## 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 of41Harlan Study Number: 41402586 Report Page2 

## CONTENT S CONTENT S . . ....................... . ........ .... ......................................................................... .. ................ . .. 2 STUDY DIRECTOR STATEMENT OF GLP COMPLIANCE ................................ . .................. .4 

### QUALI TY ASSURANCESTATEMENT ...................................................................................... 5 S IGNATURE OF CONTRIBUTING SCffiNTISTS ...................................................................... 6 SUMMARY .................................................................................................................................... 7 Introduction ............................................................................................................................ 7 Methods ............................................................................................................................... 7 Results ................. . ............ ... ......................................... ... ..................................................... 7 GENERAL INFORMATION ..... .... ....................................... ..... .................................................... 8 Schedu le .............................. . ........ . .................. ...... ..... ......... ...... .. . . . . .................. . . ... . ... ............ 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 ................ . . . ....... .. . . ................................ .. .... . .......... 1 1 3.2 Test System .......................................... .... . . ...... ...... ............................... ... ... .. ......... 1 1 3.3 Test Water ........................................... ..... . . ..... ....... ............................. ..... .. ... .. ... .... 1 1 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 Co ncentrations ........................................................ 15 4.2.2 Immobilization Data ................................................................ . .... .. ... . ..... l5 4.2.3 Validation Criteria ................................................................. . . .. ... . .... .. .... 16 4.2.4 Water Quality Criteria ........................................................... . ..... . ..... . ..... 1 6 4.2.5 Observations on Test Item Solubility ...................................................... 16 5 CONCLUS ION .................................................................................................................... 17 6 REFERENCES .................................................................................................................... 18 TABLES . . ................................................................................ . .. .. ................................................. 19 FIGURES . .......................................................................... . ... ........ .................... . .......................... 22 APPENDICES . ... . ................................................................... .......... ......................................... . ... 23Harlan Study Number: 41402586 Report Page 3 

## LIST OF TABLES Table I Tab le 2 Tab le 3 Cumulative Immobilization Data in the Range-finding Test.. .................................... 1 9 Cumulative Immobilization Data in the Definitive Test.. ........................................... 20 Water Quality Measurements ......................................................................... . .... ... .... 21 

## LIST OF FIGURES Figur e 1 Testo Temperature Logger Data .............................................. ........ .... ....... . .... .. ... .. .... 22 

## LIST OF APPENDICES Append ix 1 Append ix 2 Appendix 3 Appendix 4 Appendix 5 Appendix 6 Certificate of Analysis ........................................................................................... 23 Reconstituted Water
- E l endt M7 Medium ....................... ........... ... ...... . .... .. .... .. ... 24 Positive Control ................ .. .............. . ................................. ........ ...... .... .. .... .. .... ... .. 25 Reconstituted Water- ISO Medium ............................................... ..... .. ... ... ... .. .... 26 Analyt i cal Investigations ................................................................... ... .. ... ... ... .... .. 27 Monitoring Authority Statement of GLP Compliance ............................ .. ........... .4 1Harlan Study Number : 4 1402586 Report Page4 

## STUDY DIRECTOR STATEME NT OF GLP COMPLIANCE Harlan Laboratories Ltd., Shardlow Business Park, Shardlow, D erbysh ire, DE72 2GD, UK Harlan Study Number: 4 1402586 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 (S I 1999/3106 as amended by SI 2004/0994)). These Regulations are in 

### accordance with GLP standards published as OECD Principles o n Good Laboratory Practice (revi sed 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- l 5Harlan Study Number: 4 1402586 Report Page 5 

## QUALITY ASSURANCE STATEMENT H arl an Study N umber: 41 4 02586 Study Title: So dium Glucoheptonate CAS No. 31138-65-5: Daphnia sp., 48-Hour Acute Immob i lization Test T he general facilities and activities are inspected at least once a year and the results are reported to the relevant responsible person and management. Study - re lated procedures co nducted at the test facility were audited and inspected. T he details of these aud its and in spections are given below. R eported to th e D ates and Types of QA Ins pections re le vant Study D i rector and Tes t Facility Management Date of Inspection Type of Inspection Phase Inspected Report Date 22 J uly 2014 Study Plan 

### NIA 22 Ju ly 2014 Verificati o n 13 January 2015 Process
- based Test Item Preparation 13 January 2 01 5 13 J anuary 2015 Process
- based Test System 13 January 2015 Preparation and Exposure 15 January 20 1 5 P rocess - based Assessment of 15 January 2015 Response 30 January 2015 Pr ocess - B ased 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, Derb yshire, D E72 2GD , UK


# • • ••• •••••••• Date: 

## 2 8

**M~~ ·**  

## Z0 15Harlan Study N urn ber: 414025 86 Report Page 7


## SUMMAR Y In t roducti o n 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 Guide l ines 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) . 

### M ethods 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. 

### Res ult s 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 INTROD U CTION AND PURPO SE This report contains a description of the methods used and r es ult s 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 G uidelines I 

### Re g ula tion s This study was designed to be compatible with the procedures indicated by the follow ing 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 Guidel ine 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 -6 5-5 Amber colored liquid T-461 39.94% Not supplied Room temperature in the dark Not suppliedHarlan 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% immobili zation 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 contro l (test water only).

The test was carried out using 1st instar 

### Daphnia magna derived from in-house laboratory cultures. Adult daphnia were maintained in 1 50 mL g lass beakers containing Elendt M7 medium (see Appendix 2) in a temperature controlled room at approximately 20 °C. The li ghting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each c ulture 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 inte grity 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 20 1 5 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 CaC0 3 

### and an alkalinity of 27.6 mg/L as CaC0 3. 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 d eviation to the study plan, given that no adverse effects were observed in t h

e test, this deviation was co nsidered no t to have had an effec t on the outcome of the test. 

### 3 . 4 Proced u re 3.4 . 1 Ran ge-finding T es t 

### The test concentration to be used in the definitive test was determined by a prel i minary range fmding test. 

### In the range-find ing 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 Spo nsor, all test concentrations were corrected for a test item purity of39.94%. A nominal amount oftest item (250.4 mg) was dissolved in test water and the volume adjusted to l 

### li t er 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 . Eac h prepared co ncent ration was inverted several ti mes to ensur e 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 perio ds. 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 condit ion s but not exposed t o 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 concentrat i ons within the range to be used for the definitive test wer e analyzed. 3.4.2 Definitive Tes t 

### B ased on the resu lts of the range-fin ding test a "Limit test" was conducted at a concentr ati on of 1 00 mg ai/L to confinn that no immobi lization 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 contro l 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 wer e 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 Eva luation s 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 Cr i teria Water temperature was recor ded 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 Verifica tion of Test Concentrations Sam ples were taken fTom the contr o l and the 100 mg ai /L test group from the bulk t est prep aration at 0 ho u rs an d from the pooled repl icates at 4 8 hours for qu anti tative analys is. All samp les were stor ed frozen prior to analysis. Dup licate samples were take n and stored frozen for further analysis if necessary. The 0 -Hour samples were not stored frozen for the initial2 days after samp ling due to a technical error, however, given that near nominal concentrations were ac hi eved , this was considered not to have had an impact o n the outcome of the test. The met hod of analys is, recover y and test preparation analyses are desc ribed in Appendix 5. 

### 3 . 4.4 Statistical Anal y sis An estima t e of the EC 5

### o values was given by inspection of the immobil ization data. 

### 3.5 Va li datio n Criteria The res ul ts of th e test are co nsi de r ed va lid if the following performance c ri teria 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 disso lved 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 g iven in Table 1. No s ignificant immobilization was observed at the test concentrations of 0.1 0, 1.0, 10 and 

### I00 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 i nformation, 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 te st item durin g the definitive test are g iven 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 immobi lization 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 Cr iteria The results of the water quality measurements are given in Table 3. T emperature was maintained at 2 1 °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 Solubili ty Throughout the duration of the test, the test preparations were observed to be clear colorless solutions.Harlan Study Number: 41402586 Report Page 17 

## 5 CONCL US IO N The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC 5

### o value of greater than 100 mg ai/L. The No Observed Effect Concentration was 100 mg a i /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 To tal Hardness. In: Field and On-site Test Methods for the Analysis ofWaters , (BS 1427), pp 35-36.Harlan Study Number: 41402586 Report Page 19 

## TABLES Tablet Cumul ative Immobilizatio n Data in the Range-finding Test
No minal C umulat ive Immo bili ze d D a phni a Con ce n t r a ti o n { I nitia l Pop ulation: 10 Pe r R ep li ca te) ( mg a i!L ) 24 H o urs 48 H o urs 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% i mmobilization was observed.Harlan Study Number: 41402586 Report Page 20



### Table 2 Cumulative Immobilization D a t a in th e Defmitive Test C umul ativ e Immobilized D a phni a Nomi n a l (Initia l Population: 5 Per Replicate) Conce ntration 24 Hours 48 Hours (mg ai!L) No. Per No. P er Replicate Tota l % Replicate Total % Control R , 0 0 R2 0 0 0 0 0 0 R

3 0 0

**~** 0 0 100 R, 0 0 R2 0 0 0 0 0 0 RJ 0 0

**~** 0 0

R 1

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



### Tab le 3 W a t er Q u ality Measureme n ts
Nomin a l 0 Ho urs 24 Ho u rs 4 8 Hou rs Co n ce ntr ation

( mg a i /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 2 1 R

2 7 . 6 9.0 21 21 7 . 6 9 . 0 21 Control R 3 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 R

2 8.2 9.0 21 21 7.6 8.5 21 100 R l 8.3 9 . 0 21 21 7 . 5 8.4 21

**~** 8.2 9 . 1 21 21 7 . 5 8.4 21

R 1-

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

## FIGURES Figure 1 Testo Temperature Logger Data

20. 8

20.7

20.6 

## g 20. 5 

# i i 20.4I20. 3

20.2 Page 22

**20 1~**  **~--~~--~~~~--~~--~~--~~--~~--~--~~--~~--~~--~~~** 0 2 4 6 8 10 12 1 4 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 C ertific ate of Analysi s 

## Harcros Chemicals, Inc. Dalton Location 3452 Corporat e Drive Dalton , GA 

## 30720 Certificate of Analy s is Mother Liquor CAS# 31138 .. 65-5 DaLe: 8!2612014 

## ••• •••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••••• •

Beta Sodium Gluc-ahcptonate CAS#- 30090-50..3 Alpha Sodium Gl ucohep lonatc CAS# 13007-85-7 Polysaccharide or Degradatio n ProductS Cyanide Ammonia CASfl 7664-41-7 pH l % Water% Specific Gravity %Solids % De xtro se 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 1 062 9-11 TM 1079 35 -5 0 TM 1075 1.272 1.300 TM 10 79 35 

## - so TM 13 35 0
- trnce Ie<>t Re su lts 2j,78%
14.16 %
10.96 % 0 . 3 ppm I 113 ppm 9.1 49.1 1.293 50.9% 0/ none detected Page 23Harlan Study Number: 41402586 Report 

### Appendix 2 Recon stituted Water - Elendt M 7 Medium

Ing redient H3 B 03 MnChAH2 0 LiC I R bC I SrCh.6H20 NaBr Na2Mo04.2H20 CuCl2.2H20 ZnCh CoCh.6H20 

### KI Na2Se03 NH4V03 Na2EDTA.2 H20 FeS04.1H20 CaCh.2H20 NaHC03 MgS04.1H20 Na2Si03.9H20 KCl NaN03 

### K2HP04 KH2 P04 Th iamine hydrochloride Cyanocobalamine (vitamin B 12 ) 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 24Harlan Study Number: 41402586 Report Page 25
Appendix 3 Positive Contro l A positive control (Har l an 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 probit analysis using linear maximum likelihood regression based on the nominal test concentrations gave the following results:
No Obs erved Lowest Observed Ti me Po i nt ECso 95% Confide n ce Li mi ts Effect Effect

(Hours) (mg!L) (mg!L) Co n ce n tration Co nce n tratio n (N OE C) (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 Effec t Concentration is based upon less than I 0% immobilization at thi s concentratio n. The results from the positive control with potassium dichromate were within the normal range for this reference item.

*Due to the nature ofthe data, 95% confidence limits could not be determined. ** TOXRATPROFESS10NAL VERSION2.10.06. TOXRAT®SOLUTIONS GMBH 2001-2010Harlan Study Number: 41402586 Report
Appendix 4 Reconstituted Water - ISO Medium

Ingredient CaCh.2H20 MgS04.1H20 NaHC 0 3 KCI Fina l 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 dis so lved oxygen concentration was approximately air-saturation value . The reconstituted water had an approximate theoretical total hardness of250 mg/L as CaC0 3 .Harlan Study Number: 41402586 Report Page 27 

### App endix 5 Anal y tical Inves ti gations 

## 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 externa l standard. 

### All test sample concentrations were corrected for a purity content of 39.94%, and are r eported as mill ig r ams of active ingredient (ai) per litre . The method was deve l oped by the D epartment of Analytical Services, H arl an Laboratories Ltd , Shardlow, UK. 

### 1.2 Test Item The test item is described in the biological part of this study. 

### 1.3 Anal y tical Standard T he test item described in the biological part of this study was also used as the analytical standard. 

### 1.4 A na lytical Procedure 1.4.1 Storage The samples were stored frozen prior to analysis. 

### 1.4.2 Rea gents a nd Solvent s 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. So l vents: methano l , Romil, Ultra gradient grade. Reagents: ammonia, Fisher Scientific analytical reagent grade.Harlan Study Number: 41402586 Report Page28 Analytical L nvestigations 

### 1 . 4 .3 Pr ep a r ation of Calibra tion S tand a rd s The test item (nominal 250 mg) was dissolved in diluent" (1 00 mL) to prepare a stock so lution with a concentration of l 000 mg ai/L. This stock solution was further diluted with diluent• to produce a so lution 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 tes t item ( nominal 250 m g) was d isso l ved in diluent• (1 00 mL) to prepare a stock so lutio n with a concentration of 1000 mg ai/L. This stock solution was furthe r di l uted with di luent* 

### to pr

oduce a solution of l 0 mg ai/L. Defined vo lumes of this solut i on were then di l uted with diluent" to obtain calibrat ion 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 Pr eparation of Spiked Recovery S ampl es 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 initial ly disso lved 

### 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 sam ples . In addition, test medium w ithout the addition of the active ingred ient (synthetic contro l) was also analyzed. 

### 1.4.6 Preparation of Test Samples The test samples were thawed with the ai d of sonication. They were di l uted with test medium to a nominal concentration of 10 m g ai/L and then further diluted into th e calibration range w i th methanol: water: ammoni a (50:50:0.1 0 v/v/v) as shown in Tab l e 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: Mobi l e phase: Flow rate: Injection volume: Ionization mode: Polarity : Capillary vo ltage: 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 

### I OOV 12 Llmin Single Ion Monitoring (SIM) 225.0 m/z Page 29Harlan Study Number: 41402586 Report Page 30 Analytical Investigations 

### 1.4.8 Calculations A calibration curve was prepared using a quadratic funct ion 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+~b **2** -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 sample s was calcu l ated 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] samp le 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 equat ion: 

### 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 P age 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 cn om (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 ofrange-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 concentratio n 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 equiva lent to ten times the baseline noise . Using this method the limit of quantifi cation was determined to be 0. 14 mg ai!L .Harlan Study Number: 41402586 Report Page 33 Analytical lnvestigations 

### 2.2.2 T est Sa mp les 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 In vestigations 

## TAB LES Table 1 Preparation of Test Sam p les Sample Sample Fi n a l No min al No min al Vo lum e F inal Volu me Prepa r ation C on cen tration of Co n ce n tration 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 

### Tab le 2 Ca libration Standard Data Concentration Mean Area [mg ai/L) 

### [co un ts] 0 0 0.109 3.140 X 103 0.263 7. 11 5 X 103 0 .789 2.056 X 10 4

1. 09 2.712 X 1 04 1. 58 3 .9 7 5 X 104Harlan Study Number: 41402586 Report Page 36 Analytical Investigations 

### Table 3 Results for Range-Finding Samples Measured Sample Determined Concentrat ion Concentration Nomi nal of Active Preparation of Active Concentration Ingredient in Factor Ingredient in %of Nomina l Time Point Samp le Vial Range-Finding Concentration Cnom Samp le 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 Activ e of Active Preparation of Active Recovery Precision Active Ingredient in Ingredient in Factor Ingredi ent in Rate (Relative Standard Ingredient the Spiked the Sample the Sp iked Deviation of Sample Vial Sample Recov e ry) Cn om 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 r etrospectively.Harlan Study Number: 41402586 Report Analytical Investigations 

### Tab le 5 Resu lts for Test Samples Mea s ured Samp le Concentration Nom inal of Active Preparation Time Point

(hours) 0 48
LOQ Co n centration Ingredient in Sample Vial Cnom X [m g ai!L] [mg ai!L] Control < LOQ 100 0.86 4 Co ntro l < LOQ 100 0.800 Limit ofQuantification not applicable For reporting purpo se s, x has bee n calculated from c retro spectiv el y. Factor
F 10 100 10 100 Page 37

Determined Concentration of Active In gredie nt in %of Nominal Test Sa mpl e Concen tration

c [m g ai!L) [%]

< LOQ 

### -

86.4 86 < LOQ 

## -

80.0 80H arl an S tudy Numbe r : 414025 86 Report Analytical Investigations 

## FIGURES Figure 1 Calibration Plot

...... J!! ·o; 4 5000 ;:::)
....... c: e 40000 <


### .... c cf 35000 30000 25000 20000 15000 10000 5000 0 0.0 y =

**-46 1.50**  **lx~+** 25,6563572x + 1 4 1.0804 R' '" 0.9995

0.2 0.4 0.6 

### o. s 1.0 Page 3 8



### •

l.1 1.4 1. 6 l.S Con = tratiOD( m ga£1.)Harlan Study Number: 41402586 Report P age 39 Analytical Investigations 

### Figure 2 Chromatogram of Calibration Solu tion (1.0 mg ai/L) MS01TIC , MS Ae(S :\ANASERVS\41402586\ECAQl11'€CI'0011-40'019-1901 . 0) ES.AA. S I M

, 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 ofBiological 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 LOQHarlan Study Number: 41402586 Report Analytical Investigation s Figure 4

3500

2500
:2000
1 500

1000 Chromatogram of Test Sample (100 mg 

### ai!L) MSD1 TlC , MS Fie (S :IANASERVS>41402586\ECAal11'€CJl0011 · 40'017
- 170 1.0 ) ES-API, SIM, Frag: 100 8

**~,.,**

**-~** Page40

**500 ~--------~~**  **== ==**  **~-- --------------------------------**  **-- ----**  **----** 0 . 2 0.4 0.6 0 .8 1 . 2 1 . 6 1.8 minHarlan Study Number: 41402586 Report



### A pp endix 6 M onitorin g Authori ty Sta tem ent of GL P Co mpli a n ce 

# i 

# n H) 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 Env ironmental Fate Environmental Toxicity Phys .Chem. Testing Mutagenicity

Toxicology

12 to 14 March 2014 An inspection for compl iance with the Principles of Good Laboratory Practice was carr i ed 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 i n compliance wi th 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

. Andrew J . Gray



## ~ Head, UK GLP Mon itori ng Authority



## ~ Page41This st udy was s ubmitted to th e U.S. Environmental P rotectio n Agency (EPA) by Harcro s Chemicals as pan of a TSCA Sectio n 4 test mle. S ub m itt al of the st udy to E PA does not auth orize any entity t o c ite to or refer to the s llldy without express authorizat io n from Harcros C h emicals. Harcros Chemicals retains data compen satio n right s und er all statutory and reg ulatory prog ram s, including , but not limi ted to , EU REA CH and Korea-REAC H . Possess ion o f a copy of the study , in who le or in part , does not cons titute " l egi ti mate po ssess io n " of the study and does no t crea te any ri g hts to u se the study o r th e data p r esented her e in for any c he mi c al registra ti on (s uc h as , but not limite d to, the REAC H reg i stratio n program) or any oth er co mmercial

purpose. I n addition , Harcros C he mi cals expressly retain s all rights fo r first publicatio n of th e data and th e in for mati o n prese nted herein . I n th e eve nt an yone seek s the right to cite or refer to the data, please con tact Harcros C he mi ca l s .