen prior to analysis. Duplicate samples were taken and stored frozen for 

further analysis if necessary. 

The 0-Hour samples were not stored frozen for the initial2 days after sampling due to a technical 

error, however, given that near nominal concentrations were achieved, this was considered not to 

have had an impact on the outcome of the test. 

The method of analysis, recovery and test preparation analyses are described in Appendix 5. 

3.4.4 Statistical Analysis 

An estimate of the EC5o values was given by inspection of the immobilization data. 

3.5 Validation Criteria 

The results of the test are considered valid if the following performance criteria are met: 

• No more than 10% of the control daphnids show immobilization or other signs of disease 

or stress (e.g. discoloration or unusual behavior such as trapping at the surface water). 

• The dissolved oxygen concentration at the end of the test is ?:3 mg/L in the control and 

test vessels. 
Harlan Study Number: 41402586 Report Page 15 

4 RESULTS 

4.1 Range-finding Test 

Cumulative immobilization data from the exposure of Daphnia magna to the test item during the 

range-finding test are given in Table 1. 

No significant immobilization was observed at the test concentrations of 0.1 0, 1.0, 10 and 

I 00 mg ai/L. A single immobilized daphnia was observed in both the 1 0 and 1 00 mg ai/L test 

groups, however, as no more than 10% immobilization occurred, this was considered to be due 

to natural causes and was considered not to have had an impact on the outcome of the test. 

Based on this information, a single test concentration of four replicates, of 100 mg ai/L was 

selected for the definitive test. This experimental design conforms to a "Limit test" to confirm 

that no immobilization or adverse reactions to exposure were observed at this concentration. 

Chemical analysis of the l 00 mg ai/L test preparations at 0 and 48 hours (see Appendix 5) 

showed that measured concentrations of 93% and 87% of nominal were obtained respectively 

indicating that the test item was stable tmder test conditions. 

4.2 Definitive Test 

4.2.1 V t:rification of Test Concentrations 

Analysis of the test preparations at 0 and 48 hours (see Appendix 5) showed measured test 

concentrations to range from 86% to 80% of nominal value and so it was considered justifiable 

to estimate the ECso values in terms of the nominal test concentrations only. 

4.2.2 Immobilization Data 

Cumulative immobilization data from the exposure of Daphnia magna to the test item during the 

definitive test are given in Table 2. 

There was no immobilization in 20 daphnids exposed to a test concentration of l 00 mg aiJL for a 

period of 48 hours. Inspection of the immobilization data gave the following results: 

Time (h) ECso (mg ai/L) 

24 >100 

48 >100 

The No Observed Effect Concentration after 24 and 48 hours exposure was 100 mg ai/L. 
Harlan Study Number: 41402586 Report Page 16 

It was considered unnecessary and unrealistic to test at concentrations in excess of 1 00 mg ai/L. 

4.2.3 Validation Criteria 

The test was considered to be valid given that none of the control daphnids showed 

immobilization or other signs of disease or stress and that the oxygen concentration at the end of 

the test was 2:3 mg!L in the control and test vessels. 

4.2.4 Water Quality Criteria 

The results of the water quality measurements are given in Table 3. Temperature was 

maintained at 21 °C throughout the test, while there were no treatment related differences for 

oxygen concentration or pH. Results from the Testo temperature logger are given in Figure 1. 

4.2.5 Observations on Test Item Solubility 

Throughout the duration of the test, the test preparations were observed to be clear colorless 

solutions. 
Harlan Study Number: 41402586 Report Page 17 

5 CONCLUSION 

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been 

investigated and gave a 48-Hour EC5o value of greater than 100 mg ai/L. The No Observed 

Effect Concentration was 100 mg ai/L. 
Harlan Study Number: 41402586 Report Page 18 

6 REFERENCES 

BRITISH STANDARDS INSTITUTION (1993) Determination of Alkalinity. In: Field and Onsite Test Methods for the Analysis of Waters, (BS 1427), pp 33-35. 

BRITISH STANDARDS INSTITUTION (1993) Determination of Calcium and Total Hardness. 

In: Field and On-site Test Methods for the Analysis of Waters, (BS 1427), pp 35-36. 
Harlan Study Number: 41402586 Report Page 19 

TABLES 

Tablet Cumulative Immobilization Data in the Range-finding Test 

Nominal 

Cumulative Immobilized Daphnia 

Concentration {Initial Population: 10 Per Replicate) 

(mg a i!L) 24 Hours 48 Hours 

Control 0 0 

0.10 0 0 

1.0 0 0 

10 0 I* 

100 0 I* 

* Single immobilized daphnia considered to be due to natural causes given that no more than 10% 

immobilization was observed. 
Harlan Study Number: 41402586 Report Page 20 

Table 2 Cumulative Immobilization Data in the Defmitive Test 

Cumulative Immobilized Daphnia 

Nominal (Initial Population: 5 Per Replicate) 

Concentration 24 Hours 48 Hours 

(mg ai!L) No. Per No. Per 

Replicate 

Total % Replicate 

Total % 

Control R, 0 0 

R2 0 0 

0 0 0 0 

R3 0 0 

~ 0 0 

100 R, 0 0 

R2 0 0 

0 0 0 0 

RJ 0 0 

~ 0 0 

R1 -~=Replicates I to 4 
Harlan Study Number: 41402586 Report Page 21 

Table 3 Water Quality Measurements 

Nominal 0 Hours 24 Hours 48 Hours 

Concentration 

(mg ai/L) pH mg 0 2/L T °C T°C pH mgOiL T°C 

R• 7.6 9.0 21 21 7.7 9. 1 21 

R2 7.6 9.0 21 21 7.6 9.0 21 

Control 

R3 7.7 9.1 21 21 7.6 9.0 21 

~ 7.7 9.0 21 21 7.6 9.0 21 

R• 8.2 9. 1 2 1 2 1 7.6 8.6 21 

R2 8.2 9.0 21 21 7.6 8.5 21 

100 

Rl 8.3 9.0 21 21 7.5 8.4 21 

~ 8.2 9.1 21 21 7.5 8.4 21 

R1- ~ = Replicates I to 4 
Harlan Study Number: 41402586 Report 

FIGURES 

Figure 1 Testo Temperature Logger Data 

20.8 

20.7 

20.6 

g 20.5 

i 

i 20.4 I20.3 

20.2 

Page 22 

20 1~~--~~--~~~~--~~--~~--~~--~~--~--~~--~~--~~--~~~ 

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 38 38 40 42 44 46 48 

T1me (Hours) 
Harlan Study Number: 41402586 Report 

APPENDICES 

Appendix 1 Certificate of Analysis 

Harcros Chemicals, Inc. 

Dalton Location 

3452 Corporate Drive 

Dalton, GA 30720 

Certificate of Analysis 

Mother Liquor CAS# 31138 .. 65-5 

DaLe: 8!2612014 

•••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• 

Beta Sodium 

Gluc-ahcptonate 

CAS#- 30090-50..3 

Alpha Sodium 

Gl ucoheplonatc 

CAS# 13007-85-7 

Polysaccharide or 

Degradation ProductS 

Cyanide 

Ammonia 

CASfl 7664-41-7 

pH l% 

Water% 

Specific Gravity 

%Solids 

% Dextrose 

CAS# 50-99-7 

Specifications 

Record Onl) by HI'I.C 

Record Only by HPLC 

Record Only by 11PI.C 

'IM 1322 0 - IOppm 

TM 1321 0 - 2500 ppm 

TM 1062 9-11 

TM 1079 35 -50 

TM 1075 1.272 1.300 

TM 1079 35 -so 

TM 1335 0 - trnce 

Ie<>t Results 

2j,78% 

14.16 % 

10.96 % 

0.3 ppm 

I 113 ppm 

9.1 

49.1 

1.293 

50.9% 

0/ none detected 

Page 23 
Harlan Study Number: 41402586 Report 

Appendix 2 Reconstituted Water - Elendt M7 Medium 

Ingredient 

H3B03 

MnChAH20 

LiCI 

RbCI 

SrCh.6H20 

NaBr 

Na2Mo04.2H20 

CuCl2.2H20 

ZnCh 

CoCh.6H20 

KI 

Na2Se03 

NH4V03 

Na2EDTA.2H20 

FeS04.1H20 

CaCh.2H20 

NaHC03 

MgS04.1H20 

Na2Si03.9H20 

KCl 

NaN03 

K2HP04 

KH2P04 

Th iamine hydrochloride 

Cyanocobalamine (vitamin B12) 

D(+) biotin (vitamin H) 

Final Concentration 

(mg/L) 

0.715 

0.090 

0.077 

0.018 

0.038 

0.004 

0.016 

0.004 

0.013 

0.010 

0.0033 

0.0022 

0.00058 

0.625 

0.249 

293.8 

64.8 

123.3 

10 

5.8 

0.274 

0.184 

0.143 

0.075 

0.0010 

0.00075 

The pH of the prepared media was 7.9 ± 0.3 and stored at approximately 21 °C. 

Page 24 
Harlan Study Number: 41402586 Report Page 25 

Appendix 3 Positive Control 

A positive control (Harlan Study Number 41500252) used potassium dichromate as the reference 

item at concentrations of 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L. 

Exposure conditions for the positive control were similar to those in the definitive test. 

Analysis of the immobilization data at 24 and 48 hours by pro bit analysis using linear maximum 

likelihood regression based on the nominal test concentrations gave the following results: 

No Observed Lowest Observed 

Time Point ECso 95% Confidence Limits Effect Effect 

(Hours) (mg!L) (mg!L) Concentration Concentration 

(NOEC) (mg!L) (LOEC) (mg/L) 

24 1.1 • 0.56 1.0 

48 0.75 0.69 - 0.82 0.56 1.0 

All analysis was carried using the ToxRat Professional computer software package (ToxRat) **. 

The No Observed Effect Concentration is based upon less than I 0% immobilization at this 

concentration. 

The results from the positive control with potassium dichromate were within the normal range 

for this reference item. 

*Due to the nature of the data, 95% confidence limits could not be determined. 

** TOXRATPROFESS10NAL VERSION2.10.06. TOXRAT®SOLUTIONS GMBH 2001-2010 
Harlan Study Number: 41402586 Report 

Appendix 4 Reconstituted Water - ISO Medium 

Ingredient 

CaCh.2H20 

MgS04.1H20 

NaHC0 3 

KCI 

Final Concentration 

(mg!L) 

294 

123 

65 

5.8 

Page 26 

The reconstituted water had a pH of7.8 ± 0.2 adjusted (if necessary) with NaOH or HCI and was 

aerated until the dissolved oxygen concentration was approximately air-saturation value. 

The reconstituted water had an approximate theoretical total hardness of250 mg/L as CaC03. 
Harlan Study Number: 41402586 Report Page 27 

Appendix 5 Analytical Investigations 

1 MATERIALS AND METHODS 

1.1 Introduction 

The active ingredient concentration in the test samples was determined by direct injection into 

the mass spectrometer (HPLC-MS) using an external standard. 

All test sample concentrations were corrected for a purity content of 39.94%, and are reported as 

milligrams of active ingredient (ai) per litre. 

The method was developed by the Department of Analytical Services, Harlan Laboratories Ltd, 

Shardlow, UK. 

1.2 Test Item 

The test item is described in the biological part of this study. 

1.3 Analytical Standard 

The test item described in the biological part of this study was also used as the analytical 

standard. 

1.4 Analytical Procedure 

1.4.1 Storage 

The samples were stored frozen prior to analysis. 

1.4.2 Reagents and Solvents 

Water: prepared using an ELGA Pure Lab Option R-15 water purification system. 

Fisher Scientific, LC-MS grade. 

Test medium: as described in the biological part of this study. 

Solvents: methanol, Romil, Ultra gradient grade. 

Reagents: ammonia, Fisher Scientific analytical reagent grade. 
Harlan Study Number: 41402586 Report Page28 

Analytical Lnvestigations 

1.4.3 Preparation of Calibration Standards 

The test item (nominal 250 mg) was dissolved in diluent" (1 00 mL) to prepare a stock solution 

with a concentration of l 000 mg ai/L. This stock solution was further diluted with diluent• to 

produce a solution of l 0 mg ai/L. Defined volumes of this solution were then diluted with 

diluent" to obtain calibration standards in the range of O.l 0 to 1.0 mg ai/L. A second series of 

calibration standards was similarly prepared in the range of 0.25 to 1.5 mg ai/L. These solutions 

were used to determine the recovery and test sample concentrations. 

1.4.4 Preparation of Linearity Standards 

The test item (nominal 250 mg) was dissolved in diluent• (1 00 mL) to prepare a stock solution 

with a concentration of 1000 mg ai/L. This stock solution was furthe r diluted with diluent* to 

produce a solution of l 0 mg ai/L. Defined volumes of this solution were then diluted with 

diluent" to obtain calibration standards in the range of 0. 10 to 1.0 mg ai/L. A second series of 

calibration standards was similarly prepared in the range of 0.25 to 1.5 mg ai/L. These standards 

were used to evaluate the linearity of the analytical system. 

1.4.5 Preparation of Spiked Recovery Samples 

To demonstrate the validity of the analytical procedure, volumes of test medium were spiked 

with the test item and the recovery was assessed. The test item (nominal 250 mg) was initially 

dissolved in test medium to prepare a stock solution with a concentration of 1000 mg ai/L. 

Defined volumes of this stock solution were diluted with test medium to obtain spiked recovery 

samples at a concentration of l 00 mg ai/L. Five replicates were prepared and subjected to the 

same treatment as the test samples. In addition, test medium without the addition of the active 

ingredient (synthetic control) was also analyzed. 

1.4.6 Preparation of Test Samples 

The test samples were thawed with the aid of sonication. They were diluted with test medium to 

a nominal concentration of 10 mg ai/L and then further diluted into the calibration range with 

methanol: water: ammonia (50:50:0.1 0 v/v/v) as shown in Table 1. 

·diluent: methanol: water: ammonia: test medium (50:40:0.10:10 v/v/v/v) 
Harlan Study Number: 41402586 Report 

Analytical Investigations 

1.4.7 Instrumental Parameter 

Autosampler: 

Pump: 

Detector: 

Column: 

Mobile phase: 

Flow rate: 

Injection volume: 

Ionization mode: 

Polarity: 

Capillary voltage: 

Gas temperature: 

Nebulizer pressure: 

Fragmentor voltage: 

Drying gas flow: 

Data acquisition mode: 

SIM ions: 

Agilent 1200 Series 

Agilent 1200 Series 

Agilent 1200 Series MSD 

none, direct injection 

methanol: LC-MS water: ammonia (50:50:0.1, v/v/v) 

0.5 mL/rninute 

10 ~L 

ESI 

negative 

4000V 

350 oc 

40 psi 

IOOV 

12 Llmin 

Single Ion Monitoring (SIM) 

225.0 m/z 

Page 29 
Harlan Study Number: 41402586 Report Page 30 

Analytical Investigations 

1.4.8 Calculations 

A calibration curve was prepared using a quadratic function of the form shown in equation (1 ). 

The calibration data is presented in Table 2 and Figure 1. From the calibration curve, the 

concentration "x" of the active ingredient in an injected sample was calculated using the 

following equation: 

-b+~b2 -4·a·(c- y) --...!.....-----=--~....:... 

(1) 

2·a 

where x: 

y: 

a, b, c: 

concentration of the active ingredient in the injected sample [mg ai!L] 

average peak area of the active ingredient in the injected sample [counts] 

Constants 

The concentration of the active ingredient in the test samples was calculated using the following 

equation: 

X 

c =--·F 

R/100 

where c: 

x: 

F: 

R: 

(2) 

concentration of the active ingredient in the test sample [mg ai/L] 

concentration of the active ingredient in the injected sample solution [mg 

ai!L] 

sample preparation factor 

recovery rate [%] (If not correcting for recovery then R is taken as 1 00) 

The recovery rate (R) of the spiked recovery samples was calculated using the following 

equation: 

R =-c-·100 

Crort 

where R: 

c: 

(3) 

recovery rate [%] 

determined concentration of the active ingredient in the spiked recovery 

sample [ mg aiJL] 

fortified concentration of the active ingredient in the spiked recovery 

sample [ mg ai/L] 
Harlan Study Number: 41402586 Report Page 31 

Analytical Investigations 

The percentage of nominal concentration of the active ingredient in the test samples was 

calculated using the following equation: 

%nominal = _c_ ·1 00 

cnom 

(4) 

where 

Cnom: nominal concentration in the test sample [mg ai!L] 
Harlan Study Number: 41402586 Report Page 32 

Analytical Investigations 

2 RESULTS 

2.1 Range-Finding Test 

The analytical methodology was not validated prior to the analysis of range-finding samples; full 

validation was later conducted specifically at the test concentration employed in the definitive 

test. The results for the range-finding test samples are summarized in Table 3. 

2.2 Definitive Test 

2.2.1 Validation of the Analytical Method 

Specificity 

The biological control samples and an analyzed analytical blank showed no significant 

interfering response at the retention time of the active ingredient. The standard solutions 

contained a peak specific for the active ingredient whose area changed accordingly with known 

concentration, hence the specificity of the method by retention time was confirmed. 

Linearity 

The calibration data for the calibration standards of the active ingredient is given in Table 2 and 

Figure 1. The data was found to have a quadratic correlation within the calibration range of 

0 lo 1.6 mg ai/L. The R2 fit of the calibration curve to the data was 0.9995, and was considered 

to be acceptable. 

Accuracy (Recovery) and Precision 

A set of recovery samples accurately fortified at a relevant concentration of active ingredient was 

prepared five-fold and analyzed. The results obtained for the concentration of the active 

ingredient in the recovery samples are presented in Table 4. 

The method was considered to be sufficiently accurate and precise for the purposes of this test. 

The test sample results were not corrected for recovery. 

Limit of Quantification (LOQ) 

The limit of quantification (LOQ) was determined by calculating the sample concentration that 

gave a peak equivalent to ten times the baseline noise. Using this method the limit of 

quantification was determined to be 0. 14 mg ai!L. 
Harlan Study Number: 41402586 Report Page 33 

Analytical lnvestigations 

2.2.2 Test Samples 

The results obtained for the concentration of the active ingredient found in the test samples are 

presented in Table 5. 

Typical chromatograms are shown in Figures 2 to 4. 
Harlan Study Number: 41402586 Report Page 34 

Analytical Investigations 

3 DISCUSSION 

The detection system was found to have acceptable correlation to the fit of a quadratic equation. 

The analytical procedure was found to have acceptable recoveries of active ingredient in test 

medium. The method of analysis was validated and proven to be suitable for use. 
Harlan Study Number: 41402586 Report Page 35 

Analytical Investigations 

TABLES 

Table 1 Preparation of Test Samples 

Sample Sample Final Nominal 

Nominal Volume 

Final Volume Preparation Concentration of 

Concentration Factor Active Ingredient 

[mg ai!L] [mL] [mL] F [mg ai/L) 

Control 2 20 10 -

100 2 

20 

then 2 ~ 20 

100 1.0 

not applicable 

Table 2 Calibration Standard Data 

Concentration Mean Area 

[mg ai/L) [counts] 

0 0 

0.109 3.140 X 103 

0.263 7. 11 5 X 103 

0.789 2.056 X 104 

1.09 2.712 X 104 

1.58 3.975 X 104 
Harlan Study Number: 41402586 Report Page 36 

Analytical Investigations 

Table 3 Results for Range-Finding Samples 

Measured Sample Determined 

Concentration Concentration 

Nominal of Active Preparation of Active 

Concentration Ingredient in Factor Ingredient in %of Nominal Time Point Sample Vial Range-Finding Concentration 

Cnom Sample 

F 

X c 

[hours) [mg ai/L] [mgai/L) fmg ailL) [%) 

0 100 0.933 100 93.3 93 

48 100 0.867 100 86.7 87 

Table 4 Results for Spiked Recovery Samples 

Nominal Fortified Measured Sample Determined Mean 

Concentration Concentration Concentration Concentration Analytical 

of of Active of Active Preparation of Active Recovery Precision 

Active Ingredient in Ingredient in Factor Ingredient in Rate (Relative 

Standard Ingredient the Spiked the Sample the Spiked Deviation of Sample Vial Sample Recovery) 

Cnom Crort X F c R 

[mg ailL) [mg ailL) [mg ailL) [mg ailL] [%] [%J 

0.916 100 91.6 

0.985 100 98.5 

100 104 0.932 100 93.2 90 2.8 

0.941 100 94.1 

0.934 100 93.4 

Acceptance Target 80-120 <10 

For reporting purposes, x has been calculated from c retrospectively. 
Harlan Study Number: 41402586 Report 

Analytical Investigations 

Table 5 Results for Test Samples 

Measured Sample Concentration 

Nominal of Active Preparation 

Time Point 

(hours) 

0 

48 

LOQ 

Concentration Ingredient in 

Sample Vial 

Cnom 

X 

[mg ai!L] [mg ai!L] 

Control <LOQ 

100 0.864 

Control <LOQ 

100 0.800 

Limit of Quantification 

not applicable 

For reporting purposes, x has been calculated from c retrospectively. 

Factor 

F 

10 

100 

10 

100 

Page 37 

Determined 

Concentration 

of Active 

Ingredient in %of Nominal 

Test Sample Concentration 

c 

[mg ai!L) [%] 

<LOQ -

86.4 86 

<LOQ - 80.0 80 
Harlan Study Number: 41402586 Report 

Analytical Investigations 

FIGURES 

Figure 1 Calibration Plot 

...... 

J!! 

·o; 45000 ;:::) 

....... 

c: 

e 40000 < 

.... c 

cf 35000 

30000 

25000 

20000 

15000 

10000 

5000 

0 

0.0 

y = -46 1.50 lx~+ 25,6563572x+ 14 1.0804 

R' '" 0.9995 

0.2 0.4 0.6 o.s 1.0 

Page 38 

• 

l.1 1.4 1.6 l.S 

Con= tratiOD(mga£1.) 
Harlan Study Number: 41402586 Report Page 39 

Analytical Investigations 

Figure 2 Chromatogram of Calibration Solution (1.0 mg ai/L) 

MS01TIC, MS Ae(S:\ANASERVS\41402586\ECAQl11'€CI'0011-40'019-1901 .0) ES.AA. SIM, Frag: 100 

a 

4000 

3500 

3000 

2500 

2000 

1500 

1000 

500 

0.2 0.4 0.6 0.8 1.2 1.4 1.6 1.8 min 

Figure 3 Chromatogram of Biological Control Sample • 

MSD111C. MS Ae(S:IANASERV$14140?!ii!61ECACXl11'EC.AQ011·40'00s.o602.D) ES.AA, SIM. Frag: 100 

3000 

2500 

2000 

1500 

1000 

500 +-~==~--------------------------------------------------------------------

0.2 0.4 0.6 0.8 1.2 1.4 1.6 1.8 min 

• Less than the LOQ 
Harlan Study Number: 41402586 Report 

Analytical Investigations 

Figure 4 

3500 

2500 

:2000 

1500 

1000 

Chromatogram of Test Sample (100 mg ai!L) 

MSD1 TlC, MS Fie (S:IANASERVS>41402586\ECAal11'€CJl0011· 40'017-1701.0) ES-API, SIM, Frag: 100 

8 ~,., 

-~ 

Page40 

500 ~--------~~====~--------------------------------------------

0.2 0.4 0.6 0.8 1.2 1.6 1.8 min 
Harlan Study Number: 41402586 Report 

Appendix 6 Monitoring Authority Statement of GLP Compliance 

i nH) Department 

\:..: of Health 

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT 

OF THE UNITED KINGDOM 

GOOD LABORATORY PRACTICE 

STATEMENT OF COMPLIANCE 

IN ACCORDANCE WITH DIRECTIVE 2004/9/EC 

TEST FACILITY 

Harlan Laboratories Limited 

Shardlow Business Park 

London Road 

Shard low 

Derbyshire 

OE722GO 

DATE OF INSPECTION 

TEST TYPE(S) 

Analytical/Clinical Chemistry 

Environmental Fate 

Environmental Toxicity 

Phys.Chem. Testing 

Mutagenicity 

Toxicology 

12 to 14 March 2014 

An inspection for compliance with the Principles of Good Laboratory Practice was 

carried out at the above test facility as part of the UK Good Laboratory Practice 

Compliance Monitoring Programme. 

This statement confirms that, on the date of issue, the UK Good Laboratory 

Practice Monitoring Authority were sat1sfied that the above test facility was 

operating in compliance with the OECD Principles of Good Laboratory Practice. 

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

UK Good Laboratory Practice Regulations 1999) 

~~c--'b-

~;:l,, It¥ 

Or

Head, 

. Andrew 

UK GLP 

J. Gray 

Monitoring Authority 

~ 

~ 

Page41 
This study was submitted to the U.S. Environmental Protection Agency (EPA) by 

Harcros Chemicals as pan of a TSCA Section 4 test mle. Submittal of the study to 

EPA does not authorize any entity to cite to or refer to the sllldy without express 

authorization from Harcros Chemicals. Harcros Chemicals retains data compensation 

rights under all statutory and regulatory programs, including, but not limited to, EU 

REACH and Korea-REACH. Possession of a copy of the study, in whole or in part, 

does not constitute "legitimate possession" of the study and does not create any rights 

to use the study or the data presented herein for any chemical registration (such as, 

but not limited to, the REACH registration program) or any other commercial 

purpose. In addition, Harcros Chemicals expressly retains all rights for first 

publication of the data and the information presented herein. In the event anyone 

seeks the right to cite or refer to the data, please contact Harcros Chemicals.