p . 1



### Harcro s Chemical s Inc . 520 0 Speake rRoa

d Kansa s City , K S 6610 6 

## RECEIVE D OPP T CBI C 

# 2 Telephone : 913/621-774

7 Fax : 913/621-774

6 E-mail : OrganicsCS@Harcros.co m 

## 2M

3 JU N IU AMIO-t

* CBI C Contro l Numbe r Dat e 06/12/1 3 

# 35471 9 Documen t Contro l Offic e (DCO ) (7407M ) Offic e o f Pollutio n Contro l an d Toxic s (OPPT ) U.S
. Environmenta l Protectio n Agenc y (EPA ) 120 0 Pennsylvani a Ave
. N W Washington , D.C
. 2046 0 (202-564-8930 ) Attn : TSC A Sectio n 4 4 0 CF R § 799.508 7 (Docke t # EPA-HQ-OPPT-2007-0531 ) RE : Daphnia , 4 8 hou r Acut e Immobilizatio n Tes t CASR N 31138-65- 5 T o Who m I t Ma

y Concern : I n accordanc e wit h EPA' s fina l rule , Testin g o f Certai n Hig h Productio n Volum e Chemicals ; Secon d Grou p o f Chemicals , publishe d i n th e Federa l Registe ro n Januar y 7,201 1 at 7 6 fed
. Reg
. 1067 , pleas e fin d enclose d th e fina l repor t fo rth e Daphnia , 4 8 hou rAcut e Immobilizatio n Tes t o n D-gluco Heptoni c acid , monosodiu m salt , (2.xi.) - [CAS # 31138-65-5)
. Thi s stud y wa s conducte d t o fulfil l th e Acut e Toxicit y t o Daphni a requirement s o f testin g categor y C a s se t fort h i n Tabl e 2 o f 4 0 CF R 799.5087(j)
. I f an y furthe rinformatio n i s required , pleas e d o no t hesitat e t o contact m e a t (913 ) 621-780 2 o rat asavini@harcros.com
.

An n Mari e Savin i Harcro s Chemical s Inc
. Regulator y Specialis t cc : Pete rRadford Jame s Grantha m Rand y Buckne r Joh n Mile s Fil e Regards ,

## REPOR T 

# harla n

Sodiu m Glucoheptonate : Daphni a sp. , 48-Hou

r Acut e Immobilizatio

n Tes t
Dat a Requirements/Tes

tGuidelines : • OEC D Guideline

s fo r Testin g o f Chemical s (Apri l 2004

) N o 202 , "Daphni asp. , Acut e Immobilisatio

n Test " • Metho d C. 2 o f Commissio

n Regulatio

n (EC ) No . 440/200

8 • U S EP A Draf t Ecologica l Effect s Tes t Guidelin e OPPT S 850.101 0 "Aquati

c Invertebrat e Acut e Toxicit y Test , Freshwate

r Daphnids " Stud y Director : S Harri s Tes tFacility : Harla n Laboratorie

s Ltd . Shardlo w Business Par

k Shardlo w Derbyshir e DE7 2 2G D UK Sponsor : Harcro s Chemical

s Inc . 3452 Corporat

e Driv e DaltonG A 3072 0 UNITE D STATE S O FAMERIC

A Harla n Stud y Number: 4110469 5 Stud y Completio

n Date : 1 Ma y 201 3

Pag e 1 o f 4 1P . 3 Harla n Stud y Number : 4110469 5 Repor

t Pag e 2 

## CONTENT S CONTENT S 2 STUD Y DIRECTO RSTATEMEN T O F GL P COMPLIANC E 4 QUALIT Y ASSURANC E STATEMEN T 5 SIGNATUR

E OF CONTRIBUTIN G SCIENTIST S 6 SUMMAR Y 7 Introductio n 7 Method s 7 Results 7 GENERAL INFORMATIO N 8 Schedul e 8 Archivin g 8 1 INTRODUCTIO N AN D PURPOS E 9 1. 1 Guideline s /

Regulation s 9 2 TES T ITE M 9 3 MATERIAL S AN D METHOD S 10 3. 1 Definition s an

d Abbreviation s 1 0 3. 2 Tes t

Specie s 10 3. 3 Tes t

Water . 1 0 3. 4 Procedur

e 1 1 3.4. 1 Range-findin g Test 1 1 3.4. 2 Definitiv e Test

1 1 3.4. 3 Post-Stud

y Experimen

t 1 3 4 RESULT S 1 5 4. 1 Range-findin g Tes t 1 5 4. 2 Definitiv e Tes t

1 5 4.2. 1 Immobilizatio n Dat a1 5 4.2. 2 Observation s o n Test Ite mSolubility 1 5 4.2. 3 Physico-Chemica l Measurement

s 1 6 4.2. 4 Verificatio n o f

Test Concentration s 1 6 4. 3 Post-Stud

y Experimen

t 1 6 5 CONCLUSIO N 1 6 TABLE S 1 7 Tabl e 1 Cumulativ e Immobilizatio n Dat ai n th e Range-findin g Tes t

1 7 Table 2 Cumulativ e Immobilizatio n Dat ai n th e Definitiv e Tes t

1 8 Table 3 Cumulativ e Immobilizatio n Dat ai n th e Post-Stud

y Experimen

t 1 9 FIGURES 2 0 Figur e 1 Test

o Temperatur

e Dat a2 0 APPENDICE S 2 1 Appendix 1 Reconstitute d Wate r - Elend t M 7 Mediu m2 1 Appendix 2 Positiv e Contro l 2 3 Appendix 3 Reconstitute d Wate r 2 4 Appendi x 4 Analytica l Investigation s 2 5P- 4 Harla n Stud y Number : 4110469 5 Repor

t Pag e 3
Appendix 5 Physico-Chemica l Measurement

s 4 0 Appendix 6 Monitorin g Authority Statemen t o f GL P Complianc e 4 1p . 5 Harla n Stud y Number : 4110469 5 Repor

t Pag e 4 

## STUD Y DIRECTO RSTATEMEN T O F GL P COMPLIANC E Harla n Laboratorie s Ltd. , Shardlow Busines s Park, Shardlow , Derbyshire , DE7 2 2GD , U K

Th e stud y described wa

s performe d i n complianc e wit h U K GL P standards (Schedul

e 1 , Goo d Laborator y Practic e Regulation s 199 9 (S I 1999/3106 a

s amende d b y S I 2004/0994)) . Thes e Regulation s ar

e i n accordanc

e wit h GL

P standard

s publishe d a

s OEC D Principle s o

n Goo d Laborator y Practic e (revise d 1997 , ENV/MC/CHEM(98)17) . Thes e principle s ar

e compatibl e wit h Good Laborator y Practic e regulation s specifie d b y regulator y authoritie s throughou

t th e Europea n Community , th

e Unite d States (EP

A an d FDA) , an d Japa n (MHLW , MAF F an

d METI) .

Thi s repor

t full y an d accuratel

y reflect s th

e procedure s use

d an d dat agenerated . Ther

e wer e n o circumstance s considered to hav e affecte d th

e integrit y o f th e stud y o r the validity o f

th e data . Stud y Director : S Harri s Harla n Stud y Number : Stud y Title : Sodiu mGlucoheptonate : Daphni asp. , 48-Hou

r Acut e Immobilizatio n Tes

t 4110469 5

Date : 

## 0 1 MA

Y 201 3Harla n Stud y Number : 4110469 5 Repor

t Pag e 5 

## QUALIT Y ASSURANC E STATEMEN T Harla n Laboratorie s Ltd. , Shardlow Busines s Park, Shardlow , Derbyshire , DE7 2 2GD , U K Harla n Stud y Number : Stud y Title : 4110469 5 Sodiu mGlucoheptonate : Daphni asp. , 48-Hou

r Acute Immobilizatio n Tes t Th e genera l facilitie s an

d activitie s ar

e inspecte d a t least onc e ayea r an d th e results ar

e reporte d t o th e relevan t responsibl e perso

n an d management. Study-related procedure s conducte d a t th e tes t facilit y wer

e audited an

d inspected . Th

e detail s o f these audits an

d inspection s ar

e give n below . Date s and Type s o

f Q A Inspection

s Reported t o th e relevan tStud y Directo r an

d Tes t Facility Managemen

t Dat e o f Inspectio n Typ e o f Inspectio n Phas

e Inspected Repor t Dat e 0 2 Augus t 201 1 Genera

l Stud y Pla n Verificatio n N/ A 02 Augus t 201 1 1 0 Januar y 201 3 Proces s - base d Test

ItemPreparation 1 0 Januar y 201 3 1 0 Januar y 201 3 Proces s - base d Test

Syste m Preparation 1 0 Januar y 201 3 1 5 Januar y 201 3 Proces s - base d Exposur

e 1 5 Januar y 201 3 1 0 Januar y 201 3 Proces s - base d Assessmen

t o f Respons e 1 0 Januar y 201 3 1 1 Januar y 201 3 Proces s - base d Chemica

l Analysi s 1 1 Januar

y 201 3 0 8 Apri l 201 3 Repor t Audi t N/ A 0 8 Apri l 201 3 Th e complet

e Stud y Pla n (includin g Stud

y Specifi c Supplement

) wa s verifie dfo r

GL P complianc e a

t repor t audit . Thi s statemen

t confirm s tha

t thi s repor

t reflect s th

e ra w dat aan d th e procedure s followed . Qualit y Assurance : 

## Date : 3 0 AP R201 3Harla n Stud y Number : 4110469 5 Repor

t Pag e 6 

## SIGNATUR

E O F CONTRIBUTIN G SCIENTIST S Analytica l Chemistry : D M Mulle e Harla n Laboratorie s Ltd. , Shardlow Busines s Park, Shardlow , Derbyshire , DE7 2 2GD , U K

Date :P . 8 Harla n Stud y Number : 4110469 5 Repor t Pag e 7 

## SUMMAR Y Introductio n A stud y wa s performe

d t o asses s th e acut e toxicit y o f th e tes t ite mt o Daphniamagna

. Th e metho d followe dwa

s designe d t o b e compatibl

e wit h th e U S EP A Draf t Ecologica l Effect s Tes t Guideline s OPPT S 850.101 0 (Apri l 1996

) an d th e OEC D Guidelines fo r

Testin g o f Chemical s (Apri l 2004

) N o 202 , "Daphni

asp. , Acut

e Immobilisatio

n Test " reference

d a s Metho d C.2 o f Commissio

n Regulatio

n (EC) No . 440/2008 . 

### Method s Followin g apreliminar

y range-findin

g test , twent y daphnid s ( 4 replicates o f

5 animals ) wer e expose d t o a n aqueou s solutio n o f th e tes t ite ma t concentration

s o f 100 , 180 , 320, 56 0 an d 100 0 m g activ e ingredien

t (ai)/ L fo r 4 8 hour s a t atemperatur

e o f 2 0 ° C t o 2 1 ° C unde

r stati c tes t conditions . Th

e numbe r o f immobilize

d Daphni awer e recorde d afte r 2 4 an d 4 8 hours . Result s Th e 48-Hou r ECs o fo r th e tes t ite mt o Daphni amagn abase d o n nomina l tes t concentration

s wa s greate r tha n 100 0 m g ai/L . Th e N o Observe d Effec t Concentratio

n wa s 100 0 m g ai/L . Analysi s o f

th e tes t preparation

s a t 0 an d 4 8 hour s showe d measure d tes t concentration

s t o rang e fro m80 % t o 83 % o f nomina l valu e an d s o th e result s ar e base d o n nomina l tes t concentration

s only .

Thi

s stud y wa s submitte d t o th e U.S . Environmenta

l Protectio n Agenc y (EPA ) b y Harcro s Chemical s a s par t o f aTSC A Sectio n 4 tes t rule . Submitta

l o f th e stud y t o EP A doe s no t authoriz e an y entit y t o cit e t o o r refe r t o th e stud y withou t expres s authorizatio

n fromHarcro

s Chemicals . Harcro

s Chemical s retain s dat acompensatio

n right s unde r al l statutor y an d regulator y programs, including , bu

t no t limite dto , E U REAC H an d Korea-REACH. Possessio n o f acop y o f th e study, in whol

e o r in part , doe s no t constitut e "legitimat

e possession

" o f th e stud y an d doe s no t creat e an y right s t o us e th e stud y o r th e dat apresente d herei n fo r an y chemica l registratio

n (suc h as, bu t no t limite dto , th e REAC H registratio

n program ) o r an y othe r commercia

l purpose. I n addition, Harcro

s Chemical s expressl y retain s al l right s fo r firs t publicatio n o f th e dat aan d th e informatio

n presente d herein . I n th e even t anyon e seek s th e righ t t o cit e o r refe r t o th e data , pleas e contac t Harcro s Chemicals .p . 9 Harla n Stud y Number : 4110469 5 Repor

t Pag e 8 

## GENERA L INFORMATIO N Schedul e Experimenta

l Startin g Date : Experimenta

l Completio n Date : 2 1 Februar y 201 2 1 4 Marc h 201 3 Archivin g Unles s instructe d otherwis e b y th e Sponsor

, al l original dat

aan d th e final repor

t wil l b e retaine d i n th e Harla n Laboratorie s Ltd , Shardlow , U K archive s fo r

five year

s afte r whic h instruction s wil l b e sough t a s to furthe r

retentio n o

r disposal . Furthe

r retentio n o

r retur n o f th e dat awil l b e chargeabl e to th e Sponsor .p . 1 0 Harla n Stud y Number : 4II0469 5 Repor t Pag e 9 

## 1 INTRODUCTIO N AN D PURPOS E 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. Daphni amagn

ai s afreshwate

r invertebrat

e representativ

e o f awid e variet

y o f natura l habitats , an d ca n therefor e b e considered a

s a n importan

t non-targe t organis mi n freshwate

r ecosystems . 1. 1 Guideline s /

Regulation s Thi s stud y wa s designed to b

e compatible wit h th

e procedure s indicate d b y th e followin g internationall y accepte

d guideline s an

d recommendations :

• OECD Guideline s fo r

Testing o f

Chemical s (Apri l 2004 ) N o 20 2 "Daphni asp. , Acut e Immobilisatio n Test " • Metho d C. 2 o f

Commissio n Regulatio n (EC ) No . 440/200 8 • US EP A Draf t Ecologica

l Effect s Tes

t Guidelin e OPPT

S 850.101 0 "Aquati c Invertebrat

e Acut e Toxicit y Test, Freshwate r Daphnids "

Th e integrit y o f th e supplie d dat

arelatin g to th

e identity , purity an

d stability o f

th e test ite mi s th e responsibilit

y o f th e Sponsor . 

## 2 

## TES T ITE M

Identification : Description : Sodiu mGlucoheptonat

e dar k brow n liqui d Batch : Purity : Expir y / retest date : Storag e conditions : 921000100 49.5 % w/w 9 Februar y 201 5 roo mtemperatur

e i n th e dar kp . 1 1



### Harla n Stud y Number : 4110469 5 Repor

t Pag e 1 0 

## 3 MATERIAL S AN D METHOD S 3. 1 Definition s an d Abbreviation s Immobilization : Thos e organism s whic h ar e no t abl e t o swi mwithi n 1

5 second s afte r gentl e agitatio n o f th e tes t containe r ar e considere

d t o b e immobile . EC50 : Th e calculate d concentratio

n o f tes t ite mwhic h result s i n 50 % immobilizatio

n within th e exposur e period . NOE C (N o Observe d Effec t Th e highes t tes t concentratio

n o r loadin g rat e a t whic h n o Concentration) : significan t toxic effect

o n th e tes t organism s i s observe

d within th e exposur e period . Treatment: Comprise s tes t ite mtreatments , contro

l (tes t wate r only ) an d solven t contro l (i f applicable) . 3. 2 Tes tSpecie s Th e tes t wa s carrie d ou t usin g 1s t insta r Daphni amagn aderive d fromin-hous

e laborator

y cultures . Adul t Daphniawer

e maintaine

d i n 15 0 m L glass beaker

s containin

g Elend t M7 mediu

m(se e Appendi x 1 ) i n atemperatur

e controlle

d roo ma t approximatel

y 2 0 °C . Th e lighting cycl

e wa s controlle d t o giv e a1 6 hour

s ligh t an d 8 hour s darknes s cycl e wit h 2 0 minut e daw n an d dus k transitio n periods . Eac

h cultur e wa s fe d dail y wit h amixtur e o f alga l suspensio

n (Desmodesmu

s subspicatus

) an d Tetramin ® flak e foo d suspension

. Cultur e conditions ensure

d tha t reproductio

n wa s b y parthenogenesis . Gravi

d adult s wer e isolate d th e da y befor e initiatio n o f

th e test , suc h tha t th e youn g daphnid s produce d overnigh

t wer e less tha n 2 4 hour s old . Thes

e youn g wer e remove d fromth e cultures an

d use d fo r testing . Th

e die t an d diluen t wate r ar e considere

d no t t o contai n an y contaminan

t tha t woul d affect th e integrit y o r outcom e o f th e study . A positiv e control (Harla

n Stud y Number: 41203341 ) use d potassiu mdichromat

e a s th e referenc e item . Detail s o f

th e positiv e contro l ar e give n i n Appendi

x 2 . Th e positiv e contro l wa s conducte d betwee n 6 Jun e 201 2 an d 8 Jun e 2012 . 3. 3 Tes tWate r Fo r bot h th e rang e findin g an

d definitive tests , reconstitute d wate r wa s use d (Appendi x 3) . Th

e theoretica

l hardnes s o f reconstitute

d wate r i s 250 mg/

L a s CaCC>3 . Therefore , i n orde

r t o compl y wit h th e wate r characteristic s o f

th e OPPT S guideline , th

e prepare d wate r wa s diluted i n arati o o f

50:5 0 wit h deionize d revers e osmosi s wate r prio r t o us e t o reduc e th e tota l wate r hardness .p . 1 2 Harla n Stud y Number : 4110469 5 Repor

t Pag e 1 1

A t th e star t o f th e definitiv e test

th e diluen t ha d atota l wate

r hardnes s o f

148 mg/ L a s CaC0 3 an d a n alkalinit

y o f 2 6 mg/ L a

s CaCC>3 . Th e alkalinity wa

s determined usin g the methods describe d i n Fiel d an

d On-sit e Tes t Method s fo r

th e Analysi s o f

Water s (Britis h Standard

s Institution , 1993) . 3. 4 Procedur e 3.4. 1 Range-findin g Tes t Th e test concentration s to b

e use d i n th e definitiv e test

wer e determine d b y apreliminar y rangefinding test . I n th e range-findin g tes t

Daphni amagn

awer e expose d to aserie s o f

nomina l tes t concentration s o f 0.10 , 1.0 , 10 , 10 0 an d 100 0 m g ai/L . Th

e tes t ite mwa s dissolve d directl y i n reconstituted water . A n amoun

t o f tes t ite m(198 0 mg ) wa

s dissolved i n reconstitute d wate r an d th e volum e adjusted t o 1 liter to giv e th

e 100 0 m g ai/ L tes t concentration . Seria

l dilution s wer

e mad e i n reconstituted wate r t o giv e the remainde

r o f th e test concentrations . I n error , the tes t concentration wa

s corrected fo r

awate r conten t o f 49.5 % instea

d o f 50.5% , however , a

s thi s differenc e wa

s s o small , thi s erro

r wa s considered no t

to hav e ha d a n effec t o n the outcom

e o f th e test . Eac h prepare

d concentratio n wa

s inverte d severa

l time s t

o ensur e adequat

e mixin g an

d homogeneity , an

d th e p H o f eac h wa s adjusted t

o p H 7 . I n th e range-findin g tes t

1 0 daphnids wer

e place d i n eac h test an d contro l vesse

l an d maintaine

d i n atemperatur

e controlle d roo ma

t 2 1 ° C wit h aphotoperio d o f 1 6 hours ligh t

an d 8 hour s darknes s fo r

aperio d o f

4 8 hour s wit h 2 0 minut e daw n an d dusk transitio n periods . Eac h 25 0 m L test

an d contro l vesse

l contained 200 mL o f

tes t medi aan

d wa s covere d t o reduc e evaporation . Afte r

2 4 and 4 8 hour

s th e numbe r o f immobilize d Daphni amagn awer e recorded . Th e contro l grou

p wa s maintaine d unde

r identica l condition s bu

t no t expose d to th e tes t item . A sampl e o f eac h tes t concentratio n wa

s take n fo r chemica l analysi s a

t 0 an d 4 8 hour s i n orde r t o determin e th e stability o f

th e test ite munde

r test conditions . Al l sample s wer e store d a t approximatel

y -20 ° C prio r

to analysis . Only concentration s withi n th e rang e to b e use d fo r th e definitiv e tes t

wer e analyzed . 3.4. 2 Definitiv e Tes t Base d o n th e result s o f

the range-findin g test

th e followin g tes t

concentration s wer

e assigne d t o th e definitiv e test : 100 , 180 , 320 , 56 0 an d 100 0 m g ai/L . Al l test

sampl e concentration s wer

e corrected fo r

awate r conten t o f 50.5% .Harla n Stud y Number : 4110469 5 Repor

t p . 1 3 Pag e 1 2

3.4.2. 1 Experimenta

l Preparation Fo r th e purpos e o f

th e definitiv e tes t

th e test ite mwa s dissolve d directl y i n reconstitute d water . A n amoun

t o f tes t ite m(1010 0 mg ) wa

s dissolved i n reconstitute d wate r an d th e volum e adjuste d t o 5 liter s t

o giv e th e 100 0 m g ai/ L tes t concentration . Aliquot s (100 , 180 , 32 0 an d 56 0 mL ) o f th e 100 0 m g ai/L tes t concentratio n wer

e adde d to afina l volum

e o f 1 lite r t o giv e th e remainin

g test concentration s o f

100 , 180,32 0 an

d 56 0 m g ai/L . Eac h stoc k prepared concentratio n wa s inverte d severa

l time s t o ensur e adequat e mixin g an

d homogeneit

y an d th e p H o f eac h wa s adjusted to p H 7 . Th e concentration an

d stability o f

th e test ite mi n th

e test preparation s wer

e verifie db y chemica

l analysi s a t 0 an d 4 8 hours (se

e Appendi x 4) . 3.4.2. 2 Exposur

e Condition s I n th e definitiv e test

25 0 m L glas s jar s containin g 200 m L o f tes t preparatio n wer

e used . A t

th e star t o f th e tes t 5 daphnid s wer

e place d i n eac h test an d contro l vesse

l a t random , i n th

e tes t preparations . Fou

r replicat e test an d contro l vessel s wer

e prepared . Th

e tes t vessels wer

e the n covere d to reduce evaporatio n an d maintained i n atemperatur e controlle d roo ma

t 2 0 ° C t o 2 1 ° C wit h aphotoperio d o f 1 6 hours ligh t

an d 8 hours darknes s wit h 2 0 minut e daw n an d dus k transitio n period s wit h aligh t intensit y ranging fro m64

5 to 68 2 lux . Th

e daphnids wer

e no t individuall y identified , receive d no foo d durin g exposur e an d th e tes t vessel s wer

e not aerated . Th e contro l grou

p wa s maintaine d unde

r identica l condition s but

no t expose d to th e test item . Th e test preparation s wer

e not renewe d durin g th

e exposur e period . An y immobilizatio n o r advers e reaction s to exposur

e wer e recorde d a t 2 4 an d 4 8 hours afte r

the star t o f exposure . Th

e criterio n o f

effec t use d wa s tha t Daphni awer

e considere

d to be immobilize d i f

the y wer e unabl e t o swi mfo r

approximately 1 5 second s after gentl e agitation . 3.4.2. 3 Physico-Chemica l Measurement

s Wate r temperatur

e an d ligh t intensit y wer e recorded dail y throughout th e test . Dissolve d oxyge

n concentration s an

d p H wer

e recorded a

t th e star t an d terminatio n o f

the test . Th

e p H an d dissolved oxyge

n concentratio n wer

e measure

d using aHac

h HQ30 d Flex i handhel d mete r whils t th e temperatur

e was measure

d usin g aHann

aInstruments H I 9351 0 digita l thermometer .Harla n Stud y Number : 4110469 5 Repor

t p . 1 4 Pag e 1 3

3-4.2. 4 Verificatio n o f

Test Concentration s Wate r sample s wer

e take n fromth e contro l an

d eac h test grou p (replicate s R i - R

* pooled ) a t 0 an d 4 8 hours fo r

quantitativ e analysis . Sample s wer e stored a

t approximately -2

0 ° C prior

t o analysis . Duplicat e sample s wer

e take n an d store d a t approximately -20 ° C fo r furthe r analysi s i f necessary . Sample s a t th e N o Observe

d Effec t Concentration an

d abov e onl y wer

e analyzed . Th e metho d o f

analysis , recover

y an d tes t preparation analyse s ar

e described i n Appendix 4 . 3.4.2. 5 Evaluatio n o f

Data A n estimat

e o f the EC5 0 value s wa

s give n b y inspectio n o f

th e immobilizatio n data . 3.4. 3 Post-Study Experimen t Whils t significan t

immobilizatio n wa

s observe d to hav e occurred a

t 100 0 m g ai/ L i n th e rang e findin g test , n o immobilizatio n occurred a t an y tes t concentration employed i n th e definitiv e test . I t wa s therefor e considered appropriat

e t o conduc t apost-stud

y experimen

t i n orde r t o confirm th e results obtained . I n th e post-stud

y experimen

t Daphni amagn

awer e expose d to aserie s o f

nomina l tes t concentration s o f

0.10 , 1.0 , 10, 10 0 an d 100 0 m g ai/L . Th

e tes t ite mwa s dissolve d directl y i n reconstitute d water . A n amoun

t o f tes t ite m(198 0 mg ) wa

s dissolve d i n reconstitute d wate r an d th e volum e adjusted t o 1 lite r t o giv e th e 100 0 m g ai/ L tes t concentration . Seria

l dilution s wer

e mad e i n reconstituted wate r to giv e th e remainde

r o f th e test concentrations . Eac h prepare

d concentratio n wa

s inverte d severa

l time s to ensur

e adequat

e mixin g an

d homogeneity , an

d th e p H o f eac h wa s adjusted t

o p H 7 . I n th e post-stud

y experimen

t 1 0 daphnid s wer

e place d i n eac h test an d contro l vesse

l an d maintained i n atemperatur e controlle d roo ma

t 2 1 ° C wit h aphotoperio d o f 1 6 hour s ligh t an d 8 hour s darknes s fo r

aperio d o f

4 8 hours wit h 2

0 minut e dawn an

d dus k transitio n periods . Eac

h 25 0 mL test

an d contro l vesse

l contained 25 0 mL o f

tes t medi aan

d wa s covere d t o reduc e evaporation . Afte r

2 4 an d 4 8 hour s th e numbe r o f immobilize d Daphni amagn awer e recorded . Th e contro l grou

p was maintaine d unde

r identica l condition s but

no t expose d t o th e tes t item .p . 1 5 Harla n Stud y Number ; 4110469 5 Repor

t Pag e 1 4

A sampl e o f eac h test concentration wa

s take n fo r chemica l analysi s a

t 0 an d 4 8 hours i n orde

r t o determin e th e stability o f

th e tes t ite munde

r test conditions . Al l samples wer e store d a t approximatel

y -2 0 ° C prio r

to analysis . Onl y concentration s o f th e NOEC an

d abov e wer e analyze dp . 1 6



### Harla n Stud y Number : 4110469 5 Repor

t Pag e 1 5

4 RESULT S 4. 1 Range-findin g Tes t Cumulativ e immobilization dat

afro mth e exposur e o f Daphniamagn

at o th e tes t ite mdurin g th e range-findin

g tes t i s give n i n Tabl e 1 . N o immobilizatio

n wa s observe d a t th e tes t concentration

s o f 0.10 , 1.0 , 10 an d 10 0 m g ai/L . However , immobilization wa

s observe d a t 100 0 m g ai/L . Base d o n thi s information tes

t concentration

s o f 100 , 180 , 320 , 56 0 an d 100 0 m g ai/ L wer e selecte d fo r th e definitiv e test. Chemica l analysis o f

th e tes t preparation

s a t 0 an d 4 8 hour s (se e Appendi

x 4) showe

d tha t measure d concentration

s betwee n 83 % an d94 % o f nomina l wer e obtaine d indicatin g tha t th e tes t ite mwa s stabl e unde r tes t conditions . 4. 2 Definitiv e Tes t 4.2.1 Immobilizatio

n Dat a Cumulativ e immobilization dat

afro mth e exposur e o f Daphniamagn

at o th e tes t ite mdurin g th e definitive tes

t ar e give n i n Tabl e 2 . Ther e wa s n o immobilization i n 2

0 daphnid s expose d t o arang e o f tes t concentration

s of 100 , 180 , 320, 56 0 an d 100 0 m g ai/ L fo r aperio d o f 4 8 hours . Inspectio

n o f th e immobilizatio

n dat a gav e th e followin g results : Tim e (h ) EC 5 0

### (m g ai/L) 2 4 >100 0 4 8 >100 0 Th e N o Observe d Effec t Concentratio

n afte r 2 4 an d4 8 hour s exposur e wa s 100 0 m g ai/L . 4.2. 2 Observation

s o n Tes tIte m Solubilit

y Throughou

t th e duratio n o f th e test , th e tes t preparation

s wer e observe d t o rang e froma n extremel y pal e ambe r colore d solutio n i n th e 10 0 m g ai/ L tes t grou p t o adar k ambe r solutio n i n th e 100 0 m g ai/ L tes t group .

### p . 1 7

Harla n Stud y Number : 4110469 5 Repor

t Pag e 1 6

4.2. 3 Physico-Chemica

l Measurement

s Th e result s o f th e physico-chemica

l measurement

s ar e give n i n Appendi

x 5 , Temperatur

e wa s maintaine d a t approximatel

y 2 0 ° C t o 2 1 ° C throughou

t th e test. Th e wate r temperatur

e whic h wa s als o recorde d i n th e control vesse

l approximatel

y ever y hou r usin g aTest o temperatur

e logge r (se e Figur e 1 ) range d fro m2 0 ° C t o 2 1 °C . 4.2. 4 Verificatio

n o f Tes tConcentration

s Analysi s o f

th e tes t preparation

s a t 0 an d 4 8 hour s (se e Appendi

x 4) showe

d measure d tes t concentration

s t o rang e fro m80 % t o 83 % o f nomina l valu e an d s o i t wa s considere

d justifiable t o estimat e th e EC$ o value s i n term s o f th e nomina l tes t concentration

s only . 4. 3 Post-Stud y Experimen t Cumulativ e immobilization dat

afro mth e exposur e o f DaphniaMagn

at o th e tes t ite mdurin g th e post-stud y experimen

t ar e give n i n Tabl e 3 . N o immobilizatio

n wa s observe d a t th e tes t concentration

s o f 0.10 , 1.0

, 10 , 10 0 an d 100 0 m g ai/L . Chemica l analysis o f

th e 100 0 m g ai/ L tes t preparatio

n a t 0 an d 4 8 hour s (se e Appendi

x 4 ) showe d nea r nomina l concentration

s wer e obtaine d indicatin g tha t th e tes t syste mwa s correctl y dosed . I t wa s therefor e considere

d tha t th e immobilizatio

n observe d i n th e rang e findin g tes

t wa s du e t o contaminatio

n rathe r tha n atru e toxic effect . 

## 5 CONCLUSIO N Th e acut e toxicit y o f th e tes t ite mt o th e freshwate

r invertebrat

e Daphniamagn

aha s bee n investigate d an d gav e a48-Hou

r EC5 0 o f greate r tha n 100 0 m g ai/L . Th

e N o Observe d Effect Concentratio

n wa s 100 0 m g ai/L .Harla n Stud y Number : 4110469 5 Repor

t Pag e 1 7 

## TABLE S Tabl e 1 Cumulativ e Immobilizatio n Dat a i n th e Range-finding Tes t Nomina l Concentratio n (m g ai/L ) Cumulative Immobilized Daphni a (Initia l Population : 1

0 Pe r Replicate ) Nomina l Concentratio n (m g ai/L ) 2 4 Hours 4 8 Hours Contro l 0 0 0.1 0 0 0 1. 0 0 1 1 0 0 0 10 0 0 0 100 0 5 1 0Harla n Stud y Number : 4110469 5 Repor

t Pag e 1 8

Tabl e 2 Cumulativ e Immobilization Dat a i n th e Definitiv e Tes t
Nomina l Concentratio n (mg/L ) Cumulative Immobilize d Daphni a (Initia l Population : 5 Pe r Replicate ) Nomina l Concentratio n (mg/L ) 2 4 Hours 48 Hours Nomina l Concentratio n (mg/L ) R i R 2 R s R < Tota l % R i R 2 R 3 R 4 Total % Contro l 0 0 0 0 0 0 0 0 0 0 0 0 10 0 0 0 0 0 0 0 0 0 0 0 0 0 18 0 0 0 0 0 0 0 0 0 0 0 0 0 320 0 0 0 0 0 0 0 0 0 0 0 0 56 0 0 0 0 0 0 0 0 0 0 0 0 0 100 0 0 0 0 0 0 0 0 0 0 0 0 0

R i -R t = Replicate

s 1 t o 4Harla n Stud y Number : 4110469 5 Repor

t p . 2 0 Pag e 1 9

Tabl e 3 Cumulativ e Immobilization Dat a i n th e Post-Stud

y Experimen t Nomina ] Concentratio n (m g ai/L ) Cumulative Immobilize d Daphni a (Initia l Population : 1

0 Pe r Replicate ) Nomina ] Concentratio n (m g ai/L ) 2 4 Hour s 4 8 Hour s Contro l 0 0 0.1 0 0 0 1. 0 0 0 1 0 0 0 10 0 0 0 100 0 0 0p . 2 2 Harla n Stud y Number : 4110469 5 Repor

t Pag e 2 1 

## APPENDICE S Appendi x 1 Reconstituted Wate r - Elend tM7 Mediu m Solutio n Concentration o f stoc k solutio n (mg/L ) (I ) H3B0 3 5719 0 (II ) MnCl 2 .4H 2 0 721 0 (HI ) LiC I 6120 (IV ) RbC l 142 0 (V ) SrCI 2 .6H 2 0 304 0 (VI ) NaB r 32 0 (VII ) Na 2 Mo0 4 .2H 2 0 126 0 (VIII ) CuCI 2 .2H 2 0 33 5 (IX ) ZnCl 2 260 (X ) CoCl 2 .6H 2 0 200 (XI ) K I 6 5 (XII ) Na 2 Se0 3 43. 8 (XIII ) NH4VO 3 11. 5 (XIV ) Na 2 EDTA.2H 2 0 500 0 FeS0 4 .7H 2 0 199 1 A n aliquo t

(dependan

t o n the volum

e o f mediumrequired) o f

eac h stoc k solutio n wa

s adde d t o a fina l volum

e o f deionize d reverse osmosi s wate r t o giv e stoc

k solutio n A an

d store d a t approximately 2 1 °C .Harla n Stud y Number : 4110469 5 Reconstitute d Wate r - Elend t M 7 Repor

t p . 2 3 Pag e 2 2

Macr o NutrientStock Solution s Solution Concentration o f stoc k solutio n (g/L ) (XV ) CaCl 2 .2H 2 0 293.8 0 (XVI ) NaHC0 3 64.8 0 (XVII ) MgS0 4 .7H 2 0 246.6 0 (XVIII ) Na 2 Si0 3 .9H 2 0 50.0 0 (XIX ) KC 1 58.00 (XX ) NaN0 3 2.74 (XXI ) K 2 HP0 4 1.8 4 (XXII ) KH 2 P0 4 1.4 3 Vitami n Nutrient

s Solutio n Concentration o f stoc k solutio n (mg/L ) (XXIII ) Thiamin e hydrochlorid e 75 0 Cyanocobalamin

e (vitami n Bi 2 ) 10 D(+ ) bioti n (vitami n H ) 7. 5 Th e fina l mediumwa

s prepare d b y addin

g a n aliquo t o f stoc k solutio n A along wit h aliquots o f eac h individua l Macr

o Nutrien t

Stoc k Solutio n an

d a n aliquo t o f th e vitami n nutrien

t t o th e require d amoun

t (fina l volume ) o f

deionized revers e osmosi s water . Th e p H o f th e prepared medi awa

s 8. 0 ± 0. 2 an

d store d a t approximately 2 1 °C .Harla n Stud y Number : 4110469 5 Repor

t p . 2 4 Pag e 2 3
Appendi x 2 Positive Contro l A positiv e contro l (Harla n Stud y Number ; 41203341 ) use d potassiu

mdichromat

e a s th e referenc e ite ma

t concentration s o f

0.32,0.56 , 1.0

, 1.8 an d 3. 2 mg/L . Exposur e condition s fo r

th e positiv e contro l wer

e simila r to thos e i n th

e definitiv e test . Analysi s o f

th e immobilizatio n dat

ab y th e geometric mea

n metho d a

t 2 4 hours an

d th e trimmed Spearman-Karbe

r metho d (Hamilto n e

t a l 1977 ) a

t 4 8 hours base

d o n th e nomina

l tes t concentration s gav

e the followin g results : 

### Tim e (h ) ECs

o (mg/L ) 95% Confidenc e limit s (mg/L ) 2 4 0.7 5 0.5 6 - 1. 0 4 8 0.4 5 0.4

2 - 0.4

8 Th e N o Observe

d Effec t Concentration s after

2 4 an d 4 8 hours wer

e 0.5 6 an

d 0.3 2 mg/

L respectively . Th

e N o Observe

d Effec t Concentration i s base

d upo n zer

o immobilizatio n a

t thi s concentration . Th e result s fromth

e positiv e contro l wit h potassiu mdichromat

e wer e withi n th

e norma l rang e fo r thi s referenc e item .

* HAMILTON

, M A , RUSSO

, RC AN D THURSTON

, RV (1977

) Trimme d Spearman-Karbe

r Metho d fo r Estimatin g Media n Letha l Concentratio

n i n Toxicit y Bioassays

. Enviro n Sc i Techno l U , 714-719.p . 2 5 Harla n Stud y Number : 4110469 5 Repor

t Pag e 2 4
Appendi x 3 Reconstituted Wate r Stoc k Solutions a ) b ) c ) d ) CaCl 2 .2H 2 0 MgS0 4 .7H 2 0 NaHC0 3 KC 1 11.7 6 g/ L 4.9 3 g/ L 2.5 9 g/ L 0.2 3 g/ L ii ) Preparatio n A n aliquo t

(2 5 mL ) o f

eac h o f solution s a-d wa

s adde d to eac h lite r (fina l volume ) o f deionize d wate

r wit h aconductivit

y o f <5 ji S cm"' . Th

e reconstitute d wate

r ha d ap H o f 7. 8 ± 0. 2 adjuste d (i f

necessary

) wit h NaO H o

r HC 1 an d wa s aerate d unti l th e dissolve d oxyge n concentratio n wa

s approximatel

y air-saturatio n value . Th e reconstitute d wate

r ha d a n approximat

e theoretica

l tota l hardnes s o f

25 0 mg/ L a s CaC0 3 .p . 2 6 Harla n Stud y Number : 4110469 5 Repor

t Pag e 2 5
Appendi x 4 Analytica l Investigation s 

## Analytica l Investigation

s CONTENT S CONTENT S 2 5 1 MATERIAL S AN D METHOD S 2 6 1. 1 Introductio n 26 1. 2 Test

Ite m26 1. 3 Analytica l Standar d 26 1. 4 Analytica l Procedur e 2 6 1.4. 1 Storag

e 26 1.4. 2 Reagent

s an d Solvent s 26 1.4. 3 Preparation o f

Calibratio n Standards 2 7 1.4. 4 Preparation o f

Linearit y Standard

s 2 7 1.4. 5 Preparation o f

Spike d Recover

y Samples 2 7 1.4. 6 Preparation o f

Tes t Sample s 2 7 1.4.7 Instrumenta

l Parameter

s 2 8 1.4. 8 Calculation s 2 9 2 RESULT S 3 1 2. 1 Range-Findin g Tes t 3 1 2. 2 Definitiv e Tes t

3 1 2.2. 1 Validatio n o f

th e Analytica l Metho d 3 1 2.2. 2 Tes t

Samples 3 1 3 DISCUSSIO N 3 3 TABLE S 3 4 Table 1 Preparatio n o f Test Samples 3 4 Table 2 Calibratio n Standar d Dat a3 4 Table 3 Results fo r Range-Finding Samples 3 5 Table 4 Results fo r Post-Stud y Range-Findin

g Samples 3 5 Tabl e 5 Result s fo r Spiked Recover

y Sample s 3 5 Table 6 Results fo r Test Samples 3 6 FIGURE S 3 7 Figur e 1 Calibratio n Plo t 37 Figur e 2 Chromatogra

mo f Calibratio n Solutio n (1. 0 m g ai/L ) 3 8 Figur e 3 Chromatogra

mo f Biologica l Contro l Sampl e 3 8 Figur e 4 Chromatogra

mo f Tes t Sampl e (1000 m

g ai/L ) 3 9p . 2 7 Harla n Stud y Number : 4110469 5 Repor

t Pag e 2 6 Analytica l Investigation

s 

## 1 MATERIAL S AN

D METHOD S 1. 1 Introductio n Th e tes t ite mconcentratio n i n th

e tes t sample s wa s determined b y direc t

injectio n into th

e mas s spectromete

r (HPLC-MS ) usin g a

n externa l standar d to calibrat

e th e system . Al l test sampl e concentration s wer

e corrected fo r

awate r conten t o f 50.5% , an

d ar e reporte d a s milligram s o f

activ e ingredient

(ai ) pe r liter . Th e metho d wa

s developed b y th

e Departmen

t o f Analytica l Services , Harla n Laboratorie s Ltd , Shardlow , UK . 1. 2 Tes tIte m Th e tes t ite mi s described i n th

e biological par

t o f thi s study . 1. 3 Analytica l Standar d Th e test ite mdescribed i n th

e biological par

t o f thi s stud y wa s als o use

d a s th e analytica

l standard . 1. 4 Analytica l Procedur e 1.4. 1 Storag

e Th e samples wer

e store d a t approximately -2

0 ° C prio r

to analysis . 1.4. 2 Reagent

s an d Solvent

s Water : prepared usin g a n ELG A Pur

e La b Optio n R-1 5 wate

r purificatio n system . Tes t medium : a

s describe d i n th

e biological par

t o f thi s study . Solvents : methanol , Fishe r Scientific , HPLC grade . Reagents : ammonia , Fishe r Scientific , Analytica l reagent .Harla n Stud y Number : 4110469 5 Analytica l Investigation

s Repor t p . 2 8 Pag e 2 7

1.4. 3 Preparatio n o

f Calibratio n Standards Th e test ite m(nominal 200 mg ) wa s dissolve d i n diluent*(100 mL ) to prepar e astoc k solutio n wit h aconcentratio n o f

100 0 mg ai/L . Thi s stoc

k solutio n wa

s furthe r dilute d wit h diluent * t

o produce asolutio n o f

1 0 mg/L . Define d volume s o f thi s solutio n wer

e the n dilute d wit h diluent * to obtai n calibratio n standards i n th e rang e o f 0.1 0 t o 1. 0 m g ai/L . A secon

d serie s o f

calibratio n standard s wa s similarl y prepare

d i n th e rang e o f 0.2 5 to 1. 5 mg ai/L . These solution s wer e use d t o determin

e th e recover y an d tes t sampl e concentrations . 1.4. 4 Preparatio n o

f Linearity Standards Th e test ite m(nomina

l 20 0 mg ) wa s dissolved i n diluent*(100 mL ) to prepar e astoc k solutio n wit h aconcentratio n o f

100 0 m g ai/L . Thi s stoc

k solutio n wa

s furthe r dilute d wit h diluent * t

o produce asolutio n o f

1 0 mg/L . Define d volume s o f thi s solutio n wer

e the n dilute d with diluent * to obtai n calibratio n standards i n th e rang e o f 0.1 0 to 1. 0 m g ai/L . A secon

d serie s o f

calibratio n standard s wa s similarl y prepare

d i n th e rang e o f 0.2 5 t o 1. 5 m g ai/L . These standard

s wer e use d t o evaluat e th e linearity o f

th e analytica

l system . 1.4, 5 Preparatio n o

f Spiked Recover

y Sample s To demonstrat

e th e validit y o f

th e analytica

l procedure , volume s o f

test mediu mwer e spike d wit h th e tes t ite man d it s recover

y wa s assessed

. Th e tes t ite m(nomina

l 200 mg ) wa

s initiall y dissolved i n tes t

mediumto prepar

e astoc k solutio n wit h aconcentratio n o f 100 0 m g ai/L . Define d volume s o f

thi s stoc k solutio n wer

e dilute d wit h test

mediumto obtai n spiked recover y samples a t concentration s o f

10 0 mg ai/L . Fiv e replicate s a t eac h concentratio n leve l wer e prepare d an d subjected to th

e sam e treatmen

t a s th e test samples . I n addition , tes t mediu m withou t th e additio n o f

th e tes t ite m(syntheti c control ) wa s als o analyzed . 1.4, 6 Preparatio n o

f Tes tSample

s The. test sample s wer

e thawed wit h th

e ai d o f sonication . The

y wer e dilute d int o th

e calibratio n rang e initiall y i n tes t

mediuman

d the n further dilute d wit h methanol:water:ammoni a(50:50:0.1 0 v/v/v ) a s shown i n Tabl e 1 , an

d then analyze

d directl y using th

e instrumenta

l procedure .

* Diluent : methanol:water:ammonia:tes

t mediu m(50:40:0.10:1 0 v/v/v/v

)p . 2 9 Harla n Stud y Number : 4110469 5 Repor

t Pag e 2 8 Analytica l Investigation

s 1.4. 7 Instrumental Parameter s Autosampler : Agilen t 120 0 Series Pump : Agilen t 120 0 Serie s Detector : Agilen t 120 0 Serie s MS D Column : none , direc t injectio n Mobil e phase : waterrmethanol.-ammonia(500:500:1 , v/v/v ) Flo w rate : 0. 5 mL/minute Injectio n volume : 1 0 uJ L Colum n temperature : roo mtemperatur e Ionizatio n mode : ES I Polarity : negative Capillar y voltage : 400

0 V Ga s temperature : 35 0 ° C Nebulize r pressure : 4 0 ps i Fragmento

r voltage : 10

0 V Dryin g ga s flow : 1 2 L/mi n Dat aacquisitio n mode : Single Io n Monitorin g (SIM ) SI M ions : 22 5 m/ zp . 3 0 Harla n Stud y Number : 4110469 5 Repor

t Pag e 2 9 Analytica l Investigation

s 1.4.S Calculation s A calibratio n curv e wa

s prepare d using aquadratic functio n o f the for mshown i n equatio n (1) . Th e calibratio n dat

ai s presente

d i n Table 2 an

d Figur e I . Fro mthe calibratio n curve , th e concentration "x " o f

th e tes t ite mi n a

n injecte d sampl

e wa s calculate d usin g th

e followin g equation :

x = _-b + ^b 2 -4-a-(c-y)

2-a 

## 0 ) wher e x :ya, b, c: concentratio n o f th e test ite mi n injecte d sampl

e [m g ai/L ] averag e pea k are ao f th e tes t ite mi n injecte d sampl

e [counts ] constants Th e concentratio n o f

th e test ite mi n th

e test samples wa

s calculate d usin g th

e followin g equation :

c = R/10 0 wher e c x F R • F (2 )

concentratio n o f

th e test ite mi n th

e tes t sampl e [m g ai/L ] concentratio

n o f th e tes t ite mi n th e injecte d sampl

e solutio n [m

g ai/L ] sampl e preparation facto r recover y rat e [% ] (I f no t correctin g fo r

recover y the n Ri s take

n a s 100 ) Th e recover y rat e (R ) o f th e spike d recover

y samples wa

s calculate d using th

e followin g equation : R10

0 (3 ) 'for t wher e R : recover

y rat e [% ] c : determine

d concentratio

n o f th e tes t itemi n th e spiked recover

y sampl e [m g ai/L ] Cf 0rt : fortifie dconcentration o f th e test ite mi n th

e spike d recover

y sampl e [m g ai/L ] Th e percentag

e o f nominal concentration o f th e tes t ite mi n th

e tes t sample s wa

s calculated using th e followin g equation :p . 3 1 Harla n Stud y Number : 4110469 5 Repor

t Pag e 3 0 Analytica l Investigation

s % nomina l = — —• 10 0 (4 ) wher e Cnom : nominal concentration i n th e test sampl e [m g ai/L ]Harla n Stud y Number : 4110469 5 Analytica l Investigation

s Repor t p . 3 2 Pag e 3 1

2 RESULT S 2. 1 Range-Findin g Tes t Th e analytica

l methodolog y wa

s no t validate d prio r

to th e analysi s o f

range-findin g samples , ful l validatio n wa

s late r conducted specificall y ove r th e tes t concentratio n rang

e employed i n th

e definitiv e test . Th

e result s fo r

th e range-findin g test

samples ar

e summarized i n Table 3 . 2. 2 Definitiv e Tes t 2.2. 1 Validatio n o

f th e Analytical Metho

d Specificit y Th e biological contro l sample s an d a n analyze

d analytica

l blan k showe d n o significant interferin g response a

t th e retentio n tim e o f

th e tes t item . Th

e standar d solution s containe

d a pea k specifi c fo r

th e tes t ite mwhose are

achange d accordingly wit h know n concentration , henc e th e specificity o f

th e metho d b

y retentio n time wa

s confirmed . Linearit y Th e calibration dat

afo r th e calibratio n standard

s o f th e tes t itemi s given i n Tabl

e 2 an d Figur e 1 . Th e dat awa s foun d to hav

e aquadratic correlatio n withi n th e calibratio n rang

e o f Ot o 1. 6 m g a i /L . The R 2 fi t

o f th e calibratio n curve t

o th e dat awa s 0.9985 , an

d wa s considere

d t o b e acceptable . Accuracy (Recovery ) an d Precisio n A se t o f recover y sample s accuratel

y fortifie da

t arelevan

t concentratio n o f

test itemwa s prepare d five-foldan

d analyzed . Th

e results obtained fo r

th e concentratio n o f

th e test ite mi n th e recover y sample s ar

e presente d i n Table 5 . Th e method wa

s considered to b

e sufficientl y accurat

e an d precis e fo r

the purpose s o f

thi s test . Limi to f Quantitatio n (LOQ ) Th e limi t o f quantitatio n (LOQ ) wa

s determined b y calculatin g th e sampl e concentration tha

t gav e apea k equivalen

t to te n time s th

e baselin e noise . Usin g thi s metho d th e limi t o f quantitatio n wa

s determine d t

o b e 0.04 7 m g ai/L . 2.2.2 Tes tSamples Th e result s obtaine d fo r

th e concentration o f

th e test ite mfoun d i n th

e tes t sample s ar

e presente d i n Table 6 .p . 3 3 Harla n Stud y Number : 4110469 5 Repor

t Pag e 3 2 Analytica l Investigation

s Typica l chromatogram s ar

e show n i n Figure s 2 to 4 . 2. 3 Post-Stud y Experimen t 2.3. 1 Tes tSample

s Th e results obtaine d fo r

th e concentration o f

th e test itemfoun di n the tes t

sample s ar

e presente d i n Table 6 .p . 3 4



### Harla n Stud y Number : 4110469 5 Repor

t Pag e 3 3 Analytica l Investigation

s 

## 3 DISCUSSIO N Th e detectio n syste mwa s foun d t o hav e acceptabl

e correlatio

n t o th e fi t o f aquadrati

c equation. Th e analytica

l procedur e wa s foun d t o hav e acceptabl

e recoverie

s o f tes t ite mi n tes t medium . Th e metho d o f analysi s wa

s validate d an d prove n t o b e suitabl e fo r use .p . 3 5 Harla n Stud y Number : 4110469 5 Repor

t Pag e 3 4 Analytica l Investigation

s 

## TABLE S Tabl e 1 Preparatio n o f Tes tSample

s Nomina l Concentration o

f Tes tItem Sampl e Volum e Fina l Volume Sample

Preparatio n Facto r Fina l Nomina l Concentration o

f Tes t Ite m [m g ai/L ] [mL ] [mL ] F [m g ai/L ] Contro l 1 100 , the n 5 - > 5 0 100

0 - 100 0 1 100 , the n 5 - > 50 100

0 1.

0

Tabl e 2 Calibratio n Standard Dat a Concentratio n o

f Tes tIte m Are a [m g ai/L ] [counts] 0 4.16 1 x 10 2 0.010 4 1.074xl0 4 0.27 1 2.23 5 xlO 4 0.522 3.98 8 x 10 4 0.81 2 5.97 5 x 10 4 1.0 4 8.01 7 xlO 4 1.6 2 1.16 7 x10 sp . 3 6 Harla n Stud y Number: 4110469

5 Repor t Pag e 3 5 Analytica l Investigations 

### Tabl e 3 Result

s fo r Range-Findin

g Sample s

Tim e Point Nomina l Concentratio n o f Tes tIte m i n Range-Findin g Sampl e Measured

Concentratio n o f Tes tIte m i n Sample Via l Sample

Preparatio n Facto r Determined

Concentratio n of Tes tIte m i n Range-Findin g Sampl e % o

f Nomina l Concentration X F c [hours ] (m

g ai/L ] [m

g ai/L ] [m g ai/L ] 

### t%

l 0 10

0 0.93 7 10 0 93. 7 9 4 100 0 0.90

7 100

0 907 9 1 48 10

0 0.93 7 10 0 93. 7 9 4 100 0 0.83 2 100 0 83 2 8 3 Tabl e 4 Result s fo r Post-Stud y Range-Findin g Sample s

Tim e Point Nomina l Concentratio n o f Tes tIte m i n Range-Findin g Sampl e Measure d Concentratio n of Tes tIte m i n Sample Via l Sample

Preparatio n Facto r Determined

Concentratio n o f Tes tIte m i n Range-Findin g Sampl e % o

f Nomina l Concentration
X F c [hours ] [m

g ai/L ] [mg ai/L] {m g ai/L ] [%

] 0 100

0 0.91 6 100 0 91 6 9 2 4 8 100

0 0.88

4 100

0 88

4 8

8 

### Tabl e 5 Result

s fo r Spike d Recover y Sample s Nomina l Concentratio n of Tes tIte m Fortifie d Concentratio n o f Tes tIte m i n th e Spike d Sampl e Measure d

Concentratio n o f Tes tIte m i n th e Sampl e Via l X Sample

Preparatio n Facto r

F Determined Concentratio n of Tes tIte m i n th e Spike d Sampl e c Analytica l

Recover y Rat e

R Precisio n

(Relativ e Standar d Deviatio n o f Recovery ) [m g ai/L ] [m

g ai/L ] [m

g ai/L ] [m g ai/L ] [%

] I% ] 10 0 10

7 1.1 2 10 0 11 2 10 7 0.99 9 10 0 99. 9 10 7 0.97 4 10 0 97. 4 94 8. 2 10 7 1.0

3 10

0 10

3 10 7 0.894 10 0 89.4 Acceptanc e Targe

t 80-120 <1 0 Fo r reportin g purposes , x ha s bee n calculate d fro mc retrospectively

.p . 3 7 Harla n Stud y Number: 4110469

5 Repor t Pag e 36 Analytica l Investigations Tabl e 6 Result s fo r Tes tSample s

Tim e Point Nomina l Concentratio n of Tes tIte m i n Tes tSampl e Measured

Concentratio n of Tes tIte m i n Sampl e Via l Sample

Preparatio n Facto r Determined

Concentratio n of Tes tIte m i n Tes tSampl e % o

f Nomina l Concentration

c no m X F c [hours ] [m

g ai/L ] [m

g ai/L ] [m g ai/L ] [%

] 0 Contro l

100 0 <LO Q

0.83 0 100

0 100 0 <LO Q

83 0 83 48 Contro l

100 0 <LO Q

0.80 2 100

0 100 0 <LO Q

80 2 80 LO Q = Limi t o f Quantitatio

n not applicabl e Fo r reportin g purposes , x ha s bee n calculate d fromc retrospectively

.Harla n Stud y Number: 4110469

5 Repor t Pag e 3 7 Analytica l Investigations 

## FIGURE S Figur e 1 Calibratio n Plo t

Concentratio n (m g ai/L )Harla n Stud y Number : 4110469 5 Analytica l Investigation

s Repor t Figure 2 Chromatogra m o f Calibratio n Solutio

n (1.0 m g ai/L ) Pag e 3 8

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0. 2 0 4 0 6 0 8 1 1 2 1 4 1 8 Ij i rtiHarla n Stud y Number : 4110469 5 Repor

t Pag e 3 9 Analytica l Investigation

s Figur e 4 Chromatogra m o f Tes tSampl

e (100 0 m g ai/L ) M3D 5 TIC , M 3 Fil e (SVNi\SS? ^ 1016SeS2l£63W2-12Q 1 fy 6Si s fl,9M,Ffa g 10 0

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. , , , , , , 1 , , , 1 , T—T0 2 0 4 0 6 0 8 1 V I 1 4 W 1. 6 ni nHarlan Study Number: 41104695 Repor t p . 4 1 Pag e 4 0
Appendi x 5 Physico-Chemica l Measurement s Nomina l Concentratio

n (m g ai/L ) 0 Hours 2 4 Hour s 4 8 Hours Nomina l Concentratio

n (m g ai/L ) P H m g Oi/ L %ASV * T° C T° C p H m g Oj/ L %ASV * T° C Contro l 

### R 7. 0 8. 8 9 7 2 0 2 1 6. 8 8. 6 9 7 2 1 R 2 7. 0 9. 0 9 9 2 0 2 1 6. 9 8. 6 9 7 2 1 R 3 7. 0 9. 0 9 9 2 0 2 1 6. 9 8. 6 9 7 2 1 R < 7. 0 9. 0 9 9 2 0 2 1 6. 9 8.6 9 7 2 1 10 0 R i 7. 0 8. 9 9 8 2 0 2 1 7. 0 8. 5 9 6 2 1 R 2 7. 0 8. 9 9 8 2 0 2 1 6. 9 8. 4 9 4 2 1 R j 7. 0 8. 9 9 8 2 0 2 1 7. 0 8. 5 9 6 2 1 R < 7. 0 8. 9 9 8 2 0 2 1 7. 0 8. 5 9 6 2 1 18 0 R i 7. 0 8. 9 9 8 2 0 2 1 7. 1 8. 4 9 4 2 1 R 2 7. 0 8. 9 9 8 2 0 2 1 7. 1 8. 4 9 4 2 1 R 3 7. 0 8. 9 9 8 2 0 2 1 7.1 8. 3 9 3 2 1 R 4 7. 0 8. 8 97 2 0 2 1 7.1 8.3 93 21 32 0 R i 7. 0 8. 8 9 7 2 0 2 1 7. 2 8. 2 9 2 2 1 R 2 7. 0 8. 3 9 7 2 0 2 1 7. 2 8. 2 9 2 2 1 R 3 7. 0 8. 8 9 7 2 0 2 1 7. 2 8. 2 9 2 2 1 R i 7. 0 8. 8 9 7 2 0 2 1 7. 2 8. 2 9 2 2 1 56 0 R i 7. 0 8. 9 10 0 2 1 2

1 7. 4 8.1 91 2 1 R 2 7. 0 8. 8 9 7 2 0 2 1 7. 3 8.1 91 2 1 R 3 7. 0 8. 8 9 9 2 1 2

1 7. 3 8.1 91 2 1 R , 7. 0 8.S 9 9 2 1 2

1 7. 3 8.1 91 2 1 100 0 R

> 7. 0 8. 8 9 7 2 0 2 1 7. 4 7. 6 85 2 1 R 2 7. 0 8. 8 9 7 2 0 2 1 7. 4 7. 5 8 4 2 1 R j 7. 0 8. 8 9 7 2 0 2 1 7. 4 7. 6 85 2 1 R 4 7. 0 8. 8 9 7 2 0 2 1 7. 5 7. 6 85 2 1

*AS V = * Dissolve d oxyge n concentratio

n expresse d a s apercentag

e o f Ai r Saturatio n Valu e R i - R , = Replicate

s 1 t o 4Harla n Stud y Number : 4110469 5 Repor

t p . 4 2 Pag e 4 1
Appendi x 6 Monitorin g Authorit y Statemen

to f GL P Compliance 

### OH | Department of Health
TH E DEPARTMEN T O F HEALT H O F TH E GOVERNMEN T

O F TH E UNITE D KINGDO M GOO D LABORATOR Y PRACTIC E STATEMEN T O F COMPLIANC E I N ACCORDANC E WIT H DIRECTIV E 2004/9/E C TES T FACILIT Y TES T TYPE(S ) HarianUbpratorlMLt d Analytical/Clinica l Shardlo w Busines s Par k rh a m i«,t« , Londo n Roa d c, , C TM
. ? ? Shardlo w Environmenta l Toxicit y Derb y Environmenta l Fat e DE7 2 2G D Mutagenicit y Phys/Chem
. Test s Toxicolog y

DAT E O F INSPECTIO N 1 0 Jul y 201 2 A n inspectio n fo rcomplianc e wit h th e Principle s o f Goo d Laborator y Practic e wa s carrie d ou t a t th e abov e tes t facilit y a s par t o f th e U KGoo d Laborator y Practic e Complianc e Monitorin g Programme
. Thi s statemen t confirm s that , o n th e dat e o f issue , th e U KGoo d Laborator y Practic e Monftorin g Authorit y wer e satisfie d tha t th e abov e tes t facilit y wa s operatin g i n complianc e wit h th e OEC D Principle s o f Goo d Laborator y Practice
. Thi s statemen t constitute s a Goo d Laborator y Practic e Instrumen t (as define d i n th e U KGoo d Laborator y Practic e Regulation s 1999)
.

-3© /M / t2-. Dr
. Andre w J
. Gra y Head , U KGL P Monitorin g Authorit yp . 4 3

ORIGI NID = KCK A<913 > 621-773 3 JAK E BOORE M HARCRO S CHEMICAL

S IN C 520 0 SPEAKE

R R D KANSA S CITY . K

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