### p. 1 CBI C Contro l Numbe r 

# HUNTSMA N 35639 9 •n-ti n • CTI^IJ^IM 

# RECEIVED Enrichin g live s throug h innovatio n 

# 20I3OC T1 8 AHIhi» 7 Octobe r 11 , 201 3 Documen t Contro l Offic e (DCO ) (7407M ) ed 

### &P« ceSse< i , n 

### Offic e o fPollutio n Preventio n an d Toxic s (OPPT )
_ A s 

### " e u ysHM* 0 

### * 1 

## U S EP AV C 

### 120 0 Pennsylvani a Ave. , N W Washington , D C 20460-000 1 Attention : Mr
. Joh n Schaeffe r TSC ASectio n 4 4 0 CF R 799.508 9 Docke t I D Numbe rEPA-HQ-OPPT-2009-011 2 RE : Testin g o fCertai n Hig h Productio n Volum e Chemicals ; Thir d Grou p o fChemical s 2-Oxiranemethanamine , N-[4-(2-oxiranylmethoxy)phenylJ-N-(2-oxiranylmethyl) - (CA S No
. 5026-74-4 ) Dea rMr
. Schaeffer : Enclose d pleas e fin d th e fina l report s fo rth e followin g studie s o n 2-Oxiranemethanamine , N-[4-(2 - oxiranylmethoxy)phenylj~N-(2-oxiranylmethyl) - {CA S No
. 5026-74-4) : Determinatio n o fPhysico-Chemica l Propertie s Read yBiodegradabilit y(OEC D 30 1 B ) Fres h Wate rAlga l Growt h Inhibitio n Tes t(OEC D 201 ) Daphni a magna , Reproductio n Tes t (OEC D 211 ) 96-hou rAcut e Toxicit yStud yi n Car p (OEC D 203 ) Pseudomona s Cel l Multiplicatio n Inhibitio n Tes t Huntsma n previousl ycommitte d to performin g bot h a Reproduction/Developmenta l Toxicit yScreenin g Tes t an d a Repeate d Dos e 28-Da yOra l Toxicit yStud yi n rodents , however , becaus e preliminaryresult s fro m th e Repeate d Dos e 28-Da yOra l Toxicit yStud yi n rodent s o n a surrogate , (m-(2,3-epoxypropoxy)-N N-bis(2,3-epoxypropyl)aniline , CA S # 71604-74-5 , resulte d i n a N o Observe d Advers e EffectLeve l (NOAEL ) o f5 0 mg/kg/da ybase d o n atroph yo fth e femal e reproductiv e organ s observe d a t15 0 mg/kg/day , Huntsma n determine d th e R e production/Develo p menta l Toxicit yScreenin g Tes twa s no t needed
. Th e dat a generate d fro m th e 28-da ystud yi s bein g use d a s rea d acros s fo rth e tes trul e substance , 5026-74-4
. Th e fina l reportwil l b e sen tonc e received
. Enclose d i s a lette rsen tt o th e TSC A 8(e ) Coordinato rfo ryou rreview
. Pleas e d o no t hesitat e t o contac t m e a tth e numbe rliste d belo wi fadditiona l informatio n i s required
. Sincerely ,
Lynn e Oreschnig g RegulatoryComplianc e Specialis t Huntsma n Corporatio n Phone : 281-719-302 1 Fax : 281-719-305 0 lynne_oreschnigg@huntsman.co m HUNTSMA N CORPORATIO N

1000 3 Woodloc h Fores tDrive , Th e Woodlands , Texa s 77380 , US A Tel.:+1-281-719-600 0 Fa x.:+1-281-719-641 6 www.huntsman.co m

# HUIMTSMAIV I Enrichin g lives throug h innovatio n

Apri l 30 , 201 3 Vi a Overnigh t Courie r TSC AConfidentia l Busines s Informatio n Cente r(7407M ) EP AEas t - Roo m 642 8 U S Environmenta l Protectio n Agenc y 120 1 Constitutio n Ave , N W Washington , D C 20004-330 2 Attention : TSC ASectio n 8(e ) Coordinato r Draf t Fina l Repor t o fa Repeate d Dos e 2 8 Da yOra l Toxicit yStud ywit h m-(2,3 - epoxypropoxy)-N,N-bis(2,3-epoxypropyl ) aniline , CAS #71604-74-5 . Dea rSi ro rMadam : Th e Europea n offic e o fHuntsma n Advance d Material s (Huntsman ) ha s receive d a draf tfina l repor t fro m a Repeate d Dos e 2 8 Da yOra l Toxicit yStud ywit h m-(2,3-epoxypropoxy)-N,N bis(2,3-epoxypropyi)aniline ) 

### CAS # 71604-74-5
. Thi s stud ywa s conducte d usin g a standar d OEC D 40 7 protoco l b yWi LResearc h Europ e B.V
. i n th e Netherlands
. Th e result s o fthi s repeate d dos e stud yhav e establishe d a N o Advers e Effec tLeve l (NOAEL ) a t5 0 mg/kg/day , base d o n observe d atroph yo ffemal e reproductiv e organ s a tth e nex thighes tdos e level , 15 0 mg/kg/day , Huntsma n i s submittin g thi s informatio n pursuan tt o Sectio n 8(e ) o fth e Toxi cSubstance s Contro l Ac t (TSCA)
. Huntsma n ha s no tmad e a determinatio n a s t o .whethe ra significan tris k o f injur yt o huma n healt h o rth e environmen ti s actuall ypresente d b ythes e findings
. Stud yDesign : Th e tes t substanc e wa s administered , b ygavage , t o group s o f5 mal e an d 5 femal e Wista rrat s ove r2 8 consecutiv e days
. Dos e level s use d i n thi s study , formulate d i n propylen e glycol , wer e 0 , 50 , 150 , an d 45 0 mg/kg/day , wit h th e untreate d contro l grou p receivin g onl ypropylen e glycol
. Th e followin g stud yparameter s wer e evaluated : dail yclinica l signs ; functiona l observatio n batter ya t stud ywee k 4 , bodyweight s an d foo d consumptio n determine d weekly ; clinica l patholog yan d macroscopi cevaluatio n a tstud ytermination , an d orga n weight s an d histopatholog yevaluation s o n specifie d tissues
. Stud yFindings : •N o mortalit yoccurre d durin g thi s study
. However , a poo rhealt h statu s wa s note d fo ral l animal s a t th e highes t dos e leve l o f45 0 mg/kg/day
. Althoug h th e functiona l observatio n batter y(FOB ) reveale d n o effect s o n hearing , pupi l reflex , rightin g refle xan d gri p

860 0 Goslin g Road , Th e Woodlands , Texa s 7738 1 Tel. : 1-281-719-740 0 Fax. : 1-281-719-750 0 www.huntsman.co m

### p . 3



### strength , th e moto ractivitymeasuremen tshowe d lowe ractivity{tota l movement s an d ambulations ) at 45 0 mg/kg/day
. •Animal s administere d 45 0 mg/kg/dayshowe d lowe rbod yweigh tgain s an d reduce d bodyweight s compare d t o contro l animals
. A s a resul to fth e poo rhealt h statu s o fth e animal s at 45 0 mg/kg/day , change s were observe d i n hematology , clinica l biochemistry , orga n weight s an d histopathology
. Thes e finding s ar e considere d t o b e a secondar y effect unrelate d t o an yspecifi csystemi ctoxicityofth e tes tsubstance
. •Atroph yofth e uterus , cervi xan d vagin a wer e observe d a t15 0 mg/kg/dayaccompanie d byth e macroscopicail yvisibl e reduce d siz e ofovarie s an d uteru s at 45 0 mg/kg/day ; I n male s a t 45 0 mg/kg/day , th e prostat e glan d wa s considere d t o be smal l bu t wit h norma l histology
. At thi s level , th e content s i n th e semina l vesicle s an d coagulatin g gland s were considere d to b e reduced
. Alowe rabsolut e and/o rrelative-prostat e an d semina l vesicl e weigh t wa s note d a

t th e lowes t dos e leve l o f5 0 mg/kg/day , bu tthes e observation s were not supporte d b

yan yhistologica l findings , an d are notconsidere d t o be toxicologicail y relevant
. •Th e effect s o n live ran d kidneywere note d at 45 0 mg/kg/day : centrilobula rhypertroph y i n th e liver , discoloratio n ofkidneyan d adrenal s an d enlarge d adrena l gland s i n females , wit h microscopicall yobserve d vacuolizatio n ofth e zon a fasciculat a i n th e adrena l glands
. •Effect s were observe d i n th e gastrointestina l tract an d associate d lymphati csystem , bu t thes e effect s ar

e considere d t o be secondar yfinding s relate d t o th e physica l propertie s ofth e tes t materia l an d notdu e t o an yspecifi csystemi ctoxicityo fth e tes t substance
. Stud yConclusion : Base d o n th e result s ofthi s study , th e N o Observe d Advers e Effec tLeve l (NOAEL) fo rWista r rat s expose d t o 2-Propanol , 1-[bis[3-(dimethyiamino)propyl]amino] - ove r2 8 consecutive days i s considere d to be 5 0 mg/kg/day , base d o n atroph yofth e femal e reproductive organ s observe d at 15 0 mg/kg/day
. A s always , i fI ca n provid e an yadditiona l informatio n o n th e above study , pleas e cal l m e at (281 ) 719-3017 , o rcontac t m e vi a e-mai l at : Ray_Papciak@Huntsman.com
. Regards , Raymon d J
. Papcia k Manager , Produc

t Safet y

m i TTh e us e o fthes e studies/document s fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n International LL

C is unauthorize

d an d strictlyprohibited
. 

## FINA L REPOR T

Stud yTitl e 

## DETERMINATIO N O F PHYSICO-CHEMICA L PROPERTIE S O F P-(2,3-EPOXYPROPOXY)-N,N-BIS(2,3-EPOXYPROPYL)ANILIN E Validatio n o fa n analytica l metho d Meltin g an d boilin g temperatur e Densit y Vapou rpressur e Wate rsolubilit y Flash-poin t Auto-ignitio n temperatur e Hydrolysi s a s a functio n o fp H Oxidizin g propertie s Adsorptio n coefficien t Dissociatio n constan t Viscosit y

Autho r Dr
. Ir
. E
. Baltusse n

Tes t Facilit y NOTO XB.V
. Hambakenweterin g 7 523 1 D D 's-Hertogenbosc h Th e Netherland s LaboratoryProjec t Identificatio n NOTO XProjec t 49698 1 NOTO XSubstanc e 203277/A

- Pag e 1 o f7 6 -Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) ; withou t th e expres s writte n permissio n ofHuntsma n Internationa l LLC i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 1 1 . CONTENT S 1
. CONTENT S 2 2
. STATEMEN TO F GL P COMPLIANC E 6 3
. QUALIT YASSURANC E STATEMEN T7 4
. SUMMAR Y8 5
. INTRODUCTIO N 1 0 5.1
. Prefac e 1 0 5.2
. Ai m o fth e stud y1 0 5.3
. Storag e an d retentio n o frecord s an d material s 1 0 6
. MATERIAL S 1 1 6.1
. Tes tsubstanc e 1 1 6.2
. Electroni cdat a captur e 1 1 6.3
. Lis to fdeviation s 1 2 7
. VALIDATIO N O F A N ANALYTICA LMETHO D 1 3 7.1
. Guidelin e 1 3 7.2
. Reagent s 1 3 7.3
. Performanc e o fth e stud y1 3 7.4
. Analytica l metho d 1 4 7.5
. Formula s 1 4 7.6
. Result s 1 5 7.7
. Conclusio n 1 7 7.8
. Figure s , 1 8 8
. MELTIN G AN D BOILIN G TEMPERATUR E 1 9 8.1
. Guideline s 1 9 8.2
. Performanc e o fth e tes t 1 9 8.3
. Analytica l metho d 2 0 8.4
. Interpretatio n 2 0 8.5
. Result s 2 0 8.6
. Conclusio n 2 0 8.7
. Figure s 2 1 9
. DENSIT Y2 3 9.1
. Guideline s 2 3 9.2
. Reagent s 2 3 9.3
. Performanc e o fth e stud y2 3 9.4
. Interpretatio n 2 4 9.5
. Result s 2 4 9.6
. Conclusio n 2 4 10
. VAPOU R PRESSUR E 2 5 10.1
. Guideline s 2 5 10.2
. Performanc e o fth e tes t2 5 10.3
. Analytica l metho d 2 5 10.4
. Referenc e substance s 2 6 10.5
. Interpretatio n 2 7 10.6
. Result s 2 8 10.7
. Conclusio n 2 8 10.8
. Figure s 2 9 11
. WATE R SOLUBILIT Y3 0 11.1
. Guideline s 3 0 11.2
. Reagent s 3 0 11.3
. Performanc e o fth e stud y3 0 11.4
. Analytica l metho d 3 1 11.5
. Interpretatio n 3 1 11.6
. Result s 3 2 11.7
. Conclusion 3 2

Fina l report - Pag e 2 -Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d to Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LLC i s unauthorize d an d strictl yprohibite d p-(2,3-epoxypropoxy)-N 1 N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 1 12
. FLASH-POIN T3 3 12.1
. Guideline s 3 3 12.2
. Performanc e o fth e tes t3 3 12.3
. Interpretatio n 3 3 12.4
. Result s 3 4 12.5
. Conclusion 3 4 13
. AUTO-IGNITIO N TEMPERATUR E 3 5 13.1
. Guideline s 3 5 13.2
. Performanc e o fth e tes t 3 5 13.3
. Interpretatio n 3 6 13.4
. Result s 3 6 13.5
. Conclusio n 3 7 14
. OXIDIZIN G PROPERTIE S 3 8 14.1
. Guidelin e 3 8 14.2
. Statemen t3 8 15
. HYDROLYSI S A S AFUNCTIO N O F P H 3 9 15.1
. Guidelin e 3 9 15.2
. Reagent s 3 9 15.3
. Buffe rsolution s 3 9 15.4
. Performanc e o fth e stud y3 9 

### 15.5 . Analytica

l metho d4 1 15.6
. Interpretatio n 4 1 15.7
. Result s 4 3 15.8
. Conclusio n 5 3 15.9
. Figure s 5 4 16
. ADSORPTIO N COEFFICIEN T5 7 16.1
. Guideline s '
. 5 7 16.2
. Reagent s , 5 7 16.3
. Performanc e o fth e study ' 5 7 16.4
. Analytica l metho d 5 8 16.5
. Formula s 5 9 16.6
. Result s 5 9 16.7
. Conclusio n 6 0 16.8
. Figure s 6 1 17
. DISSOCIATIO N CONSTAN T- 6 2 17.1
. Guideline s 6 2 17.2
. Reagent s 6 2 17.3
. Buffer s 6 2 17.4
. Performanc e o fth e stud y6 4 17.5
. Spectrophotometr ymetho d 6 4 17.6
. Performanc e o fth e tes t 6 4 17.7
. Formula s 6 5 17.8
. Result s 6 6 17.9
. Conclusio n 6 8 17.10
. Figure s 6 9 18
. VISCOSIT Y7 4 18.1
. Guideline s 7 4 18.2
. Performanc e o fth e tes t 7 4 18.3
. Interpretatio n 7 4 ' 18.4
. Result s 7 5 18.5
. Conclusion 7 5

Fina l report - Pag e 3 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authonzatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 1 TABLE S Tabl e 1 Statistica l parameter s o fth e calibratio n curv e 1 5 Tabl e 2 Accurac ysample s 1 6 Tabl e 3 Stabilit yo fth e analytica l syste m an d en d solution s 1 6 Tabl e 4 Store d accurac ysample s 1 7 Tabl e 5 Densit yo fth e tes tsubstanc e 2 4 Tabl e 6 Result s o fth e isotherma l TG Aanalysi s 2 8 Tabl e 7 Vapou rpressur e o fth e tes tsubstanc e 2 8 Tabl e 8 Mai n stud y- wate rsolubilit yo fth e tes t substanc e 3 2 Tabl e 9 Mai n stud y- flash-pointo fth e tes tsubstanc e 3 4 Tabl e 1 0 Mai n study-auto-ignitio n temperatur e o fth e tes tsubstanc e 3 7 Tabl e 1 1 Preliminarytes t - hydrolysi s o fth e tes tsubstanc e a t p H 4 , p H 7 an d p H 9 "
. 4 3 Tabl e 1 2 Recoverie s 4 3 Tabl e 1 3 Mai n tes t - hydrolysi s o fth e tes t substanc e a t20° C 4 4 Tabl e 1 4 Mai n tes t- hydrolysi s o fth e tes t substanc e a t40° C 4 5 Tabl e 1 5 Mai n tes t- hydrolysi s o fth e tes t substanc e at50° C 4 5 Tabl e 1 6 Recoverie s 4 6 Tabl e 1 7 Statistica l parameter s o fth e regressio n curve s 4 6 Tabl e 1 8 Rat e constant s (k 0 bs ) an d half-lif e tim e (t M ) 4 6 Tabl e 1 9 Mai n tes t - hydrolysi s o fth e tes tsubstanc e a t20° C 4 7 Tabl e 2 0 Mai n tes t - hydrolysi s o fth e tes t substanc e a t40° C 4 8 Tabl e 2 1 Mai n tes t - hydrolysi s o fth e tes t substanc e a t50° C 4 8 Tabl e 2 2 Recoverie s , 4 9 Tabl e 2 3 Statistica l parameter s o fth e regressio n curve s 4 9 Tabl e 2 4 Rat e constant s (k obs ) an d half-lif e tim e (t% ) 4 9 Tabl e 2 5 Mai n tes t - hydrolysi s o fth e tes tsubstanc e a t20° C 5 0 Tabl e 2 6 Mai n tes t- hydrolysi s o fth e tes tsubstanc e a t40° C 5 1 Tabl e 2 7 Mai n tes t- hydrolysi s o fth e tes tsubstanc e a t50° C 5 1 Tabl e 2 8 Recoverie s , 5 2 Tabl e 2 9 Statistica l parameter s o fth e regressio n curve s 5 2 Tabl e 3 0 Rat e constant s (k obs ) an d half-lif e tim e (t % ) 5 2 Tabl e 3 1 K o co fth e tes t substanc e 6 0 Tabl e 3 2 Actua l p H value s an d optica l absorbance s a t24 9 n m 6 7 Tabl e 3 3 Optica l absorbance s a t 24 9 n m 6 8 Tabl e 3 4 Viscosit yo fth e tes t substanc e 7 5

Fina l report - Pag e 4 -Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
.
FIGURE S Figur e 1 HPLC-U Vchromatogra m o fa 2.0 0 mg/ l tes t substanc e solutio n [res
. id
. 1509 ] 1 8 Figur e 2 HPLC-U Vchromatogra m o fth e blan k accurac ysampl e [res
. id
. 1529 ] 1 8 Figur e 3 Regressio n line : respons e o fth e tes t substanc e a s a functio n o fconcentratio n [cal
. curv e

id
. 1497 ] 1 8 Figur e 4 TG Acurv e o fth e tes tsubstanc e 2 1 Figur e 5 DS C curv e o fth e tes tsubstanc e obtaine d wit h th e first experimen t : 2 1 Figur e 6 DS C curv e o fth e tes t substanc e obtaine d wit h th e secon d experimen t 2 2 Figur e 7 Weigh tlos s curv e o fth e tes t substanc e 2 9 Figur e 8 Plo t o flo g P To fth e tes tsubstanc e versu s 1 U 2 9 Figur e 9 Plo t o fth e logarithm s o frelativ e concentratio n agains t tim e a t 20° C [top] , 40° C [middle ]

an d 50° C -{bottom ] atpH 4 5 4 Figur e 1 0 Plo t o fth e logarithm s o frelativ e concentratio n agains t tim e at 20° C [top] , 40° C [middle ]

an d 50° C [bottom ] atpH 7 5 5 Figur e 1 1 Plo to fth e logarithm s o frelativ e concentratio n agains t tim e at 20° C [top] , 40° C [middle ]

an d 50° C [bottom ] a

tpH 9 5 6 Figur e 1 2 HPL C chromatogram s o fth e 14. 5 mg/ l tes t solutio n [top ; res . id
. 1571 ] an d correspondin g

blan k [bottom ; res . id
. 1572 ] 6 1 Figur e 1 3 Th e regressio n lin e o fth e referenc e substances : lo g k ' versu s lo g K o c6 1 Figur e 1 4 Absorptio n spectru m o fa 2 0 mg/ l solutio n o ftes tsubstanc e i n buffe rp H 0 6 9 Figur e 1 5 Absorptio n spectru m o fa 2 0 mg/ l solutio n o ftes t substanc e i n buffe rp H 1 6 9 Figur e 1 6 Absorptio n spectru m o fa 2 0 mg/ l solutio n o ftes t substanc e i n buffe rp H 2 6 9 Figur e 1 7 Absorptio n spectru m o fa 2 0 mg/ l solutio n o ftes t substanc e i n buffe rp H 3 7 0 Figur e 1 8 Absorptio n spectru m o fa 2 0 mg/ l solutio n o ftes t substanc e i n buffe rp H 4 7 0 Figur e 1 9 Absorptio n spectru m o fa 2 0 mg/ I solutio n o ftes tsubstanc e i n buffe rp H 5 7 0 Figur e 2 0 Absorptio n spectru m o fa 2 0 mg/ l solutio n o ftes t substanc e i n buffe rp H 6 7 1 Figur e 2 1 Absorptio n spectru m o fa 2 0 mg/ l solutio n o ftes tsubstanc e i n buffe rp H 7 7 1 Figur e 2 2 Absorptio n spectru m o fa 2 0 mg/ l solutio n o ftes t substanc e i n buffe rp H 8 7 1 Figur e 2 3 Absorptio n spectru m o fa 2 0 mg/ l solutio n o ftes tsubstanc e i n buffe rp H 9 7 2 Figur e 2 4 Absorptio n spectru m o fa 2 0 mg/ l solutio n o ftes tsubstanc e i n buffe rp H 1 0 7 2 Figur e 2 5 Absorptio n spectru m o fa 2 0 mg/ l solutio n o ftes t substanc e i n buffe rp H 1 1 7 2 Figur e 2 6 Absorptio n spectru m o fa 2 0 mg/ l solutio n o ftes tsubstanc e i n buffe rp H 1 2 7 3 Figur e 2 7 Absorptio n spectru m o fa 2 0 mg/ l solutio n o ftes t substanc e i n buffe rp H 1 3 7 3 APPENDICE S Appendi x1 GL P certificat e 7 6

Fina l report - Pag e 5 -Th e us e ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no tlimite d to Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 1 2
. STATEMEN TO F GL P COMPLIANC E NOTO XB.V. , 's-Hertogenbosch , Th e Netherland s Th e stud ydescribe d i n thi s report ha s bee n correctl yreporte d an d wa s conducte d i n complianc e with : Th e Organizatio n fo rEconomi cCo-operatio n an d Developmen t(OECD) , OEC D Principle s o n Goo d LaboratoryPractic e (GLP ) (a s revise d i n 1997) , ENV/MC/CHE M (98 ) 17
. Th e sponso ri s responsibl e fo rGoo d LaboratoryPractic e (GLP ) complianc e foral l tes t substanc e informatio n unles s determine d b yNOTOX
.

Dr
. Ir
. E
. Baltusse n Dr
. Ir
. T.H.M
. Noi j Stud yDirecto rHea d o fChemistry

Fina l report - Pag e 6 -p . 1 0 Th e use o

fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d to Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProject 49698 1 3 . QUALIT YASSURANC E STATEMEN T NOTO XB.V. , 's-Hertogenbosch , Th e Netherland s Thi s repor

t wa s inspecte d b

yth e NOTO XQualityAssuranc e Uni t (QAU ) t o confir m tha

t th e method s an d result s accuratel yan d completel yreflect th e ra wdata . Durin g th e on-sit e proces s inspections , procedure s applicabl e t o thi s type o

fstud ywer e inspected . Th e date s o

fQualit yAssuranc e inspection s ar e give n below .
Typ e o f Inspection s Stud y Phase/Proces s

Protoco l Protoco l Amendmen t 0 1 Analysi s Observation s /measurement s chemica l propertie s Repor t Protoco l Amendmen t 0 2 Star t Inspectio n dat e 19-Aug-1 1 15-Nov-1 1 17~Nov-1 1 17-Nov-1 1 17~Dec-1 1 11-Jan~1 2 En d

Inspectio n dat e 19-Aug-1 1 15-Nov-1 1 17-Nov-1 1 17 - Nov-1 1 18 - Dec-1 1 11-Jan-1 2 Reportin g

dat e 19-Aug-1 1 15-Nov-1 1 17-NOV-1 1 17-Nov-1 1 19-Dec-1 1 11-Jan-1 2 Analytica l an d physica l Proces s chemistr y

Tes t Substanc e Handlin g Obs e rvations/Meas u rem e nts 08-Aug-1 1 16-Aug-1 1 16-Aug-1 1
Analytica l an d physica l chemistr y31-Oct-1 1 10-Nov-1 1 10-Nov-1 1 Tes t Substanc e Handlin g Observations/Measurement s

NOTO XB.V . C.J . Mitchel l B.Sc . Hea d o fQualit yAssuranc e

Fina l repor

t - Pag e 7 -Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substances (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProject 49698 1 4 . SUMMAR Y Th e result s o fth e physico-chemica l propertie s o fth e tes t substanc e ar e give n below
. Paramete r Guideline(s ) Resul t Commen t Analytica l metho d Sanc o 302 9 successfull yvalidate d Meltin g temperatur e E C A. 1 OEC D 10 2 -39° C (234K ) Glas s transitio n
Boilin g temperatur e E C A. 2 OEC D 10 3 n o boilin g temperatur e reactio n and/o r decompositio n o fth e tes t substanc e a t > 250° C ( > 523K ) wa s observe d Densit y E C A. 3 OEC D 10 9 D l ° :1.2 2 Vapou rpressur e E C A. 4 OEC D 10 4 20° C 5. 5 x10' 7 P a = 4. 1 x10' 9m m H g 25° C 1. 3 x10 * P a = 9. 9 x10" 9m m H g isotherma l TG A

effusio n metho d

Wate rsolubilit y E C A. 6 OEC D 10 5 3.3 4 g/ l a t 20° C byth e flas k metho d Flash-poin t E C A. 9 U N p . 32.4. 1 IS O 271 9 AST M D 9 3 228° C Pensky-Marten s close d cu p metho d
Auto-ignitio n temperatur e E C A.1 5 DI N 5179 4 I E C 79- 4 410 a C a t 1012.6-1013. 9 hP a
Oxidizin g propertie s E C A.2 1 U N 0. 2 no toxidizin g statemen t Hydrolysi s a s a functio n o f P H E C C. 7 OEC D 11 1 p H 4 : t « = 2. 2 day s

p H 7 : X

%- 2. 3 day s p H 9 : tj 4 = 2. 6 day s a t 25° C

Fina l report - Pag e 8 -Th e us e o fthes e studies/document s fo rregulatoryo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n International LL

C i s unauthorize

d an d strictlyprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProject 49698 1
Adsorptio n coefficien t E C C.1 9 OEC D 12 1 lo g Ko C : 1.9 3 HPL C metho d base d o n soil-adsorption referenc e dat a Dissociatio n constan t OEC D 11 2 pK g valu e : 3.2 5 (basic ) spectr o photometri c metho d Viscosit y OEC D 11 4 IS O 310 4 AST M D 445-0 9 20°C : 840. 6 mm 2 / s

40°C : 138. 9 mm 2 / s

Fina l report - Pag e 9 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t notlimite d to Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibite d p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)ariilin e NOTO XProjec t 49698 1 5 . INTRODUCTIO N 5.1 . Prefac e Sponso r Huntsma n Advance d Material s Gmb H Klybeckstrass e 20 0 405 7 BASE L Switzerlan d Stud yMonito r Dr
. H
. Wilber s Tes tFacility NOTO XB.V
. Hambakenweterin g 7 523 1 D D 's-Hertogenbosc h Th e Netherland s Stud yDirecto r Dr
. Ir
. E , Baltusse n Stud yPla n Start Completio n 2 4 Augus t 201 1

0 1 Decembe r201 1 5.2
. Ai m o fth e stud y Th e ai m o fth e stud ywa s t o determin e th e followin g physico-chemica l propertie s fo r p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline :

•Validatio n o fa n analytica l metho d •Meltin g an d boilin g temperatur e •Densit y •Vapou rpressur e •Watersolubilit y •Flash-poin t •Auto-ignitio n temperatur e •Hydrolysi s a s a functio n o fp H •Oxidizin g propertie s •Adsorptio n coefficien t •Dissociatio n constan t •Viscosit y 5.3 . Storag e an d retentio n o frecord s an d material s Record s an d material s pertainin g t o th e stud yincludin g protocol , ra wdata , specimen s (excep t specimen s requirin g refrigeratio n o rfreezing ) an d th e fina l reportar e retaine d i n th e NOTO Xarchive s fo ra perio d o fa tleas t 1 0 year s afte rfinalizatio n o fth e report . Afte rthi s period , th e sponso rwil l b e contacte d t o determin e ho wth e record s an d material s shoul d b e handled , NOTO Xwil l retai n informatio n concernin g decision s made
. Thos e specimen s requirin g refrigeratio n o rfreezin g wil l b e retaine d b yNOTO Xfo ra s lon g a s th e qualit yo fth e specimen s permit s evaluatio n bu tn o longe rthan thre e months afte rfinalizatio n o fth e report . NOTO Xwil l retai n a tes tsubstanc e sampl e unti l th e expirydate , bu t n o longe rtha n 1 0 year s afte r finalizatio n o fth e report . Afte rthi s perio d th e sampl e wil l b e destroyed
.

Fina l report - Pag e 1 0 -



### i rTh e us e o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e 6 . MATERIAL S 6.1 . Tes t substanc e 6.1.1 . Tes t substanc e informatio n NOTO XProjec t49698 1

Identificatio n Structur e p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e O
.

Molecula rformul a Molecula rweigh t CA S Numbe r E C Numbe r Descriptio n Batc h Purity Tes tsubstanc e storag e Stabilit yunde rstorag e condition s Expirydat e C 1 5 H i g N0 4 277. 3 

### 5026-74-4 225-716- 2 Yello wliqui d AA0037340 0 

### 94.33 % I n refrigerato r(2-8°C ) i n th e dar k Stabl e 2 8 February201 3 6.1.2 . Stud yspecifi c tes t substanc e informatio n Ther e wa s n o stud yspecifi c tes t substanc e informatio n necessar yfo rthi s study . 6.2 . Electroni c dat a captur e Syste m control , dat a acquisitio n an d dat a processin g wer e performe d usin g th e followin g programmes :

- Car yWinU Vpharm a versio n 3. 1 (Varian , Mulgrave , Australia )

- Empowe rversio n 7.0 0 (Waters , Milford , MA , USA )

- Advantag e Integrit ySoftwar e versio n 3. 0 (TAInstruments , Ne wCastle , DE , USA ) Temperatur e and/o rrelativ e humidit yand/o rpressur e durin g sampl e storag e and/o rperformanc e o f th e studie s wer e monitore d continuousl yusin g th e followin g programme :

- REE S Centro n Environmenta l Monitorin g syste m versio n SQ L2. 0 (REE S Scientific , Trenton , NJ , USA )

Fina l report - Pag e 1 1 -Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substances (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProject 49698 1
6.3 . Lis t o fdeviation s 6.3.1 . Lis t o fprotoco l deviation s 1
. Chapte r1 3 "Hydrolysi s a s a functio n ofpH" , paragrap h 13. 4 "Performanc e o fth e study" : Durin g th e mai n studie s - Tie r2 a t al l p H value s a t20°C , th e temperatur e wa s controlle d withi n ±0.6° C (criterio n wa s ±0.5°C)
. Evaluation ; Th e temperatur e range s wer e slightl yhighe rtha n th e criterion
. Thi s wa s considere d no t t o b e o fsignificantlyinfluenc e o n th e determine d half-lif e tim e o fth e tes t substanc e a t th e temperatures . Th e stud yintegrit ywa s no t adversel yaffecte d b yth e deviations
. 6.3.2 . Lis t o fstandar d operatin g procedure s deviation s An ydeviation s fro m standar d operatin g procedure s (SOPs ) wer e evaluate d an d file d i n th e stud yfile
. Ther e wer e n o deviation s fro m SOP s tha taffecte d th e integrit yo fth e study
.

Fina l report - Pag e 1 2 -p . 1 6 Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d to Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 1 7 . VALIDATIO N O F A N ANALYTICA L METHO D 7.1
. Guidelin e Th e stud ywa s base d o n th e followin g guideline : Europea n Commission : Guidanc e fo rGeneratin g an d Reportin g Method s o fAnalysi s i n Support o f Pre-registratio n Dat a Requirement s fo rAnne xI I (Part A , sectio n 4 ) an d Anne xII I {Part A , sectio n 5 ) o f Directiv e 91/414 , SANCO/3029/9 9 rev . 4 (11/07/00)
. 7.2
. Reagent s Wate rTa p wate rpurifie d b ya Milli- Q wate rpurificatio n syste m (Millipore , Bedford , MA , USA ) Acetonitril e Biosoive , Valkenswaard , Th e Netherlands
. Al l reagent s wer e o fanalytica l grade , unles s specifie d otherwise
. 7.3
. Performanc e o fth e stud y Ahig h performanc e liqui d chromatographi cmetho d wit h spectrophotometri cdetectio n (HPLC-UV ) fo r th e quantitativ e analysi s o fth e tes tsubstanc e i n wate rwa s developed
. Validatio n o fth e analytica l metho d wa s performe d fo rth e followin g parameters : Specificit y Ates tsubstanc e solutio n an d blan k accurac ysampl e wer e analyse d b ysingl e injection
. Th e analytica l metho d wa s foun d t o b e specifi ci fth e blan k chromatogra m showe d n o respons e fo rth e tes t substanc e o ra respons e o f< 30 % o fth e limi to fquantification
. Linearit y Calibratio n solution s wer e analyse d i n duplicate
. Th e respons e o fth e calibratio n solution s wa s correlate d wit h concentratio n usin g regressio n analysi s wit h a 1/concentration 2weightin g factor
. A calibratio n curv e wit h a coefficien to fcorrelatio n (r) o f> 0.9 9 an d bac k calculate d accuracie s o fth e calibratio n solution s i n th e rang e 85-115 % wa s accepted
. Accurac yan d repeatabilit y Accurac ysample s wer e analyse d b ysingl e injectio n int o th e analytica l system . Th e analytica l metho d wa s considere d applicabl e fo rth e determinatio n o fth e tes t substanc e i fth e mea n accurac ywa s i n th e rang e 70-110 % an d th e coefficien to fvariatio n wa s £20%
. Limi to fquantificatio n Th e limi to fquantificatio n (LOQ ) i s define d a s th e lowes tconcentratio n leve l a t whic h a n accurac yi n th e rang e 70-110 % an d a repeatabilit yo f< 20 % i s demonstrated
. Th e LO Q wa s obtaine d fro m th e dat a o fth e accuracy - an d repeatabilit ytest . Stabilit yo fth e analytica l syste m an d en d solution s Calibratio n solution s wer e injecte d throughou t th e validatio n sequenc e includin g th e beginnin g an d end
. Th e analytica l syste m and/o ren d solution s wer e foun d t o b e stabl e i fth e coefficien t o fvariatio n o n th e response s o fth e solution s wa s £20%
. Stabilit yo fstoc k

solution s Stoc k solution s o fth e tes tsubstanc e wer e store d a t roo m temperature , fo ra t leas t 1 2 hours
. Additiona l calibratio n solution s wer e prepare d an d analyse d b ysingl e injection
. Th e stoc k solution s wer e foun d t o b e stabl e i fth e coefficien to fvariatio n o n th e respons e factor s o fth e calibratio n solution s prepare d wit h fres h an d store d stoc k

solution s wa s £10%
.

Fina l report - Pag e 13 -Th e us e o fthes e studies/documents fo rregulator yo rregistration'purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substances (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3- e poxypro poxy)-N,N-bis(2,3- e poxypropyl ) anilin e NOTO XProjec t 49698 1 Storag e stabilit yo fsample s Additiona l accurac ysample s wer e prepare d an d store d i n th e freeze rfo ra t leas t 1 6 hours
. O n th e da y o fanalysis , th e sample s wer e defroste d atroo m temperatur e an d treate d identicall ya s freshl y analyse d accurac ysamples
. Th e store d sample s wer e foun d t o b e stabl e i fth e mea n accurac ywa s i n th e rang e 70-110%
. 7.4 . Analytica l metho d 7.4.1 . Analytica l condition s Instrumen t Detecto r Colum n Colum n temperatur e Injectio n volum e Mobil e phas e Flo w U Vdetectio n 7.4.2 . Preparatio n o fsolution s Stoc k an d spikin g solution s Stoc k solution s o fth e tes tsubstanc e wer e prepare d i n acetonitril e a t concentration s o f171 2 - 242 0 mg/l
. Spikin g solution s wer e mad e u p fro m a stoc k solutio n and/o rdilution s o fthi s solution
. Th e solven t o f th e spikin g solution s wa s acetonitrile
. Calibratio n solution s Si xcalibratio n solution s i n th e concentratio n rang e o f0.0 4 - 2 mg/ l wer e prepare d fro m tw o stoc k solutions
. Th e en d solutio n o fth e calibratio n solution s wa s 30/7 0 (v/v ) acetonitrile/water
. Accurac ysample s 2 m l wate rwa s spike d wit h th e tes tsubstanc e a t a targe t concentratio n o f0. 1 o r10 0 mg/l
. Th e accurac ysample s wer e dilute d i n a 1: 1 (v;v ) rati o wit h 60/4 0 (v/v ) acetonitrile/wate ran d analysed
. I f necessary , th e sample s wer e furthe rdilute d wit h 30/7 0 (v/v ) acetonitrile/wate rt o obtai n concentration s withi n th e calibratio n range . Th e blan k

accurac ysampl e wa s prepare d an d treate d simila rt o th e accurac ysamples
. 7.5 . Formula s Respons e (R ) Pea k are a o fth e tes t substanc e [units ] Respons e facto r(R f ) R f= — C N where : C N = nomina l concentratio n [mg/l ] Calibratio n curv e R = a C N + b where : a = slop e [unit s xl/mg ] b = intercep t [units] Allianc e Separatio n Modul e 269 5 (Waters , Milford , MA , USA ) Dua l XAbsorbanc e Detecto r248 7 (Waters ) SymmetryShiel d RP-18 , 10 0 m m x4. 6 m m i.d. , d p = 3. 5 ur n (Waters ) 40°C±1° C 10 0 p i 30/7 0 (v/v ) acetonitrile/wate r 1. 0 ml/mi n 25 3 n m

Fina l report - Pag e 1 4 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t49698 1 Analyse d concentratio n (C A ) (R-b ) x d [mg/I ] a where : d = ( dilutio n facto r Accurac y x10 0 [%]
7.6 . Result s 7.6.1 . Specificit y Chromatogram s o fa tes t substanc e solutio n an d th e blan k accurac ysampl e ar e show n i n Figur e 1 an d Figur e 2 , respectively
. Severa l smal l peak s an d on e larg e pea k wer e observe d i n th e chromatogra m o fth e tes t substanc e solution
. I t wa s assume d tha t th e larg e pea k derive s fro m th e majo rcomponen t o fth e tes t substance , wherea s th e smal l peak s deriv e fro m impurities
. Th e pea k are a o fth e majo rcomponen t wa s use d a s respons e i n th e calculations
. Th e chromatogra m o fth e blan k sampl e showe d n o pea k a t th e retentio n tim e o fth e tes t substance
. Sinc e n o interference s wer e detected , th e specificit yrequirement s wer e me t an d th e analytica l metho d wa s foun d t o b e specifi cfo rth e tes tsubstance
. 7.6.2 . Linearit y Th e calibratio n lin e wa s constructe d usin g al l dat a points
. Figur e 3 illustrate s th e calibratio n curv e an d Tabl e 1 show s th e statistica l parameters . Ther e wa s a linea rrelationshi p betwee n respons e an d tes t substanc e concentratio n i n th e rang e o f0.040 1 - 2.0 0 mg/ l (i n en d solution)
. Sinc e th e coefficien t o f correlatio n (r) wa s > 0.9 9 an d th e bac k calculate d accuracie s o fth e dat a point s wer e i n th e rang e 85 - 115 % th e calibratio n lin e wa s accepted
. Tabl e 1 Statistica l parameter s o fth e calibratio n curv e Slop e Intercep t Weightin g facto r r 2.0 3 x10 5 -1.7 5 x10 3 1/concentration 2 0.99 7

Fina l report - Pag e 15 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substances (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 1 7.6.3 . Accurac yan d repeatabilit y Th e result s o fth e accurac ysample s ar e give n i n Tabl e 2
. Sinc e th e mea n accurac ya t eac h concentratio n leve l fel l i n th e criterio n 85-115 % an d th e coefficien t o fvariatio n wa s £20 % th e analytica l metho d wa s accepte d fo rth e analysi s o fth e tes tsubstanc e i n wate ri n th e targe t concentratio n rang e o f0. 1 -10 0 mg/l
. Tabl e 2 Accurac ysample s Targe t concentratio n [mg/l l Nominal

concentratio n [mg/l l Analyse d

concentratio n 

## rmg/ n Accurac y



## r% i Mea n

accurac y [% 1 Coefficien t o f

variatio n [% l 0. 1 0.099 5 0.10 9 10 9 10 7 2. 4

0.099 5 0.10 4 10 4 0.099 5 0.10 3 10 4 0.099 5 0.10 8 10 8 0.099 5 0.10 7 10 8 10 0 99. 5 10 5 10 6 10 1 3. 3

99. 5 98. 7 9 9 99. 5 97. 7 9 8 99. 5 10 4 10 4 99. 5 99. 4 10 0
7.6.4 . Limi t o fquantificatio n Th e limi to fquantificatio n (LOQ ) wa s assesse d at0. 1 mg/ l i n water . 7.6.5 . Stabilit yofth e analytica l syste m an d en d solution s Th e result s o fth e stabilit yo fth e analytica l syste m an d en d solution s ar e give n i n Tabl e 3
. Sinc e th e coefficien t o fvariatio n a tboth concentratio n level s wa s < 20 % th e analytica l syste m an d en d solution s wer e stabl e ove ra t leas t a 26. 7 hou rtim e interval
. Tabl e 3 Stabilit yo fth e analytica l syste m an d en d solution s Nominal concentratio n Elapse d tim e Coefficien t o fvariatio n [mq/ n [hours l 

### f%l 0.040 1 28. 3 1 7 (n=6 ) 0.090 1 28. 1 1 4 (n=6 ) 2.0 0 26. 7 6. 2 (n=6 )
7.6.6 . Stabilit yo fstoc k solution s Th e coefficien to fvariatio n o n th e respons e factor s o fth e calibratio n solution s prepare d wit h fres h an d store d stoc k

solution s wa s 2.2%
. Sinc e th e valu e wa s £10 % th e stoc k solution s wer e stabl e whe n store d a t roo m temperatur e fo ra t leas t1 6 days
.

Fina l report - Pag e 1 6 -p . 2 0 Th e use o

fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d to Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProject 49698 1 7.6.7 . Storag e stabilit yo fsample s Th e result s o fth e storag e stabilit yo fth e tes tsubstanc e i n th e freeze rar e give n i n Tabl e 4
. Sinc e th e mea n accurac yo fth e froze n sample s fel l i n th e criterio n 70-110 % th e sample s wer e stabl e whe n store d i n th e freezer
. Tabl e 4 Store d accurac ysample s Targe t concentratio n rmg/l l Nomina l

concentratio n fmg/l l Analyse d

concentratio n 

### fmg/l l Accurac y

[% l Mea n accurac y



## r% i 0. 1 0.099 5 0.076 6 7 7 7 7

0.099 5 0.076 1 7 6 10 0 99. 5 80. 8 8 1 8 0

99. 5 78. 3 7 9

7.7
. Conclusio n Th e analytica l metho d wa s validate d fo rth e followin g parameters : Specificit y specifi c Linearit y r= 0.99 7 Accurac y 10 1 an d 107 % Repeatabilit y 2. 4 an d 3.3 % Limi to fquantificatio n 0. 1 mg/ l Stabilit yanalytica l syste m an d en d solution s stabl e Stabilit ystoc k

solution s stabl e Storag e stabilit y stabl e

Fina l report - Pag e 1 7 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substances (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p . 2 1
p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e 7.8 . Figure s NOTO XProjec t 49698 1

OOK h o.cc a 0.038 ' 0 024 ' 0.0) 2 0.03 0 0.033 ' 0.03 4 0.022 '

, 0.020 '
' 0 01 8 0.018 ' 0.01 4 0.01 2 0.010 ' 0.00 8 0.00 9 0.004 ' 0,00 2 0.0 W- - • o.oo o n

0JM0- ] 0.035' 0.036 ' 0.034 ' 0.032 ' 0.030
- 0.02 5 OJK O 002 4 0.02 2

, 0.020 '
' 0 01S ' 0.018 ' 0.01 4 0.012 ' 0.010 ' 0.008 ' 0.0O9 ' 0.00 4 0.00 2 O.OO J 1.0

0 ' ' 'i.bo ' " ' 2.00ihaih o t$o'4.00 ' ' ' '4.50' 'sh e ' ' s.b o 's.io ' ' 'eg

o ' ih a' '?» ' BO O ' ' MO ' ' ' V60 ' ' ' b.io ' ' ' iic o Mnu(4 f Figur e 1 HPLC-U Vchromatogra m ofa 2.0 0 mg/l tes t substanc e solutio n [res . id . 1509] .

i.oo' ' ' 'tha ' ' '2.00:.feo'

MO' 1S04,00'

Vto '5.00'

's.w' ' eh Mnuto s 7.00 7J5 0 (.0 0 5.50 8.60 »J o ' ' io!o0 0.00 os o Figur e 2 HPLC-U Vchromatogra m o fth e blan k

accurac ysampl e [res . id . 1529] .
3C0C001 4SOCC 0 400000- 3W0OO30CO0O -

• 2OXO0
- 150KO
1CO0OO
9000 0

IO

O ' b.i o 0.2

0 ' oh o o. W' ' b.io ' ' ' o. w ' ' bjo ' ' ' oh o ' ' bi o 1^

0 n o ' 1.20

' ' ' " 1 i o i. « ' i.bo 'ie o ' 1.7 0 ' 1.80

' 'i.oo ' 2.0

0 Aitaurt Figur e 3 Regressio n line : respons e o fth e tes t substanc e a s a functio n o fconcentratio n [cal .

curv e id . 1497] .
Fina l report - Pag e 1 8 -Th e us e ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 1 8 . MELTIN G AN D BOILIN G TEMPERATUR E 8.1 . Guideline s Organizatio n fo rEconomi cCo-operatio n an d Developmen t(OECD) , OEC D Guideline s fo rth e Testin g o fChemical s no . 102 : "Meltin g Poin t / Meltin g Range" , Jul y27 , 1995
. Europea n Communit y(EC) , E C no . 440/2008 , Par tA : Method s fo rth e Determinatio n o fPhysico Chemica l Properties , Guidelin e A.1 : "Melting/Freezin g Temperature" , Officia l Journa l o fth e Europea n Unio n no . L142 , Ma y31 , 2008
. Organizatio n fo rEconomi cCo-operatio n an d Developmen t (OECD) , OEC D Guideline s fo rth e Testin g o fChemical s no . 103 : "Boilin g Point" , Jul y27 , 1995
. Europea n Communit y(EC) , E C no . 440/2008 , Part A : Method s fo rth e Determinatio n o fPhysico Chemica l Properties , Guidelin e A.2 : "Boilin g Temperature" , Officia l Journa l o fth e Europea n Unio n no . L142 , Ma y31 , 2008
. 8.2 . Performanc e o fth e tes t Th e meltin g an d boilin g temperatur e o fth e tes t substanc e wer e determine d usin g differentia l scannin g calorimetry(DSC)
. Thi s techniqu e record s th e differenc e i n hea tflo wt o tw o crucibles , on e fille d wit h tes tsubstanc e an d th e othe rfille d wit h a n inertreferenc e material , whil e thes e crucible s ar e subjecte d t o a controlle d temperatur e programme . Whe n th e tes t substanc e undergoe s a transition , suc h a s meltin g o revaporation , i t i s indicate d b ya deviatio n fro m th e bas e lin e o fth e heat-flo wrecord
. Becaus e meltin g an d evaporatio n ar e processe s i n whic h hea ti s consumed , fo rthes e processe s th e deviatio n fro m th e bas e lin e i s i n th e endothermi cdirection
. Th e meltin g temperatur e o rboilin g temperatur e (i fany ) ofth e tes t substanc e i s evaluate d fro m th e DS C curv e a s th e extrapolate d onse t temperatur e o fth e endothermi cmeltin g o revaporatio n peak
. A s a safet yprecautio n fo rth e DS C cel l a preliminarytes twa s performe d usin g a thermogravimetri c analyze r(TGA)
. Preliminarytes t 6.6 9 m g tes t substanc e wa s heate d wit h a rat e o f20°C/minut e fro m 25° C t o 432°C
. A t thi s poin t 70 % weigh tlos s o fth e tes tsubstanc e wa s observed
. Afte rth e experiment , th e sampl e wa s coole d t o 50° C an d th e consistenc yofth e tes t substanc e wa s determined
. Mai n stud y A n overvie wwit h th e experimenta l condition s o fth e mai n stud yi s give n beiow
. Experimen t Firs t Secon d Sampl e amoun t(mg ) 6.9 7 5.9 7 Initia l temperatur e (°C ) 2 5 2 5 Rat e (°C/minute ) 5 5 Temperatur e 2 (*C ) -9 0 -9 0 Rat e (°C/minute ) 2 0 2 0 En d temperatur e (°C ) 34 5 5 0 Th e experiment s wer e performe d unde ra flo wo fnitroge n an d at atmospheri cpressure .

Fina l report - Pag e 1 9 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d to Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 1 8.3 . Analytica l metho d Instrumen t Q10 0 differentia l scannin g calorimete r(TAInstruments , Ne w Castle , DE , USA ) aluminum ; close d wit h a li d i n whic h a smal l hol e wa s drille d empt ysampl e containe r Sampl e containe r Referenc e Preliminarytes t Instrumen t Sampl e containe r Q5 0 thermogravimetri canalyze r(TGA ) (TAInstruments ) aluminum ; close d wit h a li d i n whic h a smal l hol e wa s drille d 8.4 . Interpretatio n Th e meltin g temperatur e i s define d a s th e temperatur e a twhic h th e phas e transitio h fro m soli d t o liqui d stat e take s plac e a t norma l atmospheri cpressure . Thi s temperatur e ideall ycorrespond s t o th e solidificatio n o rfreezin g temperature . Th e standar d boilin g temperatur e i s define d a s th e temperatur e a t whic h th e vapou rpressur e o fa liqui d i s th e sam e a s th e standar d pressur e (1013.2 5 hPa)
. 8.5 . Result s 8.5.1 . Preliminar ytes t Th e TGA-curv e o fth e tes t substanc e i s show n i n Figur e 4
. Fro m 325° C upward s th e weigh t o fth e sampl e decrease d significantly
. At345° C th e sampl e weigh tha d decrease d b y25%
. Afte rth e experiment , blac k residu e wa s observe d o n th e li d o fth e sampl e containe ran d a blac k molte n residue remaine d i n th e sampl e containe r(origina l colour : yellow)
. Th e chang e o fth e colou r indicate d reactio n and/o rdecompositio n ofth e tes tsubstance
. 8.5.2 . Mai n stud y Th e DSC-curv e obtaine d wit h th e firs t experimen ti s show n i n Figur e 5
. Durin g cooling , a glas s transitio n wa s observe d betwee n -40° C an d -60° C (result s ar e archive d i n th e ra wdata)
. Durin g heating , a glas s transitio n betwee n -50° C an d -25° C wa s observe d (se e Figur e 5)
. Th e inflectio n poin to fth e glas s transitio n (meltin g point ) o fth e tes t substanc e wa s -39.42°C
. A n exothermi cpea k

wa s observe d fro m 250'C
. Th e effec twa s du e t o reactio n and/o rdecompositio n o f th e tes t substance
. Afte rth e experiment , dar k brow n residu e wa s observe d o n th e li d o fth e sampl e containe ran d a dar k

brow n molte n residu e remaine d i n th e sampl e container
. Th e chang e o fth e colou rindicate d reactio n and/o rdecompositio n o fth e tes t substance
. Th e secon d experimen t wa s performe d t o determin e th e duplicat e glas s transitio n temperatur e o fth e tes t substance
. Figur e 6 show s th e DSC-curv e obtained
. Simila rresult s t o th e firs t experimen t wer e obtained
. Th e inflection poin to fth e glas s transitio n wa s -39.53°C
. Afte rth e experiment , th e sampl e appeare d t o hav e bee n unchanged , I.e
. n o decompositio n and/o revaporatio n wer e observed
. 8.6 . Conclusio n Th e meltin g an d boilin g temperatur e o fp-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e wer e determine d usin g DSC
. Th e meltin g temperatur e (glas s transition ) o fth e tes t substanc e wa s -39° C (234K)
. Reactio n and/o rdecompositio n o fth e tes tsubstanc e wa s observe d a t temperature s o f> 250° C ( > 523K)
. Boilin g o fth e tes tsubstanc e wa s no t observe d belo wth e temperatur e a t whic h reactio n and/o r decompositio n started
. Base d o n this , th e tes tsubstanc e ha s n o boilin g temperature . Fina l report - Pag e 2 0 -

Ti n T i1p . 2 4 ilu a Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorized"an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e 8.7 . Figure s NOTO X Projec t 49698 1

File
. NOTOX201 0 1320 1 S471 3

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432.23 " C 70.00 % Los » 20 0 30 0 Temperatur e (°C ) 40Q 50 0 Univsrw tV*.7 AT AInalnimflnto Figur e 4 TG Acurv e o fth e tes t substance .
Fil a NOTOX201 0 1320 2 £667 3

10 0 20 0 Temperatur e (°C ) 40 0 Unlvtm l V4.7

AT AImtnimwit i Figur e 5 DS C curv e o fth e tes t substanc e obtaine d wit h th e firs t experiment .

Fina l report - Pag e 2 1 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substances (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO X Projec t 49698 1

Fife
. NOTOX201 0 1320

2 2662 2
1

0 -

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. . 1 , , , 1 , , , 1 -10 0 -3 0 -60 -4 0 -2

0 0 2 D 4

0 6

0 E>ou p Temperatur e ('C ) univarw i W AT Airatrumont s Figur e 6 DS C curv e ofth e tes t substanc e obtaine d wit h th e secon d experiment .

Fina l report - Pag e 2 2 -

### p . 2 6 Th e use o

fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d to Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibite d p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 1 9 . DENSIT Y 9.1 . Guideline s Organizatio n fo rEconomi cCo-operatio n an d Developmen t(OECD) , OEC D Guidelin e fo rth e Testin g o fChemical s no . 109 : "Densit yo fLiquid s an d Solids" , Jul y27 , 1995
. Europea n Communit y(EC) , E C no . 440/2008 , Par t A : Method s fo rth e Determinatio n o fPhysico Chemica l Properties , Guidelin e A.3 : "Relativ e Density" , Officia l Journa l o fth e Europea n Unio n no . L142 , May31,2008
. 9.2 . Reagent s Wate rTa p wate rpurifie d b ya Milli- Q wate rpurificatio n syste m (Millipore , Bedford , MA , USA)
. 9.3 . Performanc e o fth e stud y Th e density(p ) an d relativ e densit y(DTM ) o fth e tes tsubstanc e wer e determine d usin g a pycnomete r wit h a volum e o f1 0 ml
. Wit h thi s metho d th e densityo fth e tes t substanc e i s calculate d fro m th e differenc e i n weigh tbetwee n th e ful l an d empt ypycnomete ran d it s volume
. Th e volum e o fth e pycnomete rwa s determine d first
. Th e pycnomete rwa s weighe d empty . The n i t wa s fille d wit h water , drie d o n th e outsid e an d weighe d again
. Th e temperatur e o fth e wate rwa s measured
. Th e procedur e wa s performe d i n duplicate
. Th e mea n volum e o fth e pycnomete rwa s calculated
. Th e pycnomete rwa s fille d wit h tes tsubstance , drie d o n th e outsid e an d weighed
. Th e temperatur e o f th e tes t substanc e wa s measured
. Th e tes twa s performe d i n duplicate
. Th e mea n densityan d relativ e densityo fth e tes tsubstanc e wer e determined
. Handling s wer e performe d a t 20. 0 ±0.5°C
. Weighing s wer e performe d o n a n analytica l balanc e wit h a n accurac yo f0. 1 mg .

Fina l report - Pag e 2 3 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d to Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substances (REACH) , withou t th e expres s writte n permissio n o fHuntsma n internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 1 9.4 . Interpretatio n Th e density(p ) o fa substanc e i s th e quotien t o fit s mas s an d it s volum e an d i s expresse d i n S I unit s o f kg/m 3
. Th e relativ e density(DTM ) i s th e rati o betwee n th e mas s o fa volum e o fa substanc e t o b e examined , determine d a t20°C , an d th e mas s o fth e sam e volum e o fwater , determine d a t 4° C (pjj a 0= 100 0 kg/m 3 )
. Th e relativ e densityha s n o dimension
. Volum e o fth e pycnomete r(V ) V= —— —[cm 3 ] P 2 0 "H 2 O where : A= weigh to fth e empt ypycnomete r[g ] B = weigh t o fth e pycnomete rwit h wate r[g ] p ^ 0= densityo fwate ra t 20° C i.e
. 0.998 2 g/cm 3(CR C

Handboo k

o fChemistryan d Physics , 1981 ) Densit y(p ) p = ° " Amaa n [g/cm 3 ] ^meai i where : C = weigh t o fth e pycnomete rwit h tes tsubstanc e [g ] 9.5 . Result s Th e result s o fth e test s o n th e determinatio n o fth e densityo fth e tes t substanc e ar e give n i n Tabl e 5
. Th e individua l measurement s wer e obtaine d wit h a n accurac yo f< 0.0 1 g/cm 3
. Accordin g t o th e guideline , th e densityo fth e tes t substanc e i s give n a s th e mea n value
. Tabl e 5 Densit yofth e tes t substanc e Test l Tes t 2 Mea n Mas s empt ypycnomete r(A ) Mas s pycnomete rwit h wate r(B ) 30.932 5 g 40.666 0 g 30.931 0 g

40.658 5 g 30.9317 5 g Volum e o fth e pycnomete r 9.751 1 cm 3 9.745 0 cm 3 9.748 1 cm 3 Mas s pycnomete rwit h tes tsubstanc e (C ) 42.775 4 g 42.778 7 g Densit y 1.21 5 g/cm 3 1.21 5 g/cm 3 1.2 2 g/cm 3 9.6 . Conclusio n Apycnomete rwa s use d fo rth e determinatio n o fth e densit yan d relativ e densityo f p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline
. Th e densityo fth e tes tsubstanc e a t20° C wa s 1.2 2 g/cm 3(1.2 2 x10 3kg/m 3 )
. Th e relativ e densit ywa s 1.22
.
Fina l report - Pag e 2 4 -Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibite d p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 1 10 . VAPOU R PRESSUR E 10.1 . Guideline s Europea n Communit y(EC) , E C no . 761/2009 , PartA : Method s fo rth e Determinatio n o fPhysico Chemica l Properties , Guidelin e A.4 : "Vapou rPressure" , Officia l Journa l o fth e Europea n Unio n no . L220 , Augus t24 , 2009
. Organizatio n fo rEconomi cCo-operatio n an d Developmen t (OECD) , OEC D Guideline s fo rth e Testin g o fChemical s no.104 : "Vapou rPressure" , Marc h 23 , 2006
. 10.2 . Performanc e o fth e tes t Th e vapou rpressur e o fth e tes tsubstanc e (P T ) wa s determine d b yth e isotherma l thermogravimetri c effusio n method , Th e metho d i s validate d i n th e rang e 10" 8-10 3P a usin g a se t o ffiv e referenc e substance s wit h know n vapou rpressure s (se e paragrap h 10.4)
. Th e validit yo fth e metho d wa s verifie d maximu m on e mont h befor e thi s stud yusin g hexachlorobenzen e (>99% , Fluk a Chemie , Buchs , Switzerland ) a s referenc e contro l substance
. Th e logarith m o fth e evaporatio n rat e a t 20° C (lo g v T , 20 ) deviate d < 10 % fro m th e averag e valu e obtaine d durin g th e validatio n test . Fro m this , i t wa s possibl e t o appl yth e constant s obtaine d wit h th e validatio n tes tfo rth e determinatio n o fth e vapou r pressur e o fth e tes tsubstance
. Approximatel y8.2 4 - 13. 3 m g ofth e tes tsubstanc e wa s applie d t o th e surfac e o fa roughene d glas s plat e a s a homogeneou s layer
. Th e weigh t los s o fth e tes t substanc e wa s measure d continuousl ya s a functio n o ftime
. Th e followin g temperatur e progra m wa s used
. 

### Star t temperatur

e 100° C Isotherma l interval s 1 0 minute s Incremen t step s 10° C En d temperatur

e 240° C Th e weigh tlos s o fth e tes t substanc e wa s measure d continuousl ya s a functio n o ftim e a t define d isotherma l temperatures . Th e evaporatio n rat e o fth e tes t substanc e (v T ) wa s calculate d fro m th e weigh tlos s o fth e compound
. Th e P Tvalue s a t th e define d isotherma l temperature s wer e determine d usin g th e vapou rpressur e equatio n wit h th e constant s can d d specifi cfo rth e experimenta l arrangement . Linea rregressio n analysi s o fth e lo g P Tvalue s an d 1/ Twa s performe d usin g a leas t square s method
. Th e P Tvalue s o fth e tes t substanc e a t 20° C an d 25° C wer e determine d usin g th e vapou rpressur e regressio n curve . 10.3 . Analytica l metho d Instrumen tQ5 0 thermogravimetri canalyze r(TGA ) (TAInstruments , Ne w Castle , DE , USA ) Surfac e o fth e glas s plat e 0.5 0 cm 2 Th e experiment s wer e performe d unde ra flo wo fnitroge n an d a t atmospheri cpressure .

Fina l report - Pag e 2 5 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d to Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substances (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 1 10.4 . Referenc e substance s Validatio n o fth e metho d i s performe d a tleas t onc e everyyea r(NOTO Xprojec t 495910)
. Eac h reference s substanc e wa s measure d i n duplicat e b yTG Ausin g a temperatur e progra m tha t wa s specifi cfo rth e substance
. Plot s o flo g v Tobtaine d a televate d temperature s an d 1/ Twer e inter - o r extrapolate d t o determin e th e lo g v Tvalue s a t20° C (lo g v Tl 2o) - Th e lo g v T 2 0value s wer e plotte d agains t th e logarith m o fth e vapou rpressur e a t20° C i n Pasca l unit s (lo g PT.M) - Linea rregressio n analysi s usin g th e leas tsquare s metho d yielde d a n equatio n o flo g P T ,2 o = 1.1 6 xlo g v T ,2 o +4.34
. Th e coefficien t o fcorrelatio n (r ) wa s > 0.99 , Th e constant s can d d specifi cfo rth e experimenta l arrangemen twer e 1.1 6 an d 4.34 , respectively
. Reference ' P Tvalue s Correspondin g P Tvalue s Literatur e 20° C 25° C Referenc e substanc e [Pa l [m m Hg l fPa ] 

### rm m Hg l Benzo(ghi)perylen e 1.3 3 xitr 8 1.0 0 x10' 1 0 6.4 6 x10" 8 # 4.8 5 x10- 1 0 * 1 Chrysen e 8.4 0 x10" 7 6.3 0 x10" 9 5.7 6 x10' 7 # 4.3 2 x10" 9 # 2 Hexachlorobenzen e 1.4 7 x10" 3 1.1 0 x10" 5 4.2 1 x10' 3 # 3.1 6 x10- 5 " 1 Naphthalen e 7.5 6 x10 ° 5.6 7 x10" a 1.1 5 x10 1 8.6 1 x10" 2 3 Water 2.3 4 x10 3 1.75 x10 1 3.1 7 x10 3 2.3 8 x10 1 3 Determine d b

yth e vapou rpressur

e regressio n curve o

fth e validatio n study , N o literature valu e was available
. 1 Verschueren , K. , Handboo k o fenvironmenta l data o n organi cchemicals , 4 l h edition , WileyChemistr y 2
. Borges , H T, Forma l toxicit ysummar

yfo rChrysene , Th e Ris k Assessmen t Informatio n Syste m (RAIS) , 1994 3
. Lide , D R , CRC Handboo k o fChemistr

yan d Physics , 79" ' edition , CRC Press LLC, Boc a Raton , FL , USA , 1998

Fina l report - Pag e 2 6 -Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d to Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LLC i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)ani!in e NOTO XProjec t 49698 1 10.5 . Interpretatio n 10.5.1 . Specification s Th e vapou rpressur e i s a functio n o fth e temperatur e an d i s specifie d i n Pasca l (Pa ) o ri n m m Hg , 10.5.2 . Formula s Evaporatio n rat e (v T ) v T= [g/cm 2 /h ] where : A m = weigh t los s o fth e tes t substanc e [g ] F ~ surfac e o fth e sampl e plat e [cm 2 ] t = elapse d tim e fo rth e weigh tlos s [h ] Vapou rpressur e equatio n logP j = ciogv T+ d where : c= constan t specifi cfo rth e experimenta l arrangemen t d = constan t specifi cfo rth e experimenta l arrangemen t Vapou rpressur e regressio n curv e lo g P T= a 1/ T+ b where : T= temperatur e [K] a = slop e [Kj b - intercep t

Fina l report - Pag e 2 7 - rTh e use ofthes e studies/documents fo rregulator yc rregistratio n purposes , includin g bu tnotlimite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substances (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 1 10.6 . Result s Arepresentativ e weigh tlos s curv e o fth e tes tsubstanc e i s show n i n Figur e 7
. Th e result s o fth e isotherma l TG Aanalysi s fo rth e tes tsubstanc e an d th e vapou rpressur e a t 20° C an d 25° C ar e give n i n Tabl e 6 an d Tabl e 7
. Th e plo to fth e lo g P To fth e tes tsubstanc e a s functio n o fth e reciproca l temperature s i s show n i n Figur e 8
. Th e equatio n o fth e curv e was : lo g P T , 2 o = -657 3 x1/ T+ 16.1 6 ( r= 0.997 , n = 8)
. Tabl e 6 Result s o fth e isotherma l TG Aanalysi s Temperatur e PC I Weigh t los s

fpq/min l v T

[q/cm 2 /h l lo g v T lo g P T P T [Pa l 15 0 5.00 3 5.9 7 x10" 4 -3.2 2 0.6 1 4. 1

5.11 1 6.1 0 x10 - 4 -3.2 1 0.6 2 4. 2 16 0 10.4 2 1.2 4 x10' 3 -2.9 1 0.9 8 9. 6

11.3 7 1.3 6 x10 * -2.8 7 1.0 3 1 1 17 0 20.4 6 2.4 4 x10' 3 -2.6 1 1.32 2 1

22.5 8 2.7 0 x10" 3 -2.5 7 1.3 7 2 3 18 0 36.7 4 4.3 9 x10" 3 -2.3 6 1.6 1 4 1

41.7 5 4.8 0 x10" 3 -2.3 0 1.68 4 8
Tabl e 7 Vapou rpressur e o fth e tes t substanc e Temperatur e lo g P T P T P T 

## rc i [Pa ] [m m Hg l 2 0 -6.2 6 5. 5 x10 - 7 4. 1 x10' 9 2 5 -5.8 8 1. 3 x10 * 9. 9 x10" 9
10.7
. Conclusio n Th e isotherma l TG Aeffusio n metho d wa s applie d fo rth e determinatio n o fth e vapou rpressur e o f p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline
. Th e vapou rpressur e o fth e tes tsubstanc e a t 20° C (293K ) an d 25° C (298K ) was : 2 0 [Pa ] ° C [m m Hq ] 2 5 [Pa l ° C [mm Hg ] Tes t substanc e 5. 5 x10" 7 4. 1 x10 9 1. 3 x10 * 9. 9 x10" 9

Fina l report - Pag e 2 8 -Th e us e ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 1 

### 10.8 . Figures

File ; NOTOX201013202 Z47Z 2

640 0 -10.«|ifl/ml n 160,00* C
1SO.0C C -20.4 6 tig/m I n -5.003|ifj/ml n 17Q.O0' C

-36.74(jg/m/n lao.oo-c '

Tim e (min ) Universa l V4.7 ATAIna&urnflnt i Figur e 7 Weigh t los s curv e o fth e tes t substance .

0. 4 - 0. 2 - 0 - I 1 1 1 1 1 1 1 1 1 1 0.0021 8 0.002 2 0.0022 2 0.0022 4 0.0022 6 0.0022 8 0.002 3 0.0023 2 0.0023 4 0.0023 6 0.0023 8 1/ T[K- 1 ] Figur e 8 Plo t o flo g P Tofth e tes t substanc e versu s 1/T .

Fina l report - Pag e 2 9 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d to Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProject 49698 1 11 . WATE R SOLUBILIT Y 11.1 . Guideline s Organizatio n fo rEconomi cCo-operatio n an d Developmen t (OECD) , OEC D Guideline s fo rth e Testin g o fChemical s no . 105 : "Wate rSolubility" , Jul y27 , 1995
. Europea n Communit y(EC) , E C no . 440/2008 , PartA : Method s fo rth e Determinatio n o fPhysico Chemica l Properties , Guidelin e A.6 : "Wate rSolubility" , Officia l Journa l o fth e Europea n Unio n no . L142 , Ma y31 , 2008
. 11.2 . Reagent s WaterTa p wate rpurifie d b ya Milli- Q wate rpurificatio n syste m (Millipore , Bedford , MA , USA ) Doubl e distille d wate rWaldeck , Munster-Roxel , German y Acetonitril e VW R International , Leuven , Belgiu m Al l reagent s wer e o fanalytica l grade , unles s specifie d otherwise
. 11.3
. Performanc e o fth e stud y Tw o method s ar e availabl e fo rth e determinatio n o fth e wate rsolubilit yo fa tes t substance : 1
. Colum n elution metho d Th e metho d i s base d o n th e elution ofth e tes tsubstanc e wit h wate rfro m a colum n whic h i s charge d wit h a n inert supportmaterial , suc h a s bare silic a particles , coate d wit h a n exces s o fth e tes t substance
. I tca n b e applie d t o tes tsubstance s wit h a wate rsolubilit yo f< 10" 2g/l
. 2
. Flas k metho d Th e mas s concentratio n o fth e tes tsubstanc e i n aqueou s solution s saturate d wit h th e tes t substanc e wil l b e determine d afte rstirrin g o rshakin g a t a specifi ctemperatur e fo ra specifi cperio d o ftime
. Th e metho d ca n b e use d fo rtes tsubstance s wit h a wate rsolubilit yo f£10" 2g/l
. Apreliminarytes twa s carrie d ou ta s a range-findin g tes t prio rt o performanc e o fth e colum n elutio n metho d o rflas k

method
. Preliminarytes t Tes tsubstanc e wa s stirre d wit h doubl e distille d wate ra t a nomina l concentratio n o f2.0 2 g/ l at 20. 2 ±0.6° C fo r2 3 hour s ( 1 day)
. Afte rstirring, th e wate rphas e wa s centrifuged , treate d an d analysed
. Th e centrifugatio n metho d wa s checke d an d wa s foun d t o b e sufficien t t o remov e al l undissolve d tes t substance
. I n th e preliminarytes tth e wate rsolubilit yo fth e tes t substanc e wa s £10" 2g/l
. Therefore , th e flas k metho d wa s chose n fo rth e mai n study
. Mai n stud y Thre e aliquot s betwee n 1.44 an d 1.48 m g o fth e tes tsubstanc e wer e weighe d int o glas s vessels
. T o eac h vessel , 10 0 m l doubl e distille d wate rwa s added
. Th e vessel s wer e place d o n a magneti cstirrin g devic e i n a climat e roo m a t 19. 9 ±0.7° C wher e th e conten t o feac h vesse l wa s stirre d fo r24 , 4 8 o r 7 2 hours
. A s th e blank , 10 0 m l doubl e distille d wate rwa s stirre d fo r7 2 hours
. Afte rth e stirrin g period , th e wate rphases-wer e two-time s centrifuge d a t 1642 1 g an d 20° C fo r 5 minutes
. Th e aqueou s phase s wer e dilute d i n a 7: 3 (v:v ) rati o wit h acetonitril e an d analysed
. I f necessary , th e sample s wer e furthe rdilute d wit h 30/7 0 (v/v ) acetonitrile/wate rt o obtai n concentration s Fina l report - Pag e 3 0 -p . 3 4 Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
.

withi n th e calibratio n range . Th e p H o feac h o fth e wate rmixture s (excep t fo rth e blank ) wa s determine d afte rremova l o fundissolve d tes t substance
. 11.4 . Analytica l metho d 11.4.1 . Analytica l condition s Quantitativ e analysi s wa s performe d accordin g t o th e analytica l metho d a s validate d i n chapte r7
. 11.4.2 . Preparatio n o fth e calibratio n solution s Stoc k solution s Stoc k solution s o fth e tes t substanc e wer e prepare d i n acetonitril e a t concentration s o f171 2 - 223 6 mg/l
. Calibratio n solution s Calibratio n solution s i n th e concentratio n rang e o f0.040 1 - 2.0 0 mg/ l (preliminarystudy ) o r0.040 1 - 59. 9 mg/ l (mai n study ) wer e prepare d fro m tw o stoc k solutions
. Th e en d solutio n o fth e calibratio n solution s wa s 30/7 0 (v/v ) acetonitrile/water
. 11.4.3 . Sampl e injection s Calibratio n solution s wer e injecte d i n duplicate
. Tes t sample s wer e analyse d b ysingl e injection
. 11.4.4 . Calibratio n curve s Calibratio n curve s wer e constructe d usin g fiv e concentrations
. Fo reac h concentration , tw o response s wer e used
. Linea rregressio n analysi s wa s performe d usin g th e leas t square s metho d wit h a 1/concentration 2weightin g factor
. Th e coefficien to fcorrelatio n (r ) wa s > 0.9 9 fo reac h curve . 11.5 . Interpretatio n 11.5.1 . Specification s Th e wate rsolubilit yo fa tes t substanc e i s specifie d b yth e saturatio n mas s concentratio n o fth e tes t substanc e i n wate ra ta give n temperatur e an d i s expresse d i n g/l
. 11.5.2 . Formula s Respons e (R ) Pea k are a o fth e tes t substanc e [units ] p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t49698 1

Maximu m differenc e highes t value-lowes t valu e x1Q0 % meanvalu e where : 'mea n value ' i s th e mea n o fth e highes tan d lowes t value
. Calibratio n curv e R = a C N + b where : C N = nomina l concentratio n [mg/l ] a = slop e [unit s xl/mg ] b = intercep t [units] Analyse d concentratio n (C A ) (R-b ) xd x10 - 3 [g/i ] a where : d = ( dilutio n facto r
Fina l report - Pag e 3 1 -Th e use ofthes e studies/documents fo rregulator yorregistratio n purposes , includin g bu t no t limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 1 11.6 . Result s 11.6.1 . Preliminar ytes t Th e conten t o ftes t substanc e dissolve d i n th e wate rsampl e wa s 2.9 3 g/l
. 11.6.2 . Mai n stud y Th e result s fo rth e sample s take n a t24 , 4 8 an d 7 2 hour s ar e give n i n Tabl e 8
. Th e maximu m differenc e (MD ) o fth e concentration s determine d i n th e 24 , 4 8 an d 7 2 hou rtes t sample s wa s £15%
. Base d o n this , th e wate rsolubilit yo fth e tes t substanc e i s give n a s th e mea n valu e o fth e analyse d concentrations
. N o tes tsubstanc e wa s detecte d i n th e pretreate d sampl e fro m th e blan k wate rmixture
. Tabl e 8 Mai n stud y- wate rsolubilit yo fth e tes t substanc e Stirrin g tim e [hours ] Analyse d

concentratio n [g/i ] Mea n

[g/i ] M D

[% ] p H

2 4 3.2 7 3.3 4 3. 1 9. 5 4 8 3.3 6 9. 1 7 2 3.3 8 8. 3

11.7 . Conclusio n Th e flas k

metho d wa s applie d fo rth e determinatio n o fth e wate rsolubilit yo fp-(2,3-epoxypropoxy) - N,N-bis(2,3-epoxypropyl)aniline
. Th e wate rsolubilit yo fth e tes t substanc e a t20° C wa s 3.3 4 g/l
. Th e p H o fth e aqueou s sample s wa s 8. 3 - 9.5
.

Fina l report - Pag e 3 2 -p . 3 6 Th e use o

fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibite d

12 . FLASH-POIN T 12.1 . Guideline s Europea n Communit y(EC) , E C no . 440/2008 , Par t A : Method s fo rth e Determinatio n o fPhysico Chemica l Properties , Guidelin e A.9 : "Flash-point" , Officia l Journa l o fth e Europea n Unio n no . L142 , Ma y31 , 2008
. Unite d Nation s (UN) , U N no . ST/SG/AC.10/11/Rev.5 : Recommendation s o n th e Transport o f Dangerou s Goods , PartIII : Classificatio n Procedures , Tes tMethod s an d Criteri a Relatin g t o Clas s 2 , Clas s 3 , Clas s 4 , Divisio n 5.1 , Clas s 8 an d Clas s 9 , Paragrap h 32.4.1 : "Non-Viscou s Flammabl e Liquids" , 2009
. Th e Internationa l Organizatio n fo rStandardizatio n (ISO) , IS O Guid e 2719 : "Determinatio n o fFlas h Poin t - PenskyMarten s Close d Cu p method" , 20.02
. AST M International , AST M D 93 : "Standar d Tes tMethod s fo rFlas h Poin t b yPensky-Marten s Close d Cu p Tester" , Decembe r10 , 2002
. 12.2 . Performanc e o fth e tes t Th e flash-poin t o fth e tes tsubstanc e wa s determine d usin g a mode l APM- 7 Pensky-Marten s close d cu p automati cflash-poin t teste r(Tanak a Scientifi cLimited , Tokyo , Japan)
. 12.2.1 . Preliminar ytes t Th e tes t cu p wa s fille d wit h approximatel y7 0 m l tes t substanc e an d place d i n th e heatin g bloc k o fth e flash-poin ttester . Th e li d wit h a thermocoupl e fo rth e determinatio n o fth e flash-poin t an d a resistanc e thermomete rfo rmeasurin g th e temperatur e wa s place d o n th e cup . Th e tes t cu p wa s heate d wit h a rat e o fapproximatel y5°C/minute
. Th e sampl e wa s stirre d durin g heating
. Th e flam e ignite rwa s applie d s o tha ti twa s lowere d int o th e vapou rspac e o fth e cu p i n 0. 5 seconds , lef t i n it s lowere d positio n fo r1 second , an d quickl yraise d t o it s hig h positio n again
. Th e sampl e wa s no t stirre d whe n th e ignite rwa s applied
. Th e temperatur e o fth e tes tsubstanc e a tth e starto fth e tes t wa s 16.2°C
. Startin g a t 20°C , a n attemp t wa s mad e t o ignit e th e vapou rofth e tes t substanc e fo revery2° C temperatur e rise
. 12.2.2 . Mai n stud y Tw o test s wer e performed
. Th e temperatur e o fth e tes tsubstanc e a t th e start o fth e tes t wa s 19.3° C an d 22.rc , respectively
. Startin g at206°C , th e flam e ignite rwa s applie d fo revery2° C temperatur e rise
. Th e atmospheri cpressur e wa s 101 4 hPa
. 12.3 . Interpretatio n Th e flash-poin t o fth e tes tsubstanc e i s th e lowes ttemperature , correcte d t o a barometri cpressur e o f 101 3 hPa , a twhic h th e tes t substanc e evolve s vapour s i n suc h amoun t tha t a flammabl e vapour/ai r mixtur e i s produce d i n th e tes t vessel
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t49698 1

Flas h poin t(T c ) T c=T 0+0.025(1013-p ) where : T o = P flas h poin t o fth e tes t substanc e a tambien t atmospheri c pressur e [°C ] ambien t atmospheri cpressur e [hPa ]

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C i s unauthorize d an d strictl yprohibited
. p . 3 7
p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProject 49698 1 12.4 . Result s 12.4.1 . Preliminar ytest Th e estimate d flash-poin t o fth e tes tsubstanc e wa s 228°C
. 12.4.2 . Mai n stud y Th e result s o fth e mai n stud yo n th e determinatio n o fth e flash-poin t o fth e tes t substanc e ar e give n i n Tabl e 9
. Th e differenc e betwee n th e result s obtaine d wit h th e tw o test s wa s £6°C
. Tabl e 9 Mai n stud y- flash-poin t o fth e tes t substanc e

Tes t Flash-poin t Lowes t flash-poin t [°C 1 

## rc i 1 22 8 22 8 2 22 8

12.5 . Conclusio n Th e Pensky-Marten s close d cu p metho d wa s applie d fo rth e determinatio n o fth e flash-poin t o f p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline
. Th e flash-poin to fth e tes t substanc e wa s 228°C
.

Fina l report - Pag e 3 4 -Tri e us e ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d to Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 1 13 . AUTO-IGNITIO N TEMPERATUR E 13.1 . Guideline s Europea n Communit y(EC) , E C no . 440/2008 , Part A : Method s fo rth e Determinatio n o fPhysico Chemica l Properties , Guidelin e A.15 : "Auto-Ignitio n Temperatur e (Liquid s an d Gases)" , Officia l Journa l o fth e Europea n Unio n no . L142 , May31 , 2008
. Deutsche s Institu t fu rNormun g (DIN) , DI N Guid e 51794 : "Determinin g th e Ignitio n Temperatur e o f Petroleu m Products" , Ma y2003
. Internationa l Electrotechnica l Commissio n (IEC) , IE C standar d 79-4 : "Electrica l Apparatu s fo r Explosiv e Ga s Atmospheres , Par t 4 , Metho d ofTestfo rIgnitio n Temperature" , 1975
. 13.2
. Performanc e o fth e tes t Th e auto-ignitio n temperatur e o fth e tes t substanc e wa s determine d usin g a commerciall yavailabl e auto-ignitio n temperatur e apparatu s (Chilwort h Technology , Southampton , UK)
. Th e metho d i s applicabl e t o tes t substance s wit h a n auto-ignitio n temperatur e i n th e rang e 75° C -650°C
. I n everyignitio n test , liquefie d tes t substanc e wa s introduce d int o a preheate d tes t vesse l usin g a volumetri cpipette
. I t wa s observe d visuall ywhethe ra reactio n (clearl yperceptibl e flam e and/o r explosion ) o fth e sampl e withi n fiv e minute s afte rintroductio n too k plac e a t th e prevailin g temperature . Afte reac h ignitio n test , an yvaparizabl e component s lef ti n th e flas k wer e blow n ou t wit h ai r(Ai r Products , Amsterdam , Th e Netherlands)
. 13.2.1
. Preliminar ytes t Startin g a t 200°C , fo revery20° C temperatur e rise , 7 0 p i tes t substanc e wa s introduce d int o th e tes t vesse l unti l ignitio n o fth e tes tsubstanc e wa s firs t observe d (i.e
. a t 440°C)
. Startin g a t 455°C , fo revery5° C decrease , 7 0 p i tes tsubstanc e wa s teste d unti l n o ignitio n o fth e tes t substanc e ha d bee n observed
. Th e temperatur e a t whic h ignitio n wa s las t observe d i s th e estimate d auto-ignitio n temperature .

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C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProject 49698 1 13.2.2 . Mai n stud y Thre e test s wer e performed
. Th e sampl e volume s applie d an d temperatur e rang e o feac h tes t ar e give n below
. Startin g a t th e "hig h temperature" , fo revery2° C decrease , th e prevailin g volum e o ftes t substanc e wa s teste d unti l n o ignitio n o fth e tes tsubstanc e ha d bee n observed
. Th e temperatur e a t whic h ignitio n wa s las tobserve d i s th e auto-ignitio n temperatur e a t th e sampl e volum e applied
. Thi s procedur e wa s repeate d wit h th e othe rvolume s unti l a minimu m auto-ignitio n temperatur e fo reac h tes twa s obtained
. Wit h thes e result s th e auto-ignitio n temperatur e o fth e tes t substanc e wa s determined
. Th e atmospheri cpressur e durin g th e performanc e o fth e mai n stud ywa s betwee n 1012. 6 an d 1013. 9 hPa
. Tes t Volum e Temperatur e (°C ) 

### [ul l start en d Test l 7 0 43 2 42 0 25 0 41 0 40 4 65 0 42 5 41 7 Tes t2 8 5 42 8 41 0 17 5 42 7 40 5 35 0 42 3 41 5 Tes t3 6 0 43 1 41 9 20 0 42 0 40 8 45 0 43 0 41 6
13.3 . Interpretatio n Th e degre e o fauto-ignitabilit yi s expresse d i n term s o fth e auto-ignitio n temperature . Th e auto-ignitio n temperatur e i s th e lowes ttemperatur e a twhic h th e tes tsubstanc e ignite s whe n mixe d wit h ai runde r th e condition s define d i n th e tes tmethod
. 13.4 . Result s 13.4.1 . Preliminar ytes t Th e estimate d auto-ignitio n temperatur e wa s 425°C
. 13.4.2 . Mai n stud y Th e result s o fth e mai n stud yo n th e determinatio n o fth e auto-ignitio n temperatur e o fth e tes t substanc e ar e give n i n Tabl e 10
. Ignitio n o fth e tes t substanc e withi n 5 minute s afte rintroductio n wa s observe d wit h eac h sampl e volum e an d wit h eac h tes ta minimu m auto-ignitio n temperatur e wa s obtained
. Th e maximu m deviatio n betwee n th e thre e value s wa s < 20°C
. Accordin g t o th e guidelines , n o furthe rtestin g wa s required
. Th e lowes ttemperatur e a t whic h ignitio n ofth e tes t substanc e occurre d wa s 410°C , Thi s temperatur e wa s a multipl e o f5° C an d therefor e give n a s th e auto-ignitio n temperatur e o fth e tes t substance
.

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C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 1
Tabl e 1 0 Mai n stud y- auto-ignitio n temperatur e o fth e tes t substanc e Tes t Volum e



# ru n 

# ' Auto-ignitio n

temperatur e 

# m Ignitio n dela y



### Ts l Colou ro fth e

flam e Minimu m

auto-ignitio n temperatur e PC I 1 7 0 42 6 1 1 Orang e 41 0

25 0 41 0 1 8 Orang e 65 0 42 3 1 3 Orang e 2 8 5 41 6 1 9 Orang e 41 1

17 5 41 1 2 1 Orang e 35 0 42 1 2 7 Orang e 3 6 0 42 5 2 5 Orang e 41 4

20 0 41 4 1 6 Orang e 45 0 42 2 2 1 Orang e

s13.5 . Conclusio n Commerciall yavailabl e auto-ignitio n temperatur e apparatu s wa s use d fo rth e determinatio n o fth e degre e o fauto-ignStabilityo fp-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline
. Th e tes t substanc e i s auto-ignitabl e wit h a n auto-ignitio n temperatur e o f410° C a t 1012. 6 - 1013. 9 hPa
.

Fina l report - Pag e 3 7 -Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substances (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibite d p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 1 14 . OXIDIZIN G PROPERTIE S 14.1 . Guidelin e Europea n Communit y(EC) , E C no . 440/2008 , PartA : Method s fo rth e Determinatio n o fPhysico Chemica l Properties , Guidelin e A.21 : "Oxidizin g Propertie s (Liquids)" , Officia l Journa l o fth e Europea n Unio n no . L142 , Ma y31 , 2008
. Unite d Nation s (UN) , U N no . ST/SG/AC.10/11/Rev.5 : Recommendation s o n th e Transport o f Dangerou s Goods , PartIII : Classificatio n Procedures , Tes t Method s an d Criteri a Relatin g t o Clas s 2 , Clas s 3 , Clas s 4 , Divisio n 5.1 , Clas s 8 an d Clas s 9 , Tes t 0.2 : "Tes t fo rOxidizin g Liquids" , 2009
. Unite d Nation s (UN) , U N no . ST/SG/AC.10/11/Rev.5 : Recommendation s o n th e Transpor t o f Dangerou s Goods , Appendi x6 : "Screenin g Procedures" , 2009
. 14.2
. Statemen t Ates t substanc e i s considere d a n oxidizin g substanc e whe n a mixtur e o fth e substanc e an d cellulos e i n a 1: 1 rati o (b ymass ) spontaneouslyignite s o rth e mea n pressur e ris e tim e o fth e mixtur e i s les s tha n o requa l t o mea n pressur e ris e tim e ofa 1: 1 referenc e mixtur e (b ymass ) o f65 % aqueou s nitri c aci d an d cellulose
. Th e molecula rstructur e o fth e tes tsubstanc e i s show n i n paragrap h 6.1
. Th e tes t substanc e doe s no t contai n group s tha t ac t a s a n oxidizin g agent . Th e oxyge n atom s tha t ar e presen t i n th e molecula r structur e o fth e tes t substanc e ar e chemicall ybonde d t o carbo n o rhydrogen
. Th e molecula rstructure s o fimpuritie s a t<, 6 % wer e no ttake n int o accoun t bu tthes e ar e no t expecte d t o b e o finfluenc e o n th e oxidizin g propertie s o fth e tes tsubstance
. I n conclusion , p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e ha s n o oxidizin g properties
.

Fina l report

Ti n - Pag e 3 8 -

I rTh e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProject 49698 1 15 . HYDROLYSI S A S AFUNCTIO N O F P H 15.1 . Guidelin e Europea n Communit y(EC) , E C no . 440/2008 , Par tC : Method s fo rth e Determinatio n o fEcotoxicity , Guidelin e C.7 : "Degradatio n - Abioti cDegradation : Hydrolysi s a s a Functio n o fpH" , Officia l Journa l o f th e Europea n Unio n no . L142 , May31 , 2008
. Organizatio n fo rEconomi cCo-operatio n an d Developmen t(OECD) , OEC D Guideline s fo rth e Testin g o fChemical s no . 111 : "Hydrolysi s a s a Functio n o fpH" , Apri l 13 , 2004
. 15.2
. Reagent s Water Acetonitril e Sodiu m acetat e Aceti cacid , 100 % Potassiu m d/-hydro g e n phosphat e Bori caci d Potassiu m chlorid e Sodiu m azid e Sodiu m hydroxid e Al l reagent s wer e o fanalytica l grade , 15.3 . Buffe rsolution s Acetat e buffe rp H 4 , 0. 1 M Phosphat e buffe rp H 7 , 0. 1 M
Borat e buffe rp H 9 , 0. 1 M
15.4 . Performanc e o fth e stud y Th e rat e o fhydrolysi s o fth e tes tsubstanc e a s a functio n o fp H wa s determine d a t p H value s normall y foun d i n th e environmen t (p H 4-9)
. Preliminarytes t- Tie r1 Tes tsubstanc e solution s wer e prepare d i n th e buffe rsolution s a t a targe t concentratio n o f100 0 mg/l , afte rstirrin g fo ra t leas t6 0 minute s th e tes tsubstanc e wa s no tcompletel ydissolved
. Eac h solutio n wa s filter-sterilise d throug h a 0. 2 p m F P 30/0. 2 CA- S filte r(Whatman , Dassel , Germany ) an d transferre d int o a steril e vessel
. T o exclud e oxygen , nitroge n ga s wa s purge d throug h th e solutio n fo r 5 minutes
. Fo reac h samplin g time , duplicat e steril e vessel s unde rvacuu m wer e fille d wit h 6 m l tes t solutio n an d place d i n th e dar k i n a temperatur e controlle d environmen t a t 49.9° C ±0. 1 °C
. Ta p wate rpurifie d b ya Milli- Q wate rpurificatio n syste m (Millipore , Bedford , MA , USA ) Biosolve , Valkenswaard , Th e Netherland s Merck , Darmstadt , German y Merc k Merc k Merc k Merc k Merc k Boom , Meppel , Th e Netherland s unles s specifie d otherwise
. solutio n o f16.6 % 0. 1 M sodiu m acetat e an d 83.4 % 0. 1 M aceti c acid
. Th e buffe rcontain s 0.0009 % (w/v ) sodiu m azide
. solutio n o f0. 1 M potassiu m dihydrogenphosphat e adjuste d t o p H 7 usin g 1 0 N sodiu m hydroxide
. Th e buffe rcontain s 0.0009 % (w/v ) sodiu m azide
. solutio n o f0. 1 M bori caci d an d 0. 1 M potassiu m chlorid e adjuste d t o p H 9 usin g 1 0 N sodiu m hydroxide
. Th e buffe r contain s 0.0009 % (w/v ) sodiu m azide
.

Fina l report - Pag e 3 9 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t notlimite d to Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 1

Th e concentratio n o fth e tes t substanc e i n th e tes tsample s wa s determine d immediatel yafte r preparatio n (t=0 ) an d afte r5 days
. Th e sample s take n a t t= 5 day s wer e coole d t o roo m temperatur e usin g runnin g ta p water . Th e sample s wer e dilute d i n a 1: 1 (v:v ) rati o wit h 60/4 0 (v/v ) acetonitrile/buffe r solutio n (th e sam e buffe ra s wher e tes tsubstanc e solutio n i n wa s prepared ) an d analysed
. I f necessary , th e sample s wer e furthe rdilute d wit h 30/7 0 (v/v ) acetonitrile/buffe rsolutio n t o obtai n concentration s withi n th e calibratio n range . Blan k buffe rsolution s wer e treate d similarl ya s th e tes t sample s an d analyse d a t t-0
. Th e p H o feac h o fth e tes tsolution s (excep t fo rth e blanks ) wa s determine d a t eac h samplin g time
. Mai n study-Tie r2 Th e buffe rsolution s wer e filter-sterilise d throug h a 0. 2 ur n F P 30/0. 2 CA- S filte r(Whatman , Dassel , Germany ) an d transferre d int o a steril e vessel
. T o exclud e oxygen , nitroge n ga s wa s purge d throug h th e solutio n fo r5 minutes
. Th e tes tsubstanc e wa s spike d t o th e solution s a t a targe t concentratio n o f 50 0 mg/ l usin g a spikin g solutio n i n acetonitrile
. Th e obtaine d solutio n wa s filtere d agai n throug h a 0. 2 ur n F P 30/0. 2 CA- S filte rt o remov e possibl ypresen tundissolve d tes t substance
. Fo reac h samplin g time , duplicat e steril e vessel s unde rvacuu m wer e fille d wit h 6 m l tes t solutio n an d place d i n th e dar k i n a temperatur e controlle d environment
. Note : th e spikin g volum e wa s < 1 % o fth e sampl e volume
. Nominal concentration s wer e no t correcte d fo rth e spikin g volume
. Th e concentration s o fth e tes tsubstanc e wer e determine d immediatel yafte rpreparatio n (t=0 ) an d a t severa l samplin g point s afte rt=0
. Th e concentratio n o fth e bul k solutio n wa s als o determined
. Th e sample s take n afte rt= 0 wer e coole d t o roo m temperatur e usin g runnin g ta p wate r(excep t th e sample s store d a t 20°C)
. Th e sample s wer e dilute d t o 30/7 0 (v/v ) acetonitrile/borat e buffe rp H 9 a s tes tsubstanc e stabilit ywa s foun d t o b e optima l at p H 9
. Blan k buffe rsolution s containin g a simila rconten to fblan k spikin g solutio n wer e treate d similarl ya s th e tes t sample s an d analyse d att=0
. Th e p H o feac h o fth e tes t solution s (excep tfo rth e blanks ) wa s determine d at leas t a t th e beginnin g an d a tth e en d o fth e test . Th e stud ywa s performe d a t th e followin g temperatures : p H cod e • Temperatur e I Temperatur e I I Temperatur e II I pH 4 20.2° C ±0.6° C 40.0°C±0.1° C 49.9°C±0.1° C p H 7 20.2°C±0.6° C 40.0 Q C±0.1° C 49.9°C±0.1° C pH 9 20.2° C ±0.6° C 

## 40.o°c±o.rc 49.8° C ±0.2° C Identificatio n o fhydrolysi s product s - Tie r3 Researc h t o investigat e th e identit yo rnatur e an d rate s o fformatio n an d declin e o fhydrolysi s product s wa s no t require d sinc e th e studie s wer e conducte d accordin g t o th e testin g guideline s provide d i n Anne xVII I (volum e 10-10 0 tons/year)
.

Fina l report - Pag e 4 0 -,P,4 4 Th e use o

fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibite d

15.5 . Analytica l metho d 15.5.1 . Analytica l condition s Quantitativ e analysi s wa s performe d accordin g t o th e analytica l metho d a s validate d i n chapte r7
. 15.5.2 . Preparatio n o fth e calibratio n solution s Stoc k solution s Stoc k solution s o fth e tes t substanc e wer e prepare d i n acetontril e a t concentration s o f200 0 - 438 5 mg/l
. Calibratio n solution s Calibratio n solution s i n th e concentratio n rang e 0.0 4 - 2 mg/ l wer e prepare d fro m tw o stoc k solutions
. Th e en d solutio n o fth e calibratio n solutio n was : p H cod e Preliminarytes t Mai n tes t pH 4 30/7 0 (v/v ) acetonitrile/acetat e buffe rpH 4 30/7 0 (v/v ) acetonitrile/borat e buffe rpH 9 p H 7 30/7 0 (v/v ) acetonitrile/phosphat e buffe rpH 7 30/7 0 (v/v ) acetonitrile/borat e buffe rpH 9 p H 9 30/7 0 (v/v ) acetonitrile/borat e buffe rpH 9 30/7 0 (v/v ) acetonitrile/borat e buffe rpH 9 15.5.3
. Sampl e injection s Calibratio n solution s wer e injecte d i n duplicate
. Tes t sample s wer e analyse d b ysingl e injection
. 15.5.4 . Calibratio n curve s Calibratio n curve s wer e constructe d usin g a tleas tfou rconcentrations
. Fo reac h concentration , tw o response s wer e used
. Linea rregressio n analysi s wa s performe d usin g th e leas t square s metho d wit h a 1/concentration 2weightin g factor
. I fnecessary , th e calibratio n lin e wa s excludin g on e dat a poin t sinc e th e bac k

calculate d accurac ywa s > 15 % fro m th e nomina l concentration
. Th e coefficien t o f correlatio n (r) wa s > 0.9 9 fo reac h curve . 15.6 . Interpretatio n Respons e (R ) Pea k are a o fth e tes tsubstanc e [units] Calibratio n curv e R = a C N + b p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t49698 1

where : C N = r nomina l concentratio n [mg/l ] slop e [unit s xl/mg ] intercep t [units] a = b = Analyse d concentratio n (C A ) (R-b ) x d [mg/l ] a where : d = dilutio n facto r Recovery x100 % where : C N = nomina l concentratio n [mg/l ] Fina l report - Pag e 4 1 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LLC i s unauthorize d an d strictl yprohibited
. p . 4 5
p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 1 Degree o fhydrolysi s meanC o - C ( meanC 0 x100 % where : C 0= concentratio n a t t= 0 C t= concentratio n a t t= 5 day s Relativ e concentratio n (C r ) C r= meanC 0 •x100 % where : C 0~ concentratio n at t= 0 C t = concentratio n at t^ xhours Pseudo-firs t orde rcurv e 1 0 logC r= -ax t+ b where : t= tim e [hours ] a = slop e [hours' 1 ] b = intercep t Rat e constan t (k obs ) =-ax2.30 3 [hours' 1 ] where : 2.30 3 i s th e conversio n facto rbetwee n natura l an d bas e 1 0 logarithm s Arrheniu s equatio n Inkob s =^= p + !n A R xT where : Aploto fI n k o b sversu s 1/ Tgive s a linea rrelationshi p wit h a slop e o f- E/R an d intercep t I n A Half-lif e tim e (ty a ) ty a= [hours ] kob s

Fina l report - Pag e 4 2 -

Tir r i _ i —p . 4 6 Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LLC i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N 1 N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 1 15.7 . Result s 15.7.1
. Preliminar ytes t - Tie r1 Th e analytica l result s o fth e preliminarytes t ar e give n i n Tabl e 1 1 an d Tabl e 12
. At p H 4 , p H 7 an d p H 9 a degre e o fhydrolysi s o f£10 % afte r5 day s wa s observed
. Accordin g t o th e guideline , th e highe rTie rtes t wa s require d t o determin e th e half-lif e tim e o fth e tes t substance
. N o tes tsubstanc e wa s detecte d i n th e blan k buffe rsolution s p H 4 an d p H 7
. I n th e blan k

buffe rsolution s o fpH 9 a smal l respons e wa s observe d a t th e tes t substanc e retentio n time , th e contributio n bas e o n are a t o th e sample s a tt= 0 wa s 0.09% , thi s wa s negligible
. Th e mea n recoverie s o fth e buffe rsolution s fel l withi n th e criterio n rang e o f70-110%
. I t demonstrate d tha tth e analytica l metho d wa s adequat e t o support th e hydrolysi s stud yo n th e tes t substance
. Tabl e 1 1 Preliminar ytes t - hydrolysi s o fth e tes t substanc e a t p H 4 , p H 7 an d p H 9 p H cod e Samplin g tim e Analyse d concentratio n [mg/l ] Degre e o fhydrolysi s [%] Actua l p H p H cod e Samplin g tim e Analyse d concentratio n [mg/l ] Individua l Mea n Actua l p H
pH 4 0 hour s 78 6 4. 0 78 2 4. 0 5 day s 0.077 6 1 10 0 10 0 4. 0

0.061 6 1 10 0 4. 0 p H 7 0 hour s 87 6 7. 1 89 1 7. 1 5 day s 0.041 2 1 10 0 10 0 7. 1

0.11 5 10 0 7. 1 pH 9 0 hour s 97 8 9. 0 95 6 9. 0 5 day s 0.24 8 10 0 10 0 9. 0

n.d
. 10 0 9. 0 n.d
. no

tdetecte d Extrapolate d valu e Tabl e 1 2 Recoverie s p H cod e Nominal

concentratio n [mg/l l Analyse d

concentratio n [mg/l l Recovery

[% l Mea n

recovery [% l pH 4 112 2 78 6 7 0 7 0

112 2 78 2 7 0 pH 7 99 4 87 6 8 8 8 9

99 4 89 1 9 0 pH 9 105 5 97 8 9 3 9 2

105 5 95 6 9 1

Fina l report - Pag e 4 3 -Th e us e o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProject 49698 1 15.7.2 . Mai n stud y- Tie r2 PH 4 Th e analytica l result s o fth e mai n stud yar e give n i n Tabl e 1 3 - Tabl e 16
. N o tes t substanc e wa s detecte d i n th e blan k buffe rsolutions
. Th e mea n recoverie s o fth e buffe rsolution s relativ e t o th e stoc k solutio n fel l withi n th e criterio n rang e o f90-110%
. I t demonstrate d tha tth e analytica l metho d wa s adequat e t o support th e hydrolysi s stud y o n th e tes t substance
. Tabl e 1 3 Mai n tes t - hydrolysi s o fth e tes t substanc e a t 20° C Samplin g tim e Analyse d Relativ e Logarith m relativ e Actua l p H

concentratio n concentratio n concentratio n [hours ] [mg/] " - [%] 0 46 2 10 0 2.0 0 4. 0 0 46 1 10 0 2.0 0 4. 0 4 7 31 8 6 9 1.84 4. 0 4 7 32 3 7 0 1.8 5 4. 0 7 1 27 5 6 0 1.78 4. 0 7 1 27 6 6 0 1.7 8 4. 0 120. 5 18 6 4 0 1.6 1 4. 0 120. 5 18 9 4 1 1.6 1 4. 0 14 4 13 5 2 9 1.47 4. 0 14 4 13 6 2 9 1.47 4. 0 167. 5 11 7 2 5 1.40 4. 0 167. 5 11 7 2 5 1.4 0 4. 0 19 1 98. 0 2 1 1.3 3 4. 0 19 1 97. 7 2 1 1.3 3 4. 0 21 5 85.7- 1 9 1.2 7 4. 0 21 5 87. 1 1 9 1.2 8 4. 0 294. 5 56. 2 1 2 1.0 9 3. 9 294. 5 56. 2 1 2 1.0 9 3. 9 35 9 32. 3 7. 0 0.8 5 4. 0 35 9 31. 4 6. 8 0.8 3 4. 0

Fina l report - Pag e 4 4 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 1 Tabl e 1 4 Mai n tes t - hydrolysi s o fth e tes t substanc e a t 40° C Samplin g tim e Analyse d Relativ e Logarith m relativ e Actua l p H

concentratio n concentratio n concentratio n [hours ] [mg/] [%] 0 12 6 10 0 2.0 0 4. 0 0 12 6 10 0 2.0 0 4. 0 2 10 9 8 6 1.9 3 4. 0 2 10 9 8 6 1.9 4 4. 0 4. 5 91. 7 7 3 1.8 6 4. 0 4. 5 90. 7 7 2 1.8 6 4. 0 7 73. 0 5 8 1.76 4. 0 7 75. 5 6 0 1.78 4. 0 7. 6 73. 1 5 8 1.7 6 4. 0 7. 6 73. 3 5 8 1.7 6 4. 0 22.7 5 25. 5 2 0 1.3 1 4. 0 22.7 5 25. 2 2 0 1.3 0 4. 0 25. 5 20. 0 1 6 1.2 0 4. 0 25. 5 20. 4 1 6 1.2 1 4. 0 28.2 5 17. 0 1 3 1.1 3 4. 0 28.2 5 17. 3 1 4 1.1 4 4. 0 3 1 14. 9 1 2 1.07 4. 0 3 1 14. 3 1 1 1.05 4. 0 47. 5 4.8 2 3. 8 0.5 8 4. 0 47. 5 4.8 4 3. 8 0.5 8 4. 0
Tabl e 1 5 Mai n tes t - hydrolysi s o fth e tes t substanc e a t 50° C Samplin g tim e Analyse d Relativ e Logarith m relativ e Actua l p H

concentratio n concentratio n concentratio n [hours ] [mg/] [%] 0 47 4 10 2 2.0 1 4. 0 0 45 9 9 8 1.9 9 4. 0 1 38 5 8 3 1.92 4. 0 1 39 6 8 5 1.9 3 4. 0 2.2 5 31 0 6 6 1.82 4. 1 2.2 5 30 5 6 5 1.82 4. 0 3 26 9 5 8 1.76 4. 0 3 27 4 5 9 1.77 4. 1 4 22 8 4 9 1.6 9 4. 1 4 22 8 4 9 1.6 9 4. 1 5 19 7 4 2 1.6 3 4. 1 5 19 2 4 1 1.6 1 4. 1 6.2 5 15 1 3 2 1.5 1 4. 1 6.2 5 15 2 3 3 1.5 1 4. 1 7 13 3 2 8 1.45 4. 1 7 13 2 2 8 1.45 4. 1 23. 5 7.5 6 1. 6 0.2 1 4. 1 23. 5 7.2 6 1. 6 0.1 9 4. 1

Fina l report - Pag e 4 5 -Th e use ofthes e studies/documents fo rregulator yorregistratio n purposes , includin g bu t notlimite d to Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProject 49698 1 Tabl e 1 6 Recoverie s Temperatur e (°C ) Bul k

solutio n concentratio n frng/l l Analyse d

concentratio n rmg/l l Recovery 1

[% 1 Mea n

recovery [%1 2 0 45 3 46 2 10 2 10 2 46 1 10 2 4 0 12 2 12 6 10 3 10 3 12 6 10 4 5 0 45 5 47 4 10 4 10 2 45 9 10 1 Recover ywas determine d relative t o th e concentratio n ofth e prepare d bul k solution
. Arelativel yhig h spikin g concentratio n ha

d t o be use d t o allo

wdilutio n o

fth e sample s to th e en d solutio n {30/7 0 (v/v ) acetonitrile/borat e buffe r p H 9 ) to ensure sampl e stabilit ydurin g chromatographi canalysi s an d sufficien t analytica l sensitivity
. Fo rtestin g o fpseudo-firs torde rkinetic s th e mea n logarithm s o fth e relativ e concentration s betwee n 10 % an d 90 % wer e plotte d agains ttime
. A tal l temperature s linea rrelationship s wer e obtained
. Th e half-lif e time s o fth e tes tsubstanc e wer e determine d accordin g t o th e mode l fo rpseudo-firs t orde r reactions
. Al l logarithm s o fth e relativ e concentration s wer e correlate d wit h tim e usin g linea rregressio n analysis
. Figur e 9 illustrate s th e regressio n curve s an d Tabl e 1 7 show s th e statistica l parameters . Tabl e 1 7 Statistica l parameter s o fth e regressio n curve s Temperatur e (°C ) Slop e fhours" 1 l Intercep t Coefficien to f correlatio n 2 0 -3.2 1 x10' 3 1.9 8 0.99 6 4 0 -3.0 0 x10' 2 1.9 9 0.999 7 5 0 -7.6 5 x10' 2 2.0 0 0.9999 1

Th e rat e constan t (k ot)S ) an d half-lif e tim e o fth e tes tsubstanc e at eac h temperatur e wa s obtaine d an d th e Arrheniu s equatio n wa s use d t o determin e th e rat e constan tan d half-lif e tim e at 25° C {se e Tabl e 18)
. Tabl e 1 8 Rat e constant s (k obs ) an d half-lif e tim e (t V] ) Temperatur e 

### PC I kob s [hours" 1 1 2 0 7.3 9 x10' 3 3. 9 day s 2 5 1.33 x10" 2 2. 2 day s 4 0 6.9 0 x10" 2 1 0 hour s 5 0 1.76 x10" 1 3. 9 hour s

Fina l report - Pag e 4 6 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 1 oH 7 Th e analytica l result s o fth e mai n stud yar e give n i n Tabl e 1 9 - Tabl e 22
. N o tes tsubstanc e wa s detecte d i n th e blan k buffe rsolutions
. Th e mea n recoverie s o fth e buffe rsolution s relativ e t o th e stoc k solutio n fel l withi n th e criterio n rang e o f90-110% , I tdemonstrate d tha tth e analytica l metho d wa s adequat e t o support th e hydrolysi s stud y o n th e tes tsubstance
. Tabl e 1 9 Mai n tes t - hydrolysi s o fth e tes t substanc e a t 20° C Samplin g tim e Analyse d Relativ e Logarith m relativ e Actua l p H

concentratio n concentratio n concentratio n [hours ] [mg/] [%] 0 25 3 10 0 2.0 0 7. 1 0 25 2 10 0 2.0 0 7. 1 7 24 9 9 9 1.9 9 7. 1 7 25 6 10 1 2.0 1 7. 1 7 1 15 6 6 2 1.79 7. 1 7 1 15 5 6 1 1.79 7. 1 9 5 13 3 5 3 1.72 7. 1 9 5 13 4 5 3 1.73 7. 1 144. 5 94. 5 3 7 1.5 7 7. 1 144. 5 94. 2 3 7 1.5 7 7. 1 16 8 68. 5 2 7 1.43 7. 1 16 8 69. 5 2 8 1.44 7. 1 191. 5 59. 8 2 4 1.3 7 7. 1 191. 5 59. 9 2 4 1.38 7. 1 21 5 50. 5 2 0 1.30 7. 1 21 5 50. 5 2 0 1.30 7. 1 23 9 44. 3 1 8 1.24 7. 1 23 9 44. 9 1 8 1.2 5 7. 1 318. 5 29. 6 1 2 1.07 7. 1 318. 5 30. 1 1 2 1.08 7. 1 38 3 17. 5 6. 9 0.8 4 7. 1 38 3 17. 5 6. 9 0.8 4 7. 1

Fina l report - Pag e 4 7 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProject 49698 1 Tabl e 2 0 Mai n tes t - hydrolysi s o fth e tes t substanc e a t 40° C Samplin g tim e [hours ] Analyse d

concentratio n [mg/] Relativ e

concentratio n [% ] Logarith m relativ e

concentratio n Actua l p H

0 32 8 10 0 2.0 0 7. 1 0 32 6 10 0 2.0 0 7. 1 2.7 5 30 1 9 2 1,9 6 7. 1 2.7 5 30 6 9 3 1.9 7 7. 1 5 25 7 7 9 1.9 0 7. 1 5 26 1 8 0 1.9 0 7. 1 6. 5 25 0 7 6 1.8 8 7. 1 6. 5 24 5 7 5 1.8 7 7. 1 23.2 5 69. 2 2 1 1.33 7. 1 23.2 5 r68. 9 2 1 1.32 7. 1 25.7 5 57. 3 1 8 1.2 4 7. 1 25.7 5 58. 3 1 8 1.2 5 7. 1 28.2 5 49. 4 1 5 1.1 8 7. 1 28.2 5 50. 6 1 5 1.1 9 7. 1 30.7 5 41. 9 1 3 1.1 1 7. 1 30.7 5 41. 3 1 3 1.1 0 7. 1 47.2 5 15. 4 4. 7 0.6 7 7. 1 47.2 5 15. 4 4. 7 0.6 7 7. 1
Tabl e 2 1 Mai n tes t - hydrolysi s o fth e tes t substanc e a t 50° C Samplin g tim e [hours ] Analyse d

concentratio n [mg/] Relativ e

concentratio n 

### [% 1 Logarith m relativ e

concentratio n Actua l p H

0 43 3 10 0 2.0 0 7. 1 0 43 3 10 0 2.0 0 7, 1 1 37 3 8 6 1.9 3 7. 1 1 38 6 8 9 1.95 7. 1 2.2 5 32 8 7 6 1.8 8 7. 1 2.2 5 32 3 7 5 1.8 7 7. 1 3 28 0 6 5 1.8 1 7. 1 3 28 7 6 6 1.82 7. 1 4.25 23 3 5 4 1.7 3 7. 1 4.25 23 6 5 4 1.74 7. 1 5.2 5 19 5 4 5 1.6 5 7. 1 5.2 5 19 6 4 5 1.6 6 7. 1 6.2 5 16 2 3 7 1.5 7 7. 1 6.2 5 16 5 3 8 1.5 8 7. 1 7.2 5 13 8 3 2 1.5 0 7. 1 7.2 5 13 9 3 2 1.5 1 7. 1 24.7 5 8.0 8 1. 9 0.2 7 7. 1 24.7 5 8.0 5 1. 9 0.2 7 7. 1

Fina l report - Pag e 4 8 -Th e us e ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LLC i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 1 Tabl e 2 2 Recoverie s Temperatur e (°C ) Nomina l

concentratio n Fmg/l l Analyse d

concentratio n 

### rmg/l l Recovery 1



## r% i Mea n

recovery 

### f%l 2 0 26 3 25 3 9 6 9 6 25 2 9 6 4 0 34 2 32 8 9 6 9 5 32 6 9 5 5 0 44 2 43 3 9 8 9 8 43 3 9 8 Recover ywas determine d relative to th e concentratio n ofth e prepare d bul k solution
. Arelativel yhig h spikin g concentratio n had t o be use d t o allo wdilutio n ofth e sample s to th e en d solutio n (30/70 (v/v ) acetonitrile/borat e buffe r p H 9) t o ensur

e sampl e stabilit

ydurin g chromatographi canalysi s an d sufficien t analytica l sensitivity
. Fo rtestin g o fpseudo-firs torde rkinetic s th e mea n logarithm s o fth e relativ e concentration s betwee n 10 % an d 90 % wer e plotte d agains t time
. Atal l temperature s linea rrelationship s wer e obtained
. Th e half-lif e time s o fth e tes tsubstanc e wer e determine d accordin g t o th e mode l fo rpseudo-firs t orde r reactions
. Al l logarithm s o fth e relativ e concentration s wer e correlate d wit h tim e usin g linea rregressio n analysis
. Figur e 1 0 illustrate s th e regressio n curve s an d Tabl e 2 3 show s th e statistica l parameters . Tabl e 2 3 Statistica l parameter s o fth e regressio n curve s Temperatur e (°C ) Slop e [hours" 1 l Intercep t Coefficien to f correlatio n 2 0 -3.0 6 x10" 3 2.0 0 0.99 6 4 0 -2.9 5 x10* 2 2.0 3 0.99 8 5 0 -7.0 6 x10" 2 2.0 2 0.999 7

Th e rat e constan t(k obs ) an d half-lif e tim e o fth e tes tsubstanc e a t eac h temperatur e wa s obtaine d an d th e Arrheniu s equatio n wa s use d t o determin e th e rat e constan t an d half-lif e tim e at 25° C (se e Tabl e 24)
. Tabl e 2 4 Rat e constant s (k obs ) an d half-lif e tim e (t Vj ) Temperatur e 

### PC I kob s [hours" 1 l t « 2 0 7.0 5 x10' 3 4. 1 day s 2 5 1.2 7 x10' 2 2. 3 day s 4 0 6.7 9 x10 - 2 1 0 hour s 5 0 1.6 3 x10" 1 4. 3 hour s

Fina l report - Pag e 4 9 -Th e use ofthes e studies/documents fo rregulator yorregistratio n purposes , includin g bu t not limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 1 p H 9 Th e analytica l result s o fth e mai n stud yar e give n i n Tabl e 2 5 - Tabl e 28
. N o tes tsubstanc e wa s detecte d i n th e blan k buffe rsolutions
. Th e mea n recoverie s o fth e buffe rsolution s relativ e t o th e stoc k solutio n fel l withi n th e criterio n rang e o f90-110%
. I t demonstrate d tha tth e analytica l metho d wa s adequat e t o support th e hydrolysi s stud y o n th e tes t substance
. Tabl e 2 5 Mai n tes t - hydrolysi s o fth e tes t substanc e a t 20° C Sampling

time Analyse d Relativ e Logarithm relative Actua l p H concentratio n concentratio n concentratio n [hours ] [mg/] 

### E% ] 0 40 3 10 0 2.0 0 9. 1 0 40 3 10 0 2.0 0 9. 1 7 41 5 10 3 2.0 1 9. 0 7 41 1 10 2 2.0 1 9. 0 2 4 39 4 9 8 1.9 9 9. 0 2 4 39 7 9 9 1.9 9 9. 0 9 5 23 6 5 9 1.7 7 9. 0 9 5 23 1 5 7 1.7 6 9. 0 168. 5 14 4 3 6 1.5 5 9. 0 168. 5 14 5 3 6 1.5 6 9. 1 19 2 10 8 2 7 1.43 9. 1 19 2 10 9 2 7 1.43 9. 1 21 5 95. 2 2 4 1.37 9. 0 21 5 95. 0 2 4 1.37 9. 0 23 9 79. 0 2 0 1.2 9 9. 0 23 9 79. 8 2 0 1.30 9. 0 26 3 74. 8 1 9 1.2 7 9. 0 26 3 76. 6 1 9 1.2 8 9. 0 28 8 58. 2 1 4 1.1 6 9. 1 28 8 59. 0 1 5 1.1 7 9. 1 34 2 54. 2 1 3 1.1 3 9. 0 34 2 54. 7 1 4 1.1 3 9. 0 40 7 36. 6 9. 1 0.9 6 9. 0 40 7 36. 7 9. 1 0.9 6 9. 0

Fina l report - Pag e 5 0 -p . 5 4 Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropy!)anilin e NOTO XProjec t 49698 1 Tabl e 2 6 Mai n tes t - hydrolysi s o fth e tes t substanc e a t 40° C Samplin g tim e Analyse d Relativ e Logarith m relativ e Actua l p H

concentratio n concentratio n concentratio n [hours ] [mg/] [% ] 0 46 2 9 9 2.0 0 9. 0 0 47 1 10 1 2.0 0 9. 0 3 43 5 9 3 1.9 7 9. 0 3 42 5 9 1 1.9 6 9, 0 5.2 5 37 7 8 1 1.9 1 9, 0 5.2 5 37 3 8 0 1.9 0 9. 0 7 33 8 7 3 1.8 6 9. 0 7 34 4 7 4 1.8 7 9. 0 23. 5 12 1 2 6 1.4 1 9. 0 23. 5 12 3 2 6 1.42 9. 0 2 6 10 5 2 3 1.35 9. 0 2 6 10 6 2 3 1.36 9. 0 28. 5 94. 1 2 0 1.3 1 9. 0 28. 5 94. 9 2 0 1.3 1 9. 0 3 1 82. 1 1 8 1.2 5 9. 0 3 1 80. 6 1 7 1.2 4 9. 0 47. 5 37. 9 8. 1 0.9 1 9. 0 47. 5 37. 9 8. 1 0.9 1 9. 0
Tabl e 2 7 Mai n tes t - hydrolysi s ofth e tes t substanc e a t 50° C Samplin g tim e Analyse d Relativ e Logarith m relativ e Actua l p H

concentratio n concentratio n concentratio n [hours ] [mg/] [% ] 0 37 7 10 0 2.0 0 9. 1 0 38 1 10 0 2.0 0 9. 1 1 34 1 9 0 1.9 5 9. 1 1 33 8 8 9 1.9 5 9. 1 2 30 0 7 9 1.9 0 9. 1 2 30 3 8 0 1.9 0 9. 1 3 27 1 7 2 1.8 5 9. 1 3 26 7 7 1 1.8 5 9. 1 4 24 0 6 3 1.8 0 9. 1 4 24 4 6 5 1.8 1 9. 1 5 21 4 5 6 1.75 9. 1 5 21 8 5 8 1.76 9. 1 6 18 7 4 9 1.6 9 9. 1 6 19 0 5 0 1.7 0 9. 1 7 16 8 4 4 1.6 5 9. 1 7 16 7 4 4 1.6 5 9. 1 24. 5 19. 1 5. 0 0.7 0 9. 1 24. 5 19. 4 5. 1 0.7 1 9. 1

Fina l report - Pag e 5 1 -Th e use ofthes e studies/documents fo rregulator yorregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 1 Tabl e 2 8 Recoverie s Temperatur e (°C ) Nomina l

concentratio n [mg/l l Analyse d

concentratio n [mg/l ] Recovery 1

[% l Mea n

recovery [% l 2 0 41 0 40 3 9 8 9 8 40 3 9 8 4 0 51 4 46 2 9 0 9 1 47 1 9 2 5 0 38 7 37 7 9 7 9 8 38 1 9 8 Recover ywas determine d relative to th e concentratio n o fth e prepare d bul k

solution
. Arelativel yhig h spikin g concentratio n had to be use d to allo wdilutio n o

fth e sample s to th e en d solutio n (30/7 0 (v/v ) acetonitrile/borat e buffe r p H 9) to ensur

e sampl e stabilit

ydurin g chromatographi canalysi s an d sufficien t analytica l sensitivity
. Fo rtestin g o fpseudo-firs torde rkinetic s th e mea n logarithm s o fth e relativ e concentration s betwee n 10 % an d 90 % wer e plotte d agains ttime
. A tal l temperature s linea rrelationship s wer e obtained
. Th e half-lif e time s o fth e tes tsubstanc e wer e determine d accordin g t o th e mode l fo rpseudo-firs t orde r reactions
. Al l logarithm s o fth e relativ e concentration s wer e correlate d wit h tim e usin g linea rregressio n analysis
. Figur e 1 1 illustrate s th e regressio n curve s an d Tabl e 2 9 show s th e statistica l parameters . Tabl e 2 9 Statistica l parameter s o fth e regressio n curve s Temperatur e 

# rc ) Slop e [hours" 1 l Intercep t Coefficien t o f correlatio n 2 0 -2.7 4 x10" 3 2.0 1 0.99 2 4 0 -2.4 2 x10" 2 2.0 1 0.99 7 5 0 -5.3 0 x10" 2 2.0 1 0.999 8

Th e rat e constan t(k obs ) an d half-lif e tim e ofth e tes tsubstanc e at eac h temperatur e wa s obtaine d an d th e Arrhenius equatio n wa s use d t o determin e th e rat e constan t an d half-lif e tim e at 25° C (se e Tabl e 30)
. Tabl e 30 Rat e constant s (k obs ) an d half-life tim e (t % ) Temperatur e 

# rc i kob s [hours' 1 ] t w 2 0 6.3 0 x10' 3 4. 6 day s 2 5 1.1 1 x10" 2 2. 6 day s 4 0 5.5 8 x10' 2 1 2 hour s 5 0 1.22 x10" 1 5. 7 hour s

Fina l report - Pag e 5 2 -Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 1
15.8
. Conclusio n Th e preliminarytes t(Tie r1 ) an d mai n stud y(Tie r2 ) wer e performe d fo rth e determinatio n o fth e rat e o fhydrolysi s o fp-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e a t p H value s normall yfoun d i n th e environmen t (p H 4-9)
. Th e half-lif e time s o fth e tes t substanc e were :

pH 4 pH 7 pH 9 Temperatur e Temperatur e Temperatur e [°C ] 

### [°c i 

## r°ci 2 0 3. 9 day s 2 0 4. 1 day s 2 0 4. 6 day s 2 5 2. 2 day s 2 5 2. 3 day s 2 5 2. 6 day s 4 0 1 0 hour s 4 0 1 0 hour s 4 0 1 2 hour s 5 0 3. 9 hour s 5 0 4. 3 hour s 5 0 5. 7 hour s

Fina l report - Pag e 5 3 -Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substances (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 1 15.9 . Figure s

Tim e [hours ] Figur e 9 Plo t o fth e logarithm s o frelativ e concentratio n agains t tim e a t 20° C [top] , 40° C

[middle ] an d 50° C [bottom ] a t pH4 .

Fina l report - Pag e 5 4 -

nrr i lTh e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
.

Tim e [hours ] Figur e 1 0 Plo t o fth e logarithm s ofrelativ e concentratio n agains t tim e a t 20° C [top] , 40° C

[middle ] an d 50° C [bottom ] a t pH7 .

Fina l report - Pag e 5 5 -Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substances (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProject 49698 1



### \ 0.6 0 

### - 0.4 0 - I 

### , 1 1 . • , , 0 10 0 20 0 30 0 40 0 50 0 60 0 70 0 80 0 Tim e [hours ]

0.6 0 - I
. , , , , , 0 5 1 0 1 5 2 0 2 5 3 0 Tim e [hours ] Figur e 1 1 Plo t o fth e logarithm s o frelativ e concentratio n agains t tim e a t 20° C [top] , 40° C

[middle ] an d 50° C [bottom ] a t pH9 .

Fina l report - Pag e 5 6 -p . 6 0 Th e use o

fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 1 16 . ADSORPTIO N COEFFICIEN T 16.1 . Guideline s Organizatio n fo rEconomi cCo-operatio n an d Developmen t (OECD) , OEC D Guidelin e fo rth e Testin g o fChemical s no . 121 : "Estimatio n o fth e Adsorptio n Coefficien t (K o c ) o n Soil an d o n Sewag e Sludg e usin g Hig h Performanc e Liqui d Chromatograph y(HPLC)" , January22 , 2001
. Europea n Communit y(EC) , E C no . 440/2008 , PartC : Method s fo rth e Determinatio n o fEcotoxicity , Guidelin e C.19 : "Estimatio n o fth e Adsorptio n Coefficien t(K o c ) o n Soil an d o n Sewag e Sludg e usin g Hig h Performanc e Liqui d Chromatograph y(HPLC)" , Officia l Journa l o fth e Europea n Unio n no . L142 , Ma y31 , 2008
. 16.2 . Reagent s Wate rTa p wate rpurifie d b ya Milli- Q wate rpurificatio n syste m (Miilipore , Bedford , MA , USA ) Methano l VW R International , Leuven , Belgiu m Al l reagent s wer e o fanalytica l grade , unles s specifie d otherwise
. 16.3 . Performanc e o fth e stud y Th e principl e o fth e tes tmetho d i s simila rt o tha t o fth e OEC D guidelin e no . 117 ; "Partitio n coefficien t (n-octanol/water) , hig h performanc e liqui d chromatograph y(HPLC ) method"
. Whil e passin g throug h th e colum n alon g wit h th e mobil e phas e th e tes tsubstanc e interact s wit h th e stationaryphase
. A s a resul t o fpartitionin g betwee n mobil e an d stationaryphases , th e tes t substanc e i s retarded
. Th e dua l compositio n o fa cyanopropy l stationaryphase , havin g pola ran d non-pola rsite s allow s fo rinteractio n o fpola ran d non-pola rgroup s o fa molecul e i n a simila rwa ya s i s th e cas e fo rorgani cmatte ri n soi l o r sewag e sludg e matrices
. Thi s enable s th e relationshi p betwee n th e retentio n tim e o n th e colum n an d th e Ko co n organi cmatte rt o b e established
. Accordin g t o th e guidelines , th e determinatio n o fth e K o cfo rtes tsubstance s tha t ar e ionize d fo ra t leas t 10 % withi n p H 5. 5 t o 7. 5 shoul d b e performe d wit h both th e ionize d an d non-ionize d form
. Therefore , th e pK avalue s o fth e tes tsubstanc e wer e calculate d usin g th e Perri n calculatio n metho d (pKal c5.0 , module i n Palla s 3.0 , CompuDru g Internationa l Sa n Francisco , CA , USA)
. Base d o n th e calculations , th e HPL C analysi s wa s performe d a tneutra l pH
. Solution s o freferenc e substance s wit h know n lo g K Q Cvalue s base d o n soi l adsorptio n dat a an d th e tes tsubstanc e wer e analysed
. Th e capacit yfacto r(k 1 ) o feac h compoun d wa s calculate d fro m it s retentio n time
. Th e lo g k' value s ofth e reference s substance s wer e plotte d agains t th e know n lo g Ko c values
. Alinea rregressio n progra m wa s use d t o calculat e th e calibratio n curve . Linea rregressio n analysi s wa s performe d usin g th e leas t square s method
. Th e coefficien t o fcorrelatio n (r) wa s calculated
. Th e lo g K o cvalu e fo rth e tes t substanc e wa s calculate d b ysubstitutin g it s mea n lo g k' i n th e calibratio n curve . Th e valu e oflo g K o cobtaine d fro m duplicat e measurement s wa s withi n ±0.2 5 lo g units
.

Fina l report - Pag e 5 7 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d to Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibite d p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t49698 1 16.4 . Analytica l metho d 16.4.1 . Analytica l condition s

Colum n temperatur e Mobil e phas e Flo w Injectio n volum e U Vdetectio n Instrumen t Detecto r Colum n Allianc e Separatio n Modul e 269 5 (Waters , Milford , MA , USA ) Dua l AAbsorbanc e Detecto r248 7 (Waters ) Hypersi l BDS-CN , 15 0 m m x4. 6 m m i.d. , d p = 5 p m (Therm o Fishe rScientific , Waltham , MA , USA ) 35°C±1° C 30/7 0 (v/v ) methanol/wate r 1 ml/mi h 1 0 p i 21 0 n m 16.4.2
. Preparatio n o fth e solution s Solutio n o fth e unretaine d compoun d A4.8 1 g/ l stoc k

solutio n o fformamid e (99.8% , [75-12-7] , Acro s Organics , Geel , Belgium ) i n methano l wa s used
. 500p l o fth e stoc k solutio n + 66 7 p i wate rwa s dilute d i n a 5 m l volumetri cflask
. Th e volumetri cflas k

wa s fille d u p t o th e mar k wit h 30/7 0 (v/v ) methanol / water . Th e formamid e blan k

solutio n wa s 30/7 0 (v/v ) methanol/water
. Referenc e substanc e solution s Stoc k solution s o fth e referenc e substance s at concentration s o fapproximatel y1 g/ l i n methano l wer e used
. Th e stoc k

solution s wer e diluted
. Tw o referenc e substanc e solution s wer e made
. Referenc e solutio n containin g Acetanilide , Atrazine , Benzoi caci d phenyleste ran d Phenanthren e wa s mad e a s followed : 5 0 p i o feac h referenc e substanc e + 26 7 p i wate rwer e dilute d i n a 5 m l volumetri cflask
. Th e volumetri cflas k

wa s fille d u p t o th e mar k wit h 30/7 0 (v/v ) methanol / water . Th e secon d referenc e substanc e solutio n containin g Monuron , 2,5-Dichloroanilin e an d Fenthio n wa s mad e a s followed : 5 0 p i o feac h referenc e substanc e + 20 0 p i wate rwer e dilute d i n a 5 m l volumetri cflask
. Th e volumetri c flas k wa s fille d u p t o th e mar k wit h 30/7 0 (v/v) methanol / water . Th e blan k

solutio n fo rth e mixtur e o freferenc e substance s wa s 70/3 0 (v/v ) methanol/water
. Referenc e substanc e Purity CA S numbe r Supplie r lo g Ko c Acetanilid e > 99.9 % 103-84- 4 Sigma-Aldric h 1.2 6 Atrazin e 97.5 % 1912-24- 9 Sigma-Aldric h 1.8 1 Monuro n 99.9 % 150-68- 5 Sigma-Aldric h 1.9 9 2,5-Dichloroanilin e 99.8 % 95-82- 9 Sigma-Aldric h 2.5 5 Benzoi caci d phenyleste r 99.9 % 93-99- 2 Sigma-Aldric h 2.8 7 Fenthio n 98.3 % 55-38- 9 Sigma-Aldric h 3.3 1 Phenanthren e 98.1 % 85-01- 8 Acro s Organic s 4.0 9 "value s accordin g t o th e OEC D 12 1 guidelin e base d o n soi l adsorptio n data Acro s Organics , Geel , Belgiu m Sigma-Aldrich , Stei n heim , Germany Tes t solutio n A144 5 mg/ l stoc k

solutio n o fth e tes t substanc e wa s prepare d i n methanol
. Th e stoc k solutio n wa s dilute d t o obtai n a n en d solutio n o f30/7 0 (v/v ) methanol/water
. Th e fina l concentratio n o fth e tes t substanc e solutio n wa s 14. 5 mg/l
. Th e tes t substanc e blan k solutio n wa s 30/7 0 (v/v ) methanol/water
.

Fina l report - Pag e 5 8 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 1 16.4.3 . Injection s Th e referenc e substanc e an d tes t substanc e solution s wer e injecte d i n duplicate
. Blan k solution s wer e analyse d b ysingl e injection
. 16.5 . Formula s (t - t ) Capacit yfacto r(k' ) k = —^
_ t o where : t r= retentio n tim e t 0= mea n colum n dea d tim e Calibratio n curv e logk* = alogK o c+ b where : a = slop e b = intercep t 16.6 . Result s 16.6.1 . Calculatio n o fpK avalue s Th e followin g pK avalue s i n th e logarith m rang e o f1 -1 4 fo racidi can d basi cgroup s i n th e molecula r structur e o fth e tes t substanc e wer e calculate d usin g th e Perri n calculatio n method : Acidi c Basi c non e RARN(R1R2 ) pKa4.2 1 RO R pK a-3.2 8 RO R pK a-3.8 8 RO R pK a-4.3 7 Base d o n th e calculate d pK avalue s th e stud ywa s performed a t neutra l pH . 16.6.2 . Determinatio n o fth e K o c HPL C chromatogram s o fth e tes tsubstanc e solutio n an d correspondin g blan k ar e show n i n Figur e 12
. I n th e chromatogra m o fth e tes t solution , on e majo rpea k an d severa l smal l tes t substanc e peak s wer e observed
. Th e result s o fth e HPL C metho d ar e give n i n th e Tabl e 31
. Figur e 1 3 show s th e calibratio n curv e o f th e lo g k 1 o fth e referenc e substance s a s functio n o flo g K^
. Th e equatio n o fth e regressio n lin e was : lo g k ' = 0.50 2 xlo g K<, c- 0.93 0 ( r= 0.96 , n = 14)
.

Fina l report - Pag e 5 9 -Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substances (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropy.)anilin e NOTO XProjec t 49698 1 Tabl e 3 1 K o co fth e tes t substanc e Substanc e t r , 1 tp. 2 mea n t r lo g K o c Ko c Are a [min i [min i (n=2 ) % Formamid e (to) 1.99 7 1.99 5 1.99 6 Acetanilid e 2.85 3 2.85 3 1.2 6 Atrazin e 4.26 2 4.25 7 1.8 1 Monuron 4.14 8 4.14 7 1.9 9 2,5-Dichloroanilin e 5.38 0 5.38 2 2.5 5 Benzoi caci d phenyleste r 9.52 2 9.52 9 2.8 7 Fenthio n 21.31 4 21.34 7 3.3 1 Phenanthren e 19.63 4 19.69 1 4.0 9 Impurit yI 1.96 9 1.96 8 1.96 9 * * 0.2 8 Impurit yI I 2.94 6 2.93 9 2.94 3 1.2 1 1.6 x10 1 0.6 7 Impurit yII I 3.67 2 3.64 8 3.66 0 1.70 5. 0 x10 1 1. 1 Tes t substanc e 4.16 9 4.16 3 4.16 6 1.93 8. 4 x10 1 9 6 Impurit yI V 4.76 6 4.76 0 4.76 3 2.1 4 1. 4 x10 2 0.9 1 Impurit yV 5.00 5 5.00 4 5.00 5 2.2 1 1. 6 x10 2 0.7 7 Impurit yV I 5.17 3 5.16 9 5.17 1 2.2 6 1. 8 x10 2 0.5 2

' Since th e retentio n tim e o fImpurit yI was less tha n that o

fformamide , a K o can d lo g K 0! ; valu e coul d no t be calculated
. 16.7 . Conclusio n Th e HPL C metho d usin g soil-adsorption-referenc e dat a wa s applie d fo rth e determinatio n o fth e adsorptio n coefficien t (K o c ) o fp-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline
. Th e Ko can d lo g KoCvalu e o fth e tes tsubstanc e an d impuritie s wit h pea k area s > 1 % a t neutra l p H was : lo g Ko c Are a

% Tes tsubstanc e 8. 4 x10 1 1.93 9 6 Impurit yII I 5. 0 x10 1 1.70 1. 1

Fina l report - Pag e 6 0 -p . 6 4 Th e use o

fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibite d p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e 16.8 . Figure s NOTO X Projec t 49698 1

0.03S ' 0.0M ' 

### !

• 0.01 5 0.010
- 0.0W 0.000 0.030
- 0.OW
-
}

: O.OIt V 0.010
- o.oosOO00 - Figur e 1 2 HPL C chromatogram s o fth e 14. 5 mg/ l tes t solutio n [top ; res . id . 1571 ] an d

correspondin g blan k [bottom ; res . id . 1572] . 1. 5
1 -

o 

### 0. 5 - 0 - 

### -0. 5 -

- 1 

### 1.

5 2. 5 3 3. 5 4 4. 5 log K o c Figur e 1 3 Th e regressio n lin e o fth e referenc e substances : lo g k ' versu s lo g K o c .

Fina l report - Pag e 6 1 -Th e us e ofthes e studies/documents fo rregulator yc rregistratio n purposes , includin g bu t not limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substances (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p . 6 5
p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e 17 . DISSOCIATIO N CONSTAN T NOTO XProject49698 1

17.1
. Guideline s Organizatio n fo rEconomi cCo-operatio n an d Developmen t (OECD) , OEC D Guideline s fo rTestin g o f Chemicals , Guidelin e no . 112 : "Dissociatio n Constant s i n Water " Adopte d May , 1 2 1981
. 17.2 . Reagent s Citri caci d monohydrat e Sodiu m chlorid e (NaCI ) Triethylamin e Aceti caci d Disodiu m tetraborat e decahydrat e (Na 2 B 4 O7*10H 2 O ) Hydrochlori caci d (HCI ) Hydrochlori caci d (HCI ) Sodiu m hydroxid e (NaOH ) Sodiu m hydroxid e Potassiu m dihydroge n phosphat e (KH 2 P0 4 ) Disodiu m hydrogenphosphat e dodehydrat e (Na 2 HP0 4 *12H 2 0 ) Potassiu m chlorid e Acetonitril e Wate r Merck , Darmstadt , German y Merc k Merc k Merc k Merc k 37 % Merc k 1M , Merc k pellets , Merc k 1M , Merc k Merc k Merc k Merc k Biosolve , Valkenswaard , Th e Netherland s Ta p wate rpurifie d b ya Milli- Q wate rpurificatio n syste m (Millipore , Bedford , MA , USA)
. Al l reagent s were o

fanalytica l grade , unles s specifie d otherwise . 17.3 . Buffer s Al l buffer s wer e prepare d i n wate ran d adjuste d t o th e require d p H wit h th e chemica l writte n afte rth e sign"/"
. Buffe rp H 0 : 1 M HC I Buffe rp H 1 : 0.48 8 g HC I + 0.37 2 g KC I i n 100 0 m l water/adjuste d t o p H 1 wit h 1 M HC I Buffe rp H 2 : 3.1 7 g Citri caci d monohydrat e + 0.15 8 g HC I + 1.8 0 g NaC I i n 100 0 m l water /

adjuste d t o p H 2 wit h 1 M HC I Buffe rp H 3 : 4.2 1 g Citri caci d monohydrat e + 0.44 5 g NaO H + 1,8 1 g NaC I i n 100 0 m l wate r
Fina l report - Pag e 6 2 -p . 6 6 Th e use o

fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t49698 1 Buffe rp H 4 : 5.8 6 g Citri caci d monohydrat e + 1.35 g NaO H + 1.3 0 g NaC I i n 100 0 m i water /

adjuste d t o p H 4 wit h 1 M NaO H Buffe rp H 5 : 0. 6 g Aceti caci d i n 100 0 m l wate r/ adjuste d t o p H 5 wit h 1 M NaO H Buffe rp H 6 : 4.2 1 g Citri caci d monohydrat e + 2.0 2 g NaO H i n 100 0 m l wate r/ adjuste d t o p H 6 wit h 1 M NaO H Buffe rp H 7 : 3.5 6 g KH 2 P0 4+ 14.6 8 g Na 2 HP0 4 *12H 2 0 i n 100 0 m l wate r/ adjuste d t o p H 7 wit h 1 M HC I Buffe rp H 8 : 4.9 6 g Na 2 B4O7*10H 2 O + 0.7 8 g HC I i n 100 0 m ! wate r/ adjuste d t o p H 8 wit h 1 M HC I Buffe rp H 9 : 4.9 5 g Na 2 B 4 O7*10H2 O + 0.1 6 g HC I i n 100 0 m l wate r/ adjuste d t o p H 9 wit h 1 M HC I Buffe rp H 10 : 4.9 6 g Na 2 B 4 0 7 *10H 2 O + 0.7 6 g NaO H i n 100 0 m l wate r/ adjuste d t o p H 1 0 wit h 1 M HC I Buffe rp H 11 : 1.02 g Triethylamin e i n 100 0 m l wate r/ adjuste d t o p H 1 1 wit h 1 M HC I Buffe rp H 12 : 0.3 9 g NaO H i n 100 0 m l wate r/ adjuste d t o p H 1 2 wit h 1 M NaO H Buffe rp H 13 : 4.05 g NaO H i n 100 0 m l wate r/ adjuste d t o p H 1 3 wit h 1 M NaO H Buffe rp H 2.6 : 10 0 m l buffe rp H 3 adjuste d t o p H 2. 6 wit h 1 M HC I Buffe rp H 2.8 : 10 0 m l buffe rp H 3 adjuste d t o p H 2. 8 wit h 1 M HC I Buffe rp H 3.0 : 10 0 m l buffe rp H 3 adjuste d t o p H 3. 0 wit h 1 M HC I Buffe rp H 3.2 : 10 0 m l buffe rp H 3 adjuste d t o p H 3. 2 wit h 1 M NaO H Buffe rp H 3.4 : 10 0 m l buffe rp H 3 adjuste d t o p H 3. 4 wit h 1 M NaO H Buffe rp H 3.6 : 10 0 m l buffe rp H 3 adjuste d t o p H 3. 6 wit h 1 M NaO H Buffe rp H 3.8 : 10 0 m l buffe rp H 3 adjuste d t o p H 3. 8 wit h 1 M NaO H

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fthes e studies/documents fo rregulator yorregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibite d p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 1 17.4 . Performanc e o fth e stud y Thre e method s ar e availabl e fo rth e determinatio n o fth e dissociatio n constant(s ) (pK a ) o fth e tes t substance
. Th e principl e o feac h metho d i s describe d below : 1
. Titratio n metho d Ates t solutio n o fth e tes tsubstanc e i s titrate d wit h standar d aci d o rbase , a s appropriate
. Th e pK a value s ar e determine d base d o n th e titratio n curv e obtained
. Th e metho d i s no t suitabl e fo rlo w solubilit ycompounds
. 2
. Spectrophotometri cmetho d Th e spectrophotometri cmetho d involve s th e determinatio n o fth e relativ e concentration s o fth e ionise d an d unionise d form s o fth e tes t substanc e an d thei rp H dependence
. Th e metho d i s onl yapplicabl e t o compound s havin g appreciabl ydifferen tUV/Vi s adsorptio n spectr a fo rth e ionise d an d unionise d forms
. Thi s metho d ma yb e suitabl e fo rlo wsolubilit ycompounds
. 3
. Calculation metho d Calculation o fth e pK avalue s fro m th e structura l formul a i s performe d usin g th e Perri n calculatio n metho d (pKal c5.0 , module i n Palla s 3.0 , CompuDru g International , Sa n Francisco , CA , USA)
. Th e spectrophotometri cmetho d wa s selecte d fo rth e determinatio n o fth e pK avalue s o fth e tes t substance
. Th e calculatio n metho d wa s use d fo ra preliminaryestimatio n o fth e P o wo fth e tes t substance
. 17.5 . Spectrophotometri c metho d 17.5.1 . Tes t syste m Spectrophotomete rSingle-beam , Varia n Gar y5 0 UVA/IS , Varian , Mulgrave , Victoria , Australi a Sli t 1. 5 n m Sca n spee d 60 0 nm/mi n Prob e Ultr a min i immersio n prob e wit h titaniu m tub e an d stainles s stee l handle
. Pris m mad e fro m quartz , pathlengt h = 1 0 m m Typ e o fcuvett e Quart z Background-correctio n Referenc e solutio n 17.6 . Performanc e o fth e tes t 17.6.1 . Searc h fo rspectr a o fth e pur e molecul e an d th e pur e anio n Th e - N grou p i n th e tes tsubstanc e ca n dissociate
. Th e pK avalu e t o b e determine d i s thu s fo ra basi c group . A438 5 mg/ l solutio n o fth e tes t substanc e wa s prepare d i n acetonitril e an d furthe rdilute d i n acetonitril e prio rt o use . Then , fourtee n tes t solution s (al l 2 0 mg/l ) wer e prepare d b ydilutin g th e stoc k solutio n wit h variou s buffer s (p H 0 , p H 1 , p H 2 , pH.3 , p H 4 , p H 5 , p H 6 , p H 7 , p H 8 , p H 9 , p H 10 , p H 11 , p H 12an d p H 13)
. Referenc e solution s wer e prepare d b ydilutin g acetonitril e th e sam e a s th e tes t solutio n wit h eac h o f th e abov e mentione d buffers
. Fro m eac h o fth e tes t solutions , a n absorptio n spectru m betwee n 20 0 an d 90 0 n m wa s recorde d wit h th e appropriat e referenc e solutio n a s reference
. Th e p H o feac h solutio n (tes t an d referenc e solution ) wa s measure d usin g a p H meter .

Fina l report - Pag e 6 4 -

nrr iip . 6 8 Th e us e o

fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 1 17.6.2 . Choic e o fa n analytica l wavelengt h Th e analytica l wavelengt h wa s chose n fro m th e absorptio n spectr a recorde d fro m th e tes t substanc e betwee n p H 0 an d p H 13
. r 17.6.3 . Searc h fo ra n approximat e pK avalu e Th e optica l absorbance s a t th e analytica l wavelengt h wer e obtaine d fro m th e absorptio n spectr a recorde d fro m 2 0 mg/ l solution s betwee n p H 0 an d p H 7
. Fro m thes e results , a n approximat e pK a valu e wa s calculated
. 17.6.4 . Exac t determinatio n o fpK a Thre e differen tstoc k

solution s ofth e tes t substanc e i n acetonitrile ; 377 0 mg/l , 438 5 mg/ l an d 1537 6 mg/ l wer e prepare d an d dilute d wit h acetonitril e prio rt o use . Nin e tes t solution s (al l 2 0 mg/l ) wer e prepare d b ydilutin g eac h stoc k solutio n wit h buffer s p H 0 , p H 2.6 , p H 2.8 , p H 3 , p H 3.2 , p H 3.4 , p H 3.6 , p H 3. 8 an d p H 6
. Referenc e solution s wer e prepare d b ydilutin g acetonitril e th e sam e a s th e tes t solutio n wit h eac h o f th e abov e mentione d buffers
. Th e optica l absorbance s at th e analytica l wavelengt h o fal l nin e tes t solution s wer e measure d afte r zero-adjustmen twit h th e appropriat e referenc e solution
. Th e p H o feac h solutio n (tes t an d reference ) wa s measured
. 17.7 . Formula s Fo ra n basi cgroup , th e followin g equatio n i s use d i fA i > Am :

A-A m pK a = p H + lo g A i - A I fA m > Ai : A m - A pKa = p H + lo g A-A i Wher e A m i s th e optica l absorbanc e o fth e neutra l molecule
. A i i s th e optica l absorbanc e o fth e io n an d Ai s th e optica l absorbanc e ofth e mixtur e o fio n an d neutra l molecule
. p H i s th e actua l pH
.

Fina l report - Pag e 6 5 -Th e use o fthes e studies/documents fo rregulator yorregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProject 49698 1 17.8 . Result s 17.8.1 . Calculatio n metho d Th e followin g pK avalues'i n th e logarith m rang e o f1 -1 4 fo racidi can d basi cgroup s i n th e molecula r structur e o fth e tes tsubstanc e wer e calculate d usin g th e Perri n calculatio n method : Acidi c Basi c non e RARN(R1R2 ) pKa4.2 1 RO R pK a-3.2 8 RO R pK a-3.8 8 RO R pK a -4.3 7
17.8.2 . Searc h fo rspectr a ofth e pur e molecul e an d th e pur e anio n Th e - N grou p i n th e tes t substanc e ca n dissociate
. Th e pK avalu e t o b e determine d i s thu s fo ra n basi cgroup . Th e spectr a recorde d a tp H 0 an d p H 1 wer e comparabl e an d s o wer e th e spectr a recorde d a t p H 5 til l p H 13
. Th e spectr a recorde d atp H 2 , p H 3 an d p H 4 ha d a for m somewher e betwee n thos e a t p H 1 an d p H 5 an d therefor e calle d intermediat e spectra . I t wa s conclude d tha t spectr a recorde d a t p H < 1 ar e o fth e ionize d tes t substance , wherea s spectr a recorde d a t p H > 5 ar e o fth e non-ionize d tes t substance
. Th e intermediat e spectr a ar e o fa combinatio n o fbot h species
. Absorptio n spectr a a t p H 0 t o 1 3 ar e show n i n Figur e 1 4 t o Figur e 27
. 17.8.3 . Choic e o fa n analytica l wavelengt h Fro m th e spectr a o fth e ionize d substanc e wit h th e spectr a o fth e non-ionize d substance , i t wa s conclude d tha t th e differenc e i n optica l absorbanc e i s maxima l a t 24 9 nm . Henc e 24 9 n m wa s chose n a s th e analytica l wavelength
. 17.8.4 . Searc h fo ra n approximat e pK avalu e Th e optica l absorbance s a t24 9 n m wer e determined
. A s wel l a s th e actua l p H value s o fth e tes t solution s an d actua l p H value s o fth e referenc e solutions
. Fro m thes e absorbance s an d th e actua l p H value , th e approximat e pK avalu e wa s calculated
. Al l result s ar e summarize d i n Tabl e 32
.

Fina l report - Pag e 6 6 -p . 7 0 Th e use o

fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 1
Tabl e 3 2 Actua l p H value s an d optica l absorbance s a t 24 9 nm . Concentratio n stoc k [mg/l ] actua l p H value s o f

th e referenc e solution s actua l p H value s o f

th e tes t solution s Absorbanc e fo rth e tes t solutio n 

### [units l pK avalu e
438 5 0.3 9 0.3 6 0.013 8

0.9 7 1.0 1 0.017 9 3.2 5 1.9 7 1.9 9 0.082 4 2.9 7 3.0 2 3.0 4 0.355 0 3.0 8 4.1 2 4.1 2 0.639 8 3.2 9 5.0 8 5.0 8 0.721 1 3.3 2 6.1 0 6.1 1 0.733 4 7.1 2 7.1 1 0.728 4 7.9 9 8.0 2 9.0 2 9.0 4 9,9 7 10.0 1 10.7 8 10. 8 11.6 3 11.6 5 12.6 8 12.7 1
Th e approximat e pK avalu e wa s calculate d t o b e 3.18
. 17.8.5 . Exac t determinatio n ofth e pK avalu e Th e actua l p H value s o fth e referenc e solution s wer e 0.30 , 2.62 , 2.81 , 3.03 , 3.24 , 3.45 , 3.64 , 3.8 4 an d 5.70
. Th e optica l absorbance s a t 24 9 n m o fal l nin e tes tsolution s an d th e seve n calculate d pK a value s ar e summarize d i n Tabl e 33
.

Fina l report - Pag e 6 7 -Th e us e o fthes e studies/documents fo rregulator yc rregistratio n purposes , includin g bu t no t limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substances (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 1
Tabl e 3 3 Optica l absorbance s a t 24 9 nm . Concentratio n stoc k [mg/l ] Actua l p H Absorbanc e fo r th e tes t solutio n 

### funits l pK

g valu e

438 5 0.3 0 0.017 0 2.6 2 0.166 2 3.2 3 2.8 1 0.232 8 3.2 1 3.0 4 0.313 7 3.2 3 3.2 4 0.394 5 3.2 4 3.4 5 0.478 3 3.2 6 3.6 4 0.548 8 3.2 7 3.8 4 0.613 4 3.2 7 5.7 0 0.773 2
377 0 0.2 9 0.023 0 2.6 2 0.171 9 3,2 4 2.8 2 0.235 3 3,2 4 3.0 4 0.322 9 3.2 3 3.2 4 0.402 4 3.2 5 3.4 5 0.492 5 3.2 5 3,6 4 0.568 0 3.2 5 3.8 4 0.630 4 3.2 6 5.7 5 0.789 6
1537 6 0.2 9 0.023 0 2.6 2 0.184 4 3.2 2 2.8 2 0.246 5 3.2 3 3.0 4 0.331 6 3.2 4 3.2 4 0.414 4 3.2 6 3.4 6 0.507 3 3.2 8 3.6 4 0.580 6 3.2 8 3.8 4 0.648 6 3.2 9 5.7 8 0.824 0
Th e twent yon e pK avalue s wer e averaged , resultin g i n th e pK avalu e o f3.25
. Th e standar d deviatio n i s 0.02
. Th e tes t wa s performe d i n a roo m wher e th e temperatur e (20. 4 ±0.6°C ) wa s measure d continuousl y usin g a validate d computerise d syste m (Ree s Scientific , NJ , USA)
. 17.9 . Conclusio n Th e pK avalu e fo rp-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e wa s determine d t o b e 3.2 5 usin g th e spectrophotometri cmethod
. Thi s pK g valu e correspond s t o dissociatio n o fa basi cgrou p i n th e molecule
. Fina l report - Pag e 6 8 -p . 7 2 Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LLC i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e 17.10.Figure s NOTO XProject49698 1

< 

### 1.5 - 1.0 - 

### 0.5 - 0.0 - 20 0 40 0 60 0 Wavelengt h (nm ) 80 0 Figur e 1 4 Absorptio n spectru m o fa 2 0 mg/l solutio n oftes t substanc e i n buffe rp H 0 .

1.5 -

< 1.0 - 

### 0.5 - 0.0 - 20 0 40 0 60 0 Wavelengt h (nm ) 80 0 Figur e 1 5 Absorptio n spectru m o fa 2 0 mg/ l solutio n o ftes t substanc e i n buffe rp H 1 .

< 

### 1.5 - 1.0 - 0.5 - 0.0 - 20 0 40 0 60 0 Wavelengt h (nm ) 80 0 Figur e 1 6 Absorptio n spectru m o fa 2 0 mg/l solutio n o ftes t substanc e i n buffe rp H 2 .

Fina l report - Pag e 6 9 -Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p . 7 3
p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProject 49698 1

1.5 - 1.0 - 0.5 - 0.0 - 20 0 40 0 60 0 Wavelengt h (nm ) 80 0 Figur e 1 7 Absorptio n spectru m o fa 20 mg/ l solutio n o ftes t substanc e i n buffe rp H 3 .


### 1.5 -

< 1.0 - 

### 0.5 - 0.0 - 20 0 — i 1

40 0 60 0 Wavelengt h (nm ) 80 0 Figur e 1 8 Absorptio n spectru m o fa 20 mg/l solutio n oftes t substanc e i n buffe rp H 4 .
1.5 -

£ 1 

### < 1.0 - 

### 0.5 - 0.0 - 20 0 40 0 60 0 Wavelengt h (nm ) 80 0 Figur e 1 9 Absorptio n spectru m o fa 2 0 mg/l solutio n o ftes t substanc e i n buffe rp H 5 .

Fina l repor

t - Pag e 7 0 -



### 1Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d to Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProject 49698 1

20 0 40 0 60 0 80 0 Wavelengt h (nm ) Figur e 2 0 Absorptio n spectru m o fa 2 0 mg/ l solutio n o ftes t substanc e i n buffe rp H 6 .


### 1.5 - 1.0 - 0.5 - 0.0 - 20 0 40 0 60 0 Wavelengt h (nm ) 80 0 Figur e 2 1 Absorptio n spectru m ofa 2 0 mg/ l solutio n o ftes t substanc e i n buffe rp H 7 .

< 

### 1.5 - 1.0 - 

### 0.5 - 0.0 - 20 0 40 0 60 0 Wavelengt h (nm ) 80 0 Figur e 2 2 Absorptio n spectru m o fa 2 0 mg/l solutio n o ftes t substanc e i n buffe rp H 8 .

Fina l repor

t - Pag e 71 -Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p . 7 5
p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropy!)anilin e NOTO XProjec t 49698 1

V ) < 1.5 - 1.0 - 0.5 - 0.0 - 20 0 40 0 60 0 Wavelengt h (nm ) 80 0 Figur e 2 3 Absorptio n spectru m o fa 2 0 mg/l solutio n o ftes t substanc e i n buffe rp H 9 .
1.5 -
S3 < 1.0 - 0.5 - 0.0 - 20 0 40 0 60 0 Wavelengt h (nm ) 80 0 Figur e 2 4 Absorptio n spectru m o fa 2 0 mg/ l solutio n oftes t substanc e i n buffe rp H 10 .


### 1.5 -
« 1.0 H < 

### 0.5H 0.0 - 20 0 40

0 60

0 Wavelengt

h (nm ) 80

0 Figur e 2 5 Absorptio n spectru m o fa 2 0 mg/ l solutio n o ftes t substanc e i n buffe rp H 11 ,

Fina l report - Pag e 7 2 -p . 7 6 Th e us e ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t49698 1

40 0 60 0 Wavelengt h (nm ) 80 0 Figur e 2 6 Absorptio n spectru m o fa 2 0 mg/l solutio n o ftes t substanc e i n buffe rp H 12 .


## 1.5 -
« 1.0-1 X I 

### < 

## 0.5 J 0.0 - 20 0 40

0 60

0 Wavelengt

h (nm ) 80

0 Figur e 2 7 Absorptio n spectru m o fa 2 0 mg/ l solutio n o ftes t substanc e i n buffe rp H 13 .

Fina l report - Pag e 7 3 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d to Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProject 49698 1 18. VISCOSIT Y 18.1 . Guideline s Organizatio n fo rEconomi cCo-operatio n an d Developmen t (OECD) , OEC D Guideline s fo rth e Testin g o fChemical s no . 114 : "Viscosit yo fLiquids" , Ma y12,1981
. Internationa l Organizatio n fo rStandardizatio n (ISO) , IS O 3104 : "Petroleu m Product s - Transparen t an d Opaqu e Liquid s - Determinatio n o fKinemati cViscosit yan d Calculation o fDynami cViscosity" , 1994
. AST M International , AST M D 445-09 : "Standar d Tes t Metho d fo rKinemati cViscosit yo fTransparen t an d Opaqu e Liquid s (an d Calculation o fDynami cViscosity)" , Jul y1 , 2009
. 18.2 . Performanc e o fth e tes t Th e viscosit yo fth e tes t substanc e wa s determine d a t 20° C an d 40° C usin g a glas s capillary viscomete r(Tamso n Instruments , Bleiswijk , Th e Netherlands)
. Base d o n appearanc e o fth e tes t substance , th e Ubbelohd e viscometer s wit h constant s 0.465 5 an d 0.468 5 wer e selected
. Th e viscomete rwa s charge d wit h th e test'substanc e b ytiltin g i t abou t 30 ° fro m th e vertica l position
. Whe n th e viscomete rwa s returne d t o th e vertica l th e meniscu s o fth e tes t substanc e wa s betwee n th e fillin g mark s ofth e reservoi rbulb
. Th e viscomete rwa s place d i n th e thermostati cwate rbat h an d remaine d i n th e bath fo ra t leas t 3 0 minute s t o reac h th e tes t temperature . Th e tes t substanc e wa s draw n b yvacuu m throug h th e timin g tub e t o abou t 5 m m abov e th e uppe r timin g mark
. Th e vacuu m wa s released
. Th e tim e require d fo rth e meniscu s t o pas s fro m th e uppe rt o th e lowe rtimin g mar k

wa s measure d an d th e kinemati cviscosit yo fth e tes t substanc e wa s determined
. Th e kinemati cviscosit yo fth e tes tsubstanc e wa s determine d a t 2 0 + 0. 1 ° C an d 4 0 ±0. 1 °C
. Eac h tes t wa s performe d i n duplicate
. 18.3 . Interpretatio n 18.3.1 . Specification s Th e kinemati cviscosit yi s define d a s th e resistanc e t o flo wo fa flui d unde rgravity
. Th e kinemati c viscosit yi s expresse d i n mm 2 /s
. 18.3.2 . Formula s Kinemati cviscosit yo = C xt where : C = calibratio n constan t o fth e viscomete r((mm 2 /s)/s | t= flo wtim e [s ]

Fina l report - Pag e 7 4 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 1 18.4 . Result s Th e result s fo rth e kinemati cviscosit yo fth e tes tsubstanc e a t20° C an d 40° C ar e give n i n Tabl e 34
. Tabl e 3 4 Viscosit yo fth e tes t substanc e Temperatur e PC ] Tes t Calibratio n constan t [(mm 2/s)/s ] Flo wtim e [s ] Kinemati cviscosit y

[mm 2 /s ] Mea n

[mm 2 /s ] 2 0 1 0.468 5 1790. 9 839. 1 840. 6

i l 0.465 5 1809. 1 842. 2 4 0 1 0.465 5 294. 5 137. 1 138. 9

I I 0.468 5 300. 4 140. 7

18.5 . Conclusio n Th e viscosit yo fp-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e wa s determine d usin g a glas s capillaryviscometer
. Th e kinemati cviscosit yo fth e tes tsubstanc e was : Kinemati c [mr r 20° C viscosit y

i 2 /s ] 40° C Tes t substanc e 840. 6 138. 9

Fina l report - Pag e 7 5 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d to Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 1 Appendi x1 GL P certificate



## ENDORSEMEN TO F COMPLIANC E

WIT H TH E OEC D PRINCIPLE S O F GOO D LABORATOR YPRACTIC E

Pursuan tto th e Netherland s GL P Complianc e Monitorin g Programm e an d accordin g t o Directiv e 2004/9/EC , th e conformit ywit h th e OEC D Principle s o fGL P wa s assesse d o n 2 1 - 2 5 March , 201 1 at

I ti s herewit h confirme d tha t th e afore-mentione d tes t facilityi s currentl yoperatin g i n complianc e wit h th e OEC D Principle s o fGoo d Laborator yPractic e i n th e followin g are a o fexpertise : Physical-chemica l testing , Toxicitystudies ; Mutagenicity studies ; Environmenta l toxicitystudie s o n aquati can d terrestria l organisms ; Studie s o n behaviou ri n water , soil , an d air , bioaccumulation ; Residu e studies ; Analytica l an d clinica l chemistr

ytesting ; Kineti can d metabolis m studies , SafetyPharmacology
.

NOTO XB.V
. Hambakenweterin g 7 523 1 DD' s Hertogenbosc h

De n Haag , 1 9 May201 1

Foo d and ConsumerProduc

t Safet yAuthorit y(VWA ) Catharijnesmge l 5 9 3511G G Utrecht
. Th e Netherlands

Fina l report - Pag e 7 6 -

—nrr - i — i

### p . 8 0 Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LLC i s unauthorize d an d strictl yprohibited
.



# REPOR T

DETERMINATIO

N O F 'READY * BIDDEGRADABILITY : CARBO N DIOXID E (C0 2 

## ) EVOLUTIO

N TES T (MODIFIE

DSTUR M TEST ) WIT H T K12759 , ARALDIT

E M Y0510

NOTO XProjec t 169875 NOTO XSubstanc

e 59652 Cib a Projec t 96462

5

- Pag e 1 of 13 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LLC i s unauthorize d an d strictl yprohibited
. 

## p . 8 1
T K 12759 , ARALOIT

E M Y 051 0 NOTO X Projec t 16987 5 Cib a Projec t 96462 5 REPOR T APPROVA

L

STUD Y DIRECTOR : Ing . M.O.E . Desmares-Koopman s

MANAGEMENT : Ing . E.J . va n d e Waar t Sectio nHead , Geneti c & Eco-Toxicolog

y

# rC . Enninga

;a l directo r Date :

- Pag e 2 -

### p . 8 2 Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d to Registration , Evaluation,
. Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LLC i s unauthorize d an d strictl yprohibited
. 

## T K 12759 , ARALDIT

E M Y 051 0 NOTO X Projec t 16987 5 Cib a Projec t 96462 5 SUMMAR Y T K 12759 , ARALDIT

E M Y 051 0 wa s teste dfo r it s read

yblodegradablllt yi n the Carbo n Dioxide (C0 2 

## ) Evolutio n Tes t (Modifie d Stur mTest ) a t ca . 3 9 m

g per 2 litres , correspondin

g t o ca . 1 2 m

g TOC/1 . Th e stud yprocedure wa

s base d o n EE C directiv e 92/69 , C.4-C , Decembe r 1992 , an d OEC D guidelin e No . 30 1 B Jul y17 , 1992 . Th e Theoretica l C0 2 

## productio n (ThCOo ) O f T K 12759 , ARALDIT

E M Y 051 0 (MF : C 15 

## H 1 

## gN0 4 

## , MW

: 27 7 g ) wa

s calculate dt o b

e 2.38 3 m g C0

2

## /mg . Th e relativ e degradation value s calculate dfro mthe measurement s performed during the tes t perio d revealed n o significan t degradation o f T K 12759 , ARALOIT E M Y 051 0 i n tes t bottl e A(significant : >10*) . I ntes t bottl e B 30 ^ degradation wa s measure d o n da y29 . Thi s resul t wa

s no t comparabl e t o the result s o f tes t bottl e Aan d the toxicit ycontrol , an d i t wa s assume d tha t the produce

d C0 2 

## i n tes t bottl e B wa s no t tes t substanc e related , bu t the resul t o f a technica l failure . Therefore , th e degradation wa

s base d o n the dat a o f da

y2 7 an d considered a

s no t significant . Sinc e al l furthe r acceptabilit ycriteri a wer e met , thi s stud

ywa s considered t o b

e valid . I nconclusion , T

K 12759 , ARALDITE . M

Y 051 0 wa s no t readil ybiodegradabl e unde r the condition s o f the modifie d Stur mtes t presentl yperformed .

- Pag e 3 -Th e us e o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LLC i s unauthorize d an d strictl yprohibited
. 

## T K 12759 , ARALOIT

E M Y 051 0 NOTO X Projec t 16967 5 Cib a Projec t 96462 5 PREFAC E Sponso r Ciba-Geig yLtd . Polymers Divisio n R-1006.52 0 CH-4002 BASE

L Switzerlan

d Stud yMonito r Dr . E.G. Semaden i Testing Facilit y NOTO X B.V. Facilit Hambakenweterin g 3 523 1 D O 's-Hertogenbosc

h Th e Netherland s Stud yDirecto r Ing . M.3.E . Desmares-Koopman s Technica l Coordinato r Mrs . C.J . va n Leeuwen-va

n Drune n Stud yPla n Start : Februar y14 , 199 6 Completion : Marc

h14 , 199 6 TES T SUBSTANC

E Identificatio n T K 12759 , ARALDIT

E M Y 051 0 Descriptio n Clear yellowliqui d{Determine d a t NOTOX ) Batc h 50008 M Purit y > 95% 

## Tes t substanc e storag e I nfreeze r i n th e dar k Stabilit yunde

r storag e condition s Stabl e Expir ydat e Ma y01 , 199 8 Stabilit yi n water A t leas t 9 6 h Th e sponso r i s responsibl e fo r the completenes s an d GL P Compliance o f al l tes t substanc e data . PURPOS E Th e purpos e o f th e stud

yi s t o evaluat e a non-volatil e tes t substanc e fo r i t s read ybiodegradabilit yi n a

n aerobi c aqueou

s mediu mwit hmicrobia l activit yintroduce db

yinoculatio no f activate dsludge .

- Pag e 4 -

### p . 8 4 Th e us e ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d to Registration , Evaluation , Authorizatio n an d Restriction s Chemica l Substance s (REACH) , withou t th e expres s wntte n permissio n o fHuntsma n International LL

C i s unauthorize

d and strictlyprohibited
. 

## T K 12759 , ARALDIT

E M Y 051 0 NOTO X Projec t 16987 5 Cib a Projec t 96462 5 GUIDELINE S Th e stud yprocedur e describedi n thi s repor t wa s base d o n the followin g guidelines : Europea n Economi c Communit

y(EEC) , EE

C directiv e 92/69 , Par t C : Method s fo r the determinatio no f ecotoxicity , Publicatio nNo . L383 , Decembe r 1992 , C.4 . "Biodegradation : determinatio no f the 'ready* biodegradability , C.4-C : Carbo

n dioxid e (CO2 ) evolutio ntes t (Modifie dStur mTest) . Organisatio nfo r Economic Co-operation an d Developmen

t (OECD) , OEC

D guideline s fo r Testing o f Chemicals , Section3 , Degradation an d Accumulation , guidelin e No . 30 1 B : "Read yBiodegradability : C0 2 

## Evolutio n Test " adopte

d Jul y17 , 1992 . DEFINITION

S Readily

biodegradable: Test substances givinga result of at least 60%

yiel do f C0 2 

## (withi n2 8 days) . Thi s pas s leve l mus t b e reache d withi n1 0 day

s o f biodegradation exceedin g 1096 . ThC0 2 

## : Theoretica l carbo n dioxid e (mg ) i s the quantit yo f carbo n dioxid e calculate dt o b e produce d fro mthe know n or measure

d carbo n conten t o f the tes t substanc e whe

n full ymineralized ; als o expresse d as m g carbo n dioxid e evolved pe

r m g tes t substance . TO C : Tota l organi c carbo n o f a sampl e i s the su

mo f the organi c carbo

n i n solutio nan

d i n suspension .

- Pag e 5 -

### p . 8 5 Th e us e o

fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LLC i s unauthorize d an d strictl yprohibited
. 

## T K 12759 , ARALDIT

E M Y 051 0 NOTO X Projec t 16987 5 C1b a Projec t 96462 5 PREPARATIO

NO F TES T SOLUTION

S Afte r additio no f milli- Q wate r t o eac hweighing bottl e containin g T K 12759 , ARALDIT

E M Y 05.10 (A : 38. 7 mg , 6 : 39. 5 an d toxicit ycontrol : 39. 0 mg) , followe db

yultr a sonicatio n(1 5 min.) , th e resultin g mixtur e was quantitativel yadde

d t o th e tes t media . Th e tes t solution s were continuousl ystirre ddurin g th e test . TES T SYSTE M Sourc e

Treatmen t

Reaso n fo r selectio n Th e sourc e o f tes t organisms wa s activate d sludg e freshl yobtaine d froma municipa l sewag e treatmen t plant : 'Waterscha p d e Maaskant' , *s-Hertogenbosch , th e Netherlands . Th e sludg e wa

s kep t unde r continuou s aeratio n unti l furthe r treatment . Th e concentratio n o f suspende d solid s wa

s 3. 2 g/ l 1

n th e concentrate d sludge . Befor e use , th e sludg e wa s allowe d t o settl e fo r a t leas t 3 0 minute s an d th e liqui ddecanted fo r us e as inoculu ma t th e amoun

t o f 1 0 ml/ 1 o f minera l medium . Th e tes t ha s bee

n accepte d internationall y (EEC , OECD ) fo r determinin g th e 'ready ' biodegradabilit yo f tes t substance s unde r aerobi c conditions . TES T PROCEDUR

E AN D CONDITION

S Tes t duratio n Tes t vessel s Milli- Q wate r

Stoc k solution s o f minera l component s 2 8 days (las t C0 2

## ~measurement o n th e 29t h day) . 2 litr e all-glas s brow n coloure d bottles . Tap-wate r purifie db

yrevers e osmosi s an

d subsequentl ypassed ove r activate dcarbo n an d ion-exchang e cartridge s (Millipor e Corp, , Bedford , Mass. , USA) . A ) 8.5 0 g KH 2 

## P0 4 

## 21.7 5 g K 2

## HP0 4 

## 67.2 0 g Na 2



## HP0 4 

## .12H 2 

## 0 0.5 0 g NH 4 

## C 1 dissolve di n 1 1 Milli- Q water , p H 7.4+0. 2 B ) 22.5 0 g MgS0 4 

## .7H 2 

## 0 dissolve d i n 1 1 Milli- Q water . C ) 36.4 0 g CaCl 2 

## ..2H 2 

## 0 dissolve d i n 1 1 Milli- Q water . D ) 0.2 5 g FeCl 3 

## .6H 2 

## 0 dissolve d 1 n 1 1 Milli- Q water . Pag e 6 -

i n ii

### p . 8 6 Th e us e o

fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. 

## T K 12759 , ARALDIT

E M Y 051 0 NOTO X Projec t 16987 5 Cib a Projec t 96462 5 Minera l mediu

m Bariu mhydroxide , 0.012 5 M
C0 2 

## -fre e ai r

Tes t concentratio n 1 1 minera l mediu mcontains : 1 0 m

l o f solutio n(a) , l m

l o f solution s (b ) t o (d ) an d Milli- Q water . 4 g Ba(0H) 2 

## .8H 2 

## 0 pe r litr e Milli- Q water wa s filtere dthroug

hfilte r pape

r an d store d i na sealed vesse l t o preven t absorptio no f C0 2 

## fro mthe air . Amixtur e o f oxyge

n (21% ) an d nitroge n(79% ) wa s le d throug

ha bottle , containing 0. 5 - 1 litr e 0.012 5 M Ba(0H) 2 

## solutio nt o tra p C0 2 

## whic

hmight b e presen t i nsmal l amounts . Th

e C0 2 

## -fre e ai r wa

s sparge d throug h the scrubbin

g solution s a t a constan t rate . Th e tes t substanc e wa

s teste d i nduplicat e a t ca . 3 9 m g per 2 litres , correspondin g t o ca . 1 2 m g TOC/l . Th

e organi c carbo

n conten t wa

s base d o n the molecular formula . Preparationo f bottles : Pre-incubationmediu

m

Typ e an d numbe r o f bottle s

Preparatio n Minera l components , Milli- Q water (ca . 80 % tota l volume ) an

d Inoculu m(1 % fina l volume ) wer e adde d t o eac hbottle . Thi s mixtur e wa s aerated wit hC0 2 

## -fre e ai r overnigh t t o purg e the syste mo f C0 2 

## . Tes t suspension : containing tes t substanc e an d inoculu m ( 2 bottles) . Inoculu mblank : containing onl yinoculu m( 2 bottles ) Positiv e control : containing referenc e substanc e (ca . 4 0 mg/ l sodiu macetat e (Merc

k art . 6268 , batch04 9 TA933768) , T0C = 1 2 mg/l ) an d inoculu m ( 1 bottle) . Toxicit ycontrol : containing tes t substance , referenc e substanc e an

d inoculu m( l bottle) . Th e tes t substanc e an

d positiv e contro l wer

e adde d t o the bottles . Th e volume s o f suspensions wer e mad e u p i n a l l bottle s b

ythe additio no f Milli- Q water previousl yaerated wit hC0 2 

## *-free air , resultin g i nthe minera l mediu mdescribed before . Three C0 2 

## -absorber s (bottle s fille dwit h10 0 m l 0.012 5 M Ba(0H) 2 

## ) wer

e connecte

d i nserie s t o the exi t ai r lin e o f eac htes t bottle . Star t o f the incubatio nTh e tes t wa s starte db

ybubbling C0 2 

## -fre e ai r throug hthe solutio na t a rat e o f approximatel y1-2 bubble s per secon d (ca . 30-10 0 ml/min) .

- Pag e 7 -Th e us e o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. 

## T K 12759 , ARALDIT

E M Y 051 0 NOTO X Projec t 16987 5 Cib a Projec t 96462 5 p . 8 7

DETERMINATIO

NO F C0 2 

## Experimenta l C0 2 

## productio nTh e C0 2 

## produce

d i neac htes t bottl e reacte d wit hth e bariu

mhydroxide I n the ga

s scrubbin g bottl e an

d precipitate dou t as bariu mcarbonate . Th

e amoun t o f C0 2 

## produce

d wa s determine

d b ytitratin g th e remainin g Ba(0H) 2 

## wit h0.0 5 M standardized HCI . Measurements Titration s wer e mad e ever ysecon d o r thir d da yduring the firs t 1 0 days , an d thereafte r a t leas t ever

yfift hda

yunti l the 28t hday . Eac htim e th e C0 2 

## -absorbe r neares t t o th e tes t bottl e wa

s remove d fo r titration ; eac

h o f th e remainin

g tw o absorber s wa

s move d on e positio ni nthe directio no f the tes t bottle . Ane wC0 2

## ~absorber wa

s placed a t th e fa r en

d o f the series . Phenolphthalei nwa s use d as pH-indicator . O n th e 28t hday , the p

H o f the tes t suspensions wa

s measure d an d 1 m l o f concentrated HC

I wa s adde d t o eac hbottle . Th e bottle s wer

e aerated overnigh t t o driv e of f C0 2 

## presen t i nth e tes t suspension . Th e fina l titratio nwa

s mad e o n da y29 . Theoretica l C0 2 

## productio nTh e theoretica l C0 2 

## productio nwa s calculate d fro mth e molecular formula .

- Pag e 8 -

### p . 8 8 Th e us e ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. 

## T K 12759 , ARALOIT

E M Y 051 0 NOTO X Projec t 16987 5 Cib a Projec t 96462 5 DAT AEVALUATIO

N ThC0 2 

## , expresse

d a s m g C0

2

## /m g tes t substance , tha t ca n b e generated b

ya tes t substanc e wa

s calculate d as follows : No . o f carbo

ni ntes t substanc e x M

W C0 2 

## ThC0 2 

## = M W tes t substanc e Th e firs t step i ncalculatin g the amoun t o f C0 2 

## produce

d i s t o correc t fo r backgroun d (endogeneous ) C0 2 

## production . Thu s the amoun

t o f C0 2 

## produce

d b ya tes t materia l i s determine d b ythe differenc e (i nm

l o f titrant ) betwee n the experimenta l an

d blan k Ba(0H) 2 

## traps . Th e amoun t o f 0.0 5 NHC

I titrate di s converted int o m g o f C0 2 

## produced : (0.0 5 xm l HC I titrated ^ m g C0 2 

## « 2 x 4 4 = l. i x m l o f HC I titrated . Calculation s wer

e base d o n the actua l normalit ywithou t roundin g off . Relativ e degradationvalue s wa s calculate d fro

mthe cumulative C0 2 

## productio nrelativ e t o the tota l expecte d C0 2 

## productio nbase

d o n the tota l carbo

nconten t o f the amoun

t o f tes t materia l presen t i nthe tes t bottles . The

ywer e plotte dversu s tim

e togethe r wit hthe relativ e degradationo f the positiv e control . ACCEPTABILIT

Y O F TH E TES T Th e result s o f the biodegradatio ntes t wer e considered t o b

e vali dwhen :

- th e tota l C0 2 

## evolutio ni nthe inoculu mblan k a t the en

d o f the tes t di d no t normall yexcee

d 4 0 mg/l . I f value s greate r tha n7 0 m g C0 2 

## / 1 ar e obtained , the dat a an

d experimenta l technique shoul d b e examine d critically .

- the differenc e o f duplicat e value s fo r the %-degradationo f the tes t substanc e a t the plateau , a t the en d o f the tes t o r a t the en d o f the 10-da ywindow , as appropriate , wa s les s tha

n20 .

- the percentage degradationo f the referenc e substanc e reache d the leve l fo r read ybiodegradabilit y(60% ) b y1 4 days . Because o f the stringencyo f th e method , lo wvalue s d

o no t necessaril ymea

n tha t the tes t substanc e i s no t biodegradabl e unde r environmenta l conditions , bu t indicate s mor e wor k wil l b e necessar

yt o establis h biodegradability . Toxicit ycontrol : i f les s tha n35 % degradation(base

d o n DOC ) o r les s than 25 * degradation(base

d o n ThC0 2 

## ) occurred i n1 4 days , the tes t substanc e ca nb e assume d t o b e inhibitory .

- Pag

e 9 -Th e us e o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
.



## RESULT S Theoretica l C0 2 

## productio n Th e Theoretica l C0 2 

## productio n(ThC0 2 

## ) o f T K 12759 , ARALDIT

E M Y 051 0 (MF : Ci 5 

## H 19 

## N04 , MW

: 27 7 g ) wa

s calculate d t o b

e 2.38 3 m

g C0 2 

## /mg . Th e concentratio nwas 38. 7 (A ) an d 39. 5 m g (B ) T K 12759 , ARALDIT

E M Y 051 0 i n2 litre s tes t medium . Hence , th e theoretica l C0 2 

## productio nfollowin g complet e degradatio nwa s 92. 2 m g fo r Aan

d 94. 1 m g pe r 2 litre s fo r B . Th e positiv e contro l containe d 80. 1 m g sodiu macetat e (ThC0 2 

## * 1,07 3 m g C0 2 

## /mg ) resultin g i na theoretica l C0 2 

## productio nfollowin g complet e degradatio no f 85. 9 m

g pe r 2 litres . Th e toxicit ycontro l containe d 80. 1 m g sodiu m acetat e an

d 39. 0 m g T K 12759 , ARALOIT E M Y 051 0 i n2 litre s o f tes t medium . Hence , th e theoretica l C0 2 

## productio nfollowin g complet e degradatio no f T K 12759 , ARALDIT

E M Y 051 0 plu s sodiu

macetat e wa

s 178. 9 m

g pe r 2 litres . Biodegradatio n Th e result s ar e summarized 1 n Table s 1-4 , an

d Figur e 1 . Th e relativ e degradatio nvalue s calculate d fromth e measurement s performe d durin g th e tes t perio d reveale d n o significan t degradatio no f T K 12759 , ARALOIT E M Y 051 0 i ntes t bottl e A(significant : >10%) . I ntes t bottl e 6 30

% degradatio nwa

s measure d o n da y29 . Thi s resul t wa

s no t comparabl e t o th e result s o f tes t bottl e Aan d th e toxicit ycontrol , an d i t wa s assume d tha t th e produced C0 2 

## i ntes t bottl e B wa s no t tes t substanc e related , bu t th e resul t o f a technica l failure . Therefore , th e degradatio nwas based o n th e dat a o f da

y2 7 an d considere d as no t significant . I nth e toxicit ycontro l mor e than25 % degradatio noccurre d i n1 4 days (based o n ThC0 2 

## ) . Therefore , th e tes t substanc e wa s assume d t o b e no t inhibitory . Monitorin g o f temperatur e an d p H Th e temperatur e recorde d durin g th e stud yvarie d between 2 0 an d 23°C . Th e p H value s o f th e differen t tes t medi a were : T K 12759 , ARALDIT

E M Y 051 0 NOTO X Projec t 16987 5 Ciba Projec t 96462 5

Jus t befor e th e star t o f th e test : O

n da y28 : 81an k contro l (A ) Blan k contro l (B ) Positiv e contro l 7. 5 7. 5 7. 5 7. 6 7. 6 7. 6 7. 5

7. 5 7. 8 7. 7 7. 6 7. 9 T K 12759 , ARALDIT

E M Y 051 0 (A ) T K 12759 , ARALDIT

E M Y 051 0 (B ) Toxicit ycontro l

- Pag e 1 0

### p . 9 0 Th e us e ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibite d 

## T K 12759 , ARALOIT

E M Y 051 0 NOTO X Projec t 16987 5 Cib a Projec t 96462 5 Acceptabilit yo f the tes t Th e positiv e contro l substanc e wa s degrade d 79 % i n 1 4 day

s (see Tabl e 1 an

d Figur e 1) . Th e tota l CQ 2 

## releas e I n the blan

k reache d a tota l value o f 2 0 m

g C0 2 

## pe r 2 litre s o f mediu

m(see Tabl e 4) . Except fo r da

y29 , the differenc e o f duplicat e value s fo r ^-degradation o f T K 12759 , ARALDIT

E MY.0510 wa

s alway s les s tha

n 20 . Sinc e the degradation o n da y2 9 i n tes t bottl e B wa s considered t o b

e du e t o a technica l failure , an d the fina l judgemen

t fo r thi s bottl e wa

s base d o n the dat a o f da

y27 , the differenc e o f duplicat e value s o n da y2 9 ha d n o effec t o

n the outcom

e o f thi s study . T K 12759 , ARALDIT

E M Y 051 0 wa s foun d t o b e no t inhibitory . CONCLUSIO N T K 12759 , ARALOIT

E M Y 051 0 wa s no t readil ybiodegradabl e unde r the condition s o f the modifie d Stur mtes t presentl yperformed .

- Pag e 1 1 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibite d 

## T K 12759 , ARALDIT

E M Y 051 0 NOTO X Projec t 16987 5 C1b a Projec t 96462 5 Tabl e 1 : CO , evolutio nan d percentag e o f biodegradatio no f the positiv e contro l substance . Da y m l titran t m l titran t Produce d C0 2 Normalit y Produce d Cumulativ e Degradatio n Blan k (mean ) C0 2 tra p i nm l HC I HC I m g C0 2 n g C0 2 

### % 2 49.0 7 40.2 7 6.8 0 0.04 8 9.2 9 9.2 9 10. 8 5 48.9 0 22.8 6 26.0 2 0.04 8 27.4 8 36.7 7 42. 8 7 47.9 4 33.5 7 14.3 7 0.04 8 15.1 7 51.9 4 60. 5 9 46.8 5 40.73 . 6.1 5 0.04 8 8.6 1 60,5 5 70. 5 1 4 47.2 3 40.3 6 6.6 7 0.04 8 7.2 5 67.8 0 78. 9 1 9 47.9 0 41,4 6 6.4 2 0,04 9 6.9 2 74.7 2 87. 0 2 3 48.5 1 41.5 9 6.9 2 0.04 9 7.4 6 82.1 8 93. 7 2 7 45.6 0 38.1 5 7.4 5 0.04 9 8.0 3 90.2 1 105. 0 29 43.9 5 42.1 2 1.8 3 0.04 9 1.9 7 92.1 6 •107. 3 29 45.2 0 44.8 0 0.4 0 0.04 9 0.4 3 92.6 1 107. 6 29 48.2 6 46.5 0 0.0 0 0.04 9 0.0 0 92.6 1 107. 6 Theoretica l C0 2 productio nsodiu macetate ; 83. 9 » g C0 2 /2 1 Tabl e : 2a : C0 2 evolutio nan d percentag e o f biodegradatio no f th e tes t substanc e (bottl e A ) Oa y m l titran t (n l titran t Produce d C0 2 Normalit y Produce d Cumulativ e Degradatio n Blan k (mean ) C0 2 tra p m l HC I N tn g C O 2 m

g C0 2 3

1 2 49.0 7 48.3 2 0.5 5 0.O4 8 0.5 6 0.5 8 0. 6 5 48,9 0 48.7 0 0.2 0 0.04 8 0.2 1 0.7 9 0. 9 7 47.9 4 48.7 1 0.0 0 0.04 8 0.0 0 0.7 9 0. 9 9 48.8 8 48.7 4 0.1 4 0.04 8 0.1 5 0.9 4 i . o 1 4 47.2 3 47.8 4 0.0 0 0.04 8 0.0 0 0-9 4 1. 0 ) 9 47.9 0 47.9 6 0.0 0 0.04 9 0.0 0 0.9 4 1- 0 2 3 48.5 1 47.8 9 0.6 2 0.049 ' 0.6 7 1.6 1 1. 7 2 7 43.6 0 45.2 6 0.3 4 0,04 9 0.3 7 1.9 8 2, 1 29 43.9 5 43.7 7 0.0 0 0.04 9 0.0 0 1.9 6 2. 1 29 45.2 0 44.1 5 1.0 3 0.04 9 1.1 3 3.1 1 3. 4 29 48.2 6 46.6 2 0.0 0 0.04 9 0.0 0 3.1 1 3. 4 Theoretica l C0 2 productio ntes t substance : 92. 2 n g C0 2 121 Tabl e 2b : co 2 evolutio nan d percentag e o f biodegradeitio no f th e tes t substanc e (bottl e B ) Da y m l titran t m l titran t Produce d CO , Normalit y Produce d Cumulativ e Degradatio n Blan k (mean ) CQ 2 tra p m l HC I N m g co 2 m

g C0 2 

### % 2 49.0 7 48.6 9 0.3 8 0.04 8 0.4 0 O.A O 0. 4 5 48.9 0 49.0 0 0.0 0 0.04 8 0.0 0 O.A O 0. 4 7 47.9 4 48.4 9 0.0 0 0.04 6 0.0 0 O.A O 0. 4 9 4S.8 8 47.3 3 1.3 3 0.04 8 1,6 2 2.0 2 2. 1 1 4 4 7 .2 3 48.6 3 0.0 0 0.04 8 0.0 0 2.0 2 2. 1 3 9 47.9 0 SB.2 6 0.0 0 0.04 9 0.0 0 2.0 2 2. 1 2 3 48.5 1 48.3 7 0.1 4 0.04 9 0.1 5 2.1 7 2. 3 2 7 45.6 0 45.2 8 0-3 2 0,04 9 0.3 4 2.3 1 2. ? 2 9 43.9 5 37.6 9 6.2 6 0.04 9 6.7 5 9-2 6 9. 8 2 9 45.2 0 29.4 7 15.7 3 0.04 9 16.9 6 26.2 2 27. 9 2 9 46.2 6 46.3 3 1.91 0.04 9 2.0 6 28.2 6 30. 1 Theoretica l CO 2 productio ntes t substance : 94.1 »gCO2 /2 1



## - Pag e 1 2 -

### p . 9 2 Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu tno tlimite d t o Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou tth e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. 

## T K 12759 , ARALDIT

E M Y 051 0 NOTO X Projec t 16987 5 Cib a Projec t 96462 5

Tabl e 3 : C0 2 evolutio n en d percentag e o f biodegradatio n o f th e toxicit ycontrol . Da y m l titran t m l titran t Produce d C0 2 Normalit y Produce d Cumulativ e Degradatio n Blan k (mean ) C0 2 tra p » 1 HC I N Bi g C0 2 

### »

g co 2 X 2 49.0 7 3 3 ,0 1 14.0 6 0.04 8 14.8 5 14 . B 3 6. 3 5 48.9 0 26.7 0 22.2 0 0.04 6 23.4 4 38.2 9 21. 4 7 47.9 4 38.7 7 9.1 7 0.04 8 9.6 8 47.9 7 26. 8 9 48.8 8 44.5 8 4.3 0 0.04 6 4.5 4 52.3 1 29. 4 1 4 47,2 3 42.4 8 4.7 3 0.04 6 5.0 2 37.5 3 32. 2 19 • 47.9 0 42.9 6 4,9 4 0.04 9 3.3 3 62.8 6 35. 1 2 3 . 48.3 1 41.7 6 6.7 5 0.04 9 7.2 6 70.1 4 39. 2 2 7 45.6 0 32.3 6 13.2 4 0.04 9 14,2 7 64.4 1 47. 2 2 9 43.9 5 41.8 2 2.1 3 0.04 9 2.3 0 86.7 1 48. 5 2 9 43.2 0 44.6 1 0.3 9 0.04 9 0.6 4 67.3 5 48. 8 2 9 46.2 6 48.4 2 0.0 0 0.04 9 0.0 0 87.3 3 48. 6 Theoretica l co 2 productio n tes t substance : 92. 9 m g C0 2 /2 1 Theoretica l C0 2 productio n sodiu macetate : 65. 9 n

g C0 2 /2 l Theoretica l C0 2 productio n tes t substanc e plu s sodiu macetate : 176. 9 m g C0 2 /2 1 Note : Sinc e calculation s Mara performe d withou t roundin g of f a deviatio n I n th e las t figur e o f th e theoretica l C0 2 productio n pe r 2 litre s ma yb e observed . Tabl e 4 : co 2 evolutio nI n th e blank . Da y Stand . m l titran t Produce d C0 2 Normalit y Produce d 

### cumulativ e Ba(0H) 2 Blan k » 1 HC I t l m g C0 2 

### *g co 2 m l HC I * 5 (mean value ) 2 9.9 0 49.0 7 0.4 3 0.04 6 0.4 5 0-4 5 5 9.8 1 48.9 0 0.1 3 0.04 8 0.1 6 0.6 1 7 9.8 1 47.9 4 1.1 1 0.04 8 . 1.1 7 1.7 9 9 ,9.8 3 48.8 8 D.2 7 0.04 8 0.2 9 2.0 7 1 4 9.8 1 47.2 3 1.6 2 0.04 8 1.9 2 3.9 9 1 9 9.8 4 47.9 0 1.3 0 0.04 9 1.4 0 5.39 . 2 3 9.8 4 46.3 1 0.6 9 0.04 9 0.7 4 6.1 3 2 7 9.8 5 45.6 0 3,5 3 0.04 9 3.8 3 9.9 6 2 9 9.8 0 43.9 5 3.0 5 0.04 9 5.4 4 15.4 0 2 9 9.8 0 45.2 0 3.8 0 0.04 9 4.1 0 19.3 0 2 9 9.8 0 48.2 6 0.7 4 0.04 9 0.6 0 20.3 0 Figur e 1 ; Biodegradatio n o f T K 12759 , ARALDIT

E H Y 051 0 I n th e modifie d Sturmtest :

- : positiv e contro l o : T K 12759 , ARALDIT

E M Y 031 0 (A) ' [ ] : Toxicit ycontro l x: T K 12739 , ARALDIT

E H Y 051 0 (B )

0 2 4 6 « 10 12 14 16 IS20 22 24 2C 7.0 C0UK5 E O F OlQOtGRAQAHO

NINTIM

E (DAYS )



## - Pag e 1 3 -Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n



### »

Mi l II~ ~Th e us e ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
.



## FINA L REPOR T

Stud yTitl e 

## FRES H WATE R ALGA L GROWT H INHIBITIO N TES T WIT H P-(2,3-EPOXYPROPOXY)-N,N-BIS(2,3-EPOXYPROPYL)ANILIN E
Autho r Ir
. LM
. Bouwma n

Tes t Facilit y NOTO X B.V
. Hambakenweterin g 7 523 1 D D 's-Hertogenbosc h Th e Nethedand s LaboratoryProjec t Identificatio n NOTO X Projec t 49698 7 NOTO X Substanc e 203277/A

- Pag e 1 o f3 3 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e express writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibite d p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProject 49698 7 1 . CONTENT S 1 CONTENT S 2 2
. STATEMEN TO F GL P COMPLIANC E 4 3
. QUALIT YASSURANC E STATEMEN T5 4
. SUMMAR Y6 5
. INTRODUCTIO N 7 5.1
. Prefac e 7 5.2
. Ai m o fth e stud y7 5.3
. Guideline s 7 5.4
. Storag e an d retentio n o frecord s an d material s 7 5.5
. Definition s 8 6
. MATERIAL S AN D METHOD S •8 6.1
. Tes t Substanc e 8 6.1.1
. Tes t substanc e informatio n 8 6.1.2
. Stud yspecifi ctes t substanc e informatio n 8 6.1.3
. Referenc e substanc e 9 6.2
. Tes tSyste m 9 6.3
. Fres h wate ralga e cultur e 9 6.4
. Preparatio n o ftes t solution s 1 0 6.5
. Combine d limit/range-findin g tes t 1 0 6.6
. Fina l tes t 1 0 6.6.1
. Tes tconcentration s 1 0 6.6.2
. Tes t procedure s an d condition s 1 0 6.6.3
. Samplin g fo ranalysi s o ftes tconcentration s 1 1 6.6.4
. Measurement s 1 1 6.6.5
. Recordin g o fcel l densitie s 1 1 6.7
. Electroni cdat a captur e 1 1 6.8
. Interpretatio n 1 2 6.8.1
. Dat a handlin g i 1 2 6.8.2
. Acceptabilit yo fth e tes t1 1 3 6.9
. Lis t o fdeviation s 1 3 6.9.1
. Lis t o fprotoco l deviation s 1 3 6.9.2
. Lis to fstandar d operatin g procedure s deviation s 1 3 7
. RESULT S 1 3 7.1
. Combine d limit/range-findin g tes t1 3 7.2
. Fina l tes t 1 4 7.2.1
. Measure d tes tsubstanc e concentration s 1 4 7.2.2
. Mea n cel l densitie s 1 4 7.2.3
. Reductio n o fgrowt h rat e an d inhibition o fyiel d 1 5 7.2.4
. Determinatio n o feffec t concentration s 1 6 7.2.5
. Experimenta l condition s 1 6 8
. CONCLUSIO N 1 7

- Pag e 2 -p . 9 6 Th e use o

fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProject 49698 7 TABLE S Tabl e 1 Mea n cel l densitie s (x10 4cells/ml ) durin g th e combine d limit/range-findin g tes t 1 4 Tabl e 2 Percentag e reductio n o fgrowt h rat e an d inhibitio n o fyiel d durin g th e combine d

limit/range-findin g tes t 1 4 Tabl e 3 Mea n cel l densitie s (x10 4cells/ml ) durin g th e fina l tes t :..1 5 Tabl e 4 Percentag e reductio n o fgrowt h rat e (tota l tes tperiod ) an d percentag e inhibition

o fyiel d durin g th e fina l tes t : 1 6 Tabl e 5 Percentag e reductio n o fgrowt h rat e a t differen ttim e interval s durin g th e fina l test....1 6 Tabl e 6 Effec t parameter s 1 6 Tabl e 7 p H level s recorde d durin g th e fina l tes t1 7 Tabl e 8 Individua l cel l densitie s 1 8 Tabl e 9 Calculatio n o fgrowt h rat e an d yiel d 1 9 Tabl e 1 0 Calculation o fgrowt h rat e (section-by-section ) 2 0 Tabl e 1 1 EC-value s fo rgrowt h rat e reductio n 2 3 Tabl e 1 2 EC-value s fo ryiel d inhibition 2 4 FIGURE S Figur e 1 Growt h curve s a tdifferen t concentration s o fp-(2,3-epoxypropoxy>-N,N-bis(2,3 -

epoxypropyl)anilin e 1 5 Figur e 2 Percentag e reductio n o fgrowt h rat e a s functio n o fth e lo g concentratio n (mg/l ) o f

p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e 2 3 Figur e 3 Percentag e inhibition o fyiel d a s functio n o fth e lo g concentratio n (mg/l ) o fp-(2,3 -

epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e 2 4 APPENDICE S APPENDI X1 WORKSHEE TDATA1 8 APPENDI X2 STATISTICS : GROWT H RAT E (0-7 2 HOURS ) 2 1 APPENDI X3 STATISTICS : YIELD (0-7 2 HOURS ) 2 2 APPENDI X4 EC-VALUE S 2 3 APPENDI X5 REFERENC E TES T2 5 APPENDI X6 ANALYTICA LREPOR T2 6

- Pag e 3 -Th e us'eofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. 

### p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline NOTO XProjec t 49698 7 2 . STATEMEN TO F GL P COMPLIANC E NOTO XB.V. , 's-Hertogenbosch , Th e Netherlands Th e stud ydescribe

d i n thi s repor

t has bee n correctl yreporte d an d was conducte

d i n complianc

e with : Yh e Organization fo

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d b yNOTOX.
NOTO XB.V.

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### p . 9 8 Th e us e o

fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. 

### p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t49698 7 3
. QUALIT

YASSURANC E STATEMEN T NOTO XB.V. , 's-Hertogenbosch , Th e Netherland s Thi s repor

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yth e NOTO

XQualit yAssurance Uni tt o confir m thatth e method s and
. result s accuratel yan d completel yreflec tth e ra wdata
. Durin g th e on-sit e process inspections , procedure s applicabl e t o thi s typ e ofstud ywere inspected
. Th e date s ofQualit

yAssurance inspection s are give n below
. Typ e o f Inspection s Phase/Proces s Star t Inspectio n dat e En d

Inspectio n dat e Reportin g

dat e Stud y Protoco l

Protoco l Amendmen t 0 1 Report 02-Aug-1 1 09-Aug-1 1 24-Nov-1 1 02-Aug-1 1

09-Aug-1 1 24-Nov-1 1 02-Aug-1 1

09-Aug-1 1 24-Nov-1 1 Proces s Environmenta l Toxicolog y

Tes tSubstanc e Handlin g Exposur e Observations/Measurement s 18-JuM I 22-JU.I-1 1 22-Jul-1 1
Analytica l an d physica l chemistr y 08-Aug-1 1 16-Aug-1 1 16-Aug-1 1 

### Test Substance Handlin g Observations/Measurements
NOTO XB.V
. C.J
. Mitchel l B.Sc
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t - Pag e 5 -Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p . 9 9
p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 7 4 . SUMMAR Y Pseudokirchnerieil a subcapitata , Fres h WaterAlga l Growt h Inhibitio n Tes t wit h p-(2,3-epoxypropoxy) - N,N-bis(2,3-epoxypropyl)aniline
. Th e stud yprocedure s describe d i n thi s report wer e base d o n th e OEC D guidelin e No
. 201 , 2006
. I n addition , th e procedure s wer e designe d t o mee tth e tes tmethod s o fth e Commissio n Regulatio n (EC ) N o 440/2008 , Par

t C.3 , 2008 ; Amende d byE C No
. 761/2009 , th e IS O Internationa l Standar d 8692 , 200 4 an d th e OEC D serie s o n testin g an d assessmen t numbe r23 , 2000
. Th e batc h ofp-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e teste d wa s a yello wliqui d wit h a purit yo f94.33 % an d th e substanc e wa s notcompletel ysolubl e i n tes t mediu m a t loadin g rate s o f 1. 0 mg/ l an d higher
. Afina l tes t wa s performe d base d o n th e result s o fa precedin g combine d limit/range-findin g test . Al l tes t solution s wer e prepare d separatel yapplyin g tw o day s o fmagneti cstirrin g t o reac h maximu m solubilit yo fth e tes t substanc e i n th e tes tmedium
. Th e resultin g aqueou s mixture s wer e lef t t o stabiliz e fo r2 hour s wher e afte rth e Wate rAccommodate d Fraction s (WAFs ) wer e siphone d of fan d use d fo r testing
. Th e fina l tes t solution s wer e al l clea ran d colourless
. Thre e replicates o fexponentiall ygrowin g alga l culture s wer e expose d t o WAF s prepare d a t loadin g rate s o f4.6 , 10 , 22 , 4 6 an d 10 0 mg/l
. Simultaneously, si xreplicates wer e expose d i n a contro l group . Th e initia l cel l densit ywa s 10 4cells/m l an d th e tota l tes t perio d wa s 7 2 hours
. Sample s fo ranalytica l confirmatio n o factua l exposur e concentration s wer e take n a tth e start , afte r2 4 hour s o fexposur e an d a t th e en d o fth e test . A t th e starto fth e test , th e actua l tes tconcentration s wer e 3.2 , 3.3 , 5.5 , 3 3 an d 3 4 mg/ l i n th e WAF s prepare d a t loadin g rate s o f4.6 , 10 , 22 , 4 6 an d 1Q 0 mg/l , respectively
. Thes e concentration s remaine d stabl e durin g th e firs t2 4 hour s o fexposur e (82-87 % o finitial ) bu t decrease d t o 59-66 % o f initia l a t th e en d o fth e tes t period
. Not e tha tactua l concentration s bein g almos t simila ri s relate d t o mediu m solubilit yan d th e preparatio n o fWAFs
. Base d o n thes e results , th e averag e exposur e concentration s wer e calculate d t o be : 2.5 , 2.6,4.2 , 2 6 an d 2 6 mg/l
. Th e stud yme t th e acceptabilit ycriteria prescribe d byth e protoco l an d wa s considere d valid
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e reduce d growt h rat e o fthi s fres h wate ralga e specie s significantlya t a n averag e exposur e concentratio n o f2 6 mg/l
. Th e EC 5 0fo rgrowt h rat e reductio n (E R C 5 0 : 0-72h ) wa s 1 3 mg/ l wit h a 95 % confidenc e interva l rangin g fro m 8. 3 t o 1 9 mg/l
. Th e EC 5 0fo ryiel d inhibition (E Y C 5 0 : 0-72h ) wa s 7. 0 mg/ l wit h a 95 % confidenc e interva l rangin g fro m 5. 3 t o 9. 3 mg/l
. Th e NOE C fo rgrowt h rat e reductio n wa s 4. 2 mg/l , whil e th e NOE C fo ryiel d inhibitio n could onl yb e estimate d t o b e <2. 5 mg/l
.

- Pag e 6 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)ani!in e 5 . INTRODUCTIO N 5.1 . Prefac e NOTO XProjec t 49698 7

Sponso r

Stud yMonito r(To xan d ecotox ) Tes tFacilit y

Stud yDirecto r Technica l Coordinato r Principa l Scientis t Stud yPla n 5.2 . Ai m o fth e stud y Huntsma n Advance d Material s Gmb H Klybeckstrass e 20 0 405 7 BASE L Switzerlan d Dr
. P
. Dollenmeie r NOTO XB.V
. Hambakenweterin g 7 523 1 D D 's-Hertogenbosc h Th e Netherland s Ir
. L.M
. Bouwma n R.W.A.M
. Coole n Dr
. Ir
. E
. Baltusse n Start Completio n : 1 9 Septembe r20f l

: 1 3 Octobe r201 1
Th e purpos e o fth e stud ywa s t o evaluat e th e tes t substanc e fo rit s abilit yt o generat e toxi ceffect s i n Pseudokirchneriell a subcapitat a durin g a n exposur e perio d o fa t leas t 4 8 an d a t mos t 9 6 hour s and , i f possible , t o determin e th e EC 5 0fo rboth reductio n o fgrowt h rat e an d inhibition o fyield
. 5.3
. Guideline s Th e stud yprocedure s describe d i n thi s report wer e base d o n th e Organizatio n fo rEconomi cCo operatio n an d Developmen t(OECD) , OEC D guideline s fo rTestin g o fChemicals , guidelin e No
. 201 : "Freshwate rAlg a an d Cyanobacteria , Growt h Inhibitio n Test" , Adopte d Marc h 23 , 2006
. I n addition , th e procedure s wer e designe d t o mee tth e tes tmethod s prescribe d b yth e followin g guideline s an d guidanc e document : •Commissio n regulatio n (EC ) No
. 440/200 8 o f3 0 May2008 , Par t C : Method s fo rth e determinatio n o fecotoxicity , Publicatio n No
. L142 , C3 : "Alga l Inhibitio n Test" ; Amende d b yE C No
. 761/200 9 o f 2 3 Jul y2009 , Publicatio n No
. L220
. •IS O Internationa l Standar d 8692 : "Wate rqualit y- Freshwate ralga l growt h inhibitio n tes t wit h unicellula rgree n algae" , Secon d edition , 0 1 Octobe r2004
. •Guidanc e documen t o n aquati ctoxicit ytestin g o fdifficul tsubstance s an d mixtures , OEC D serie s o n testin g an d assessmen t numbe r23 , Decembe r14 , 2000
. 5.4 . Storag e an d retentio n ofrecord s an d material s Record s an d material s pertainin g t o th e stud yincludin g protocol , ra wdata , specimen s (excep t specimen s requirin g refrigeratio n o rfreezing ) an d th e fina l report ar e retaine d i n th e NOTO Xarchive s fo ra perio d o fa t leas t1 0 year s afte rfinalizatio n o fth e report . Afte rthi s period , th e sponso rwil l b e contacte d t o determin e ho wth e record s an d material s shoul d b e handled
. NOTO Xwil l retai n informatio n concernin g decision s made
. Thos e specimen s requirin g refrigeratio n o rfreezin g wil l b e retaine d b yNOTO Xfo ra s lon g a s th e qualit yo fth e specimen s permit s evaluatio n bu tn o longe rthan thre e months afte rfinalizatio n o fth e report .

- Pag e 7 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s wntte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p . 10 1
p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 7 NOTO Xwil l retai n a tes t substanc e sampl e unti l th e expirydate , bu t n o longe rtha n 1 0 year s afte r finalizatio n o fth e report . Afte rthi s perio d th e sampl e wil l b e destroyed
. 5.5 . Definition s Cel l densit yi s th e numbe ro fcell s pe rmillilitre
. Growt h rat e i s th e increas e i n cel l densitype runi ttime
. I t i s derive d fro m th e slop e o fth e growt h curv e i n a logarithmi cplot
. Followin g fro m th e mathematica l natur e o fexponentia l growth , th e measur e o fth e specifi cgrowt h rat e i s preferabl e ove rbiomas s o ryield
. Th e ERCSO i s th e concentratio n o ftes t substanc e tha tresult s i n a 50 % reductio n i n growt h rat e relativ e t o th e control
. Yiel d i s define d a s th e biomas s a t th e en d o fth e exposur e perio d minu s th e biomas s a t th e start o fth e exposur e period
. Th e E y C 5 0i s th e concentratio n o ftes tsubstanc e tha t result s i n a 50 % inhibitio n o f yiel d relativ e t o th e control
. N o Observe d Effec tConcentratio n (NOEC ) i s th e highes t concentratio n teste d a t whic h th e measure d parameter(s ) show(s) n o significan t effec to n alga l growt h relativ e t o contro l values
. 6 . MATERIAL S AN D METHOD S 6.1 . Tes t Substanc e 6.1.1 . Tes t substanc e informatio n Identificatio n Structur e p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e O
. 0

Molecula rformul a Molecula rweigh t CA S Numbe r E C Numbe r Descriptio n Batc h Purity Tes t substanc e storag e Stabilit yunde rstorag e condition s Expirydat e C 1 5 H 1 9 N0 4 277. 3 

### 5026-74-4 225-716- 2 Yello w liquid AA0037340 0 

### 94.33 % I n refrigerato r(2-8°C ) i n th e dar k Stabl e 2 8 February201 3 6.1.2 . Stud yspecifi c tes t substanc e informatio n Volatile Stabilit yi n wate r Solubilit yi n wate r N o N o N o

- Pag e 8 -p . 10 2 Th e use o

fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 7 6.1.3 . Referenc e substanc e Thi s reportinclude s th e result s ofth e mos t recen t referenc e tes twit h potassiu m dichromat e (APPENDI X5)
. 6.2 . Tes t Syste m Specie s Pseudokirchneriell a subcapitata , strain : NIVACH L1 Sourc e In-hous e laboratoryculture
. Reaso n fo rselection Thi s syste m i s a n unicellula ralga l specie s sensitiv e t o toxi c substance s i n th e aquati cecosyste m an d ha s bee n selecte d a s a n internationall yaccepte d species
. 6.3 . Fres h wate ralga e cultur e Stoc k cultur e Alga e stoc k culture s wer e starte d b yinoculatin g growt h mediu m wit h alga l cell s fro m a pur e cultur e o n agar . Th e suspension s wer e continuousl yaerate d an d expose d t o ligh t i n a climat e roo m a t a temperatur e o f21-24°C
. Ligh t intensit y6 0 t o 12 0 uE/m 2 / s whe n measure d i n th e photosyntheticall y effectiv e wavelengt h rang e o f40 0 t o 70 0 nm . Stoc k cultur e mediu m M1 ; accordin g t o th e NP R 650 5 ("Nederlands e Praktij k Richtlij n no . 6505" ) formulate d usin g Milli-R O wate r(tap wate rpurifie d byrevers e osmosis ; Millipor e Corp. , Bedford , Mass. , USA ) wit h th e followin g composition : NaN0 3 50 0 mg/ l K 2 HP0 4 .3H 2 0 5 2 mg/ l MgSO,.7H a O 7 5 mg/ l Na a CO 3 .10H 2 O 5 4 mg/ l C 9 H a 0 7 .H 2 0 6 mg/ l NH4NO 3 33 0 mg/ l CaCI 2 .2H 2 0 3 5 mg/ l C e H 6 Fe0 7 .xH : 0 6 mg/ l H 3 B0 3 2. 9 mg/ l MnCI 2 .4H z O 1.8 1 mg/ l ZnCI ; 0.1 1 mg/ l CuS0 4 .5H 2 0 0.0 8 mg/ l (NH<) s Mo 7 024.4H 2 0 0.01 8 mg/ l Pre-cultur e 3 day s befor e th e start o fth e test , cell s fro m th e alga l stoc k cultur e wer e inoculate d i n cultur e mediu m a ta cel l densityo f 1 x10 4cells/ml
. Th e pre-cultur e wa s maintaine d unde rth e sam e condition s a s use d i n th e test . Th e cel l densitywa s measure d immediatel ybefor e use . Pre-cultur e mediu m M2 ; accordin g t o th e OEC D 20 1 Guideline , formulate d usin g Milli- Q wate r(ta p wate rpurifie d b yrevers e osmosi s (Milli RO ) an d subsequentl ypasse d ove ractivate d carbo n an d ion-exchang e cartridges : Milli- Q water ; Millipor e Corp. , Bedford , Mass. , USA ) preventin g precipitatio n an d wit h th e followin g composition : NR.C I 1 5 mg/ l MgCh.6Hi O 1 2 mg/l CaCI 2 2H 2 0 1 8 mg/l MgSO,.7H 2 0 1 5 mg/l KH 2 PO , 1. 6 mg/l FeCl a .6H a O 64 (jg/ l Na a EDTA.2H 2 0 100 pg/l H 3 B0 318 5 pg/l

- Pag e 9 -Th e us e ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substances (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProject49698 7 MnCl 2 .4H 2 0 ZnCI 2 CoCI 2 .6H 2 0 CuCI 2 .2H 2 0 Na 2 Mo0 4 .2H 2 0 NaHC0 3 Hardness (Ca+Mg ) p H 41 5 pg/l 3 pg/l 1.5 pg/l 0.0 1 pg/l 7 ug/i 50 mg/l 0.24 mmol/l (24 m g CaCOs/l ) 8. 1 ±0. 2 6.4 . Preparatio n oftes t solution s Th e standar d tes t procedure s require d generatio n o ftes tsolutions , whic h containe d completel y dissolve d tes tsubstanc e concentration s o rstabl e an d homogeneou s mixture s o rdispersions
. Th e testin g o fconcentration s tha twoul d distur b th e tes tsyste m wa s prevente d a s muc h a s possibl e (e.g
. fil m o fth e tes t substanc e o n th e wate rsurface)
. Th e batc h o fp-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e teste d wa s a yello wliqui d wit h a purit yo f94.33 % an d th e substanc e wa s not completel ysolubl e i n tes t mediu m a t loadin g rate s o f 1. 0 mg/ l an d higher
. Al l tes t solution s wit h loadin g rate s o f1. 0 mg/ l an d highe rwer e prepare d separatel yapplyin g tw o day s o fmagneti cstirrin g t o reac h maximu m solubilit yo fth e tes t substanc e i n th e tes t medium
. Th e resultin g aqueou s mixture s wer e lef tt o stabiliz e fo r1-2 hour s wher e afte rth e Wate rAccommodate d Fraction s (WAFs ) wer e siphone d offan d use d fo rtesting
. On e lowe rtes t concentratio n fo rth e combine d limit/range-findin g tes twa s prepare d b ysubsequen t dilutio n o fth e 1. 0 mg/ l WA F i n tes t medium
. Th e fina l tes t solution s wer e al l clea ran d colourless
. Afte rpreparation , volume s o f5 0 m l wer e adde d t o eac h replicat e o fth e respectiv e tes t concentration
. Subsequently , 1 m l o fa n alga ! suspensio n wa s adde d t o eac h replicat e providin g a cel l densityo f 10 4cells/ml
. | 6.5 . Combine d limit/range-findin g tes t Th e stud ystarte d wit h a combine d limit/range-findin g test . Si xreplicate s o fexponentiall ygrowin g alga e wer e expose d t o a contro l an d a WA F prepare d at a loadin g rat e o f10 0 mg/l
. Tes t procedur e an d condition s wer e simila rt o thos e applie d i n th e fina l tes t wit h th e followin g exceptions : •Thre e replicate s pe rconcentratio n wer e expose d t o WAF s prepare d a t 1. 0 an d 1 0 mg/ l an d t o a 10 % dilutio n o fth e WA F prepare d a t 1. 0 mg/l ; •On e extr a tes tvesse l pe rconcentratio n withou talga e wa s use d a s backgroun d fo rth e determinatio n o fth e alga l cel l densitya teac h tim e interval ; •p H wa s onl ymeasure d i n th e contro l an d th e highes t tes t concentration
. 6.6 . Fina l tes t 6.6.1 . Tes t concentration s p-(2,3-epoxypropoxy) - N,N-bis(2,3-epoxypropyl)anilin e Control s Replicate s WAF s prepare d a t loadin g rate s o f4.6 , 10 , 22 , 4 6 an d 10 0 mg/ l Tes t mediu m withou t tes tsubstanc e o rothe radditive s 3 replicate s o feac h tes t concentration ; 6 replicate s o fth e control ; 1 extr a replicat e o fth e contro l an d eac h tes t concentratio n fo rsamplin g purposes ; 2 replicates o fth e highes t concentratio n withou t algae
. 6.6.2 . Tes t procedure s an d condition s Tes t duratio n 7 2 hour s

- Pag e 10 -p . 10 4 Th e us e ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 7 Tes t typ e Stati c Tes tvessel s 10 0 ml , all-glass , containin g 5 0 m l o ftes t solutio n Mediu m M 2 Cel l densityA n initia l cel l densityo f1 x10 4cells/ml
. Illuminatio n Continuousl yusin g TLD-lamp s o fth e typ e 'Cool-white ' o f 3 0 Watt , wit h a ligh t intensit ywithi n th e rang e o f7 7 t o 9 0 uE.rTf 2 .s' 1
. Incubatio n Cappe d vessel s wer e distribute d a t rando m i n th e incubato r an d a s suc h wer e dail yrepositioned
. Durin g incubatio n th e alga l cell s wer e kep t i n suspensio n b ycontinuou s shaking
. 6.6.3 . Samplin g fo ranalysi s oftes t concentration s Sample s fo rpossibl e analysi s wer e take n fro m al l tes t concentration s an d th e contro l accordin g t o th e schedul e below
. Th e metho d o fanalysi s i s describe d i n th e appende d Analytica l Report (APPENDI X6)
. Frequencya t t= 0 h , t=2 4 h an d t=7 2 h Volum e 2 m l Storag e Sample s wer e store d i n a freeze runti l analysis
. A t th e en d o fth e exposur e period , th e replicate s wit h alga e wer e poole d a t eac h concentratio n befor e sampling
. Compliance wit h th e Qualit ycriteria regardin g maintenanc e o factua l concentration s wa s demonstrate d b yrunnin g a tes tvesse l a t th e highes tsubstanc e concentratio n bu t withou t alga e an d sample s fo ranalysi s wer e take n a tth e start , afte r2 4 hour s o fexposur e an d a t th e en d o fth e tes t period
. Additionally , reserv e sample s o f2 m l wer e take n fro m al l tes tsolution s fo rpossibl e analysis
. I fno t alread yused , thes e sample s wer e store d i n a freeze rfo ra maximu m o fthre e months afte rdeliveryo f th e draf treport , pendin g o n th e decisio n o fth e sponso rfo radditiona l analysis
. 6.6.4 . Measurement s p H A t th e beginnin g an d a t th e en d o fth e test . Th e p H o fth e solution s shoul d preferabl yno t deviat e by mor e tha n 1. 5 unit s durin g th e test'
. Temperatur e o fmediu m Continuousl yi n a temperatur e contro l vessel
. 6.6.5 . Recordin g o fcel l densitie s A t th e beginnin g o fth e test , cell s wer e counte d usin g a microscop e an d a countin g chamber
. Thereafte rcel l densitie s wer e determine d b yspectrophotometri cmeasuremen t o fsample s a t 72 0 n m usin g a spectrophotomete rwit h immersio n prob e (pathlengt h =2 0 mm)
. Alga l mediu m wa s use d a s blank
. 6.7 . Electroni c dat a captur e Observations/measurement s i n th e stud ywer e recorde d electronicall yusin g th e followin g programme(s) :

- Shimadz u Spectrophotomete rUV-180 0 includin g UVProb e 2.3 3 softwar e (Shimadzu , Kyoto , Japan) : Alga l cel l density
.

- Pag e 1 1 -Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p . 10 5
p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProject 49698 7

- REE S Centro n Environmenta l Monitorin g syste m versio n SQ L2. 0 (REE S Scientific , Trenton , NJ , USA) : Temperature
. 6.8 . Interpretatio n 6.8.1 . Dat a handlin g Calibratio n curv e Quantificatio n o fcel l densitie s wa s base d o n a calibratio n curve . Cel l densitywa s plotte d versu s extinctio n usin g spectrophotometri cmeasurement s o fa minimu m o fsi xdilution s o fa n alga l suspensio n wit h differen t cel l densities
. Th e calibratio n curv e wa s compose d usin g linea rregression
. Th e softwar e automaticall ycalculate s th e cel l densitie s base d o n thi s curv e fo rth e spectrophotometri c measurement s a tth e variou s point s i n tim e durin g th e tes tperiod
. Compariso n o faverag e growt h rate s Th e averag e specifi cgrowt h rat e fo ra specifi cperio d i s calculate d a s th e logarithmi cincreas e i n th e biomas s fro m th e equatio n fo reac h singl e vesse l o fcontrol s an d treatments : InX , -lnX f, M H = _ . '(day 1 ) Where : /J 4= th e averag e specifi cgrowt h rat e fro m tim e i t o j

X j = th e biomas s a t tim e i X j = th e biomas s a t tim e j Th e averag e growt h rat e a teac h tes t substanc e concentratio n i s the n compare d wit h th e contro l valu e an d th e percentag e reductio n i n growt h rat e i s calculated : % l r= VC-V T X1Q O Where : %l r= percen tinhibition i n averag e specifi cgrowt h rat e

f j c= mea n valu e fo raverag e specifi cgrowt h rat e i n th e contro l grou p u r= averag e specifi cgrowt h rat e fo rthetreatmen treplicat e Yiel d Th e percen tinhibitio n i n yiel d i s calculate d fo reac h treatmen treplicat e a s follows : %/ y= Yc ~ Y r x1Q 0 Where : %l y= percen t inhibition o fyiel d

Y c= mea n valu e fo ryiel d i n th e contro l grou p Y T= valu e fo ryiel d fo rth e treatmen t replicat e Determinatio n o fth e averag e exposur e concentration s 2 4 xJC t = 0xC 1=2 4+ 4 8 xJC,_2 4 xC t = „ Th e averag e exposur e concentration s wer e calculate d as: - —-——— , bein g th e Tim e Weigh t Averag e (TWA ) ofth e concentration s o fp-(2,3-epoxypropoxy)-N , N-bis(2,3 - epoxypropyl)anilin e measure d i n th e sample s take n a t th e start(C l=0 ) , afte r2 4 hours (C t= 24 ) an d th e en d o fth e tes t(Ct =72 )
. Determinatio n o fth e NOE C an d calculatio n o fthe 1 ECTM Fo rdeterminatio n o fth e NOE C an d th e EC 5 0th e approache s recommende d i n th e OEC D guidelin e 20 1 wer e used
. A n effec twa s considere d t o b e significan ti fstatistica l analysi s o fth e dat a obtaine d fo r th e tes tconcentration s compare d wit h thos e obtaine d i n th e negativ e contro l reveale d significan t

- Pag e 1 2 -Th e us e ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 7 reductio n o fgrowt h rat e o rinhibitio n o fyiel d (ANOVA , Bonferron i t-Test , TOXSTA TReleas e 3.5 , 1996 , D.D
. Gulley , A.M
. Boelter , H.L
. Bergman)
. Additionally , th e EC 1 0wa s determine d t o mee t th e recommendation s as'pu t dow n i n " ARevie wo fStatistica l Dat a Analysi s an d Experimenta l Desig n i n OEC D Aquati cToxicolog yTes tGuidelines " b yS
. Pack , Augus t1993
. Calculation o fth e EC 5 0an d EC1 0 value s wa s base d o n log-linea rregressio n analysi s o fth e percentage s o fgrowt h rat e reductio n an d th e percentage s o fyiel d inhibitio n versu s th e logarithm s o fth e correspondin g averag e exposur e concentration s o fth e tes t substance
. 6.8.2 . Acceptabilit yo fth e tes t 1
. I n th e control , cel l densityincrease d b ya n averag e facto ro f>1 6 withi n tw o days
. 2
. Th e mea n coefficien to fvariatio n fo rsection-by-sectio n specifi cgrowt h rate s i n th e contro l culture s di d no texcee d 35%
. 3
. Th e coefficien to fvariatio n ofaverag e specifi cgrowt h rate s durin g th e whol e tes t perio d i n replicat e contro l culture s di d no t excee d 7%
. 6.9 . Lis t o fdeviation s 6.9.1 . Lis t o fprotoco l deviation s 1
. Combine d limi t range/findin g test ; Afte r4 8 hour s o fexposur e on e ou t o fth e si xreplicat e vessel s fo rth e highes ttes tconcentratio n brok e an d could therefor e n o longe rb e use d fo rmeasuremen t o falga l cel l density
. Evaluation : Th e combine d limit/range-findin g tes tcould stil l b e use d a s a range-findin g findin g tes tt o determin e th e rang e o fconcentration s use d i n th e fina l test . Result s wer e base d o n fiv e instea d o fsi xreplicates
. 2
. Fina l test : Afte r7 2 hour s o fexposur e on e ou to fth e si xreplicat e vessel s fo rth e contro l grou p brok e an d coul d therefor e no tb e use d fo rmeasuremen t o falga l cel l densit ya t th e en d o fth e test . Evaluation : Result s wer e base d o n fiv e instea d o fsi xreplicates
. Accordin g t o th e OEC D guidelin e th e numbe ro freplicat e vessel s fo rth e contro l grou p shoul d b e a t leas t three
. Therefore , th e tes t ca n stil l b e use d t o determin e th e effec t parameter s (EC 5 0an d NOEC)
. 3
. Fina l test : N o microscopi cobservation s o fth e alga l cell s wer e mad e a t th e en d o fth e test . Evaluation : Th e stud ycoul d stil l b e use d t o determin e EC 5 o values , whic h wa s th e mai n goal
. Th e stud yintegrit ywa s no t adversel yaffecte d b yth e deviations
. 6.9.2 . Lis t o fstandar d operatin g procedure s deviation s An ydeviation s fro m standar d operatin g procedure s wer e evaluate d an d file d i n th e stud yfile
. Ther e wer e n o deviation s fro m standar d operatin g procedure s tha t affecte d th e integrit yo fth e study
. 7 . RESULT S 7.1 . Combine d limit/range-findin g tes t Th e mea n cel l densitie s measure d durin g th e combine d limit/range-findin g tes t ar e presente d i n Tabl e 1
. Tabl e 2 present s th e percentage s growt h rat e reductio n an d yiel d inhibitio n pe r concentration
. Base d o n thes e result s sample s take n fro m th e WAF s prepare d a t 1. 0 an d 10 0 mg/ l wer e analysed
. Th e initia l concentration s wer e 0.7 2 an d 6 0 mg/l
. Thes e concentration s remaine d stabl e durin g th e firs t2 4 hours o fexposur e (80-8 1 % o finitial ) bu t decrease d t o 45-62 % o finitia l a t th e en d o fth e tes tperio d (se e als o Tabl e 2 o fth e appende d Analytica l Report)
. Th e expecte d EC 5 0fo r bot h growt h rat e reductio n an d yiel d inhibition wa s betwee n concentration s obtaine d i n WAF s prepare d a t 1 0 an d 10 0 mg/l
. Al l tes tcondition s wer e maintaine d withi n th e limit s prescribe d b yth e protocol
.

- Pag e 13 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e express writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p . 10 7
p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProject 49698 7 Tabl e 1 Mea n cel l densitie s (x10 4cells/ml ) durin g th e combine d limit/range-findin g tes t

. Loadin g rate 1 Tes t substance 2 (ma/I ) Exposur e tim e (hours )
. Loadin g rate 1 Tes t substance 2 (ma/I ) 0 2 4 4 8 7 2 contro l 1. 0 11. 0 54. 0 227, 2 10 % O f1. 0 1. 0 11. 2 59. 2 239. 5 1. 0 1. 0 10. 6 53. 3 219. 2 1 0 1. 0 10, 9 48. 0 197. 7 10 0 1. 0 5. 7 8. 5 6. 4 WA F prepare d at th e give n loadin g rat e 2Th e test substance i s p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e Tabl e 2 Percentag e reductio n ofgrowt h rat e an d inhibitio n o fyiel d durin g th e combine d

limit/range-findin g tes t Loadin g rate 1 Tes t substance 2 (mg/l ) Mea n growt h rat e Yiel d (0-7 2 h ) Loadin g rate 1 Tes t substance 2 (mg/l ) u (0-7 2 h ) Reductio n (% ) x10 4cells/m l Inhibitio n (% ) contro l 0.0753 2 226.1 9 10 % o f1. 0 0.0760 8 -1. 0 238.5 0 -5. 4 1. 0 0.0748 2 0. 7 218.2 4 3. 5 1 0 0.0734 2 2. 5 196.7 0 13. 0 10 0 0.0257 6 65. 8 5.4 1 97. 6 WA F prepare d a

tth e give n loadin g rate 2Th e test substance i s p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e 7.2 . Fina l tes t 7.2.1 . Measure d tes t substanc e concentration s Th e result s o fanalysi s o fth e sample s take n durin g th e fina l tes t ar e describe d i n Tabl e 3 o fth e appende d Analytica l Report
. At th e start o fth e test , th e actua l tes tconcentration s wer e 3.2 , 3.3 , 5.5 , 3 3 an d 3 4 mg/ l i n th e WAF s prepare d a tloadin g rate s o f4.6 , 10 , 22 , 4 6 an d 10 0 mg/l , respectively
. Thes e concentration s remaine d stabl e durin g th e firs t 2 4 hour s ofexposur e (82-87 % o finitial ) bu t decrease d t o 59-66 % o f initia l a tth e en d o fth e tes tperiod
. Not e tha tactua l concentration s bein g almos t simila ri s relate d t o mediu m solubilit yan d th e preparatio n o fWAFs
. Base d o n thes e results , th e averag e exposur e concentration s wer e calculate d t o be : 2.5 , 2.6 , 4.2 , 2 6 an d 2 6 mg/l
. 7.2.2 . Mea n cel l densitie s Tabl e 3 show s mea n cel l densitie s measure d a t 24-hou rinterval s a t th e differen t concentration s o fp - (2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline
. Th e respectiv e growt h curve s ar e show n i n Figur e 1 (se e APPENDI X1 fo rth e cel l densitie s pe rreplicate)
. Not e tha t th e mea n cel l densityo fth e contro l wa s base d o n 5 replicates a tth e en d o fth e tes t du e t o th e fac t tha t on e vesse l fal l an d broke
.

- Pag e 1 4 -

Tin
-Th e us e ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis{2,3-epoxypropyl)anilin e NOTO XProjec t 49698 7 Tabl e 3 Mea n cel l densitie s ( x10 4cells/ml ) durin g th e fina l tes t Loadin g rate 1 Tes t substance 2 (mg/l ) Exposur e tim e (hours ) Loadin g rate 1 Tes t substance 2 (mg/l ) 0 2 4 4 8 7 2 contro l 1. 0 8. 7 38. 4 158. 5 4.6(2.5 ) 1. 0 8. 2 34. 2 131. 7 10(2.6 ) ' 1. 0 9. 0 36. 6 145. 6 2 2 (4.2 ) 1. 0 8. 7 31. 5 108. 7 4 6 (26 ) 1. 0 6. 2 7. 1 4. 2 10 0 (26 ) 1. 0 5. 5 7. 6 3. 5 WA F prepare d atth e give n loadin g rate 2Th e testsubstance i s p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e ( ) Averag e exposur

e concentratio n 180

Exposure tim e (hours ) Figur e 1 Growt h curve s at differen t concentration s o fp-(2,3-epoxypropoxy)-N,N-bis(2,3 -

epoxypropyl)anilin e 7.2.3 . Reductio n o fgrowt h rate an d inhibitio n o fyiel d Tabl e 4 show s th e calculatio n o fth e percentage s o fgrowt h rat e reductio n (tota l tes t period ) an d th e percentage s o fyiel d inhibition
. Tabl e 5 show s th e calculatio n o fth e percentage s o fgrowt h rat e reductio n a tdifferen t tim e interval s (se e APPENDI X1 fo rth e value s o fgrowt h rat e an d yiel d pe r replicate)
. Statistica l analysi s o fth e dat a i s show n i n APPENDI X2 an d APPENDI X3
. Growt h rate s wer e i n th e rang e o fth e contro l a t 2.5/2. 6 mg/ l durin g th e 72-hou rtes t period , wherea s th e growt h rat e o falga e expose d t o 4. 2 an d 2 6 mg/ l wer e reduced
. Statisticall ysignifican t reductio n o f growt h rat e wa s foun d a ta tes t concentratio n o f2 6 mg/ l (Bonferron i t-Test , a = 0.05) , tha t wa s measure d i n WAF s prepare d a tbot h 4 6 an d 10 0 mg/l
. Inhibitio n o fyiel d increase d wit h increasin g concentratio n o fp-(2,3-epoxypropoxy)-N,N-bis(2,3 - epoxypropyljanilin e fro m 2.5/2. 6 mg/ l upward s resultin g i n almos t complet e inhibition a t 2 6 mg/l
. Statisticall ysignifican tinhibition o fyiel d wa s foun d a t al l tes tconcentrations , excep t fo r2. 6 mg/ l measure d i n th e WA F prepare d a t1 0 mg/ l (Bonferron i t-Test , a = 0.05)
. N o explanatio n ca n b e give n fo rth e relativel yhig h inhibition a tth e lowes tloadin g rate . Statistica l analysi s doe s no t giv e a clea r outpu to n th e NOE C fo ryiel d inhibition
.

- Pag e 1 5 -Th e us e o fthes e studies/documents fo rregulator yo rregistran t i n Authorizatio n an d Restrictio n o fChemica l Substances (REAC1-) ) Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)an i in e Tabl e 4 Percentag e reductio n ofgrowt h

o fyiel d durin g th e fina l tes t Loadin g rate 1 Tes t substance 2 

# inm contro l _4. 6 (2.5 ) 10(2.6 ) _22 (4.2 ) _4 6 (26 ) 10 0 (26 ) Mea n growt h rat e

u (0-7 2 h ) Reductio n (%) 0.0703 0 0.0677 4 0.0691 7 0.0651 2 0.0197 7 0.0168 3 3. 6

1. 6 7. 4 71. 9 76. 1 x10 * cells/m l

15 7 13 0 14 4 10 7 2.£ 0 WA F prepare d a

t th e give n loadin g rat e 2Th e tes t substance i s p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropy l Janilin e ( ) Averag e exposur

e concentratio n Tabl e 5 Percentag e reductio n o fgrowt h tes t Loadin g rate 1 Tes t substance 2 fmg/l ) contro l 4. 6 (2.5 ) _1 0 (2.6 ) _22 ( 4 2 ) 4 6 (26 ) _10 0 (26) M(0-24h ) Reductio n (% > u (24-4 8 h ) Reductio n (% ) p (48-7 2 h ) Reductio n {%) 0.0901 1 0.0875 3 0.0912 6 0.09029 0.0757 3 0.0713 8 2. 9

-1. 3 -0. 2 16. 0 20. 8 0.0613 5

0.0593 1 0.0587 5 0.0533 9 0.0053 5 purposes , includin g bu t no t limite d t o Registration , Evaluation , , withou t th e expres s writte n permissio n o fHuntsma n p . 10 9

NOTO XProjec t 49698 7 rat e (tota l tes t period ) an d percentag e inhibitio n Yiel d (0-7 2 h )
5 0 6 6 6 1 6 9 Inhibitio n (%

) 17. 0 8. 2 31. 6 97. 9 98. 4

rat e a t differen t tim e interval s durin g th e fina l Mea n growt h rat e

0.0126 0 ' WA F prepare d a

t th e give n loadin g rat e 2Th e tes t substance i s p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e ( ) Averag e exposur

e concentratio n 7.2.4 . Determinatio n o feffec t concentration s Tabl e 6 show s th e effec t parameter s base d o n averag e exposur e concentrations , se e als o APPENDI X4
. Tabl e 6 Effec t parameter s

Paramete r Concentratio n Tes t substance 1 <m fl /l ) 95%-confidenc e interva l NOER C 4. 2 72h>E R Ci o 3. 6 2. 3 - 5. 4 72h^E R C 5 o 1 3 8.3-1 9 NOEy C <2. 5 72tvE Y C 1 0 2. 4 1.8-3. 2 72h-E Y C 5 o 7. 0 5.3-9. 3 7.2.S . Experimenta l condition s Tabl e 7 show s th e p H recorde d a t th e beginnin g s prescribe d b yth e protoco l (6.0-9.0 , preferabl yno t th e tes t mediu m wa s 22.1° C a t th e start o fth e tes t measure d i n th e incubato rwa s maintaine d betwee n th e limit s prescribe d b yth e protoco l (21-24°C , constan t



### - P £ 3. 3

4. 7 13. 4 91. 3 79. 2 0.0589 8

0.0560 9 0.0575 1 0,0516 7 0.0000 0 0.0000 0 4. 9

2. 5 12. 4 100. 0 100. 0

i-epo>ypropyl)anilin e

n d th e en d o fth e test . Th e p H wa s withi n th e limit s varyin g b ymor e tha n 1. 5 unit)
. Th e temperatur e o f Durin g th e exposur e perio d th e temperatur e 22. 4 an d 23.5°C
. Temperatur e remaine d withi n withi n 2°C)
.

g e 16 -

III I iiTh e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-{2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProject49698 7 Tabl e 7 p H level s recorde d durin g th e fina l tes t Loadin g rate 1 Tes t substance 2 (mg/l ) Exposur e tim e (hours ) Loadin g rate 1 Tes t substance 2 (mg/l ) 0 7 2 contro l 8. 1 7. 9 4. 6 (2.5 ) 8. 2 7. 9 1 0 (2.6 ) 8. 1 7. 9 2 2 (4.2 ) 8. 2 7. 8 46(26 } 8. 1 7. 9 10 0 (26 ) 8. 2 7. 9 ' WA F prepare d a

tth e give n loadin g rate 2Th e testsubstance i s p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e ( ) Averag e exposur

e concentratio n 8 . CONCLUSIO N Unde rth e condition s o fth e presen tstud ywit h Pseudokirchneriell a subcapitata , p-(2,3-epoxypro poxy) - N,N-bis(2,3-epoxypropyl)anilin e reduce d growt h rat e o fthi s fres h wate ralga e specie s significantl ya t a n averag e exposur e concentratio n o f2 6 mg/l
. Th e EC 5 0fo rgrowt h rat e reductio n (E R C 5 0 : 0-72h ) wa s 1 3 mg/ l wit h a 95 % confidenc e interva l rangin g fro m 8. 3 t o 1 9 mg/l
. Th e EC 5 0fo ryiel d inhibition (E Y C 5 0 : 0-72h ) wa s 7. 0 mg/ fwit h a 95 % confidenc e interva l rangin g fro m 5. 3 t o 9. 3 mg/l
. Th e NOE C fo rgrowt h rat e reductio n wa s 4. 2 mg/l , whil e th e NOE C fo ryiel d inhibition coul d onl yb e estimate d t o b e <2. 5 mg/l
.

- Pag e 1 7 -Th e use o fthes e studies/documents fo rregulator yo rregistrati < m Authorizatio n an d Restrictio n o fChemica l Substance s (REACh| ) Internationa l LL

C i s unauthorize d an d strictl yprohibite d p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)an i purposes , includin g bu t no t limite d to Registration , Evaluation , , withou t th e expres s writte n permissio n ofHuntsma n 

### p . 11 1

in e NOTO XProject 49698 7 APPENDI X1 WORKSHEE TDAT A
Tabl e 8 Individua l cel l densitie s Numbe ro finoculate d cell s att=0 : 1x10 " cells/m l Loadin g rate 1 Vesse l Exposur e tim e (hours ) Tes t substance 2 numbe r (mg/l ) 0 2 4 4 8 7 2 contro l 1 1.0 0 8.9 5 39.7 9 165.5 4 2 1.0 0 8.0 0 37.4 8 3 3 1.0 0 8.7 4 41.7 3 169.6 6 4 1.0 0 8.1 8 30.4 6 135.0 9 5 1.0 0 9.8 0 431 3 173.6 0 6 1.0 0 8.6 1 37.8 8 148.6 0 4. 6 (2.5 ) 1 1.0 0 7.8 3 31.6 5 122.0 4 2 1.0 0 8.4 0 34.5 0 144.6 5 3 1.0 0 8.2 9 36.5 4 128.2 8 1 0 (2.6 ) 1 1.0 0 8 6 6 35.6 9 14 5 4 6 2 1.0 0 9.6 3 38.9 7 150.0 5 3 1.0 0 8.5 6 35.2 7 141.3 1 2 2 (4.2 ) 1 1.0 0 8.8 5 31.6 0 109.2 0 2 1.0 0 8.6 8 31.4 0 107.0 3 3 10 0 8.6 7 31.3 5 109.8 6 4 6 (26 ) 1 1.0 0 5.6 6 5.6 8 3.8 6 2 1.0 0 6.3 6 7.4 7 5.4 4 3 1.0 0 6.4 8 8.0 9 3.4 2 10 0 (26 ) 1 1 0 0 5.4 3 6.6 2 2.2 0 2 1.0 0 5.7 2 7.6 9 4.1 7 3 1.0 0 5.4 9 8 4 2 4.1 3 WA F prepare d atth e give n loadin g rat e 2Th e test substance i s p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e 3N o measuremen t ofalga l cel l densit yatth e en d o

fth e test ; visse l was broke n { ) Averag e exposur

e concentratio n

- Pag e 1 8 -

iITh e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO X Projec t 49698 7 APPENDI X1 WORKSHEE TDATA - continue d - Tabl e 9 Calculatio n o fgrowt h rat e an d yiel d Growt h rat e Loadin g rate 1 Vesse l Growt h rat e Yiel d red
. Yiel d inhib
. Tes t substance 2 numbe r (M ) <x10 4cells/ml ) 

### (% ) (%

) (mg/l ) 0-7 2 h 0-7 2 h 0-7 2 h 0-7 2 h contro l 1 0.0709 6 3 164.5 4 3 /L 3 0.0713 0 16 8 6 6 4 0.0681 4 134.0 9 ' 5 0.0716 2 172.6 0 6 0.0694 6 147.6 0 mea n 0.0703 0 15 7 5 0 C V 2 % 4 6 (2.5 ) 1 0.0667 3 121.0 4 5 2 3 2 0.0690 9 143.6 5 2 9 3 0.0674 2 127.2 8 4 1 9 10(2.6 ) 1 0.0691 6 14 4 4 6 2 8 2 0,0696 0 149.0 5 1 5 3 0.0687 6 140.3 1 2 1 1 2 2 ( 4 2 ) 1 0.0651 8 108.2 0 7 3 1 2 0.0649 0 106.0 3 8 3 3 3 0.0652 7 108.8 6 7 3 1 4 6 (26 ) 1 0.0187 4 2.8 6 7 3 9 8 2 0.0235 1 4.4 4 6 7 9 7 3 0.0170 6 2.4 2 7 6 9 8 10 0 (26 ) 1 0.0109 8 1.2 0 8 4 9 9 2 0.0198 3 3.1 7 7 2 9 8 3 0.0196 8 3.1 3 7 2 9 8 WA F prepare d a

tth e give n loadin g rat e 2Th e testsubstance i s p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e 3N o measuremen t o

falga l cel l densit

ya t th e en d ofth e test ; vesse l was broke n ( ) Averag e exposur

e concentratio n

- Pag e 1 9 -Th e us e o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s {REACH,) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)an i in e APPENDI X1 WORKSHEE TDAT A - continue d -
Tabl e 1 0 Calculatio n ofgrowt h rat e (section-by-section ) NOTO X Projec t 49698 7

Loadin g rate 1 Vesse l Growt h rat e IV) Growt h rat e reductio n {% ) Tes t substance * numbe r (mq/l ) 0-2 4 h 24-4 8 h 48-7 2 h 0-2 4 h 24-4 8 h 48-7 2 h contro l 1 0.0913 1 0 0621 7 0.0594 0

2 0.0866 6 0 0643 3 3

. 3 0.0903 2 0.0651 5 0.0584 4 4 0.0875 8 0.0547 8 0.0620 6 5 0.0951 1 0.0617 3 0.0580 3 6 0.0896 9 0.0617 4 0.0569 6 mea n 0.0901 1 0.0616 5 0.0589 8 C V 3 % 6 % 3 % Th e mea n C Vfo rsection-bv-sectio n speci l c c rowt h rat e was : 2 1 % 4. 6 (2.5 ) 1 0.0857 6 0 0581 9 0.0562 3 5 6 5

2 0.0886 9 0.0588 4 0.0597 3 2 5 - 1 3 0 0881 4 0.0617 9 0.0523 2 2 0 1 1 1 0 (2.6 ) 1 0.0899 4
. 0.0590 1 0.0585 4 0 4 1

2 0.0943 9 0.0582 3 0.0561 7 - 5 6 5 3 0.0894 6 0.0590 0 0.0578 3 1 4 2 2 2 (4.2 ) 1 0.0908 3 0 0530 5 0.0516 6 - 1 1 4 1 2

2 0.0900 4 0.0535 7 0.0510 9 0 1 3 1 3 3 0.0900 1 0.0535 4 0.0522 5 0 1 3 1 1 46(26 ) 1 0.0722 0 0.0001 6 0.0000 0 2 0 10 0 10 0

2 0 0771 O 0.0066 6 0.0000 0 1 4 8 9 10 0 3 0.0778 9 0.0092 3 0.0000 0 1 4 8 5 10 0 10 0 (26 ) 1 0 0705 2 0.0082 3 0.0000 0 2 2 8 7 10 0

2 0.0726 6 0.0123 2 0.0000 0 1 9 8 0 10 0 3 0.07095 0.0178 4 0.00000 2 1 7 1 10 0

2Th e tes t substance i s p-(2,3-epoxypropoxy)-N,N-bis(2,3- ' 3N o measuremen t o falga l cel l densit ya t th e en d o

fth e test ; ( ) Averag e exposur

e concentratio n epoxyprcpyl)ani!in e vesse l was broke n

r r - Pag e 2 0 -

ri

### p. 11 4 Th e us e o

fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no tlimite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou tth e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. 

### p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t49698 7 APPENDI X2 STATISTICS : GROWT H RAT E (0-7 2 HOURS) Chi-Squar e Tes t fo r Normalit y Actua l an d Expecte d Frequencie s INTERVA L <-1. 5 -1. 5 t o <-0. 5 0. 5 t o 0. 5 >0. 5 t o 1. 5 >1. 5 EXPECTED OBSERVE D 1.340 0 0 4.840 0

7 7.640 0

5 4 .840 0

8 1.340 0

0 Chi

- Squar e ~ 6.619 4 (P
- valu e = 0.1574 ) Critica l Chi-Squar e = 13.27 7 (alph a = 0.0 1 , d f = 4 )
= 9.48 8 (alph a = 0.0 5 , d f = 4 } Dat a PAS S normalit ytes t (alph a = 0.01) . Continu e analysis .

Levane' s Tes t fo r Homogeneity o f Varianc e ANOV ATabl e SOURC E D

F S

S M

S F Betwee n 5 0.000 0 0.000 0 0.762 7 Withi n(Error ) 1 4 0.000 1 0.000 0 Tota l 1 9 0.000 1 (p-valu e = 0.5914 ) Critica l F = 4.695 0 (aloh a = 0.01 , d f = 5,14 )

= 2.958 2 (alph a = 0.05 , d f = 5,14 ) Sinc e F < Critica l F FAI L T O REJEC T Ho : Al l equa l (alph a = 0.01 ) ANOV ATabl e ' SOURC E D

F S S M S F Betwee n 5 Withi n(Error ) 1 4 0.010 6 0.000 1 0.002 1

0.000 0 346.754 3 Tota l 1 9 0.010 7 Critica l F = 4.695 0

= 2.958 2 Sinc e F > Critica l F (alph a = 0.01 , d f = 5,14 ) (alph a = 0.05 , d f = 5,14 ) REJEC T Ho : Al l equa l (alph a (p-valu e

= 0.05 ) = 0.0000 )

Bonferroni . t-Tes t TABL E 1 O F 2 Ho : Control=Treatmen t TRANSFORME

D MEA

NCALCULATED I N S1G GROU P IDENTIFICATIO N MEA N ORIGINA

L UNIT S t STA

T 0.0 5 1 contro l 0.070 3 0.070 3 2 4. 6 0.067 7 0.067 7 1 411 3 3 1 0 0.069 2 0.069 2 0 621 5 4 2 2 0.065 1 0.065 1 2 867 2 5 4 6 0.019 8 0.019 8 2 7 970 0 

### * 6 10 0 0.016 8 0.016 8 2 9 597 5 

### * Bonferron i t critica l valu e = 2.976 8 ( 2 Tailed , alph a = 0.05 , d f = 5,1 4 

### ) Bonferron i t-Tes t TABLE 2 OF 2 Ho : Contro l =Treatraen t HU M O F MI NSI G DIF F % O F DIFFERENCE GROU P IDENTIFICATIO N REPS (I NORIG . UNITS ) CONTRO L FRO M CONTRO

L 1 contro l 5 2 4. 6 3 0.005 4 7. 6 0.002 5 3 1 0 3 0.005 4 7. 6 0.001 1 4 2 2 3 0.005 4 7. 6 0.005 2 5 4 6 3 0.005 4 7. 6 0.050 5 6 10 0 3 0.005 4 7. 6 0.053 5



### - Pag e 2 1 -Th e use o fthes e studies/documents fo rregulator yorregistrati ti n Authorizatio n an d Restrictio n o fChemica l Substances (REACH, ) Internationa l LL

C i s unauthorize d an d strictl yprohibited
.
m e p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)an i APPENDI X3 STATISTICS : YIEL D (0-72 HOURS ) purposes , includin g bu tno tlimite d t o Registration , Evaluation , , withou tth e expres s writte n permissio n o fHuntsma n p . 11 5

NOTO XProject

496987

Chi-Squar e Tes t fo r Normalit y

INTERVA L EXPECTED OBSERVE D <-1. 5 1.340 0 0 Actua l an d Expecte d Frequencie s

-1. 5 t o <-0. 5 -0 . t o 0. 5 4.840 0 7 .640 0

5 >0. 5 t o 1. 5

4.840 0 >1. S

1.340 0 0 Chi-Squar e = 6.619 4 (p-va:.u e = 0.1574 ) Critica l Chi-Squar e = 13.27 7 (alph a = = 0.0 1 , d f = 4 ) = 9.48 8 (alph a f

0.0 5 , d f = 4 ) Dat a PAS S normalit ytes t (alph a = 0.01) . Co n :inu e analysis .

Levene' s Tes t fo r Homogeneity o f Varianc e ANOV ATabl e SOURC E D

F S S M S Betwee n Withi n(Error ) 5 1 4 433.251 6

741.963 3 86.650 3

52.997 4 F

1.635 0
Tota l 1 9 1175.214 8 Critica l F = 4.695 0 (alph a = 0.01 , d f =

= 2.958 2 (alph a =0.05 , d f = Sinc e F < Critica l F FAI L T O REJEC T H o (p-valu e = 0.2150 ) 5,14 ) 5,14 ) A l l equa l (alph a = 0.01 )

SOURC E D

F ANOV

A Tabl e S S M S Betwee n Withi n(Error ) 5 1 4 81995.122 2

1368.267 3 16399.024 4 97.733 4 167.793 5
Tota l 1 9 83363.389 5 Critica l F = 4.695 0 (alph a = 0.01 , d f =

= 2.958 2 (alph a = 0.05 , d f = Sinc e F > Critica l F REJEC T Ho : Al l

eqja l (p-valu e = 0.0000 ) 5,14 ) 5,14 ) (alph a = 0.05 ) Bonferron i t-Tes t TABLE 1 O F 2 Ho : Control=Treatmen t GROU P IDENTIFICATIO N

contro l 4. 6 1 0 2 2 4 6 10 0 TRANSFORME

D MEA N 157.498 0 130.656 7 144.606 7 107.696 7 3.240 0 2.500 0 MEA

NCALCULATED I NSI G ORIGINA L UNIT S t STA

T 0.0 5 157.498 0 130.656 7 144.606 7 107.696 7 3.240 0 2.500 0 3.717 8

1.785 6 6.898 0 21.366 2 21.468 7 Sonferron i t critica l valu e = 2.97 f ( 2 Tailed , alph a = 0.05 , d f = 5,14 ) Bonferron i t-Tes t TABLE 2 O F 2 Ho : Control=Treatmen t GROU P IDENTIFICATIO N NU M O F REPS MI NS

(I N I GDIF F % O F DIFFERENCE ORiTjG . UNITS ) CONTRO L FRO M CONTRO

L contro l 4. 6 1 0 2 2 4 6 10 0 1.492 0 1.492 0 1.492 0 1.492 0 1.492 0 13. 6

13. 6 13. 6 13. 6 13. 6 26.841 3

12.891 3 49.801 3 154.258 0 154.998 0



### - P s ge22 -

i ip . 11 6 Th e us e ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 7 APPENDI X4 EC-VALUE S Tabl e 1 1 EC-value s fo rgrowt h rat e reductio n Concentratio n X Y (mg/l ) Lo g cone , (mq/f ) Reductio n (% ) 2. 5 0.39 8 5. 1 2. 5 0.39 8 1. 7 2. 5 0.39 8 4. 1 2. 6 0 41 5 1. 6 2. 6 0.41 5 1. 0 2. 6 0.41 5 2. 2 4. 2 0.62 3 7. 3 4. 2 0.62 3 7. 7 4. 2 0.62 3 7. 2 2 6 1.41 5 73. 3 2 6 1.41 5 66. 6 2 6 1.41 5 75. 7 2 6 1.41 5 84. 4 2 6 1.41 5 71. 8 2 6 1.41 5 72. 0 Slope : 72.972 3 Intercept : -30.155 4 Multipl e R : 0.987 2 n = numbe ro fobservations : 1 5 Regressio n line ' Y= 72.97 X- 30.1 6 Predictio n o fXvalue s base d o n know n Yvalue s Know n Y Reductio n (% ) 1 0 Xre g (mg/l ) ^QX95% -

(mq/l ) 1 0 X9E% +

(mg/l ) 1 0 3.5 5 2.3 3 5.4 0 2 0 4.8 7 3.2 1 7.3 8 5 0 12.5 4 8.2 6 1 9 0 6

Figur e 2 Percentag e reductio n o fgrowt h rat e a s functio n o fth e lo g concentratio n (mg/l )

o fp-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline . Dashe d curve s represen tth e 95 % confidenc e limits
.

- Pag e 2 3 -Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substances (REACH) , withou t th e express writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p . 11 7
p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProject49698 7 APPENDI X4 EC-VALUE S - continue d - Tabl e 1 2 EC-value s fo ryiel d inhibitio n Concentratio n X Y (rnq/l ) Lo q cone , (mq/l ) Inhibitio n (% ) 2. 5 0.39 8 2 3 2 2. 5 0.39 8 8. 8 2. 5 0.39 8 19. 2 2 6 0.41 5 8. 3 2. 6 0.41 5 5. 4 2. 6 0.41 5 10. 9 4 2 0.62 3 31. 3 4. 2 0.62 3 32. 7 4. 2 0.62 3 3 0 9 2 6 1.41 5 98. 2 2 6 1 41 5 97. 2 2 6 1.41 5 98, 5 2 6 1.41 5 99. 2 2 6 1.41 5 98. 0 2 6 1.41 5 98. 0 Slope : 84.629 7 Intercept
. -21.565 1 Multipl e R : 0.993 8 n = numbe ro fobservations : 1 5 Regressio n line : Y

= 84.63 X- 21.5 7 Predictio n o fXvalue s base d o n know n Yvalue s Know n Y Inhibitio n (% ) 

### io Xre g

(mg/l ) ,JQX95% -

(mq/l ) (mq/l ) 1 0 2.3 6 1.7 6 3 1 7 2 0 3.1 0 2.3 1 4.1 5 5 0 7.0 1 5.2 6 9.3 3



### 1 1 0 10 0 Concentratio n (mg/l ) Figur e 3 Percentag e inhibitio n ofyiel d a s functio n ofth e lo g concentratio n (mg/l ) o fp - (2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline . Dashe d curve s represen t th e 95 % confidenc e limits
.

- Pag e 2 4 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e APPENDI X5 REFERENC E TES T NOTO XProjec t49698 7

Pseudokirchneriell a subcapitata , strain : NIVACHL-1
. Fres h wate ralga l growt h inhibitio n tes t wit h potassiu m dichromat e (NOTO XProject498136)
. Start o ffirs t exposure : 1 0 Octobe r201 1 Completio n las texposure : 1 3 Octobe r201 1 Th e stud yprocedure s describe d i n thi s report wer e base d o n th e OEC D guidelin e No
. 201 , Adopte d Marc h 23 , 200 6 an d IS O Standar d 8692 , Secon d edition , 0 1 Octobe r2004
. Thi s referenc e tes twa s carrie d ou t t o chec k th e sensitivit yo fth e tes t syste m use d b yNOTO Xt o Potassiu m dichromat e (Merck, Art
. 1.04864 , Batc h K3486976 4 607)
. Alga e wer e expose d fo ra perio d o f7 2 hour s t o K 2 Cr 2 0 7(Potassiu m dichromate ) concentration s o f 0.18 , 0.32 , 0.56,1.0 , 1. 8 an d 3. 2 mg/ l an d t o a control
. Th e initia l cel l densitywa s 1. 0 x10 4cells/ml
. Results : Overviewo f% reductio n o fgrowt h rat e an d % inhibition o fyiel d i n th e referenc e test : Nomina l cone . K 2 Cr 2 0 7 (mq/l ) Mea n growt h rat e Yiel d (0-7 2 h ) Nomina l cone . K 2 Cr 2 0 7 (mq/l ) M (0-7 2 h ) Reductio n (% ) x10 4cells/m l Inhibitio n (% ) Contro l 0.0699 7 153.2 6 0.1 8 0.0693 3 0. 9 146.59 4. 4 0.3 2 0.0668 1 4. 5 122.63 20. 0 0.5 6 0.0600 4 14. 2 74.5 6 51. 4 1. 0 0.0434 0 38. 0 21.8 9 85. 7 1. 8 0.0286 0 59. 1 6.8 6 95. 5 3. 2 0.0210 9 69. 9 3.5 7 97. 7 Potassiu m dichromat e reduce d growt h rat e o fthi s fres h wate ralga e specie s a t nomina l concentration s o f0.5 6 mg/ l an d higher
. Th e EC 5 0fo rgrowt h rat e reductio n (E R Cso : 0-72h ) wa s 1. 5 mg/ I wit h a 95 % confidenc e interva l rangin g fro m 1. 1 t o 2. 1 mg/l
. Th e historica l range s fo rgrowt h rat e reductio n li e betwee n 0.8 2 an d 2. 3 mg/l
. Hence , th e E R C 5 0 : 0-72 h fo rth e algafcultur e teste d correspond s wit h thi s range . Th e ECs o fo ryiel d inhibition (EyC^ : 0-72h ) wa s 0.5 2 mg/ l wit h a 95 % confidenc e interva l rangin g fro m 0.3 4 t o 0.7 8 mg/l
. Th e historica l range s o fth e 72h-EC 5 o fo ryiel d inhibition li e betwee n 0.4 3 an d 1. 1 mg/l
. Hence , th e E Y C 5 0 : 0-72 h fo rth e alga l cultur e teste d correspond s wit h thi s range . Th e protocol , ra wdat a an d reporto fthi s stud yar e kep ti n th e NOTO Xarchives
. Th e tes t describe d abov e wa s performe d unde rGL P condition s wit h a QA-check
.

- Pag e 2 5 -Th e use o fthes e studies/documents fo rregulator yorregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p . 11 9

p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO X Projec t 49698 7 APPENDI X 6 ANALYTICA L REPOR T



# DETERMINATIO N O F TH E CONCENTRATION S

Autho r Dr
. Ir
. E
. Baltusse n

- Pag e 2 6 -

I ITh e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p . 12 0

1 . CONTENT S 1
. CONTENT S 2 7 2
. REPOR TAPPROVA L2 8 3
. INTRODUCTIO N 2 9 3.1
. Prefac e 2 9 3.2
. Ai m o fth e stud y2 9 4
. MATERIAL S AN D METHOD S 2 9 4.1
. Reagent s 2 9 4.2
. Sample s 2 9 4.3
. Analytica l metho d 2 9 4.3.1
. Analytica l condition s 2 9 4.3.2
. Preparatio n o fth e calibratio n solution s 2 9 4.3.3
. Sampl e injection s 3 0 4.4
. Electroni cdat a captur e 3 0 4.5
. Formula s 3 0 5
. RESULTS 3 1 5.1
. Calibratio n curve s 3 1 5.2
. Sample s 3 1 5.2.1
. Procedura l recoverysample s 3 1 5.2.2
. Tes tsample s 3 1 6
. TABLE S 3 2 TABLE S Tabl e 1 Procedura l recoverysample s 3 2 Tabl e 2 Concentration s o fth e tes tsubstanc e i n tes tmediu m - combine d limit/range-findin g

tes t 3 3 Tabl e 3 Concentration s o fth e tes tsubstanc e i n tes tmediu m - fina l tes t 3 3

- Pag e 2 7 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu tno tlimite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. 

### p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline 2 . REPOR TAPPROVA L NOTO XB.V. NOTO XProjec t 49698 7

Principa l Scientis

t Analytica l Chemistr

y Dr . Ir. E . Baltusse n

Date :

Fina l Repor

t - Page 28 -Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 7 3 . INTRODUCTIO N 3.1 . Prefac e Stud ypla n analytica l phas e Start 3 0 Septembe r201 1 Completio n : 2 2 Octobe r201 1 3.2 . Ai m o fth e stud y Th e purpos e o fth e analytica l phas e wa s t o determin e th e actua l concentration s i n sample s take n fro m th e tes t solution s use d durin g th e ecotoxicit ytest . 4 . MATERIAL S AN D METHODS . 4.1 . Reagent s Wate rTa p wate rpurifie d bya Milli- Q wate rpurificatio n syste m (Millipore , Bedford , MA , USA ) Acetonitril e Biosolve , Valkenswaard , Th e Netherland s M2-mediu m se e mai n report Al l reagent s wer e o fanalytica l grade , unles s specifie d otherwise
. 4.2 . Sample s Th e sample s wer e store d i n th e freeze r( £-15°C)
. Storag e stabilit yo fsample s unde rthes e condition s wa s demonstrate d i n NOTO Xprojec t 496981
. O n th e da yo fanalysis , th e sample s wer e defroste d a troo m temperature . Th e tes t sample s wer e dilute d i n a 1: 1 (v:v ) rati o wit h 60/4 0 (v/v) acetonitrile/M2-mediu m an d analysed
. I fnecessary , th e sample s wer e furthe rdilute d wit h 30/7 0 (v/v ) acetonitrile/M2-mediu m t o obtai n concentration s withi n th e calibratio n range . 4.3 . Analytica l metho d 4.3.1 . Analytica l condition s Quantitativ e analysi s wa s base d o n th e analytica l metho d validate d fo rth e tes t substanc e i n NOTO X projec t496981
. Instrumen t Allianc e Separatio n Modul e 269 5 (Waters , Milford , MA , USA ) Detecto rDua l AAbsorbanc e Detecto r248 7 (Waters ) Colum n SymmetryShiel d RP-18 , 10 0 m m x4. 6 m m i.d. , d p = 3. 5 ur n (Waters ) Colum n temperatur e 40° C ±1° C Injectio n volum e 10 0 p i Mobil e phas e 30/7 0 (v/v ) acetonitrile/wate r Flo w1. 0 ml/mi n U Vdetectio n 25 3 n m 4.3.2 . Preparatio n o fth e calibratio n solution s Stoc k an d spikin g solution s Stoc k solution s o fth e tes tsubstanc e wer e prepare d i n acetonitril e a t concentration s o f241 5 - 382 0 mg/l
. Spikin g solution s wer e mad e u p fro m a stoc k solutio n and/o rdilution s o fthi s solution
. Th e solven t o f th e spikin g solution s wa s acetonitrile
.

- Pag e 2 9 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substances (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p . 12 3
p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 7 Calibratio n solution s Calibratio n solution s i n th e concentratio n rang e o f0.0 4 - 2 mg/ l wer e prepare d fro m tw o stoc k solutions
. Th e en d solutio n o fth e calibratio n solution s wa s 30/7 0 (v/v ) acetonitrile/M2-medium
. Procedura l recoverysample s 2 m l blan k

mediu m wa s spike d wit h th e tes tsubstanc e a t a targe t concentratio n o f0. 1 o r10 0 mg/l
. Th e accurac ysample s wer e treate d similarl ya s th e tes t sample s (se e paragrap h 4. 2 'Samples')
. 4.3.3 . Sampl e injection s Calibratio n solution s wer e injecte d i n duplicate
. Tes tsample s an d procedura l recoverysample s wer e analyse d bysingl e injection
. 4.4 . Electroni c dat a captur e Syste m control , dat a acquisitio n an d dat a processin g wer e performe d usin g th e followin g programme :

- Empowe rversio n 7.0 0 (Waters , Milford , MA , USA)
. Temperature

and/or relative humidity

during sample storage and/or performance

of the studies were monitored

continuously

using the following

programme:

- REE S Centro n Environmenta l Monitorin g syste m versio n SQ L2. 0 (REE S Scientific , Trenton , NJ , USA)
. 4.5
. Formula s Respons e (R ) Pea k are a tes tsubstanc e [units ] Calibratio n curv e R = a C N + b where : C M = nomina l concentratio n [mg/l j slop e [unit s xl/mg ] intercep t [units] a - b = Analyse d concentratio n (C A ) (R-b ) x d [mg/l ] a where : d = dilutio n facto r Recovery 10 0 [%]
Relativ e t o initia l concentratio n C A(t= xhours ) C A( t= 0 hours ) x10 0 [%]

- Pag e 3 0 -Th e us e ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d stncfJyprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProject 49698 7 5 . RESULT S 5.1 . Calibratio n curve s Calibratio n curve s wer e constructe d usin g fiv e concentrations
. Fo reac h concentration , tw o response s wer e used
. Linea rregressio n analysi s wa s performe d usin g th e leas t square s metho d wit h a 1 /concentration 2weightin g factor
. Th e coefficien t o fcorrelatio n (r ) wa s > 0.9 9 fo reac h curve . 5.2 . Sample s 5.2.1 . Procedura l recover ysample s Th e result s fo rth e procedura l recoverysample s ar e give n i n Tabl e 1
. Th e mea n recoverie s o fth e procedura l recoverysample s fel l withi n th e criterio n o f70-110%
. I t demonstrate d tha t th e analytica l metho d wa s adequat e fo rth e determinatio n o fth e tes t substanc e i n th e tes tsamples
. 5.2.2 . Tes t sample s Th e result s fo rth e tes t sample s ar e give n i n Tabl e 2 an d Tabl e 3

- Pag e 3 1 -Th e us e o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyr)anilin e NOTO XProject 49698 7 6 . TABLE S Tabl e 1 Procedura l recover ysample s Dat e o f preparatio n jdd-mm-yyj Dat e o f

analysi s [dcj-mm-yy ] Targe t

concentratio n Jmg/r j Nominal concentratio n rmavT j Analyse d concentratio n Jm.g/IJ Recovery



### f% ) Mea n recovery I% ] 30-09-1 1 30-09-1 1 0. 1 0.10 0 0.092 4 9 2 9 8 0.10 0 0.10 3 10 3 30-09-1 1 30-09-1 1 10 0 10 0 10 1 10 1 10 1 10 0 10 1 10 1 22-10-1 1 22-10-1 1 0. 1 0.099 3 0.086 7 8 7 8 4 0.099 3 0.080 8 8 1 22-10-1 1 22-10-1 1 10 0 99. 3 95. 9 9 7 9 8 99. 3 99. 6 10 0

- Pag e 3 2 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d to Registration , Evaluation , Authonzatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 7 Tabl e 2 Concentration s o fth e tes t substanc e i n tes t mediu m - combine d limit/range findin g tes t Tim e o f samplin g [hours ! Dat e o f

samplin g [dd-mm-yy l Dat e o f

analysi s 1 [dd-mm-yy l Loadin g rat e 2

rmg/l l Concentratio n

analyse d [mq/l l Relativ e t o

initia l 

## r% i 0 19-09-1 1 30-09-1 1 1 0.72 8 10 0 60. 0 100 3 56. 4 2 4 20-09-1 1 30-09-1 1 1 0.58 0 8 0 10 0 48. 8 8 1 100 3 51. 4 9 1 7 2 22-09-1 1 30-09-1 1 1 0.32 5 4 5 10 0 37. 0 6 2 100 3 33. 7 6 0 Sample s were store d i n th e freeze r( s -15°C ) unti l th e da yofanalysis
. Awate raccommodate d fractio n (WAF ) prepare d at th e loadin g rate . Withou t algae
. Tabl e 3 Concentration s o fth e tes t substanc e i n tes t mediu m - fina l tes t Tim e o f Dat e o f Dat e o f Loading rate 1 Concentratio n Relativ e t o samplin g samplin g analysis 1 analyse d initia l fhours l rdd-mm-yy l [dd-mm-yy l [mg/l l [mg/l l [%1 0 10-10-1 1 22-10-1 1 0 n.d
. 4. 6 3.2 3 1 0 3.3 0 2 2 5.4 8 4 6 32. 6 10 0 33. 8 100 3 36. 4 2 4 11-10-1 1 22-10-1 1 0 n.d
. n.a . 4. 6 2.8 2 8 7 1 0 2.7 4 8 3 2 2 4.5 9 8 4 4 6 27. 1 8 3 10 0 27. 6 8 2 100 3 28. 5 7 8 7 2 13-10-1 1 22-10-1 1 0 n.d
. n.a . 4. 6 1.8 9 5 9 1 0 2.0 3 6 1 2 2 3.2 2 5 9 4 6 21. 4 6 6 10 0 21. 3 6 3 100 3 22. 8 6 2

' Samples were store d i n th e freeze r( s -15°C ) unti l th e da yofanalysi s 2Awate raccommodate d fractio n (WAF ) prepare d at th e loadin g rate . 3Withou t algae
. n.d
. No

t detected , n.a . No

tapplicable
.

- Pag e 3 3 -Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
.



## FINA L REPOR T

Stud yTitl e 

## DAPHNI AMAGNA , REPRODUCTIO N TES TWIT H P-(2,3-EPOXYPROPOXY)-N,N-BIS(2,3-EPOXYPROPYL)ANILIN E (SEMI-STATIC )
Autho r Ir
. L.M
. Bouwina n

Tes t Facilit y NOTO X B.V
. Hambakenweterin g 7 523 1 D D 's-Hertogenbosc h Th e Netherland s LaboratoryProjec t Identificatio n NOTO X Projec t 49698 9 NOTO X Substanc e 203277/A

- Pag e 1 o f3 7 -Th e use ofthes e studies/documents fo rregulator yorregistratio n purposes , includin g bu t not limite d to Registration , Evaluation, ' Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p . 12 8
p-(2,3-epoxypropoxy)~N,N-bis(2,3-epoxypropyl)anilin e NOTO XProject 49698 3 1
. CONTENT S 1
. CONTENT S 2 2
. STATEMEN TO F GL P COMPLIANC E 4 3
. QUALIT YASSURANC E STATEMEN T, 5 4
. SUMMAR Y, , , 6 5
. INTRODUCTIO N 7 5.1
. Prefac e 7 5.2
. Ai m o fth e stud y7 5.3
. Guideline s 7 5.4
. Storag e an d retentio n o frecord s an d material s 7 5.5
. Definition s 8 6
. MATERIAL S AN D METHOD S 3 6.1
. Tes tSubstanc e 8 6.1.1
. Tes tsubstanc e informatio n 8 6.1.2
. Stud yspecifi ctes t substanc e informatio n 9 6.2
. Tes t syste m 9 6.3
. Breedin g 9 6.4
. Preparatio n o ftes t solution s 1 0 6.5
. Preliminarytes t .1 0 6.6
. Reproductio n tes t .........1 1 6.6.1
. Tes tconcentrations...
. ....1 1 6.6.2
. Tes tprocedur e an d condition s 1 1 6.6.3
. Samplin g fo ranalysi s o ftes t concentration s 1 2 6.6.4
. Measurement s an d recording s 1 2 6.7
. Interpretatio n 1 2 6.7.1
. Dat a handlin g , 1 2 6.7.2
. Electroni cdat a captur e 1 3 6.7.3
. Acceptabilit yofth e tes t1 3 6.8
. Lis to fdeviation s 1 3 6.8.1
. Lis t o fprotoco l deviation s 1 3 6.8.2
. Lis to fstandar d operatin g procedure s deviation s 1 4 7
. RESULTS 1 4 7.1
. Preliminarytes t1 4 7.2 , Reproductio n test. . 1 4 7.2.1
. Measure d concentration s 1 4 7.2.2
. Conditio n o fparenta l daphnid s 1 5 7.2.3
. Reproductio n 1 5 7.2.4
. Bod ylengt h 1 6 7.2.5
. Determinatio n o feffec t concentration s 1 7 7.2.6
. Experimenta l condition s 1 7 8
. CONCLUSIO N 1 7

- Page 2 -

Ill l ' I ITh e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p"(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropy!)anilin e NOTO XProjec t 49898 9 TABLE S Tabl e 1 Mortalit yan d reproductiv e potenc ydurin g th e preliminarytes t 1 4 Tabl e 2 Cumulativ e mortalit yo fparenta l daphnid s durin g th e 2 1 -da yexposur e perio d 1 5 Tabl e 3 Averag e bod ylength s (mm ) o fparenta l daphnid s at th e en d ofth e tes t 1 6 Tabl e 4 Effec tparameter s 1 7 Tabl e 5 Cumulativ e score s o flivin g newbor n pe rparen ti n th e contro l 1 8 Tabl e 6 Cumulativ e score s o flivin g newbor n pe rparen t a t 0.04 0 mg/ l , 1 8 Tabl e 7 Cumulativ e score s o flivin g newbor n pe rparen t a t 0.09 6 mg/ l 1 9 Tabl e 8 Cumulativ e score s oflivin g newbor n pe rparen t a t 0.4 2 mg/ l 1 9 Tabl e 9 Cumulativ e score s oflivin g newbor n pe rparen t at 1. 7 mg/ l 2 0 Tabl e 1 0 Cumulativ e score s oflivin g newbor n pe rparen t at 4. 8 mg/ l 2 0 Tabl e 1 1 Individua l value s fo rbod ylengt h o fth e survivin g parenta l daphnid s ,.2 1 Tabl e 1 2 p H value s i n freshl yprepare d medi a (F ) an d ol d medi a (O ) 2 6 Tabl e 1 3 Dissolve d oxyge n concentration s (mg/l ) i n freshl yprepare d medi a (F ) an d ol d

medi a (O ) , 2 6 Tabl e 1 4 Temperatur e ( n C ) i n freshl yprepare d medi a (F ) an d ol d medi a (O ) 2 7 Tabl e 1 5 Hardnes s (mg/ l CaC0 3 ) i n freshl yprepare d medi a (F ) an d ol d medi a (O ) 2 7 FIGUR E Figur e 1 Cumulativ e mea n numbe ro flivin g youn g pe rparen t a t th e variou s averag e

concentration s ofp-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyi)anilin e durin g th e 21-da ytes tperio d 1 6 APPENDICE S APPENDI X1 CUMULATIV E SCORE S O F LIVIN G NEWBOR N DAPHNID S PE R PAREN T1 8 APPENDI X2 PARENTA LBOD YLENGT H 2 1 APPENDI X3 STATISTICS ; PARENTA LMORTALIT Y2 2 APPENDI X4 STATISTICS ; REPRODUCTIO N 2 3 APPENDI X5 EC-VALUE S 2 4 APPENDI X6 EXPERIMENTA LCONDITION S 2 6 APPENDI X7 ANALYTICA LREPOR T2 8

- Pag e 3 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substances (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibite d 

### p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t49698 9 2
. STATEMEN TO F GL P COMPLIANC E NOTO XB.V. , 's-Hertogenbosch , Th e Netherland s Th e studydescribe d i n thi s repor t has bee n correctl yreporte d an d was conducte d i n compliance with : Th e Organizatio n fo rEconomi cCo-operatio n an d Developmen t(OECD ) Principle s ofGood Laborator y Practice (GLP ) (as revise d i n 1997) ENV/MC/CHE M (98 ) 17
. Th e sponso ri s responsibl e fo rGood Laborator yPractice (GLP ) compliance fo ral l testsubstance informatio n unles s determine d b yNOTOX
.
NOTO XB.V
.

Fina l Repor

t - Pag e 4 -

### p . 13 1 Th e us e ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d to Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
.


### 3 . QUALIT

YASSURANC E STATEMEN T NOTO XB.V. , 's-Hertogenbosch , Th e Netherland s Thi s repor

t was inspecte d b

yth e NOTO

XQualit yAssurance Uni tt o confir m thatth e method s an d results accuratel yan d completel yreflec tth e ra wdata
. Durin g the'on-sit e process inspections , procedure s applicabl e to thi s typ e o fstud ywere inspected
. Th e date s ofQualit

yAssurance inspection s are give n below
. p-{2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t49698 9

Type o f Inspection s Phase/Proces s Star t En d Inspectio n Inspectio n Reportin g date dat

e date Stud y Protoco l

Protoco l Amendmen t0 1 Protoco l Amendmen t02 Repor t 02-Aug-1 1 02-Aug-1 1

09-Aug-1 1 09-Aug~1 1 23-Dec-1 1 23~Dec-1 1 28-Feb-1 2 29-Feb-1 2 02-Aug-1 1 09-Aug-1 1 23-Dec~1 1 29-Feb-1 2 Proces s Environmenta l Toxicolog y17-OcM 1 21-Oct~1 1

Test Substance Handlin g Exposur e O bservatio n s

/ Meas u r e m e nts 21-Oct-1 1
Analytica l an d physica l chemistr y Test Substance Handlin g Observations/Measurements 31-oct- n IO-Nov-1 1 10-Nov-1 1

NOTO XB.V
. C.J
. Mitchel l B.Sc
. Hea d ofQualit

yAssurance

Date:J3.Ti^rrr:&>J. Z

Fina l Repor

t - Pag e 5 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p . 13 2
p-(2 1 3-epoxypropoxy)-N,N-bis(2,3-epoxypropy!)anilin e NOTO XProject 49698 9 4 . SUMMAR Y Daphni a magna , 21-da yreproductio n stud ywit h p~(2,3-epoxypropoxy)-N,N-bis{2,3~epQxypropyl)aniline
. Th e stud yprocedure s describe d i n thi s reportwer e base d o n th e OEC D guideline s fo rTestin g o f Chemicals : Guidelin e No , 211 , 2008
. I n addition , th e procedure s wer e designe d t o mee t th e tes t method s an d validit ycriteri a o fth e IS O Internationa l Standar d 10706 , 2000 , th e Commissio n Regulatio n (EC ) N o 440/200 8 PartC.20 , 200 8 an d th e OEC D guidanc e documen t numbe r23 , 2000
. Th e batc h o ftes t substanc e teste d wa s a yello wliqui d wit h a purit yo f94.33%
. Areproductio n tes twa s performe d base d o n th e resul to fa precedin g 7-da ypreliminarytest . Preparatio n oftes tsolution s starte d wit h a loadin g rat e o f1 0 mg/ l applyin g tw o day s o fmagneti c stirrin g t o reac h maximu m solubilit yo fth e tes tsubstanc e i n th e tes t medium
. Th e resultin g aqueou s mixtur e wa s lef t t o stabiliz e fo r1-1. 5 hours wher e afte rth e Wate rAccommodate d Fractio n (WAF ) wa s siphone d offan d use d a s th e highes ttes tconcentration , Ai l lowe rtes t concentration s wer e prepare d b ysubsequen t dilution s o fth e WA F i n tes tmedium
. Th e fina l tes t concentration s containe d 1.0 , 3.2 , 10 , 3 2 an d 100 % o fth e WA F prepare d ata loadin g rat e o f1 0 mg/l
. Th e reproductio n tes t wa s performe d i n a semi-stati csystem , include d 1 0 vessel s pe rtes t concentratio n an d 2 0 vessel s fo ra n untreate d contro l group . Eac h o fth e vessel s containe d on e neonat e (<24 h old ) Daphni a magn a i n 5 0 m l tes tmedium
. Th e stud yduratio n wa s 2 1 day s an d th e tes t solution s wer e renewe d every4 8 hours
. Th e daphnid s wer e fed o n a dail ybasi s wit h a Chlorelt a pyrenoidos a suspension
. During th e tes tsample s fo ranalytica l confirmatio n o factua l exposur e concentration s wer e take n atth e startan d th e en d o ffou rinterval s of4 8 hours
. Analysi s o fth e sampl e take n fro m th e WA F prepare d at 1 0 mg/ l at th e start o ffou rrenewa l periods showe d tha t th e actua l tes t concentratio n range d betwee n 4. 2 an d 6. 6 mg/l
. Tes t concentration s slightl ydecrease d durin g th e 4 8 hou rrenewa l period s (t o 70-82 % o finitial)
. Tes t concentration s containin g 1 0 an d 32 % o fth e WA F prove d t o hav e bee n prepare d properl yan d als o slightl ydecrease d durin g th e 4 8 hou rrenewa l periods (t o 63-79 % o finitial)
. Th e tes t concentratio n containin g 1.0 % o fth e WA F wa s belo wth e lowes tcalibratio n standar d a t al l times , whil e th e tes t concentratio n containin g 3.2 % o fth e WA F varie d betwee n bein g belo wo rjus tabov e th e lowes t calibratio n standard
. Give n thes e result s effec tparameter s wer e base d o n th e averag e exposur e concentrations : 0.040 , 0.096 , 0.42 , 1. 7 an d 4. 8 mg/l
. Thre e ou to fth e twent yparenta l daphnid s i n th e contro l grou p die d durin g th e tes t period
. Mortalit yi n th e tes tconcentration s wa s no t statisticall ysignifican twhe n compare d t o th e control
. Th e averag e cumulativ e numbe ro fyoun g pe rfemal e i n th e contro l grou p afte r2 1 day s wa s 13 6 ±16
. Th e reproductio n curve s recorde d at 0.040 , 0.09 6 an d 0.4 2 mg/ l closel yfollowe d th e curv e ofth e control
. Th e curv e recorde d at 1. 7 mg/ l staye d behin d fro m th e start an d dapnid s expose d t o 4. 8 mg/ l di d no t reproduc e a tall
. Ther e wer e som e recording s o fimmobil e youn g and/o raborte d egg s i n th e contro l an d th e three lowes t tes tconcentrations , whil e significan tamount s o faborte d egg s wer e recorde d a tth e tw o highes t tes t concentration s throughou t th e 2 1 -da ytes t period
. Statistica l analysi s showe d tha tth e reproductio n o fth e daphnid s wa s significantlyreduce d a t 1. 7 mg/ | an d higher
. Mea n parenta l bod ylengt h wa s no tsignificantlyreduce d a tan yo fth e tes t concentrations
. Th e stud yme tth e acceptabilit ycriteria prescribe d byth e protoco l an d wa s considere d valid
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e di d no taffec t reproductio n o fDaphni a magn a at a n averag e concentratio n o f0.4 2 mg/ l afte r2 1 day s o fexposur e (NOEC)
. Exposur e t o averag e concentration s o f1. 7 mg/ l an d highe rinduce d significan t inhibition o fth e reproductiv e capacit yo fth e parenta l daphnid s (LOEC)
. Th e 21d-EC 5 0fo rreproductio n wa s calculate d t o b e 1. 4 mg/ l wit h a 95 % confidenc e interva l rangin g fro m 0.9 1 t o 2. 2 mg/l
.

- Pag e 6 -p . 13 3 Th e use o

fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyi)anilin e NOTO XProjec t 49698 9 5 . INTRODUCTIO N 5.1 , Prefac e Sponso rHuntsma n Advance d Material s Gmb H Klybeckstrass e 20 0 405 7 BASE L Switzerlan d Stud yMonito r Tes t Facilit y Dr
. P
. Dollenmeie r NOTO XB.V
. Hambakenweterin g 7 523 1 D D 's-Hertogenbosc h Th e Netherland s Stud yDirecto r Technica l Coordinato r Principa l Scientis t Ir
. L.M
. Bouwma n ing
. V.E
. Carolu s Dr
. Ir
. E
. Baltusse n Stud yPla n Start: 2 0 Septembe r201 1 Completio n : 2 9 Decembe r201 1 5.2 . Ai m o fth e stud y Th e purpos e o fth e tes t wa s t o evaluat e th e effect s o fth e tes tsubstanc e o n th e mobility , th e growt h an d th e reproductiv e capacit yo fDaphni a magna . Fo rthi s purpose , tes t organism s wer e expose d t o aqueou s solution s containin g th e tes tsubstanc e at variou s concentrations
. Th e tim e o fth e firs t productio n o fyoung , th e numbe ro fyoun g born , immobilit yan d othe rsign s o fintoxicatio n observe d wer e compare d wit h correspondin g parameter s i n th e controls
. 5.3 . Guideline s Th e stud yprocedure s describe d i n thi s reportwer e base d o n th e Organizatio n fo rEconomi cCo operatio n an d Developmen t(OECD) , OEC D guideline s fo rTestin g o fChemicals , guidelin e No
. 211 : "Daphni a magna , Reproductio n Test" , Adopted : Octobe r2008
. I n addition , th e procedure s wer e designe d t o mee tth e tes t method s prescribe d b yth e followin g guideline s an d guidanc e document : •Commissio n Regulatio n (EC ) N o 440/200 8 o f3 0 Ma y2008 , Par t C : Method s fo rth e determinatio n o fecotoxicity , Publicatio n No
. L142 , C.20
. "Daphni a magn a Reproductio n Test"
. « IS O Internationa l Standar d 10706 : "Determinatio n o flon g ter m toxicit yo fsubstance s t o Daphni a magn a Strau s (Cladocera , Crustacea)" , 2000-03-30
. •Guidanc e documen t o n aquati ctoxicit ytestin g o fdifficul tsubstance s an d mixtures , OEC D serie s o n testin g an d assessmen t numbe r23 , Decembe r14 , 2000
. 5.4 . Storag e an d retentio n ofrecord s an d material s Record s an d material s pertainin g t o th e stud yincludin g protocol , ra wdata , specimen s (excep t specimen s requirin g refrigeratio n o rfreezing ) an d th e fina l report ar e retaine d i n th e NOTO Xarchive s fo ra perio d o fa tleas t1 0 year s afte rfinalizatio n o fth e report . Afte rthi s period , th e sponso rwil l b e contacte d t o determin e ho wth e record s an d material s shoul d b e handled
. NOTO Xwil l retai n informatio n concernin g decision s made
. Thos e specimen s requirin g refrigeratio n o rfreezin g wil l b e retaine d b yNOTO Xfo ra s lon g a s th e qualit yo fth e specimen s permit s evaluatio n bu tn o longe rthan three month s afte rfinalizatio n o fth e report .

- Pag e 7 -p . 13 4 Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substances (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
.

NOTO Xwil l retai n a tes t substanc e sampl e unti l th e expir ydate , bu t n o longe rtha n 1 0 year s afte r finalizatio n o fth e report . Afte rthi s perio d th e sampl e wil l b e destroyed
. 5.5 . Definition s Paren t animal s ar e thos e femal e daphnid s presen t a t th e start o fth e tes t an d ofwhic h th e reproductiv e outpu ti s th e objec t o fstudy
. Offsprin g ar e th e youn g daphnid s produce d b yth e paren t animal s i n th e cours e o fth e test . Th e Lowes t Observe d Effec t Concentratio n (LOEC ) i s th e lowes t teste d concentratio n at whic h th e substanc e i s observe d t o hav e a statisticall ysignifican t effec t o n reproductio n an d paren t mortalit y(a t p < 0.05 ) whe n compare d wit h th e control , withi n a state d exposur e period
. However , al l tes t concentration s abov e th e LOE C mus thav e a harmfu l effec t equa l t o o rgreate rthan thos e observe d a t th e LOEC
. Whe n thes e tw o condition s canno t b e satisfied , a ful l explanatio n mus t b e give n fo rho wth e LOE C (an d henc e th e NOEC ) ha s bee n selected
. Th e N o Observe d Effec t Concentratio n (NOEC ) i s th e tes tconcentratio n immediatel ybelo wth e LOEC , whic h whe n compare d wit h th e control , ha s n o statisticall ysignifican t effec t ( p < 0.05) , withi n a state d exposur e period , EC Xi s th e concentratio n o fth e tes tsubstanc e dissolve d i n wate rtha t result s i n a xpe rcen t reductio n i n reproductio n o fDaphni a magn a withi n a state d exposur e period
. Mortality
. A n anima l i s recorde d a s dea d whe n i t i s immobile , i.e
. whe n i t i s no t abl e t o swim , o ri f there i s n o observe d movemen t o fappendage s o rpostabdomen , withi n 1 5 second s afte rgentl e agitatio n o fth e tes tvessel
. 6 . MATERIAL S AN D METHOD S 6.1 . Tes t Substanc e 6.1.1 . Tes t substanc e informatio n Identificatio n p-(2,3~epoxypropoxy)-N,N-bis(2,3~epoxypropyl)anilin e Structur e O
. p~(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProject49698 9

O

Molecula rformul a Molecula rweigh t CA S Numbe r E C Numbe r Descriptio n Batc h Purity Tes t substanc e storag e Stabilit yunde rstorag e condition s Expirydat e C 1 6 H,gN0 4 277. 3 5026-74- 4 225-716- 2 Yello wliqui d AA0037340 0 

### 94.33 % I n refrigerato r(2-8°C ) i n th e dar k Stabl e 2 8 February201 3

- Pag e 8 -

m ip . 13 5 Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e 6.1.2 . Stud yspecifi c tes t substanc e informatio n NOTO XProject49698 9
Volatil e Stabilit yi n wate r Solubilit yi n wate r 6.2 . Tes t syste m Specie s
Sourc e Reaso n fo rselection Validit yo fbatc h

Characteristic s
6.3
. Breedin g Starto feac h batc h Maximu m ag e o fth e culture s Monitorin g o fth e individua l culture s Temperatur e o fmediu m Feedin g

Validit yo fth e culture s

Mediu m N o N o N o Daphni a magn a (Crustacea , Cladocera ) (Straus , 1820) , at leas tthir d generation , obtaine d b yacyclica l parthenogenesi s unde rspecifie d breedin g conditions
. In-hous e laboratorycultur e wit h a know n history
. Thi s syste m ha s bee n selecte d a s a n internationall y accepte d invertebrat e species
. Daphnid s originate d fro m a health ystock , 2 n dt o 5 , M brood , showin g n o sign s o fstres s suc h a s mortalit y>20% , presenc e o fmales , ephippi a o rdiscoloure d animal s an d ther e wa s n o dela yi n th e productio n o fth e firs tbrood
. T o initiat e th e test , youn g daphnid s < 2 4 hour s ol d wer e selected , fro m parenta l daphnid s greate rtha n tw o week s old
.
Wit h newbor n daphnids , i.e
. les s tha n 3 day s old , b yplacin g the m individuall yi n 5 0 m l M7-medium
. 4 week s Thre e time s a wee k

th e youn g ar e counte d an d th e parenta l daphnid s ar e transferre d t o ne wmedia
. 18-22 X Daily , a suspensio n o ffres h wate ralga e Historica l dat a o n th e reproductiv e capacit yar e base d o n th e number s o flivin g youn g counte d thre e time s a wee k i n th e individua l culture s an d teste d t o mee tth e validit ycriteri a fo r survival an d reproduction
. M7 , a s prescribe d b yDr
. Elendt-Schneide r (Elendt , B,-P. , 1990 : Seleniu m deficienc yi n Crustacea
. A n ultrastructura l approac h t o antenna l damag e i n Daphni a magn a Straus
. Protoplasm a 154 , 25-33)
. Compositio n o

fmediu m M 7 Adjuste d IS O medium 1 th e followin g chemical s (analytica l grade ) ar e dissolve d i n ta p wate rpurifie d b yRevers e Osmosi s (RO water , GEO N Waterbehandeling , Berkel-Enschot , Th e Netherlands ) Macr o salts
. CaCI ; ,2H; 0 211 5 mg/l

MgSOiJHi O 88. 8 mg/l NaHCO ; 46. 7 mg/l KC 1 4. 2 mg/ l

- Pag e 9 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d to Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p . 13 6

p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 9 Mediu m M7 : trace elements , macr o nutrients an d vitamin s ar e added to freshl yprepare d IS O mediu m to reac h th e followin g concentrations
. Trace elements
. B 0.12 5 rng/ l F e 0 0 5 mg/ l M n 0.02 5 mg/ l U , R b an d S r 0.012 5 mg/ l M o 0.006 3 mg/ l B r 0.002 5 mg/ l C u 0.001 6 mg/ l Z n 0.0063 mg/ l C o an d I 0.002 5 mg/ l S e 0,001 0 mg/ ! V 0.000 3 mg/ l Na.;EDTA,2H : 0 2. 5 mg/ l Macr o nutrient s Na ; ,Si0.j
. 9H;. 0 10. 0 mg/ l NaN0 3 0.2 7 mg/ l KH^PO- i 0,1 4 mg/ l K 2 HPO J 0.1 3 mg/ l Vitamins " Thiamin e 75. 0 pg/l

B t i 1.0 (jg/ l Bioti n 0.75 ug/ l Th e hardness ' 18

0 mg/l expresse d a

s CaCO ^ an d th e pH : 7, 7 t0.3
. 6.4 . Preparatio n o ftes t solution s Th e standar d tes t procedure s require d generatio n o ftes t solutions , whic h shoul d contai n completel y dissolve d tes tsubstanc e concentration s o rstabl e an d homogeneou s mixture s o rdispersions
. Th e testin g o fconcentration s tha t distur b th e tes tsyste m shoul d b e prevente d (e.g
. precipitat e o ra fil m o f th e tes tsubstanc e o n th e wate rsurface)
. Th e batc h o fp~(2,3~epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e teste d wa s a yello wliqui d wit h a purityo f94.33 % an d th e substanc e wa s not completel ysolubl e i n tes t mediu m at loadin g rate s o f 1. 0 mg/ l an d higher
. Preliminary

test Al l tes t solution s wit h loadin g rate s o f1. 0 mg/ l an d highe rwer e prepare d separatel yapplyin g tw o day s o fmagneti cstirrin g t o reac h maximu m solubilit yo fth e tes t substanc e i n th e tes t medium
. Th e resultin g aqueou s mixture s wer e lef tt o stabiliz e fo r1 hou rwher e afte rth e WaterAccommodate d Fraction s (WAFs ) wer e siphone d offan d use d fo rtesting
. On e lowe rtes t concentratio n wa s prepare d b y subsequen t dilutio n o fth e 1. 0 mg/ l WA F i n tes tmedium
. Th e fina l tes t solution s wer e al l clea ran d colourless
. Reproduction

test Preparatio n o ftes t solution s starte d wit h a loadin g rate'o f1 0 mg/ l applyin g tw o day s o fmagneti c stirrin g t o reac h maximu m solubilit yo fth e tes tsubstanc e i n th e tes t medium
. Th e resultin g aqueou s mixtur e wa s lef tt o stabiliz e fo r1-1. 5 hours wher e afte rth e Wate rAccommodate d Fractio n (WAF ) wa s siphone d of fan d use d a s th e highes ttes tconcentration
. Al l lowe rtes t concentration s wer e prepare d bysubsequen t dilution s o fth e WA F i n tes t medium
. 6.5 . Preliminar ytes t Apreliminarytes t wa s performe d t o defin e th e effect s o fp-(2,3-epoxypropoxy)~N,N-bis(2,3 - epoxypropyl)aniiin e o n mobilit yan d mortalit yo fDaphni a magn a t o b e teste d i n th e reproductio n test . Tes t procedur e an d condition s wer e simila rt o thos e applie d i n th e reproductio n tes t wit h th e fallowin g exceptions :

- Pag e 10 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)~N,N~bis{2,3-epoxypropyl)ani!in e NOTO XProjec t 49698 9

•Te n daphnid s pe rconcentratio n (tw o replicates , fiv e daphnid s pe rreplicate ) wer e expose d t o a control , WAF s prepare d a tloadin g rate s o f1.0 , 1 0 an d 10 0 mg/ l an d t o a dilutio n containin g 10 % o fth e WA F prepare d a t a loadin g rat e o f1. 0 mg/l ; •Tes t duratio n wa s 7 days ; •Tes t solution s wer e renewe d o n day s 1 , 3 an d 6 ; •N o bod ylengt h wa s recorde d a tth e en d o fth e test ; •Sample s fo ranalysi s wer e take n a tday s 0 , 1 an d 3 fro m th e freshl yprepare d solution s an d a t day s 1 , 3 an d 6 fro m th e ol d solutions ; •Sample s wer e take n fro m solution s containin g th e daphni a an d foo d (algae) , bu t als o fro m a n extr a vesse l incubate d unde rth e sam e conditions , bu twithou tdaphni a an d algae
. 6.6 . Reproductio n tes t 6.6.1 . Tes t concentration s p-(2,3-epoxypro poxy ) - N,N-bis(2,3-epoxypropyl)anilin e Control s 6.6.2 . Tes t procedur e an d condition s Tes t duratio n 2 1 day s Tes ttyp e Semi-stati c Frequencyo frenewa l Every4 8 hour s 1,0 , 3.2 , 10 , 3 2 an d 100 % o fa WA F prepare d a ta loadin g rat e o f1 0 mg/ I Tes t mediu m withou t tes t substanc e o rothe radditive s

Tes tvessel s Mediu m Volume : 6 0 m i (6x 0 3. 5 cm) , all-glas s covere d wit h a Perspe xplate
. M7 1 Experimenta l design
Ligh t
Feedin g Atth e start o fth e experimen t (nomina l da y0 ) 1 0 neonat e daphnids , les s tha n on e da yold , pe rgrou p wer e divide d ove rte n vessel s eac h containin g a minimu m o f5 0 m l tes t medium
. Th e contro l grou p consiste d o f2 0 daphnids
. 1 6 h photoperio d daily ; intensit ya t th e start : 624-70 8 lu x(8.8-1 0 uE. m 2 .s' 1 ) intensit ya t th e end : 612-71 0 lu x(8.8-9. 9 pE.rrfV 1 ) Daily , 0. 5 m l o fChlorell a pyrenoidos a suspension 2 containin g 0.40-0.5 4 x10 3cells/m l wa s adde d a s fee d fo r th e daphnids
. Thi s dail yratio n corresponde d t o 0. 2 m g C/Daphnia/day , whic h i s th e recommende d valu e fo rdail y feedin g pe rdaphni d i n th e reproductio n tes t accordin g t o th e OEC D Guidelin e 211
.

Tota l (dissolved ) organi ccarbo n o fth e M 7 mediu m use d i n th e reproductio n studywas measure d a t th e star t o fth e test . Measurements were performe d wit h a Shimadz u TOC-VCP H tota l organi ccarbo n analyze rcombine d wit h a Shimadz u ASI-Vauto sample r(Shimadz u Kyoto , Japan)
. Th e sampl e volum e was 4 0 m l an d th e numbe ro frepeats was a

t least 3
. 2 Alga e suspension s wer e mad e b yinoculatin g growt h mediu m wit h Chloreil a pyrenoidos a fro m a pur e culture
. Th e suspension s wer e continuousl yaerate d an d expose d t o ligh t (6000-1000 0 lux ; 60-12 0 uE, m .s' : ) i n a climate roo m a ta temperatur

e o f2 3 ±2" C fo r2-3 weeks
. Afte rthi s period , th e TO C concentratio n o fth e batc h alga l suspensio n was measure d befor e use a s fee d tn th e reproductio n study
. Th e exac tvolum e t o b

e added pe rtes

t vesse l was the n calculated
.

- Pag e 1 1 -Th e use ofthes e studies/documents fo rregulator yorregistratio n purposes , includin g bu t not limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyi)aniiin e 6.6.3 . Samplin g fo ranalysi s o ftes t concentration s NOTO XProject49698 9 Sample s fo ranalysi s wer e take n fro m al l tes tconcentration s an d th e control
. Th e metho d o fanalysi s i s describe d i n th e appende d Analytica l Repor t (APPENDI X7)
. Sampling : Frequency

Volum e Storag e A t th e beginnin g an d a t th e en d o ffou rinterval s o f4 8 hour s (nomina l day s 0 an d 2 , 6 an d 8 , 1 2 an d 14 , 1 8 an d 20 ) 2 m l Sample s wer e store d i n a freeze runti l analysis
. A t th e en d o fth e refreshmen tperiod , th e replicate s wer e poole d a t eac h concentratio n befor e sampling
. Additionally , reserv e sample s o f2 m l wer e take n fro m al l tes tsolution s fo rpossibl e analysis
. I fno t alread yused , thes e sample s wer e store d i n a freeze rfo ra maximu m ofthree months afte rdeliveryo f th e draf t report , pendin g o n th e decisio n ofth e sponso rfo radditiona l analysis
. 6.6.4 . Measurement s an d recording s Parenta l daphnid s Conditio n Everyworkda yan d upo n renewa l o n non-workdays , th e numbe ro fliving , immobil e an d dea d parenta l daphnid s wa s recorded
. Dea d daphnid s wer e remove d whe n observed
. Everyworkda yan d upo n renewa l o n non-workdays
. A t th e en d o fth e test . Whe n observed
. Everyworkda yan d upo n renewa l o n non-workdays , th e numbe ro fnewbor n youn g wa s counte d an d th e conditio n o f th e youn g recorded
. Thereafte rth e youn g wer e removed
. Whe n observed
. Whe n observed
. Presenc e o fegg s i n th e broo d pouc h Bod ylengt h Offsprin g Appearanc e firs tbroo d Newbor n daphnid s Presenc e o funhatche d egg s Incidenc e o fimmobilit y Test , mediu m Temperature , oxyge n an d p H
Hardnes s Ligh t 6.7 . Interpretatio n 6.7.1 . Dat a handlin g Th e value s fo rreproductio n observe d a t variou s concentration s o fth e tes t substanc e wer e expresse d a s mea n numbe ro flivin g youn g pe rparent . Th e mea n value s fo rreproductio n a t eac h concentratio n wer e compare d t o thos e recorde d i n th e contro l o n th e variou s day s o frecording
. Further , th e lengt h o f th e parenta l daphnid s (da y21 ) expose d t o th e tes tsubstanc e wer e compare d t o th e control
. Atth e start o fth e tes tan d jus t befor e an d afte reac h renewa l i n on e o fth e vessel s o feac h tes t grou p wit h survivin g daphnids
. Onc e a wee k

i n fres h an d ol d medi a fro m th e contro l an d th e highes ttes t concentratio n At th e start an d th e en d o fth e tes t

- Pag e 1 2 -p . 13 9 Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e Exposur e concentration s Th e exposur e concentration s wer e calculate d a s NOTO XProjec t49698 9

4 bein g th e mathematica l mean s o fth e concentration s o fp-(2,3-epoxypropoxy)-N,N~bis(2,3 - epoxypropyl)anilin e measure d i n th e sample s take n fro m th e freshl yprepare d solution s (Ct=o , C,= s , an d C^ s ) an d fro m th e 48-hou rol d solution s (C t =2 , C ( = a , Ct=i 4 an d C t =2n)
. I n cas e concentration s measure d wer e belo wth e lowes t calibratio n solution , th e fina l exposur e concentration(s ) wer e take n a s a facto ro f2 belo wth e lowes tcalibratio n solution
. Thi s procedur e i s base d o n th e OEC D "Guidanc e documen to n th e us e o fth e harmonise d syste m fo rth e classificatio n o f chemical s whic h ar e hazardou s fo rth e aquati cenvironment"
. Statistica l analysi s Parenta l surviva l o n da y2 1 wa s statisticall yteste d usin g Fisher' s exact tes t (TOXSTA TReleas e 3.5 , 1996 , D.D
. Gulley , A.M
. Boelter , H.L
. Bergman)
. Tota l reproductio n o n da y2 1 wa s teste d fo rnormalit y an d fo rhomogeneit yo fvarianc e an d statisticall yteste d usin g a n ANOV Ates t followe d b ya mea n compariso n tes t(Bonferron i t-Test , TOXSTA TReleas e 3.5 , 1996)
. Th e respectiv e threshol d level s o f effec t(LOEC ) an d th e respectiv e NOEC s wer e determine d o n basi s o fthes e statistics
. EC-value s Th e 21d-EC 5 Qfo rreproductio n wa s base d o n log-linea rregressio n analysi s o fth e percentage s o f reproductiv e reductio n versu s th e logarithm s ofth e correspondin g averag e exposur e concentration s of th e tes tsubstance
. 6.7.2 . Electroni c dat a captur e Observations/measurement s i n th e stud ywer e recorde d electronicall yusin g th e followin g programme(s) :

- REE S Centro n Environmenta l Monitorin g syste m versio n SQ L2. 0 (REE S Scientific , Trenton , NJ , USA )

- Shimadz u TOC-Contro l Vversio n 2.1 0 (Shimadzu , Kyoto , Japan) : carbo n analysis
. 6.7.3 . Acceptabilit yofth e tes t 1
. Th e mortalit yo fth e paren tanimal s (femal e Daphnia ) di d no texcee d 20 % at th e en d o fth e tes t (15%)
. 2
. Th e averag e cumulativ e numbe ro fyoun g pe rfemal e i n th e control s afte r2 1 day s wa s >6 0 (13 6 ±16)
. 6.8 . Lis t ofdeviation s 6,8.1 . Lis t o fprotoco l deviation s 1
. Th e conditio n o fparenta l daphnid s wa s no trecorde d o n th e firs t da yo fexposure . Evaluation : N o parenta l daphnid s die d durin g th e firs t refreshmen t period
. I n additio n n o advers e effec to n mobilit yo fth e daphnid s wer e observed
. 2
. Inadvertently , oxyge n concentration s an d p H wer e no t measure d i n th e ol d solution s o n th e secon d da yo fth e reproductio n test . Evaluation : N o parenta l daphnid s die d durin g th e firs t refreshmen t period
. I n additio n n o advers e effec to n mobilit yo fth e daphnid s wer e observed
. Th e stud yintegrit ywa s no tadversel yaffecte d b ythes e deviations
.

- Pag e 13 -Th e us e o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d to Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibite d p . 14 0
p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 9 6.8.2 . Lis t o fstandar d operatin g procedure s deviation s An ydeviation s fro m standar d operatin g procedure s wer e evaluate d an d file d i n th e stud yfile
. Ther e wer e n o deviation s fro m standar d operatin g procedure s tha taffecte d th e integrit yo fth e study
. 7 . RESULT S 7.1 . Preliminar ytes t Tabl e 1 contain s th e dail yobservation s o n mortalit yan d reproductiv e potencyo fsurvivin g parenta l daphnid s durin g th e 7-da ypreliminarytest . Al l daphnid s expose d t o th e highes t tes t concentratio n die d withi n 2 4 hour s o fexposure . N o (significant ) mortalit ywa s observe d at th e thre e lowes t tes t concentration s an d th e control
. Not e tha ta maximu m of20 % mortalit yi s acceptabl e fo rth e contro l an d therefor e no t considere d significant
. Almos t al l survivin g daphnid s i n th e contro l an d th e three lowes t tes t concentration s carrie d egg s i n thei rbroo d pouc h o n da y7 o fexposure . Overall , n o effect s o n surviva l o rtim e o feg g developmen twer e observe d a t th e thre e lowes t tes t concentration s whe n compare d t o th e control
. Analysi s o fth e sample s take n fro m th e WAF s prepare d at 1. 0 an d 1 0 mg/ l a t th e start o fth e three renewa l periods showe d tha t th e actua l tes tconcentration s range d betwee n 0.6 6 an d 0.9 7 mg/ l fo rth e 1. 0 mg/ l WA F an d betwee n 6. 6 an d 7. 3 mg/ l fo rth e 1 0 mg/ l WAF
. Thes e tes t concentration s remaine d (relatively ) stabl e durin g th e 2 4 an d 4 8 hou rrenewa l periods (79-90 % o finitial) , bu t decrease d t o 61 - 68 % o finitia l durin g th e 7 2 hou rrenewa l period
. Se e als o Tabl e 2 o fth e appende d Analytica l Report , Tes tcondition s wer e maintaine d withi n th e limit s prescribe d b yth e protocol
. Base d o n th e result s ofthi s preliminarytest , i t wa s decide d t o perfor m a reproductio n tes t wit h a WA F prepare d at a loadin g rat e o f1 0 mg/ l a s th e highes ttes tconcentration
. Tabl e 1 Mortalityan d reproductiv e potenc ydurin g th e preliminar ytes t Loadin g rate 1 Tes t substance 2 (mg/l ) Numbe r

Daphni a expose d Cumulativ e numbe ro fdea d parenta l daphnid s an d parent s

wit h egg s i n th e broo d pouc h betwee n parenthese s Loadin g rate 1 Tes t substance 2 (mg/l ) Numbe r

Daphni a expose d Da y1 Da y2 Da y3 Da y4 Da y5 Da y6 Da y7 contro l 1 0 0(0 ) 0(0 ) 0(0 ) 0(0 ) 0(0 ) 0(1 ) 0(10 ) 10 % o f1. 0 1 0 0(0 ) 0(0 ) 0(0 ) 0(0 ) 0{0 ) 0(1 ) 0(9 ) 1. 0 1 0 0(0 ) 0(0 ) 0(0 )

0 (0 ) 0(0 ) 

## o (b ) 1 0 0(0 ) 0(0 ) 1 (5 ) 1 (9 ) 0 (10 ) 1 0 1 0 0(0 ) 0(0 ) 0(0 )

0 (0 ) 0(0 ) 

## o (b ) 1 0 0(0 ) 0(0 ) 0(2 ) 1 (9 ) 0 (10 ) 10 0 1 0 10(0 ) 1 0 0(0 ) 

## o (b ) 1 0 1 0 1 0 1 0 1 0 I WA F prepare d atth e give n loadin g rat e " Th e test substance i s p-(2,3-epoxypropoxy)-N,N-bis(2 l3-epoxypropyl)anilin e 7.2 . Reproductio n tes t 7.2.1 . Measure d concentration s Th e result s o fanalysi s o fth e sample s take n durin g th e reproductio n tes t ar e describe d i n Tabl e 3 o f th e appende d Analytica l Report
. Analysi s o fth e sampl e take n fro m th e WA F prepare d a t 1 0 mg/ l a t th e start o ffou rrenewa l periods showe d tha tth e actua l tes t concentratio n range d betwee n 4. 2 an d 6, 6 mg/l
. Tes t concentration s slightl ydecrease d durin g th e 4 8 hou rrenewa l periods (t o 70-82 % ofinitial)
. Tes t concentration s containin g 1 0 an d 32 % o fth e WA F prove d t o hav e bee n prepare d properl yan d als o slightl ydecrease d durin g th e 4 8 hou rrenewa l periods (t o 63-79 % o finitial
. Th e tes t concentratio n containin g 1.0 % o fth e WA F wa s belo wth e lowes t calibratio n standar d a tal l times , whil e th e tes t concentratio n containin g 3.2 % o fth e WA F varie d betwee n bein g belo wo rjus tabov e th e lowes t calibratio n solution
. Give n thes e result s effec t parameter s wer e base d o n th e averag e exposur e concentrations : 0.040 , 0,096 , 0.42 , 1. 7 an d 4, 8 mg/l
.

- Pag e 1 4 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy}~N,N-bis(2,3-epoxypropyl)8nilin e NOTO XProjec t 49698 9 7.2.2
. Conditio n o fparenta l daphnid s Thre e ou t o fth e twent yparenta l daphnid s i n th e contro l die d durin g th e tes t perio d (se e Tabl e 2)
. Hence , parenta l mortalit ydi d no t excee d 20 % i n th e control
. Mortalit yi n al l p~(2,3-epoxypropoxy)-N,N bis(2,3-epoxypropyl)anilin e concentration s teste d wa s lowe ro rslightl yhighe rtha n i n th e contro l bu t no tstatisticall ysignifican t(Fisher' s exac ttest , a=0.05 , se e APPENDI X3)
. I n th e contro l group , th e presenc e o fegg s i n th e broo d pouc h wa s recorde d fo rth e firs t tim e o n da y4 - 6 an d th e firs t broo d appeare d betwee n da y7 an d da y9
. Th e firs t recordin g o fth e presenc e o fegg s i n th e broo d pouc h wa s simila rt o th e contro l i n treate d solution s u p t o an d includin g 4. 8 mg/l
. Hence , n o dela yi n appearanc e o fth e firs tbroo d wa s observe d i n th e treatment s whe n compare d t o th e control
. Tabl e 2 Cumulativ e mortalit yo fparenta l daphnid s durin g th e 21-da yexposur e period
. Averag e cone . Tes tsubstance 1 (mg/l ) Numbe r

expose d Cumulativ e numbe ro fdea d parenta l daphnid s o n day : Mortalit y 

### % Averag e cone . Tes tsubstance 1 (mg/l ) Numbe r

expose d 2 4 7 1 0 1 1 1 2 1 4 1 6 1 8 1 9 2 0 2 1 Mortalit y 

### % contro l 2 0 0 0 0 0 0 0 1 1 2 2 3 3 1 5 0,04 0 1 0 0 0 0 0 0 0 0 0 0 ' 1 1 1 1 0 0.09 6 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.4 2 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 0 1. 7 1 0 0 0 0 2 2 2 2 2 2 3 3 3 3 0 4. 8 1 0 0 0 0 1 1 1 1 1 1 1 1 1 1 0 1 Th e testsubstance i s p-(2,3-epoxypropDxy)-N.N-bis(2,3-epoxypropyl)anilin e 7.2.3
. Reproductio n Record s o fcumulativ e mea n numbe ro flivin g offsprin g produce d pe rparen t durin g th e tes t perio d ar e summarise d i n Figur e 1 wit h a dat a table
. Th e dat a recorde d pe rparen t ar e presente d i n APPENDI X1
. Th e averag e cumulativ e numbe ro fyoun g pe rfemal e i n th e contro l grou p afte r2 1 day s wa s 13 6 wit h a coefficien to fvariatio n o f1 1 %
. Th e reproductio n curve s recorde d a t 0.040 , 0.09 6 an d 0.4 2 mg/ l closel y followe d th e curv e ofth e control
. Th e curv e recorde d a t1. 7 mg/ l staye d behin d fro m th e start an d daphnid s expose d t o 4. 8 mg/ l di d no treproduc e atall
. Ther e wer e som e recording s o fimmobil e youn g and/o raborte d egg s i n th e contro l an d th e thre e lowes ttes t concentrations , whil e significan t amount s o faborte d egg s wer e recorde d a tth e tw o highes t tes tconcentration s throughou t th e 21-da ytes t period
. Statistica l analysi s showe d tha tth e reproductio n o fth e daphnid s wa s significantlyreduce d a t 1. 7 mg/ l an d highe r(Bonferron i t-Test , a=Q.05 , se e APPENDI X4)
.

- Pag e 1 5 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d to Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substances (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p . 14 2
p-(2,3-epoxypropoxy)-N,N~bis(2,3-epoxypropyt)anilm e NOTO XProjec t49698 9

16 0

c o £ 3 3 C C ro

Day 7 Day8,Dav1 C Da y1 1 'Da y1 2 Dayl 3 Day1 4 Day1 5 Day16jDay! 8 Da y1 3 | Da y2 0 Day2 1 -•-Contro l 1. 3 4. 1 25. 5 29. 2 49, 1 5 1 3 Sfe. 3 3 2 5 84~2 ' 10 9 6 118. 8 126, 3 13 6 2 —13—0.0-1 0 mg/ l 3. 3 12, 0 2S. 9 33. 2 43. 3 S3. 8 S2. 1 76. 1 82. 1 10Q. 4 110. 3 125, 0 131. 6 - H - 0.03 6 mg/ l 4, 4 3. 1 23. 9 28. 3 44. 4 56. 7 60. 4 8 1 3 93. 0 103. 1 117. 1 124. 6 137, 9 -v- 0 4 2 mg/ l 8. 1 9, 5 31, 1 34. 2 47. 1 61 A 54, 9 3 5 1 ' 109. 1 124. 9 123, 3 13 5 2 --•-1. 7 mg/

l 1. 3 1 3 "ia, a 20. 4 32. 4 " 3 3 4 " 35. 4 46 , S 48. 3 " 62. 0 " " S2A SA 6 56,6 " -»! •-4. 9 rmj/ l 0, 0 0. 0 0, 0 0, 0 0. 0 0 0 0. 0 0. 0 0 0 0. 0 0. 0 0 0 o'. o Days ofexposur e Figur e 1 Cumulativ e mea n numbe roflivin g youn g pe rparen t a t th e variou s averag e

concentration s ofp-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e durin g th e 2 1 -da ytes t perio d 7.2.4 . Bod ylengt h Th e averag e bod ylength s ofth e survivin g daphnid s pe rconcentratio n measure d a t th e en d o fth e tes t an d th e relativ e reductio n o fbod ylength s compare d t o th e contro l ar e summarise d i n Tabl e 3
. Se e APPENDI X2 fo rth e individua l bod ylength s o fth e survivin g parenta l daphnids
. Statistica l analysi s o f th e dat a wa s no t neede d a s th e % reductio n recorde d wa s no tbiologicall ysignifican t (<10%)
. Tabl e 3 Averag e bod ylength s (mm ) ofparenta l daphnid s a t th e en d ofth e tes t Concentratio n 4,4'-DD S (mg/l ) Averag e

lengt h (mm ) S D % Reductio n contro l 4.3 2 (n=17 ) 0.1 2 0.04 0 4.2 9 (n=9 ) 0,1 1 1 0.09 6 4.4 0 (n-10 ) 0,1 0 - 2 0.4 2 4.3 9 (n=9 ) 0.1 0 - 2 1. 7 4.3 1 (n=7 ) 0.1 2 0 4. 8 4.1 8 (n=9 ) 0.1 4 4 Th e tes t substance i s p-(2,.3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilm e n = numbe ro

fdaphnid s survivin g th e test perio d SD= standar

d deviatio n

- Pag e 16 -Th e us e ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d to Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49898 9 7.2.5 . Determinatio n ofeffec t concentration s Tabl e 4 show s th e effec tparameter s base d o n mea n measure d concentrations , se e als o APPENDI X5
. Tabl e 4 Effec t parameter s Paramete r Concentratio n 95% - Tes t substance 1 confidenc e (mg/l ) interva l 21-da yLCso (parenta l mortality ) >4. 8 21-da yNOE C fo rparenta l mortality 3 4. 3 21-day EC50 for reproduction 2 1. 4 0.9 1 -2. 2 21-da yLOE C fo rreproduction 4 1. 7 21-da yNOE C fo rreproduction 4 0.4 2 21-da yLOE C fo rparenta l bod ylengt h >4. 8 21-da yNOE C fo rparenta l bodylengt h 4. 8 th e test substance i s p-(2,3~epoxypropaxy]-N.N-bi5(2, ^ 2Log-linea rregressio n analysi s 3Fisher' s exac

t test(Toxstat) 4Bonferron i t -Test (Toxstat) 7.2.6
. Experimenta l condition s Th e p H value s recorde d durin g th e tes tar e presente d i n APPENDI X6 , Tabl e 12
. Th e p H remaine d withi n th e rang e o f7. 5 t o 8. 4 throughou tth e tes t an d thu s wa s maintaine d withi n th e limit s prescribe d byth e protoco l (6.0-9.0 , constan twithi n 1. 5 units)
. Th e dissolve d oxyge n concentration s measure d durin g th e stud yar e presente d i n APPENDI X6 , Tabl e 13
. Th e oxyge n concentratio n i n al l tes t solution s remaine d withi n th e rang e o f8. 2 t o 9. 7 mg/ l durin g th e exposur e perio d an d thu s complie d t o th e requirement s a s lai d dow n i n th e protoco l (> 3 mg/l)
. Temperature s recorde d i n th e tes t medi a ar e show n i n APPENDI X6 tTabl e 14
. Th e temperature s i n th e tes tmedi a varie d betwee n 19. 4 an d 20.5°C
. Th e temperatur e continuousl ymeasure d i n a temperatur e contro l vesse l varie d betwee n 19. 3 an d 20.3° C durin g th e test , an d complie d wit h th e requirement s a s lai d dow n i n th e protoco l (18-22°C , constan t withi n 2°C) , Th e result s o fth e measurement s o ftota l hardnes s ar e presente d i n APPENDI X6 , Tabl e 15
. Tota l hardnes s varie d betwee n 17 9 an d 21 4 m g calciu m carbonat e pe rlitre , an d thu s complie d t o th e requirement s a s lai d dow n i n th e protoco l (>14 0 m g CaG0 3pe rliter)
. Th e tota l dissolve d organi ccarbo n conten t o fth e M 7 mediu m wa s 0.4 2 mg/l
. Thi s complie d wit h th e requirement s a s lai d dow n i n th e guideline s (TO C < 2 mg/l)
. 8 . CONCLUSIO N Unde rth e condition s o fth e presen tstud yp-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e di d no t affec t reproductio n o fDaphni a magn a a ta n averag e concentratio n o f0.4 2 mg/ l afte r2 1 day s o f exposur e (NOEC)
. Exposur e t o averag e concentration s o f1, 7 mg/ l an d highe rinduce d significan t inhibition o fth e reproductiv e capacit yo fth e parenta l daphnid s (LOEC)
. Th e 2 1 d-EC S Qfo rreproductio n wa s calculate d t o b e 1, 4 mg/ l wit h a 95 % confidenc e interva l rangin g fro m 0.9 1 t o 2. 2 mg/l
.

- Pag e 1 7 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substances (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p . 14 4
p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO X Projec t 49698 9 APPENDI X1 CUMULATIV E SCORE S O F LIVIN G NEWBOR N DAPHNID S PE R PAREN T Tabl e 5 Cumulativ e score s o flivin g newbor n pe rparen t i n th e contro l Paren t Da

y7 Da y8 _Day_10

_ Day1 1 Da

y1 2 Day1 3 Day1 4 Da y1 5 Da y1 6 Day1 8 Da

y1 9 Day2 0 Day2 1 P 1 0 2 7 3 1 3 1 3 4 (2i ) + P 2 0 0 3 1 3 1 5 6 5 6 5 6 9 0 9 0 9 0 12 6 12 6 12 6 P 3 2 5 2 9 

### ~JzL 6 3 6 3 8 7 8 7 8 7 11 9 11 9 16 1 16 1 16 1 P 4 0 0 2 7 2 7 4 8 4 8 4 8 8 4 8 4 10 9 12 2 12 2 12 2 P5
- 0 0 2 0 2 0 4 5 4 5 4 5 3 1 3 1 11 7 11 7 11 7 14 0 P 6 0 0 2 9 2 9 5 6 5 6 5 6 8 9 8 9 12 9 12 9 12 9 12 9 P 7 0 0 3 3 3 3 5 2 5 2 5 2 8 4 8 4 + P 8 0 0 2 6 2 6 6 6 6 6 6 6 10 1 10 1 14 1 14 1 14 1 16 8 P 9 0 1 3 1 3 3 9 3 9 3 9 7 2 7 2 7 2 10 0 10 0 13 5 13 5 P1 0 0 0 1 9 1 9 4 1 4 1 4 1 6 3 6 8 8 9 8 9 8 9 10 5 P1 1 0 1 8 1 8 4 1 4 1 4 1 7 0 7 0 7 0 9 9 9 9 13 7 13 7 P1 2 0 0 2 2 2 2 4 8 4 8 4 8 8 2 8 2 8 2 11 7 11 7 11 7 P1 3 0 0 27 2 7 5 2 52 5 2 8 8 8 8 12 6 12 6 12 6 15 2 P1 4 0 0 25 2 5 5 5 5 5 5 5 9 1 (1i ) 9 1 12 9 12 9 12 9 12 9 P1 5 0 0 2 9 2 9 6 2 6 2 6 2 9 3 9 3 9 3 13 3 13 3 13 3 P1 6 0 0 2 0 2 0 5 0 5 0



## _ _5 

## ? 85 

## J1' L 8 5 12 1 12 1 12 1 14 7 (1i ) P1 7 0 0 2 4 2 4 5 3 5 3 5 3 3 5 (1i ) 3 5 12 5 12 5 12 5 14 4 P1 8 0 1 9 3 0 3 0 3 0 5 8 6 3 6 3 6 3 9 2 9 2 13 6 13 6 P1 9 0 0 2 2 2 2 4 5 4 5 4 5 7 7 (1i ) 7 7 10 9 10 9 + P2 0 0 0 2 5 2 5 4 8 4 8 4 8 7 7 7 7 10 3 10 3 10 3 13 4 Mean 1. 3 4. 1 25. 5 29. 2 49, 1 51. 8 56. 3 82. 5 84, 2 109. 6 118. 8 126, 3 13 6 2 S D 5. 6 8. 5 10, 3 9. 9 9. 7 11, 4 11, 4 9. 5 12. 6 17. 0 18. 1 15, 7 15, 5 n 2 0 2 0 2 0 2 0 2 0 2 0 1 9 1 9 1 9 1 8 1 8 1 7 1 7 + Parenta l daphni d was foun d dead ( ) Numbe ro

fimmobilise d youn g (i ) o n th e respectiv e da y(no t cumulative ) Tabl e 6 Cumulativ e score s oflivin g newborn pe rparen t a t 0.04 0 mg/ l Paren t Da

y7 Day 8 Da

y1 0 Da y1 1 Da y1 2 Da y1 3 Da y1 4 Day1 5 Da

y1 6 Da y1 8 Da y1 9 Da y2 0 Da y2 1 P1 _ 1 8 1 8 4 7 4 7 4 7 7 8 7 3 7 8 10 6 10 6 10 6 13 8 13 8 P 2 0 0 1 2 1 2 3 9 3 9 3 9 7 8 7 8 10 6 10 6 10 7 10 7 P 3 0 2 3 2 3 5 0 5 0 5 0 7 8 7 8 7 8 9 7 9 7 13 0 13 0 P 4 1 5 1 5 2 3 2 3 2 3 6 3 6 3 6 3 9 5 9 5 12 4 12 4 12 4 P 5 0 0 2 5 2 5 5 0 5 0 5 0 8 5 8 5 11 8 11 8 11 8 11 8 P 6 0 2 4 2 4 4 6 4 6 4 6 6 8 6 8 6 8 10 0 P 7 0 1 6 3 9 3 9 3 S 6 8 7 2 7 2 7 2 10 2 10 2 13 9 13 9 P 8 0 0 2 8 2 8 5 3 5 3 5 3 8 5 (1i l 8 5 12 4 12 4 12 4 15 4 P 9 0 2 4 2 4 4 8 4 8 4 8 7 7 7 7 7 7 10 8 10 8 13 7 13 7 P1 0 0 0 1 4 1 4 4 3 4 3 4 3 7 7 7 7 10 3 10 8 10 8 13 7 Mean 3. 3 12, 0 25. 9 33. 2 43. 8 53, 8 62, 1 76. 1 82. 1 106. 4 110, 3 125. 0 131. 6 S D 7. 0 10, 8 10. 4 14. 6 8. 7 12, 2 14. 9 6. 9 11. 3 .3-0 9. 5 12. 2 13. 7 n 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 9 9 9 + Parenta l daphni d was foun d dead ( ) Numbe ro

fimmobilise d youn g (i ) o n th e respectiv e da y(no t cumulative )

- Pag e 18 -

I ITh e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)ani!in e NOTO X Projec t 49698 9 APPENDI X1 CUMULATIV E SCORE S O F LIVIN G NEWBOR N DAPHNID S PE R PAREN T-

continue d - Tabl e 7 Cumulativ e score s oflivin g newbor n pe rparen t a t 0.09 8 mg/ l Paren t Da

y7 Day S Day1 0 Da y1 1 Day1 2 Day1 3 Da y1 4 Day1 5 Da

y1 6 Da y1 8 Da y1 9 Day2 0 Day2 1 P 1 0 0 2 8 2 8 6 5 6 5 6 5 10 3 10 3 13 7 13 7 13 7 15 4 P 2 0 0 2 5 2 5 4 9 4 9 5 0 •8 5 8 5 8 5 11 3 11 3 11 3 P 3 0 1 1 3 6 3 6 3 6 7 3 7 3 7 3 10 1 10 1 10 1 14 3 14 3 P 4 1 6 1 6 3 7 3 7 3 7 6 2 6 2 6 2 8 3 8 8 8 8 12 1 12 1 P 5 0 0 1 9 1 9 4 5 4 5 4 5 9 3 9 3 11 9 11 9 11 9 14 7 P 6



## __? o 2 0 4 9 4 9 4 9 8 2 8 2 3 2 11 4 11 5 14 9 (1e ) 14 9 14 9 P 7 8 8 

### • • 2 6 2 6 2 6 5 4 5 4 5 4 8 4 8 4 11 2 11 2 11 2 P 8 0 2 6 2 6 5 6 4 3 4 3 7 9 7 9 7 9 11 6 11 6 11 6 14 8 P 9 0 0 2 0 2 0 4 5 4 5 4 5 9 0 9 1 12 4 12 4 12 4 15 2 Pt O 0 0 23 _ 2 3 4 9 4 9 4 9 9 2 9 2 11 2 11 2 11 2 14 0 Mean 4. 4 8. 1 28. 9 28. 9 44. 4 56. 7 60. 4 81. 3 93. 0 108. 1 117. 1 12 4 6 13 7 9 S D 7. 6 9. 8 9. 2 9. 2 10. 3 13. 3 13. 9 14. 9 10. 4 18, 0 17. 1 13. 6 16, 2 n 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 1 0 ( ) Numbe r o f unhatche d egg s (e ) on the respective da y(no t cumulative ) 

### Tabl e 8 Cumulativ e score s o

f livin gnewbor nper parent at 0.4

2 mg/ l Paren t Day7 Da

y8 Da y1 0 Da y 1 1 Da y1 2 Da y1 3 Day1 4 Da

y1 5 Da y1 6 Da y1 8 Da y1 9 Day2 0 Da

y2 1 P 1 0 0 2 9 + P 2 0 0 2 4 2 4 5 6 5 6 5 6 9 1 9 1 12 9 12 9 12 9 15 9 P 3 0 0 2 3 2 3 4 9 4 9 4 9 8 8 8 8 8 3 11 1 11 1 11 1 P 4 0 0 2 6 2 6 5 7 5 7 5 7 9 9 9 9 13 5 13 5 13 5 13 5 P 5 1 7 1 7 4 0 4 0 4 0 6 7 6 7 6 7 9 5 9 5 12 0 12 4 12 4 P 6 1 8 1 8 3 9 3 9 3 9 72(11 ) 7 2 7 2 10 0 10 0 13 2 (2e ) 13 2 13 2 P T 0 0 2 3 2 3 5 0 5 0 5 0 8 2 8 2 10 5 10 5 10 5 13 7 P 8 2 1 2 1 4 7 4 7 4 7 3 0 

### a o 8 0 12 0 12 0 14 9 14 9 14 9 P 9 0 1 4 1 4 4 0 4 0 4 0 7 1 7 1 7 1 10 0 10 0 12 7 12 7 P1 0 2 5 2 5 4 6 4 6 4 6 8 2 8 2 (1e j 11 0 11 0 14 3 Mean 8. 1 9. 5 3 1 1 34. 2 47. 1 61. 4 64. 9 81. 3 95. 1 109. 1 124. 9 128. 3 135. 2 S D 10. 7 10. 4 11. 2 10. 1 6. 7 14. 6 12. 4 10. 3 14. 6 15. 9 17, 0 14. 0 14. 2 

### n 1 0 1 0 1 0 9 9 9 9 9 9 9 9 9 9 + Parenta l daphni d was foun d dead ( ) Numbe ro

fimmobilise d youn g (i ) o runhatche d egg s (e) o n th e respective da y(notcumulative )

- Pag e 19 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d to Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p . 14 6
p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyt)anilin e NOTO XProjec t 49698 9
APPENDI X1 CUMULATIV E SCORE S O F LIVIN G NEWBOR N DAPHNID S PE R PAREN T-

continue d ~ Tabl e 9 Cumulativ e score s o flivin g newbor n pe rparen t a t 1. 7 mg/ l Paren t Da

y7 Da y8 Da y1 0 Day1 1 Da

y1 2 Da y1 3 Da y1 4 Da y1 5 Day1 6 Day1 8 Day1 9 Da y2 0 Da

y2 1 P 1 0 0 + P 2 0 0 P 3 0 0 0 1 3 1 3 1 3 2 9 2 9 2 9 3 6 3 6 62( 1 e ) 6 2 P 4 1 3 1 3 1 5 1 5 1 5 2 2 (2 0 2 2 2 2 3 4 (4e ) 3 4 3 4 3 7 (10i , 9e ) 3 7 P 5 0 0 2 6 2 6 4 8 4 9 4 9 6 8 6 8 6 3 6 8 (10e ) 6 8 (1i ) 6 8 P 6 0 0 2 7 2 7 4 3 4 3 4 8 7 0 7 0 7 8 (7e ) 7 8 7 8 8 5 (2i , 8e ) P 7 0 0 2 3 2 3 2 7 2 7 2 7 4 9 4 9 5 1 (11e ) 5 1 5 1 5 1 (1e ) P S 0 0 2 3 2 8 4 0 4 0 4 0 4 8 (9i ) 4 8 5 8 5 8 (7i , 3e ) 5 3 5 8 P 9 0 0 1 4 1 4 3 1 3 1 3 1 3 9 (6i , 4e 1 3 9 4 2 (13e ) 4 2 4 2 4 9 (3t , 7e ) P1 0 0 0 1 7 1 7 3 7 3 7 3 7 4 9 4 9 4 9 (10e ) + Mean 1. 3 1. 3 18 6 20. 4 32. 4 33. 4 35. 4 46. 8 48. 3 52. 0 52. 4 56. 6 53. 6 S D 4. 1 4, 1 9. 4 6. 3 13, 5 12. 6 9. 8 16. 9 14. 3 15. 4 16. 6 14. 4 15. 3 n 1 0 1 0 8 

## a 8 8 8 8 8 7 7 7 + Parenta l daphni d was foun d dead ( ) Numbe ro

fimmobilise d youn g (i ) o runhatche d eggs (e) o n th e respective da y(no t cumulative )
Tabl e 1 0 Cumulativ e score s oflivin g newbor n pe rparen t a t 4. 8 mg/ l Paren t Da

y7 Da y3 Da y1 0 Day1 1 Da

y1 2 Da y1 3 Day1 4 Da

y1 5 Day1 6 Da

y1 8 Da y1 9 Da y2 0 Day2 1 P 1 0 0 0 0( 1 Se ) 0(3e ) 0(3e ) 0 (11 , 2 1 e ) 0 0 0 (23e ) 0 0 (11 , 27* ) 0 P 2 0 0 0 0 0 (9e ) 0 (18e ) 0 0 0 (34e ) 0 0 0 (111 , 14e ) 0 P 3 0 0 0 0 (17e ) 0 0 0 (I9e ) 0 0 0 (24e ) 0{2e ) 0 (2i , 13e ) 0 P 4 0 0 0 0 0(16e ) 0 0 (4e ) 0 (45e ) 0 0 (31e ) 0(7e ) 0(1i ) 0 P 5 0 0 0 0 0 (22e ) 0 0 0 (24e ) 0 0 (34e ) 0 0|1e ) 0 (11e ) P 6 0 (17e ) 0 0 0 0(4e ) 0 (23e ) 0 0 0 (43e ) 0 0(8e ) 0 (5i , 16e ) 0 P 7 0 0 0 0 0 0 (2i , 25e ) 0 0 0 (23e ) 0( 1 Oe ) 0 (32e ) 0 0 P 8 0 0 0 0 (22e ) 0(2e ) 0(2e ) 0(17e ) 0 0 0 (33e ) 0(16 ) 0 (2i , 23e ) 0 P 9 0 0 0 0 (12e ) 0 0(1e ) 0 (1i , I6e ) 0 0 0 (28e ) 0 0 (28e ) 0(1e ) P1 0 0 (10e ) 0 + Mean 0. 0 0. 0 0. 0 0. 0 0, 0 0. 0 0. 0 0. 0 0. 0 0. 0 0. 0 0, 0 0, 0 S D 0. 0 0. 0 0. 0 0. 0 0, 0 0, 0 0. 0 0. 0 0. 0 0. 0 0. 0 0. 0 0. 0 n 1 0 1 0 9 9 9 9 9 9 9 9 9 9 9 + Parenta l daphni d was foun d dead ( ) Numbe ro

fimniobilise d youn g (

0 o runhatche d eggs (e) o n th e respective da y(not cumulative )

- Pag e 2 0 -p . 14 7 Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d to Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)~N,N-bis(2,3-epoxypropyl)anilin e NOTO X Projec t 49698 9 APPENDI X2 PARENTA L BOD YLENGT H
Tabl e 1 1 Individua l value s fo rbod ylengt h o fth e survivin g parenta l daphnid s
Paren t Contro l

(mm ] 0.04 0 mg/ l

(mm ) 0.09 6 mg/ l

(mm ) 0,4 2 mg/ l

(mm ) 1. 7 mg/ l

(mm ) + 4. 8 mg/ l

(mm ) P 1 + 4.4 5 4.3 0 + 1. 7 mg/ l (mm ) + 4.3 8 92 4.4 5 4,3 0 4.2 2 4.3 8 + 4.1 4 P 3 4.3 8 4.3 8 4.4 5 4,2 2 4.0 6 4.3 0 P 4 4.1 4 4.2 2 4,4 5 4.4 5 4 3 8 4.1 4 P5 4.2 2 4.1 4 4.4 5 4.4 5 4.4 5 3.9 1 P 6 4,4 5 + 4.5 3 4.4 5 4.3 8 4.2 2 P 7 + 4.1 4 4.3 8 4.3 0 4.3 0 4.0 6 P 8 4 3 0 4,3 0 4.4 5 4.3 0 4.3 0 4.2 2 P 9 4 , O S 4.3 0 4.3 0 4.4 5 4.3 0 4.0 6 P1 0 4.4 5 4.3 8 4.4 5 4.5 3 + + P1 1 4.4 5 P1 2 4.2 2 P1 3 4.2 2 P1 4 4,3 0 P1 5 4.3 8 P1 6 4.3 0 P1 7 4.4 5 P1 8 4.3 0 P1 9 + P2 0 4,3 8 Mea n 4.3 2 4.2 9 4,4 0 4.3 9 4.3 1 4.1 6 S D 0.1 2 0.11 * 0.1 0 0.1 0 0.1 2 0.1 4 n 1 7 9 1 0 9 7 9 + Parenta l daphni d was foun d dead

- Pag e 2 1 -Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substances (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p . 14 8

p-{2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)ani!in e NOTO X Projec t 49698 9 APPENDI X3 STATISTICS : PARENTA L MORTALIT Y
Summary

of Fisher' 3 Exac t

NUMBER NUMBE R 3I G GROU P I DEN

T ] F. E CATIO N EXPOSE D DEA D 0.0 5 CONTROL 2 0 ; ? 1 0.04 0 1 0 1 2 0.09b 1 0 

### o

0.4 2 1 0 1 4 i . 7 3 0 3 S 4. 8 i ' l 1

I'l l - Pag e 2 2 -

iI 1Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o f Chemica l Substance s (REACH) , withou t th e expres s writte n permissio n o f Huntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibite d 

### p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 9 APPENDI X4 STATISTICS : REPRODUCTIO N Chi-Squar e Tes t fo r Normalit y Actua l an d Expecte d Firequenc J e s INTERVA L < - i . 5 -1. 5 t o -0. 5 0. 5 t o 0. 5 >0. 5 t o 1. 5 >1. 5 EXPECTE D 4.0*37 0 1-5. 7 62 0 2 3 . 302 0 1-1.762 0 4.037 0 OBSERVE D 5 1 0 2 9 1 2 5 Chi-Squar e =

- 3.354 2 {p-valu e - 0.4261 ) Critica l Chi-Squar e - 13.27 7 £alph a - 0.0 1 , d f - 4 )

- 9.43 d (alph a - - 0.0 5 , d f - 4 ) Dat a PASS normalit ytes t (alph a - 0.01) . Continue

- analysis . Leven e s Tea t fa r Homogeneit y o f Varianc e AN'OV ATabl e SOURCE D F M S F 954.939 0 190.997 8 2.013 5 WH " hi . n [Error ) '3 5 521.7 . 3 3 8 S 9-1. 8 60 7 Tota l 6 0 617 2 . 327 9 (p-valu e - 0.0910 ) C r i r. 3 ca l F --

- i . 370 0 (alph a 0.01 , d f = - 5,55 )
= 2.3&2 8 (alph a - 0.05 , d f - 5,55 ) S. i nu e F <

- Critica l F FAI L TO PEJEC T H o A l l equa l (alph a - • 001. ) ANOV ATabl e SOURCE : D F S 3 M S i

r Betwee n W J t hLn (Srsoi ) 5 5 5 l I :.60o5.202 9

10739.862 7 ,U2 1 3 19 5 0 4 O o 252 0 159.860 2
Tota l S O 166804.065 6 (p-valu e - 0.0000 ) Critica l F~ 3 . 3 700 (alph a= 0.01 , d f - 5,55 )

- 2.3S2 9 (alph a= 0.05 , d £~ 5,55 ) Sinc e F> Critica l FREJECTHo : Al l equa l (.alph a- 0.05 ) Bot r erron i L-Tes t TABL E i O F 2 Ho : Control
- Tre a inti'-in t TRANSFORME D MEA NCALCULATE D I N GROU P IDENTIFICATIO N MEA N ORIGINA L UNIT S t STAT 0,0 5 1 contco l .1.36.176 5 13c.1 7 6 5 2 0.04 0 131.iy.5 6 U l . 555 6 0 802 2 3 0.09 6 1.37.T-00 0 137.500 0 0 309 5 4 0 . 4 2 135.222 2 135.222 2 0 165 7 1 . 7 S&.571 4 bS. b H 4 1 2 366 9 * S 4. 8 0.000 0 0.000 0 2 3 *4Q 9 * Boi-iCetri.o ni t cri t ica l va l u e = • 2 , 668 2 [ 2 Tailed , alph a ~ 0.05 , d f * 5,55 ) Bonferron i t-Tes t TABL E 2 O F 2 Ho : Contro l -Tre a tmen t NU NO F 3 DIF F ? , O F IHFFKRENCH : GROU P I DEN T 1FICATIO H REP S [ I No : . I' G •NIT S CONTRO L FRO M CONTRO L 1 contro l 1 7 2 0.O4 0 9 1 5 11. 3 4.620 9 3 0. 0 9 6 .1 0 1 4 858 5 10. 9 1.723 5 4 0,4 2 9 1 5 3 6P 5 11. 3 0.95 4 2 5 I . 7 7 1 " 7 4 3 7 12.3 7"' . SO5 0 6 4. 8 Q 1 5 .36SS 11. 3 L36.176 5



### - Pag e 2 3 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substances (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. P-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniliri e NOTO X Projec t 49698 9 APPENDI X5 EC-VALUE S Concentratio n X Y (mg/l ) Lo g cone , (mg/l ) Reductio n (% ) 0.04 0 -1. 3 0.04 0 21. 4 0.04 0 

### * 4. 5 0.04 0 8. 9 0.04 0 

### * 13. 3 0.04 0 

### - -2. 1 0.04 0 -13. 1 0.04 0 

### - -0. 6 D.04 0 

### * -0. 6 0.09 S 

### * -13. 1 0.09 6 

### - 17. 0 0,09 6 

# • -5. 0 0.09 6 

# • 11. 1 0.09 6 

### • -7. 9 0.09 6 

### * -9. 4 0.09 6 

### * 17. 8 0.09 6 

### • -8. 7 0.09 6 -11. 6 0.09 6 

### • -2. 8 0.4 2 -0.37 7 -16. 8 0.4 2 -0.37 7 18. 5 0.4 2 •0.37 7 0. 9 0.4 2 - 0 37 7 8. 9 0.4 2 -0.37 7 3. 1 0.4 2 -0.37 7 -0. 6 0.4 2 -0.37 7 -9, 4 0.4 2 -0.37 7 6. 7 0 4 2 -0.37 7 -5. 0 1. 7 0.23 0 54. 5 1. 7 0,23 0 72. 8 1. 7 0.23 0 50. 1 1. 7 0.23 0 37. 6 1, 7 0.23 0 62. 5 1. 7 0.23 0 57. 4 1. 7 0.23 0 64. 0 4. 8 0.68 1 100. 0 4. 8 0.63 1 100. 0 4. 8 0.6S 1 100. 0 4. 8 0.68 1 100, 0 4. 8 0,68 1 100, 0 4. 8 0.68 1 100, 0 4. 8 0.63 1 100. 0 4. 8 0.63 1 100. 0 4. 8 0.68 1 100. 0 Slope - 93.801 8 Intercept : 35.374 2 Multipl e R : 0.982 2 n = numbe ro fobservation s 2 5 Regressio n line
. 93.80 X 35.8? Predictio n o fX value s base d o n know n Yvalue s Know n Y Reductio n (% } 1 0 X r O g (mg/l ) 1 0 X3S% -

(mg/l ) (mg/l ) 1 0 0.5 3 0.3 4 0.8 3 2 0 0.6 8 0.4 4 1.0 5 5 0 1.4 1 0.9 1 2.1 9

- Pag e 2 4 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2 1 3-epoxypropyl)anilin e NOTO XProjec t 49698 9 APPENDI X5 EC-VALUE S - continue d -



### Concentratio n (mg/l )

- Pag e 2 5 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substances (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bss(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 9 APPENDI X6 EXPERIMENTA L CONDITION S Tabl e 1 2 p H value s i n freshl yprepare d medi a (F ) an d ol d medi a (O ) Nomina l da y 0 2 2 4 4 6 6 8 

### a 1 0 1 0 1 2 1 2 Grou p (mg/l ) F O F O F O F O F 0 F O F contro l 8. 0 7. 9 3. 1 7. 9 8, 0 7, 9 7. 9 3, 0 7. 8 8. 0 7. 9 7. 9 0.04 0 8, 0 7. 9 8. 1 7. 9 8. 1 7. 9 7. 9 8. 0 7. 9 8. 0 7. 9 7. 9 0.09 6 8. 0 7. 9 8. 2 7. 9 8. 1 7. 9 7. 9 

### a. o 7. 8 8. 0 7. 9 7. 9 0,4 2 8. 1 7. 9 8. 1 7. 9 8. 0 7. 9 7, 9 8. 0 7. 8 7. 9 7. 9 7. 9 1. 7 _Jil
_ 7. 9 

### JA 8, 0 7. 9 7. 9 8. 0 7. 8 7. 9 7, 9 7, 9
_ 4. 8 8. 1 7. 9 8. 1 7. 9 8. 1 7. 9 7. 9 3. 0 8. 0 7. 9 8. 0 8. 0 Nomina ! da y 1 4 1 4 1 6 1 6 1 8 1 8 2 0 2 0 Grou p (mq/l ) O F O F O F 0 . p contro l 8. 0 7. 9 7. 9 8, 0 7. 9 8. 0 7. 5 8. 4 0.04 0 8. 0 8. 0 7. 8 7. 9 7. 8 8. 0 7. 6 8. 4 0.09 6 8. 0 8, 0 7. 8 7, 9 7. 8 8. 0 7. 6 8. 3 0.4 2 8. 0 3. 0 7. 7 8. 0 7. 8 8. 0 7. 6 8. 3 1. 7 8. 0 8. 0 7. 3 3. 0 7. 8 8. 0 7. 7 8. 2 4 8 8. 1 8. 0 7. 8 8, 0 7. 8 8 0 7 8 8. 2

Tabl e 1 3 Dissolve d oxyge n concentration s (mg/l ) i n freshl yprepare d medi a (F ) an d ol d

medi a (0 } Nomina l da y 0 2 2 4 4 6 6 8 8 1 0 1 0 1 2 1 2 Group (mg/l) F



# __

°
. JL
_ O F O F O F 0 F O F contro l 9. 1 3. 3 9. 2 9. 0 9. 2 9. 4 8. 7 9. 0 8. 4 9. 1 8. 6 9. 2 0.04 0 9. 0 8. 9 9. 2 9. 0 9. 4 9 3 8. 6 9. 0 8. 6 9. 1 8. 8 9. 1 0.09 6 9. 0 8. 8 9. 4 9, 0 9. 5 9. 3 8. 6 9. 0 8. 7 9. 1 8. 8 9, 1 0.4 2 8 9 8, 9 9. 1 8. 9 9, 4 9. 3 8. 6 9 0 8. 5 9. 1 8. 8 9. 0 1. 7 8. 8 8. 9 9. 0 9. 0


### . 9- 1 9,3
_ 8, 7 9. 0 S. 5 9. 0 8. 9 9. 1 4, 8 8. 7 9. 0 9. 0 9. 0 9. 7 9. 3 8. 7 9. 0 9. 0 9. 0 9. 1 9. 0 Nomina l da y 1 4 1 4 1 6 1 6 1 8 1 8 2 0 2 0 Grou p (mg/l ) O F O F 0 F O F contro l 9. 3 9. 2 8. 9 9, 4 8. 4 9, 2 8, 7 9. 3 0,04 0 I H .9,2
_ _a3
_ 

### .. . *L 4



### _9 

### : 1 8 3 9. 2 8. 5 9. 3 0.09 6 9. 3 9. 3 8. 3 9. 4 8. 5 9. 2 3. 6 9. 3 0.4 2 9. 3 9. 2 8. 5 9. 4 3. 3 9. 2 8. 7 9, 2 1, 7 9. 6 9 2 3. 2 9. 3 8. 7 9. 2 8. 9 9. 2 4. 8 9. 7 9. 1 8. 5 9. 2 8. 6 9, 1 9, 0 9, 1

- Pag e 2 6 -Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d to Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibite d p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProjec t 49698 9 APPENDI X6 EXPERIMENTA L CONDITION S - continue d -
Tabl e 1 4 Temperatur e ( D C ) i n freshl yprepare d medi a (F ) an d ol d medi a (O ) Nomina l da y 0 2 2 4 4 6 6 8 8 1 0 1 0 1 2 1 2 Grou p (mg/l ) F O F O F O F O F O F O F contro l 19, 8 19. 6 19. 7 19. 8 19. 6 20. 0 19. 9 19. 9 19. 9 19. 9 19. 7 19. 5 19, 8 0,04 0 19. 9 19. 8 19. 8 19. 6 19, 5 20. 0 19. 9 19. 9 19, 9 19, 9 19. 8 19. 8 19. 9 0.09 6 19. 9 19. 8 19. 8 19. 5 19. 4 20. 0 19. 9 19. 9 19. 9 19. 9 19. 8 19. 8 19. 9 0.4 2 20. 0 19. 8 19. 8 19. 5 19. 4 20. 0 19. 9 20. 0 19. 9 19. 9 19. 9 19. 9 19. 3 1. 7 20. 0 19. 9 2 0 0 19. 5 19, 5 20. 0 20. 0 20. 1 20. 0 19. 9 19. 9 20. 0 19. 8 20. 0 4. 8 20. 2 20. 0 2 0 5 19. 5 19. 6 20. 0 20. 0 20. 1 20. 0 20. 0 19, 9 20. 1 19, 9 20. 2 Nomina l da y 1 4 1 4 1 6 1 6 1 8 1 8 2 0 2 0 Grou p (mg/t ) O F 0 F 0 F O F contro l 20. 0 19. 9 20. 2 20. 2 20. 0 19. 8 19. 5 19. 4 0,04 0 20. 2 19. 9 2 0 2 20. 3 20, 1 19. 7 19. 7 19, 4 ,0,09 6
_ 0,4 2 20. 2 20. 2 20. 0 20. 1 20. 2 20. 2 20. 1 19. 8 19, 8 19. 5 ,0,09 6
_ 0,4 2 20. 2 20. 2 20. 0 20. 1 20. 3 20. 2 20, 0 19. 8 19. 8 19. 6 1. 7 20. 2 20. 3 20. 3 20. 2 20. 0 19. 9 19. 3 19. 7 4. 8 2 0 1 2 0 5 20. 3 20. 1 20. 1 20. 1 19. 7 19 8

Tabl e 1 5 Hardnes s (mg/l CaC0 3 ) i n freshl yprepare d medi a (F ) an d ol d medi a (O ) Nomina l da y 4 4 1 2 1 2 2 0 2 0 Grou p (mg/l ) O F O F 0 F contro l 17 9 17 9 19 6 19 6 21 4 21 4 4. 8 17 9 17 9 19 6 19 6 21 4 21 4

- Pag e 2 7 -D 15 4 Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d t o Registration , Evaluation , K ' Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e express writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)~N,N-bis(2,3-epoxypropyl)aniiin e NOTO X Projec t 49698 9 

## APPENDI X 7 ANALYTICA

L REPOR T



# DETERMINATIO N O F TH E CONCENTRATION S

Autho r Dr
. ir
. E
. Baltusse n

I V - Pag e 2 8 -



### I

### "I ~Th e us e ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
.

1 . CONTENT S 1
. CONTENT S 2 9 2
. REPOR TAPPROVA L3 0 3
. INTRODUCTIO N , 3 1 3.1
. Prefac e 3 1 3.2
. Ai m o fth e stud y3 1 4
. MATERIAL S AN D METHOD S 3 1 4.1
. Reagent s 3 1 4.2
. Sample s 3 1 4.3
. Analytica l metho d 3 1 4.3.1
. Analytica l condition s 3 1 4.3.2
. Preparatio n o fth e calibratio n solution s 3 1 4.3.3
. Sampl e injection s 3 2 4.4
. Electroni cdat a captur e 3 2 4.5
. Formula s 3 2 5
. RESULTS 3 3 5.1
. Calibratio n curve s 3 3 5.2
. Sample s 3 3 5.2.1
. Procedura l recoverysample s 3 3 5.2.2
. Tes tsample s , 3 3 6
. TABLES......
. 3 4 TABLE S Tabl e 1 Procedura l recoverysample s 3 4 Tabl e 2 Concentration s o fth e tes t substanc e i n tes t mediu m - preliminarytes t 3 5 Tabl e 3 Concentration s o fth e tes tsubstanc e i n tes tmediu m - reproductio n tes t 3 6

- Pag e 2 9 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substances (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p . 15 6

p-(2,3-epoxypropoxy)-N,N-bis{2,3-epoxypropy!)anilin e NOTO X Projec t 49698 9 2
. REPOR TAPPROVA L NOTO X B.V , Principa l Scientis t E
. Baltussen , Ph D Analytica l Chemistr y



### Date: .,}2~....th*rcK .%£>j2r.

Fina l Report
- Pag e 3 0 -p . 15 7 Th e us e ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibite d p-(2,3-epoxypropoxy)-N 1 N-bis(2,3-epoxypropyl)anilin e NOTO XProject 49698 9 3 . INTRODUCTIO N 3.1 . Prefac e Stud ypla n analytica l phas e Start: 2 2 Augus t 201 1 Completio n : 0 7 January201 2 3.2 . Ai m o fth e stud y Th e purpos e o fth e analytica l phas e wa s t o determin e th e actua l concentration s i n sample s take n fro m th e tes tsolution s use d durin g th e ecotoxicit ytest . 4 . MATERIAL S AN D METHOD S 4.1 . Reagent s Wate rTa p wate rpurifie d b ya Milli- Q wate rpurificatio n syste m (Millipore , Bedford , MA , USA ) Acetonitril e Biosolve , Valkenswaard , Th e Netherland s M7-mediu m se e mai n report Al l reagent s wer e o fanalytica l grade , unles s specifie d otherwise
. 4.2 . Sample s Th e sample s wer e store d i n th e freeze r( < -15°C)
. Storag e stabilit yo fsample s unde rthes e condition s wa s demonstrate d i n NOTO Xprojec t 496981
. O n th e da yo fanalysis , th e sample s wer e defroste d a troo m temperature . Th e tes t sample s wer e dilute d i n a 1: 1 (v:v ) rati o wit h 60/4 0 (v/v ) acetonitrile/M7-mediu m an d analysed
. I fnecessary , th e sample s wer e furthe rdilute d wit h 30/7 0 (v/v ) acetonitrile/M7-mediu m t o obtai n concentration s withi n th e calibratio n range . 4.3 . Analytica l metho d 4.3.1 . Analytica l condition s Quantitativ e analysi s wa s base d o n th e analytica l metho d validate d fo rth e tes t substanc e i n NOTO X projec t496981
. Instrumen tAllianc e Separatio n Modul e 269 5 (Waters , Milford , MA , USA ) Detecto rDua l AAbsorbanc e Detecto r248 7 (Waters ) Colum n SymmetryShiel d RP-18 , 10 0 m m x4. 6 m m i.d. , d p = 3. 5 p m (Waters ) Colum n temperatur e 40° C ±1 ° C Injectio n volum e 10 0 p i Mobil e phas e 30/7 0 (v/v ) acetonitrile/wate r Flo w1.0ml/mi n U Vdetectio n 25 3 n m 4,3.2 . Preparatio n ofth e calibratio n solution s Stoc k an d spikin g solution s Stoc k solution s o fth e tes tsubstanc e wer e prepare d i n acetonitril e at concentration s o f206 4 - 257 2 mg/l
.

- Pag e 3 1 -Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p . 15 8
p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropy!)anilin e NOTO XProjec t 49698 9 Spikin g solution s wer e mad e u p fro m a stoc k solutio n and/o rdilution s o fthi s solution
. Th e solven t o f th e spikin g solution s wa s acetonitrile
. Calibratio n solution s Calibratio n solution s i n th e concentratio n rang e o f0.040 0 - 2.0 0 mg/ l wer e prepare d fro m tw o stoc k solutions
. Th e en d solutio n o fth e calibratio n solution s wa s 30/7 0 (v/v ) acetonitnle/M7-medium
. Proceduraj.recoverysample s 2 m l blan k

mediu m wa s spike d wit h th e tes t substanc e ata targe t concentratio n of0, 1 o r1 0 mg/l
. Th e accurac ysample s wer e treate d similarl ya s th e tes tsample s (se e paragrap h 4. 2 'Samples')
. 4.3,3 . Sampl e injection s Calibratio n solution s wer e injecte d i n duplicate
. Tes t sample s an d procedura l recoverysample s wer e analyse d b ysingl e injection
. 4.4 . Electroni c dat a captur e Syste m control , dat a acquisitio n an d dat a processin g wer e performe d usin g th e followin g programme :

- Empowe rversio n 7.0 0 (Waters , Milford , MA , USA)
. Temperatur e and/o rrelativ e humidit ydurin g sampl e storag e and/o rperformanc e o fth e studie s wer e monitore d continuousl yusin g th e followin g programme :

- REE S Centro n Environmenta l Monitorin g syste m versio n SQ L2. 0 (REE S Scientific , Trenton , NJ , USA)
. 4.5 . Formula s Respons e (R ) Calibratio n curv e Pea k are a tes t substanc e [units] R = aC N + b where : C N = nomina l concentratio n [mg/l ] a = slop e [unit s xl/mg ] b = intercep t [units ] Analyse d concentratio n (CA ) C A_- ( - _ b > x d [mg/l ] a where : d ~ dilutio n facto r Recovery ^x10 0 [%]
Relativ e t o nomina l concentratio n 

### • x1 o o 

### [% ]
Relativ e t o initia l concentratio n Cill^lx^ O [% ] C A(t--=0days )

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m i i iTh e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO XProject 49698 9 5 . RESULT S 5.1 . Calibratio n curve s Calibratio n curve s wer e constructe d usin g fiv e concentrations
. Fo reac h concentration , tw o response s wer e used
. Linea rregressio n analysi s wa s performe d usin g th e leas t square s metho d wit h a 1/concentration 2weightin g factor
. Th e coefficien to fcorrelatio n (r) wa s > 0.9 9 fo reac h curve . 5.2 . Sample s 5.2.1 . Procedura l recover ysample s Th e result s fo rth e procedura l recoverysample s ar e give n i n Tabl e 1
. Th e mea n recoverie s o fth e procedura l recoverysample s fel l withi n th e criterio n o f70-110 % excep t fo r th e hig h concentration s leve l o n 29-09-1 1 wher e on e o fth e duplicat e result s deviate d abov e 110%
. I t wa s assume d tha tthi s wa s probabl ya n outlie ran d sinc e i t occurre d durin g th e range-findin g tes t i tha s n o influenc e o n th e study
. Th e procedura l recoverysample s demonstrate d tha t th e analytica l metho d wa s adequat e fo rth e determinatio n o fth e tes tsubstanc e i n th e tes t samples
. 5.2.2 . Tes t sample s Th e result s fo rth e tes t sample s ar e give n i n Tabl e 2 an d Tabl e 3
.

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C i s unauthorize d an d strictl yprohibited
. p . 16 0
p-(2,3-epoxypropoxy)-N,N-bis(2 1 3-epoxypropyl)anilin e NOTO XProjec t 49698 9 6 . TABLE S Tabl e 1 Procedura l recover ysample s Dat e o f preparatio n [dd-mm-yyj Dat e o f

analysi s [dd-mm-yy l Targe t

concentratio n [rng/l ] Nominal

concentratio n


### , [mg./! l Analyse d

concentratio n [mg/l l Recovery

[% ] Mea n recovery [%] 29-09-1 1 29-09-1 1 0. 1 0.10 0 0.10 2 10 2 10 4 0.10 0 0.10 5 10 5 29-09-1 1 29-09-1 1 1 0 10. 0 12. 1 12 1 11 5 10. 0 10. 9 10 9 17-12-1 1 17-12-1 1 0. 1 0.10 0 0.078 8 7 9 8 0 0.10 0 0.082 0 8 2 17-12-1 1 17-12-1 1 1 0 10. 0 8.4 4 8 4 8 2 10. 0 7.9 8 8 0 06-01-1 2 06-01-1 2 0. 1 0.10 1 0.087 5 8 6 n.a . 0.10 1 0.048 0 4 7 1 06-01-1 2 06-01-1 2 1 0 10. 1 8.7 3 8 6 8 5 10. 1 3.5 4 8 4 Thi s resul t was identifie d a s a n outlie rbase d on a Dixon' s Q-tes L

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I ITh e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d to Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3-epoxypropoxy)-N,N~bis(2,3-epoxypropy!)aniiin e NOTO XProjec t 49698 9
Tabl e 2 Concentration s o fth e tes t substanc e i n tes t mediu m ~ preliminar ytes t Tim e o f samplin g TMJ£ayjl__
_ Dat e o f

samplin g [dd-mm-yy ] Dat e o f

analysi s 1 [dd-mm-yy ] Percentag e o f

WA F filtrate 2 

### E% ] Analyse d

concentratio n 

### fm.g/l l Relativ e t o

initia l 0 20-09-1 1 29-09-1 1 1 0.81 1 (fresh ) 1 0 7.3 4 1 21-09-1 1 29-09-1 1 1 0.73 2 - 9 0 (old ) 1 0 6.3 3 8 6 1 3 0.74 6 9 2 1 0 3 6.5 5 8 9 1 21-09-1 1 29-09-1 1 1 0.64 6 (fresh ) 1 0 6.6 0 3 23-09-1 1 29-09-1 1 1 0.50 9 7 9 (old ) 1 0 5.2 1 7 9 1 a 0.51 3 7 9 1 0 3 5.2 8 8 0 3 23-09-1 1 29-09-1 1 1 0.96 6 (fresh ) 1 0 7.1 6 6 26-09-1 1 29-09-1 1 1 0.59 3 6 1 (old ) 1 0 4.8 5 6 8 1 : i 0.65 6 6 8 1Q 3 5.1 4 7 2 Samples wer

e store d i n th e free2e r(<
. - 1 S"C ) unti l th e da

yofanalysis
. Percentag e o

fa wate raccommodate d fractio n (WAF) prepare d a t a loadin g rat e o

f1 0 mg/l
. Without algae.

- Pag e 3 5 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d to Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p . 16 2
p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniIin e NOTO XProjec t 49698 9 Tabl e 3 Concentration s ofth e tes t substanc e i n tes t mediu m - reproductio n tes t Tim e o f Dat e o f Dat e o f Percentag e o f Analyse d Relativ e t o samplin g samplin g analysi s 1 WA F filtrat e 2 concentratio n 3 initia l 

### fdays l [dd-mm-yy ] [dd-mm-yy ] 

### [% l [mg/l ] [%] 0 08-12-1 1 17-12-1 1 0 n.d
. (fresh ) 1 < 0.080 0 3. 2 0.14 5 1 0 0.56 6 3 2 2.4 2 10 0 6.5 5 2 10-12-1 1 17-12-1 1 0 n.d
. n.a . (old ) 1 < 0.080 0 n.a . 3. 2 0.12 2 8 4 1 0 0.44 3 7 8 3 2 1.9 2 7 9 10 0 5.3 5 8 2 6 14-12-1 1 17-12-1 1 0 n.d
. (fresh ) 1 < 0.080 0 3. 2 0.15 2 1 0 0.55 3 3 2 2.2 3 10 0 6.1 2 8 16-12-1 1 17-12-1 1 0 n.d
. n.a . (old ) 1 < 0.080 0 n.a . 3. 2 0.11 0 7 2 1 0 0.41 8 7 6 3 2 1.74 7 8 10 0 4.75 7 8 1 2 20-12-1 1 06-01-1 2 0 n.d
. (fresh ) 1 < 0.080 0 3. 2 < 0.080 0 1 0 0.39 0 3 2 1.45 10 0 4.1 6 1 4 22-12-1 1 06-01-1 2 0 n.d
. n.a . (old ) 1 < 0.080 0 n.a . 3. 2 < 0.080 0 n.a . 1 0 0.24 4 6 3 3 2 1.05 7 2 10 0 2.8 9 7 0 1 8 26-12-1 1
. 06-01-1 2 0 n.d
. (fresh ) 1 < 0.080 0 3. 2 0.17 2 1 0 0.50 2 3 2 1.9 1 10 0 5.2 3

- Pag e 3 6 -

I ITh e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. p-(2,3~epoxypropoxy)-N,N-bis(2,3-epoxypropyl)anilin e NOTO X Projec t 49698 9
Tabl e 3 Concentration s o fth e tes t substanc e i n tes t mediu m - reproductio n tes t

(continued ) Tim e o f Dat e o f Dat e o f Percentag e o f Analyse d Relativ e t o samplin g samplin g analysi s 1 WA F filtrat e 2 concentratio n 3 initia l |days ] [dd-mm-yy ] [dd-mm-yy ] {% ] [mg/l ] [% ] 2 0 28-12-1 1 06-01-1 2 0 n.d
. n.a . (old ) 1 < 0.080 0 n.a . 3. 2 < 0.080 0 n.a . 1 0 0.33 0 6 6 3 2 1.2 7 6 7 10 0 3.8 7 7 4 Samples wer

e store d i n th e freeze r( s -15"C ) unti l th e da yo fanalysis , Percentag e o

fa wate rsolubl e fractio n (WSF ) prepare d a

t a loadin g rate o

f1 0 mg/l
. I n severa l sample s test substance was detecte d a t a concentratio n beio

wth e lowes

t calibratio n solution
. Thes e sampfes wer e reporte d a

s < 0.0800 mg/l (i.e
. a concentratio n analyse d lowe rtha n th e lowestcalibratio n standar d take n th e dilutio n facto ro

fth e sample s (2 ) int

o account)
. n.d
. No

t detected , n.a . No

t applicable
.

- Pag e 3 7 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d to Registration , EvaliUfio i Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n internationa l LLC i s unauthorize d an d strictl yprohibited
.



# REPOR T

96-HOU R ACUT E TOXICIT

Y STUD Y I NCAR

P WIT H T K 12759 , Araldit e M

Y 051 0

NOTO X Projec t 16954 8 CIB Ates t numbe

r 96462 3

- Pag e 1 o f 9 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. 

## T K 12759 , AraTdit e M

Y 051 0 NOTO X Projec t 16954 8 •
REPOR T APPROVA

L

STUO V DIRECTOR :

MANAGEMENT :

- Pag e 2 -D 1 6 Th e us e ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. 

## T K 12759 , AraldU e M

Y 051 0 NOTO X Projec t 16954 8
SUMMAR Y 96-Hou r Acut e Toxicit yStud

yi n Car p wit hT K 12759 , AraldU e M

Y 0510 . Th e stud yprocedure s described i nthi s repor t wer e base d o n the EE C directiv e 92/69 , Par t C.l . "Acute toxicit yfo r fish" ; an d the OCC

O guidelin e No . 203 : "FishAcut e Toxicit yTest" , Adopte d 1 7 July , 1992 . Th e definitiv e tes t wa

s performe

d wit hcar

p expose d t o concentration s rangin g fro m1. 0 t o 1 0 mg/ l i n a stati c system . Aceton e wa s use d as a solven t i n the stoc k solutions . Th e 96h-LC5 0 fo r car

p i s 4. 2 mg/ l wit h0 % mortalit ya t 3. 2 ng n an d 10 0 % mortalit ya t 5. 6 mg/l . Th e BW Z o f T K 12759 , AraldU e M

Y 051 0 fo r acut e toxicit yi n fis hi s 5.4 .

- Pag e 3 -Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu tno tlimite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. 

## p . 16 7
T K 12759 , Araldit e M

Y 051 0 NOTO X Projec t 16954 8
PREFAC E Sponsor

Stud yMonito r Testin g Facilit y
Stud yDirecto r Stud yPlan Ciba-Geig yLtd . Polymer s Divisio n R-1006.52 0 P.O . Bo x CH-400 2 BASE

L Switzerlan d Dr . E.G . Semaden

i Additive s Divisio n R-1002.2.6 2 NOTO X 8.V . Hambakenweterin

g 3 523 1 D O 's-Hertogenbosch Th e Netherland s Ors . M . Soger s Star t : 2 5 March , 1996 ' Completio n ; 1 9 April , 199 6 TES T SUBSTANC

E (NOTO X Substanc e Code : 59652 ) Identificatio n Descriptio n Batch Purit y Tes t substanc e storag e Stabilit yunde r storag e condition s Expirydat e Stabilit yi n vehicl e T K 12759 , Araldit e M

Y 051 0 Clea r yello wliqui d(Determine d a t NOTOX ) 50008 M > 95 * I nfreeze r i n th e dar k Stabl e Ma y01 , 199 8 Water : a t leas t 9 6 h Acetone : a t leas t 9 6 h PURPOS E Th e purpos e o f th e stud

ywas . t o evaluat e th e tes t substanc e fo r it s abilit y t o generat e acut e toxi c effect s i n Cyprinu s carpi o durin g a n exposur e perio do f 9 6 hours .

- Pag e 4


# 1 nr r iiTh e us e ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d to Registration , Evafuati l Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LLC i s unauthorize d an d strictl yprohibited
. 

## T K 12759 , Araldit e M

Y 051 0 NOTO X Projec t 16954 8
GUIDELINE S Th e stud yprocedure s described i n thi s repor t wer e base d o n the followin g guidelines : European'Economi c Communit y(EEC) , EE

C directiv e 92/69 , Par t C : Method s fo r the determinatio n o f ecotoxicity , Publicatio nNo . L383 , Decembe r 1992 , C.l . "Acute toxicit yfo r fish" . Th e OEC D guideline s fo r Testing o f Chemicals , guidelin e No . 203 : "Fis h Acut e Toxicit yTest" , Adopte d 1 7 July , 1992 . ARCHIVIN G NOTO X 8.V. wil l archiv e th e followin g dat a fo r a t leas t 1 0 years : report , an d ra wdata . DEFINITION

S Fis hwer e considered t o b

e dea d whe n n o reactio nwa

s observe d afte r touching th e cauda l peduncl e an d visibl e breathing movements wer e absent . Th e LC5 0 i s th e concentratio n killin g 509 6 o f the fis hafte r a define d perio do f exposure . TES T SPECIE S Specie s Source Mea n lengt h Mea n weight Characteristic s Reaso n fo r selectio n Tota l fis huse

d Car

p (Cyprlnu s caroio . Teleostei , cyprinidae ) (Linnaeus , 1758 ) Zodiac , proefacc , "D

e Haa r Vissen" , L.U . Wageningen , th e Netherlands . Range-findin

g test : 1.9 0 + 0.2 7 c

m Fina l test : 1.9 6 + 0.1 0 ciii Range-findin

g test : 0.3 0 + 0.0 7 g Fina l test : 0.2 1 ± 0.0 3 c m F l fro ma singl e parent-pai r bre d i n untreate dwater . Thi s syste

mha s bee n selecte d as a

n internationall yaccepte

d species . 7 9

- Pag e 5 -Th e us e o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. 

## p . 16 9
T K 12759 , Araldit e M

Y 051 0 NOTO X Projec t 16954 8
HOLDIN G Quarantine/Acclimation Medium '

Measurement s Feedin g Contro l o f sensitivit y

' i 

## Validit yo f batch A t leas t 1 2 day

s afte r delivery . ISO-medium , formulatedusin g Milli-R o water (tap-wate r purifie db

yrevers e osmosis ; Millipor e Corp. , Bedford , Mass. , USA ) wit hthe followin g composition : Ca 2 + 

## 8 0 mg/ l 1 2 mg/ l 1 5 mg/ l 3 mg/ l 14 5 mg/ l 4 9 mg/ l 4 7 mg/ l 2 4 

## M g Na + 

## K + 

# c r S0„ 2 HC0 3 

## Hardnes s 1s 25 0 m

g CaC03/ l Oxyge n concentration , pH , nitrat e an d nitrit e concentratio nan

d ammoni a concentration : onc

e a week . Temperature ; ever yday . Dail ywit hTrouvit . Areferenc e tes t wit hpentachloropheno l (PCP , SIGMA ) i s carrie dou t withi na perio do f 3 months . Th

e result s o f thi s tes t ar e appende d t o the report . Inthe batch offish used forthe test, mortality

duringthe seven days priorto the start ofthe test was less than 5%. RANGE-FINDIN

G TES T Arange-finding tjes t wa

s performe

d t o provid e informatio nabout the rang e o f concentrations ! t o b

e use d Ir i the fina l test : thre e fis hpe

r concentratio nwer

e expose d t o a concentratio nrang e o f 0. 1 t o 1 0 mg/ l wit ha

n increasing facto r o f 10 , whil e seve n fis hwer

e expose d t o 10 0 mg/ l an

d the blan

k an d solven t control .

- Pag e 6 -Th e us e o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d to Registration , EvalJItio^i f Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa ! LL

C i s unauthorize d an d strictl yprohibited
. 

## T K 12759 , Araldit e M

Y 051 0 NOTO X Projec t 16954 8
FINA L STUDY : TES

T PROCEOUR

E AN D C0N0ITI0N

S Tes t duratio n Tes t typ e Tes t vessel s Tes t mediu

m

Numbe r o f fis h Loadin g llluminatio n Aeratio n Feedin g Introductio no f fis h Euthanasi a 9 6 hour s Stati c 5 litres , all-glass . ISO-medium , aerated unti l the dissolve doxyge n concentratio nha

d reache d saturatio nan

d the p H ha d stabilized. . Afte r aeratio nthe hardnes s wa s 25 0 m g CaCO s per litr e an

d the p H wa s 8. 0 + 0.2 . 7 fis hper concentratio nan d controls . 0.74 g fish/litre , i.e . 7 fis hper 2 litre s o f tes t medium . 1 6 hour s photoperiod dail y Th e tes t media wer

e aerated continuously . N o feeding fro

m2 4 hour s prio r t o the tes t an d during the tota l tes t period . Directl yafte r preparatio no f the tes t media . A t the en d o f the tes t the survivin g fis hwer e rapidl ykille db

yexposin g the mt o ca . 1.2 % ethylen e glyco l monophenylether i nwater . PREPARATIO

NO F TES T SOLUTION

S Stoc k solution s wer

e prepare d i naceton

e a t 100 , 56 , 32 , 1 8 an d 1 0 mg/ml . A volum e o f 0. 2 m

l wa s taken fro

meac hstock an

d mixe d wit h2 litre s o f tes t mediu mprovidin g the tes t concentration s o f 10 , 5.6 , 3.2 , 1. 8 an d 1. 0 mg/l . Th e fina l tes t solution s wer e al l clea r withou t precipitation . MEASUREMENT

S AN D RECORDING

S Mortalit yan

d othe r effect s At (2) , 24 , 48 , 7 2 an d9 6 hour s followin g the star t o f exposure . Dea

d fis hwer

e remove d whe n observed . Dissolved oxyge

n content , p H an d Temperature Daily , beginnin g a t the star t o f the test .

- Pag e 7 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d to Registration , Evaluation , Authorizatio n an d Restriction.ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa ! LL

C i s unauthorize d an d strictl yprohibited
. 

## p . 17 1
T K 12759 , Araldit e M

Y 051 0 NOTO X Projec t 16954 8
RESULT S Range-findin

g test : Incidenc e an

d tota l mortality : Concentratio n (mg/l ) initia l

numbe r o f fis h Cumulativ e mortalit y Total Mortality (%) 

### Concentratio n (mg/l ) initia l

numbe r o f fis h 2 4 h 4 8 h 7 2 h 9 6 h Total Mortality (%) 

### Blank-contro l 7 0 0 0 0 0 Solvent-contro l 7 0 0 0 0 0 0. 1 3 0 0 0 0 0 1 3 0 0 0 0 0 1 0 3 0 3 3 3 10 0 10 0 7 7 7 7 

### 7 10 0


## Definitiv e study : i 

## Incidenc e an

d tota l mortality ; Concentratio n (mg/l ) Initia l

numbe r o f fis h Cumulativ e mortalit y Total Mortality (%) 

### Concentratio n (mg/l ) Initia l

numbe r o f fis h 2 4 h 4 8 h 7 2 h 9 6 h Total Mortality (%) 

### Blank-contro l 7 0 0 0 0 0 Solvent-contro l 7 0 0 0 0 0 1. 0 7 0 0 0 0 0 1. 8 7 0 0 0 0 0 3. 2 • 7 0 0 0 0 0 5. 6 7 0 3 * 7 7 10 0 1 0 7 0 7 7 7 10 0
* Survivin g fis hshowe

d sever e disturbanc e o f swimmin g behaviour . 

## pH , dissolve doxyge

n an d temperature : ConcentrationDa

y0 Da

y1 Da

y2 Da

y3 Da

y4 (mg/l ) p

H D O p H D O p H D O p H D O p H D O Blank-contro l 8 1 9 . 1 8. 0 9. 0 7. 8 8 8 7 9 8, 9 7 9 8 9 Solvent-contro l 8 1 9 . 0 8. 0 8, 8 7. 8 8 7 7 9 8. 8 7 8 3 8 1. 0 8 1 9 . 0 8. 0 8. 8 7. 8 8 8 7 8 8. 9 7 8 8 6 1. 8 8 0 9 . 2 8. 0 8. 8 7. 8 9 0 7 8 8. 9 7 7 8 8 3. 2 8 0 9 1 7. 9 8. 7 7. 8 8 8 7 9 8. 9 7 6 8 7 5. 6 , 8 0 9 1 8. 0 8. 9 7. 8 8 9 8 0 8. 9 1 0 8 0 9 2 8. 0 8. 8 Temperatur e 2 0 . 5 2 0 . 4 2 0 5 20 . 5 2 0 4 

## Pag e 8Th e us e ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d to Registration , Evaluation , Authorizatio n anc

i Restrictio n o

fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. 

## T K 12759 , Araldit e M

Y 051 0 NOTO X Projec t 16954 8

CONCLUSIO N T K 12759 , Araldit e M

Y 0510 : Th e 96h-LC5 0 fo r car pI s 4. 2 mg/

l wit h0 %mortalit

y at 3. 2 mg/

l an d10 0 % mortalit ya t 5. 6 mg/l .



## - Pag e 9 -Th e us e o fthes e studies/document s fa rregulatoryo rregistratio n purposes , includin g bu t ne t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou tth e expres s writte n permissio n o fHuntsma n 

### p . 173 [P S + R , P O 2. 1(Kopie;



### I Ablag s '

j

IITh e us e ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu tno tlimite d t o Registration , EvalPdtn Authorizatio n an d Restrictio n ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LLC i s unauthorize d an d strictl yprohibited
.



# REPOR T

PSEUDOMOHA

S CEL L MULTIPLICATIO

NINHIBITIO

NTES T WIT H T K 12759 , ARALDIT

E M Y 051 0

NOTO X Projec t 16970 6 Cib a Projec t 96462 4 NOTO X Substance 5965 2

- Pag e l o f 1 0 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LLC i s unauthorize d an d strictl yprohibited
. 

## p . 17 5
T K 12759 , ARALDIT

E M Y 051 0 NOTO X Projec t 16970 6 Cib a Projec t 96462 4 REPOR T APPROVA

L

STUD Y DIRECTOR : I r , Y.H.M. va n Er p



# Date : WVv

^ ^ x


## MANAGEMENT : Ing , E.J . va n d e tfaart SectionHead , Geneti c & Eco-Toxicolog

y

Date : ^a ,

- Pag e 2 -

### p . 

### 17 6 Th e us e o

fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LLC i s unauthorize d an d strictl yprohibited
. 

## T K 12759 , ARALDIT

E M Y 051 0 NOTO X Projec t 16970 6 Cib a Projec t 96462 4 SUMMAR Y T K 12759 , ARALDIT

E M Y 051 0 wa s investigate dfo r it s abilit yt o inhibi t th e cel l multiplicatio no f the bacteri a specie s Pseudomona s putid a accordin

g t o DI N3841 2 Par t L 8 (Marc

h1991 ) an

d NEN-EN-IS

O 1071 2 (1996) , Suspensions o f Pseudomona s putid a wer

e expose d t o concentration s rangin g fro m 312. 5 t o 10,00 0 y

g T K 12759 , ARALOIT

E M Y 051 0 per litre . Aceton e wa s use d as vehicle . Afte r incubatio nthe extinctio nvalue s o f th e bacteria l cel l suspensions wer e measured . N

o toxicit ywa

s observed . Base d o n the result s o f th e tes t presentl yperformed , th e toxicit y threshol dvalue (EC10 ) o f T K 12759 , ARALDIT

E M Y 051 0 wa s over 1 0 mg/l . Th e "Bewertungszahl " (Assessmen t figur e o r value fo r bacteria l toxicity ) i s therefor e < 4. 9 (Bewertun g wassergefahrdender Stoffe , 1989) . Th e EC50 value fo r the referenc e substance , 3,5-dichlorophenol , teste d during th e sam

e experiment wa

s 2 0 rag/1. Sinc e thi s wa s withi nthe accepte

d rang e o f 10-3 0 mg/ l fo r the EC50 an d th e multiplicatio nfacto r o f the bacteri a withi nthe tes t wa s over 60 , i t wa

s conclude

d tha t th e tes t condition s wer

e optima l an

d th e result s obtaine d wer e valid .

- Pag e 3 -Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu tnotlimite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorize d an d strictl yprohibited
. 

## p . 17 7
T K 12759 , ARALOIT

E M Y 051 0 NOTO X Projec t 16970 6 Cib a Projec t 96462 4 PREFAC E Sponsor Ciba-Geig yLtd . Polymer s Divisio n• Ba u R-1002.2.6 0 CH-400 2 BASE

L Switzerland , Stud yMonito r Dr . E.G . Semaden i Testin g Facilit y NOTO X B.V . Hambakenweterin

g 3 523 1 D D 's-Hertogenbosch Th e Netherland s Stud yDirecto r I r . Y.H.M.-va n Er p Technica l Coordinato r A.M.C. . Berten s Stud yPlan Star t : Marc h28 , 199 6 Completed : Marc

h29 , 199 6 TES T SUBSTANCE , 

## Ident i ficatio n Descriptio n Batc h Purit y Tes t substanc e storage Stabilit yunde

r storag e condition s Expir ydat e Stabilit yi nvehicl e T K 12759 , Araldit e M

Y 051 0 Clear yellowliqui d(Determine d a t NOTOX ) 50008 M > 95% 

## I nfreeze r i nthe dar

k Stabl e Ma y01 , 199 8 Wate r ; a t leas t 9 6 h Acetone : a t leas t 9 6 h GUIDELINES • !

| 

## Th e stud yprocedure .describe

d i nthi s repor t wa

s base d o n the followin g guidelines : DI N3841 2 Par t L 8 , Marc

h1991 : Determinationo f the Inhibitio nEffec t o f Wate r Constituent s o

n Bacteri a (Pseudomona

s Cel l Multiplicatio nInhibitio n Test ) wit hminor ; modifications . NEN-EN-IS O 10712 , 1996 : Wate

r qualit y- Pseudomona

s putid a growth inhibitio ntes t (pseudomona s cel l multiplicatio ninhibitio ntest) .

- Pag e 4 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LLC i s unauthorize d an d strictl yprohibited
. 

## T K 12759 , ARALDIT

E M Y 051 0 NOTO X Projec t 16970 6 C1b a Projec t 96462 4 OBJECTIV E Purpos e o f the stud

y Th e objectiv e o f thi s stud yi s t o evaluat e the " tes t substanc e fo r it s abilit yt o inhibi t the cel l multiplicatio no f the bacteria l specie s Pseudomona

s putida . Furthermore , the toxicit ythreshol dvalue (EC10 ) an d EC50 value (i f possible ) o f the tes t substanc e fo r Pseudomona s putid a i s determined . Pseudomona

s putida . a

n aerobi c gram-negativ

e bacteria l specie s fro

mthe genu s Pseudomonas , i s a natura l inhabitan t o f surfac e waters . Thi s bacteriu m i s a model water organis minvolve di nbiologica l self
purification . Th

e inhibitio no f multiplicatio no f bacteria l cell s o f Pseudomona

s putid a 1 s the selecte dparameter fo r dissolve dtoxi c water ingredients . DEFINITION

S Effectiv e concentratio nfEClQ . EC501 : the concentratio no f tes t substanc e determined afte r a tes t perio do f 1 6 + 1 hou r fro mthe concentration-effec t relationshi p a t whic

hcel l multiplicatio ni s inhibite db y1 0 % o r 5 0 % compare d t o tha t o f the control . E C i s expresse

d i nm g tes t substanc e per litre . Formazin e nephelometri c uni t (FNU1 : Formazin e turbidit yunits . Th

e optica l densit yo f a bacteria l suspensio n a t X=43 6 nm , measure

d as FNU . Biomass : cultur e o f bacteria l cell s afte r a certai ntime , expresse d a s FNU . "Bewertunqszahl" : Assessmen t figur e o r value fo r bacteria l toxicit y expresse d as the negative logarithm s o f the EC10 . Example : EC10 : 15 0 mg/ l « 15 0 pp m» 1. 5 . 10" 4 

## = Bewertungszahl 3.8 . TES T SYSTE M Tes t syste

mPseudomona s putida . Strai nMIGULA , Berli n33/ 2 (DS M 50026) , Source BGA, Berlin , Germany . Rational e Recognize d b yinternationa l guidelines . Stock culture s o f the bacteri a are store di nliqui dnitroge n(~I96°C) .

- Pag e 5 -Th e us e ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LLC i s unauthorize d an d strictl yprohibited
. 

## T K 12759 , ARALDIT

E M Y 051 0 NOTO X Projec t 16970 6 Cib a Projec t 96462 4 TES T SUBSTANC

E PREPARATIO

N Sinc e the tes t substanc e wa s poorl ysolubl e i n water an d n o homogeneou

s suspensio n 1 n water coul d b

e obtained , a stoc k solutio n(A ) o f 10 0 g pe r litr e i n acetonewa s prepared . Thoroug hmixin g wa s performed t o enhance the dissolvin g o f the tes t substance . Solutio nAwa s dilute d1: 1 wit haceton e (stoc k B , 5 0 g/l) . I n the sam e wa ystoc k C (2 5 g/l ) t o stoc k F (3. 1 mg/l ) wer e prepared . REFERENC E SUBSTANC

E 3, 5 ~ Dichloropheno l (9 7 % , Aldrich) . Astoc k solutionlo f 10 0 m g 3,5-dichloropheno l per litr e i n Milli- Q water wa s prepared . EXPERIMENTA

L CONDITION

S AN D SOLUTION

S Vessel s i Incubatio ntemperatur e Incubatio ntim

e ;



## - preliminar ysuspensio

n culture s i

- exposure i Stoc k solutio nI

Stoc k solutio nI I

Stock solutio nII I Stoc k solutio nI

V Cultur e mediu

m(stock ) 10 0 m

l Erlenmeyer flask s 2 3 + 1° C 6 + 1 hour s 16+ 2 hour s 10. 0 g NaN0 3 

## 2. 4 g K2HPO

4 1. 2 g KH 2

## P0 4 

## 1. 0 g yeas t extrac t Dissolved i n 50 0 m

l Milli- Q water . 10. 0 g NaN0 3 

## 2. 4 g K

2 

## HP0 4 

## - 1. 2 g KH 2 

## P0 4 

## Dissolved i n 50 0 m

l Milli- Q water . 40, 0 g D(+")
- glucos e Dissolved i n 50 0 m

l Milli- Q water . 0.0 1 g F e (III ) citrat e 4. 0 g MgS0 4 

## . 7H 2 

## 0 Oissolved i n 1 1 M1111-

Q water . Nutrien t medium : 1 8 g o f aga r dissolve di n water , plu s 5 0 m

l o f solutio nI , 12 5 m

l o f solutio nII I an

d 10 0 m l o f solutio nIV , mad

e u p t o on e litr e wit hwater .

- Pag e 6 -

### p . 18 0 Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d to Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n ofHuntsma n Internationa l LLC i s unauthorize d an d strictl yprohibited
. 

## T K 12759 , ARALDIT

E M Y 051 0 NOTO X Projec t 16970 6 Cib a Projec t 96462 4 PERFORMANC

E O F TH E TES T Preliminarysuspensio

n culture s Asmal l amoun

t o f bacteri a fro

ma maximally7-da

yol d stoc k cultur e o f Pseudomona

s putid a wa

s Inoculate d i n pre-cultur e mediu m (2 5 m l o f stoc k solutio nI an

d II I plu s 5 0 m

l o f solutio nIV , mad

e u p t o on e litr e wit h water ) i n Erlenmeyer flasks . Afte r incubatio nthe fina l turbidit yvalue o f the bacteria l suspensio

n wa s adjuste d u

p t o FNU/436 n

m= 50 . Exposur e Three paralle l dilutio nserie s i n 10 0 m l Erlenmeyer flask s covered wit h aluminiu mcaps wer

e prepare d fro mthe formulate d tes t substanc e stoc k solutio nas described below . Cone . M

i 111
- Tes t Stoc k solutio n Bacteria l tota l tes t wate r solution 1 

## I I I I I I V suspensio n volum e subst . (ml ) (u l o f stock ) (ml ) (m! ) (ml ) FN07436.5 0 

# (•1 ) (pg'i ) (ml ) 10,00 0 3 2 4 A 1 1 2 4 40 2 

## 5,00 0 3 2 4 B 1 1 2 4 4 0 2,50 0 3 2 4 C 1 1 2 4 4 0 1,25 0 3 2 4 D 1 1 2 4 4 0 62 5 3 2 4 E 1 1 2 4 4 0 31 2 3 2 4 F l 1 2 4 4 0 0 3 

## 3 2 4 acetone 1 1 2 4 4 0 

## ° —Ta p water purifie db

yrevers e osmosi s an

d subsequentlypasse

d over activate dcarbo

n an d ion-exchang

e cartridge s (Millipor e Corp. , Bedford , Mass. , USA) . 1 

## = Fina l volum

e o f aceton

e wa s 0.0195 . 2 

## - p H o f on e o f the tes t vessel s wa s 8.1 . 3 

## m 1 0 contro l flask s wer e prepared . Si xflask s wer

e mad e u p identicall yas above , onl y4 m l o f pre-cultur e mediu

m wa s adde d insteado f th

e bacteria l suspensio

n (turbidit ycontrol) . Th e serie s o f the referenc e substanc e wer e prepare d b ydirec t dilution s fro

m the stoc k solutio nas follows : Cone . Milli- Q Referenc e Stoc k solutio n Bacteria l tota l referenc e wate r stoc k so l I I I I I I V suspensio n volum e subst . (ml ) (ml ) (ml ) (ml ) (ml ) FNU/436=5 0 (ml ) (mg/l ) (ml ) 4 0 16. 0 16. 0 1 1 2 4 4 0 2 8 20. 8 11. 2 1 1 2 4 4 0 2 0 24. 0 8. 0 1 1 2 4 4 0 1 4 26. 4 5. 6 1 1 2 4 4 0 1 0 28. 0 4. 0 1 1 2 4 4 0 7 29. 2 2. 8 1 1 2 4 4 0



## - Pag e 7 -

### p . 18 1 Th e us e o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu tnotlimite d to Registration , Evaluation , Authorizatio n an d Restriction ; ofChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LLC i s unauthorize d an d strictl yprohibited
. 

## T K 12759 , ARALDIT

E M Y 051 0 NOTO X Projec t 16970 6 Cib a Projec t 96462 4 Afte r a n appropriat e tim

e i na n incubato r shaker the turbidit yvalue s o f th e cel l suspensions (biomasses ) wer e measured . MEASUREMENT

S Equipmen t Calibratio nsolutio n

Turbidit yvalue ; 

## bacteria l suspensio

n UV/VI

S Spectrophotometer.Lambd a 5 (Perki nElmer , th e Netherlands) , X » 43 6 nm; 1 0 m mlaye r Formazin standard suspensio n (i nFormazin

e Turbidit yUnits ) : FNU/436 n m= 10 0 FNU/436 n m« 7 5 FNU/436 nm* 50 FNU/436 n m» 2 5 FNU/436 n

m. 1 2 FNU/436 n m. 5 0 CALCULATION

S Th e percentage o f cel l multiplicatio nInhibitio nfo r eac hteste d concentratio nwa

s calculate da

s follows : I * B e - a n * 10 0 B e - 8o I Cell multiplication inhibition in% 8 n Measure

d biomas s (extinctio n- extinctio no f turbidit ycontrol ) a t the en d o f the tes t perio dfo r th e n t h

## concentratio no f the tes t materia l B e Measure

d biomas s (extinction ) a t th e en d o f the tes t perio di nth e contro l batch . B o Measure

d biomas s (extinction ) a t the star t o f the tes t (t=0 ) i nth e contro l batch . Th e inhibitio nvalue s calculate dfo r eac hconcentratio nwer

e plotte d agains t the correspondin g concentratio no f the tes t substance . ACCEPTABILIT

Y OFj TH E TES T Th e EC50 value o f the referenc e substanc e 3,5-dlchloropheno l shoul d b e i n the accepte

d rang e o f 10-3 0 mg/l . Th e inoculu muse d i n the contro l batchshoul d b e multiplie db

ya facto r o f a t leas t 6 0 withi nthe tes t period .

I Th e precedin

g criteri a ar e no t absolut e an d othe r modifying factor s might ente r int o the fina l evaluatio ndecision .

- Pag e 8 -Th e us e o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation , Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa l LL

C i s unauthorized'an d strictl yprohibited
. 

## T K 12759 , ARALOIT

E M Y 051 0 ' NOTO X Projec t 16970 6 Cib a Projec t 96462 4 RESULT S Bacteria l toxicit y Afte r incubatio nextinctio nvalue s wer e measure d an d the cel l multiplicatio ninhibitio nfo r eac htes t concentratio nwa

s determined (se e Tabl e 1) . Sinc e n o significan t (significant : >109> ) inhibitio no f cel l multiplication , wa s observe d the toxicit ythreshol dvalue o f T K 12759 , ARALOIT

E M Y 051 0 fo r Pseudomona

s putid a wa

s ove r 1 0 mg/l . Acceptabilit yo f the tes t Th e EC50 value o f 3,5-dichloropheno l determined fo r Pseudomona s putid a a

s calculate db

ylinea r regression( Y * • 6.816 9 X -83.9824 ) wa

s 2 0 mg/ l (se e Tabl e 2) . Th e inoculu muse d i n th e contro l batchwa

s multiplie db

ya facto r 7 7 withi n the tes t period . Withrespec t t o the accepte d rang e o f 10-3 0 mg/ l fo r the EC50 o f the referenc e substanc e an

d the multiplicatio nfacto r o f the bacteri a withi n the tes t bein

g over 60 , 1 t wa

s conclude

d tha t the tes t .conditlonswer e optima l an

d the result s obtaine d wer e valid . CONCLUSIO N Base d o n the result s o f the .tes t presentl yperformed , the toxicit y threshol dvalue (EC10 ) o f T K 12759 , ARALOIT

E M Y 051 0 wa s over 10,00 0 mg/l . Th e "BewertungszahlVi s therefor e < 4, 9 (Bewertun g wassergefahrdende r Stoffe , 1989) .

- Pag e 9 -

### p . 18 3 Th e use ofthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t not limite d to Registration , Evaluation,
. Authorizatio n an d Restrictio n o fChemica l Substance s (REACH) , withou t th e expres s writte n permissio n o fHuntsma n Internationa ! LL

C i s unauthorize d an d strictl yprohibited , 

## T K 12759 , ARALDIT

E M Y 051 0 NOTO X Projec t 16970 6 Cib a Projec t 96462 4 TABL E 1 i 

## Extinctio nvalue s o f the inoculate ddilutio nserie s I , II , 111 , the noninoculate ddilutio nserie s t (turbidit ycontrol ) an d the contro l flask s 8 .

CONCENTRATIO

N o f T K 12759 , EXTINCTIO NI NFN

U 43 6 n b CORRECTE D INHIBITIO N ARALDIT E M

Y 051 0 serie s MEA N MEA N 

### % (ug'l ) I I I 

### i n I,II,I I 

### t 312. 5 

## a 29 1 1.21 9 1.29 2 1.26 7 0.00 4 1.26 3 2. 8 62 5 i 259 1.35 6 1.24 8 1.26 8 0.00 1 1.28 7 1. 0 125 0 l .23 1 1.20 1 1.17 3 1.20 2 0.00 1 1.20 1 7. 7 250 0 1.23 1 1.19 1 1.23 4 1.22 2 0.00 1 1.22 1 6. 1 500 0 1.29 1 1.39 4 1.29 4 1.32 6 0.00 1 1.32 5 -2, 0 1000 0 1 .17 2 1.29 3 1.22 2 1.22 6 0.00 2 1.22 4 5. 9
CONTRO L 1.52 0 MEA N1.29 9 6 0 0.016 8 1,17 5 1.34*5 1,20 1 1.34 9 1.25 1 1.41 0 1.37 6 1.29 7 1.26 5



## TABL E 2 Extinction values ofthe reference substance dilution series: 3,5-dichlorophenol .

CONCENTRATIO

NEXTINCTIO NI NFN U 43 6 n mINHIBITIO N 3,5-dichloropheno l sarle s r X

7 1.32 4 -1. 9 1 0 1.26 4 2. 7 I d 1.26 9 2. 4 2 0 0.42 4 68. 2 2 6 0.01 3 100. 1 4 0 0.01 6 100. 0 I



## - Pag

e 1 0 -Th e use o fthes e studies/documents fo rregulator yo rregistratio n purposes , includin g bu t no t limite d t o Registration , Evaluation ; Authorizatio n an d Restrictio n o fChemica l Substances (REACH) , withou t th e expres s writte n permissio n o fHuntsma n e d an d strictl yprohibited
. PS + Ft, POZ 1 Eing
. 3 >.^f c U J . * i

5
. !