STUDY REPORT Sponsor: Monitor: Order no.: Test Faciity: Date:

Page 1 of 24Study Report TABLE OF CONTENTS 1 OBJECTIVES .......

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4 2 TEST ITEM

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4 3 MATERIALS

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.. .. 4 3.1 Reference and Calibration Items ......................................................................................................

4 3.2 Chemicals and Materials ...................................................................................................................

4 3.3 Instruments

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... .. 5 4 METHODS ............ ...
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5 4.1 NMR Spectroscopy ............................................................................................................................

5 4.2 Mass Spectroscopy .....................................................................................

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5 5 RESULTS AND DISCUSSION

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.. ....... .. . .... ............ .... . ? 6 SUMMARY
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. ........ ... 24 7 PERSONNEL

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.. 24 8 CONFIRMATION OFTHESTUDY REPORT

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. .............. ...... .............................. 24 page2 of24Study Report FIGURES Fig. 1 Chemical structure of the test item ..............................................................................................

4 Fig. 2 Change of pH value during titration with- solution .........................

. ........................................

8 Fig. 3 Change of temperature during titration with- solution .............................................................

8 Fig.4 Suggested chemical structure at RT 9.5 min of main peak ................... ........ ...................................

9 Fig. 5 Suggested chemical structure at RT 9.5 and 16.6 min of main peak ...............................................

9 Fig. 6 Suggested reaction of- at high pH values ...............................

....

. ................... ........................

9 Fig. 7 Base Peak

( +ESI/MS), detail: V1 to V7 .............................................................................................

1 0 Fig. 8 Base Peak (-ESIIMS), detaii:V1 to V7 ..............................................................................................

11 Fig. 9 Detail: (1) Base Peak and (2) +ESIIMS at RT 9.5 min of main peak (V4) ......................................

12 Fig. 10 Detail: (1) Base Peak and (2) -ESI/MS at RT 9.5 min of main peak (V4) ...................................
12 Fig. 11 Detail:{1) Base Peak and (2) +ESIIMS at RT 14.6 min (V4) ...........................................................

13 Fig. 12 Detail: (1) Base Peak, (2) lon trace at m/z 471 and (3) -ESIIMS at RT 14.6 min (V4) ................... 13 Fig. 13 Suggested chemi:::al structure at RT 14.6 min .................................................................................

14 Fig. 14 Comparison of 13 C-NMR spectra, detailed carbonyl region, bottom blue( .. in **~0.** pH 4). XTAN reaction ... 14 Fg. 15

1 H-NMR spectrum of test item Fig. 16 **I.~C-NMR** spectrum of test item Fig. 17 ':C-NMR spectrum of test item Fig. 18 ':C-NMR spectrum of test item Fig.19 't-NMR spectrum of test item Fig. 20 ':C-NMR spectrum of test item 1"1 Fig. 21 ·c-NMR spectrum of test item Fig. 22 L'c-NMR spectrum of test item Fig. 23

1 H-NMR spectrum of test item Fig. 24

1 H-NMR spectrum of test item Fig. 25

1 H-NMR spectrum of test item Fig. 26

1 H-NMR spectrum of test item Fig.27

1 H-NMR spectrum of test item Fig.28

1 H-NMR spectrum of test Fig. 29

1 H-NMR spectrum attest item Fig. 30

1 H-NMR spectrum of test i:em

in D 2 0 .. ...
. .......... ...............

. ....................

.. .... ....... .. 15 D20 .......................................................................

15 after 1000

I additbn,0.5h .................................
16 I additbn, 24h ..................................
16 after 2000

I additbn, 1h ....................................
17 I addition, 24h ..................................
17 I addition, 1h ..... .... .............

.. ............ 18 after 2600

I addition, 24h .................................
18 after 1000

I addition, 30 min ..................................
19 after 1000

I addition, 16h ...................................
20 after 1000

I addition,24h ....................................
20 after 2000

I addition, 30 min ..............................
21 after 2000

I addition

, 24h ...........................
21 2600

I add.,30 min (pH shift2.9 ppm!) ............. 22 2600

I additbn, 24h ...................................
22 Fig. 31 HMBC spectrum of test item **,~~~~after** 1000

I addition, 24h .........................................

23 Fig. 32 HMBC spectrum of test item
I after 2000

I addition, 24h .........................................

23 TABLES Tab.1 Tab.2 Tab.3 Tab.4 Tab.5 Test item data ......................................................................................................................................

4 Chemicals actually used as reference and calibration items .................. ...... ...................................

4 Chemicals and materials used in the study .......................................................................................

4 HPLC and MS parameters ..................................................................................................................

6 Physical properties pH and temperature during titration ...................................................................

7 page 3of24StudyReport1OBJECTIVESThepurposeofanalysisistodevebpthereactionof-hydrolysisindifferentdilutions.Thepresenceofcyclicureamoleculesshouldbetested.2TESTITEMTab.1Testitemdata3MATERIALS3.1ReferenceandCalbrationItemsTab.2Chemicalsactuallyusedasreferenceandcalibrationitems•3.2ChemicalsandMaterialsTab.3Chemicalsandmaterialsusedinthestudypage4of24Study Report 3.3 Instruments NMR spectrometer Avance 111600 (Bruker, Karlsruhe, D), magnetic flux density 14.1 Tesla ONP cryo probe head; automated sample changer Bruker B-ACS 120 Computer Intel Core2 Duo 2.4 GHz under MSWindows XP and BrukerTopSpin 2.1 for acquisition Bruker TopSpin 2.1 for processing Standard operation procedure SAA-GMR028-02 Mass spectrometer Finnigan LTQ {Thermo Electron Corp.

, San Jose, CA) ±ESI modus, introduction via LC Gradient pump Rheas2000 (Flux Instruments, Basei,CH) Degassing unit Online-DEGASSER ERC-3415
a (ERC GmbH, Riemerling

, D) Autosampler HTC PAL System (CTCAnalytics AG, Zwingen, CH) Column oven L-5025 (Merck, Darmstadt, D) Diode array detector UV6000LP (ThermoFinnigan

, San Jose, CA) Computer PIV 2B GHz under Windows XP and Software Xcaibur2

. 0. Standard operation procedures SAA-GMR011-02

, -GMR012-03 and -GMR016-02 Electronic semi-micro balance Sartorius R 180 D-"•D1 (Gottingen, D) Standard operation procedure SAA-G MR005-04 4 METHODS 4.1 NMRSpectroscopy 1 H-NMR and 13 C-NMR spectra were recorded directly after sample preparation during the titration process and after different reaction times

. The actually used NMR parameters appear on the spectrum plot. 4.2 MassSpectroscopy HPLC/MS analyses (±ESI) were recorded from a second titration trial of severa

l addition steps (adding of 50 ul , 100 ul, 150 ul, 200 I-ll and 400 ul , equivalent to V1 to V5). The last titration samples were additionally heated (equivalent to V6 and V7)
. The sample solutions were diluted and introduced by LC / MS coupling with column. page 5of24Study Report Tab.4 HPLC and MS parameters Separation Column: Nucleosil120 5C18 EC,51Jm, 125x 2 mm (No. 56) Mobile Phase: A: Acetonitrile+ 0.1% HCOOH B: Water + 0.1% HCOOH Gradient:

t [min] A[%) 8[%] 0
10
90 40
90
10 45
90
10 45.1
10
90 50
10
90 Flow: 230 IJI!min Injection Volume: 10 IJI Column Temperature: 40°C Detection Wavelength: 200-600 nm (PDA) Split:

- Scan Parameter

: 50-2000 amu Number Micro Scans in Time:

5 in 200 ms Polarity and Source Type: +ESI -ESI Capillary Temperature: 275°C
275 ° C Sheath Gas Flow: 40

40 Aux Gas Flow:
0

0 Sweep Gas Flow:

0

0 Spray Voltage: 5kV 4.9kV Capillary Voltage

: 38V -34V Tube Lens

: 125V -100V page 6 of24Study Report 5 RESULTS AND DISCUSS ION 5 ml test solution of- (100 mg/ ml D.?O) was prepared. A NaOD solution (pH 13) was equipped with a magnetic stirrer and a device for monitoring pH values and temperature.
In several steps of 100 IJI each of the -solution was added, pH value and temperature were recorded. Sample preparation was done after adding 1000 IJI, 2000 IJI, 2600 IJI (pH and temperature jump) and 3000 IJ by filling approx. 600 IJI of the reaction mixture into a NMR tube. Tab. 5 shows the reaction kinetic data

. Tab.5 Physical properties pH and temperature during titration Comments

: Samples taken at yellow highlighted titration steps The titration curves are shown in Fig. 2 and Fig. 3 . The step by step addition of - solution changes the pH only slightly until approx. 2300 IJI were added. The buffer capacity was reached at 2500 IJI. At 2600 IJI addition the pH dropped down comparable to a titration point. Further addition of - solution shows linear strong decrease of the pH value. After this titration point the reaction mixture became cloudy which is an indicator of intermolecular polymerisation

. The reaction mixtures taken before stayed clear even after 24h. The temperature increases nearly linear indicating an exothermic reaction

. At the titration point the temperature dropped down and rises up in the same linear way after further adding of - solution. The NMR spectra ndicate a first and fast chemical reaction of- with NaOD at high pH values

, the splitting of sulfonic acid and the formation of the carbaminic acid. This molecule dominants the NMR spectra after short reaction times. The further degradation
to the amine is a slower reactbn and can be observed over hours and days at these high pH values (see comparison e.g. of Fig. 17 and Fig
. 18).

page 7 of24Fig, 3 10,.50 10,1.10 27 **26,~** 26 2S..S p 25 .s ,l4,5 #! **~ ~.s**  23 u.s 22 Study Report pH Variation durlns Titration ... ·--.. ...... ..... .. **·~** 0 Temp. Variation during Titration ..... ........ ....... .......

y ......... ...... ...... .... !• 0 500 Altar the caneu.mption of the **~** NaOO the 9H dropped down and the eeconcfary lll8diorl etarta fcrmJnQ the emlno fllnction by loss of **~** followed by 1t1e tttltd ructiOn atep of forming the utea 811uctln$. .. The tnttamalar:ular reac:ttan d is e GOmpetltlve reaction 10 the lnlemlolec:Uiat **~~.** The end producta are molecules or partial cyclic **a~ructum** (d. Ag. 4 and Fig. 5). The Sll\letuMA were confirmed by the HPLCIMS **anal~** of **se~** reac1ton mblt.na. page 8 of24Study Report .. s l !
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( 1) Base P e ak and (2) -ESIIMS at RT 9.5 min of main peak (V4) page 12 of 24• I

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: (1) Base Peak,(2) lontrace at m/z471 and (3)-ESI/MSat RT 14.6 min (V4) page 13of 24IStudyReportnMass=426.331839DaFig.13SuggestedchemicalstructureatAT14.6min..................Iuu••..-..apage14of24\ l'Ol.."''::';O at: ..... !>rnmCfo'CflP1t<t "'"' **t:~'\36** '""' I. 7 CI'J.:!6 **;-a** If: **o:~~H: 'Jt~:f·&N.:**  10 **.-.~"'\!.a.uc** . ..,_ **~01";** ttX'JO(t)(IIJOs .... 1 **lftA.,..~tH•** .. ,. IH 1.:0CIU"AC. ....... **11'01t~m1U\V** t.OC!.7:!10Sif.H.- """'"' **t't.O~.W7l"'r.tlt'** FU 0 (130>1.- • 100 Study Report p) .... l Fig.15

1 H-NMR spectrum of test item -in

0 2 0 • D• -· -- . ·- " . --. -- **~.'** -· . . ··- **'A~** Fig.16

•• II page 15 of 24Study Report **l·'~ ~r"** 

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13 C-NMR spectrum of test item-after 1000 1-11 addit

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13 C-NMR spectrum of test item -after 1000 1-11 addition, 24h page 16of24·-· ..... . , . .... Fig.19 .... , .. **~..** ..... . -· ...... ,""" ·-u• ''"'"' '1) .. _.,,,. .. , .... . **~..:**
... **~:lll'··~···· '","i,~l,.""·:::**  ·.-. ···:--r_: .... Fig . 20 Study Report __ **,.C'M.2mi~Ufrod~n** 13 C - NMR spectrum of test item -after 2000 IJI addition, 1 h ........ 13 C-NMR spectrum of test item--after 2000 JJI addition, 24h page 17 of24Fig. 21 . . '

. ,. .. ···" Study Report 13 C-NMR spectrum of test item- after 2600 **~I** addition, 1 h -. ..... page 18 of24Study Report **~1~** 1:":\t\ !.rY'I'tltmHr,,, :;:• 1 C!o!...'-'1 **1:7'~** If. **14'~!-;'f'oH:** U 181:":'!-H: :-r.5('o.l...::-t'lt« 11.0 40.5J.iUWw." **C.~~Atr<.** 7..6a,... 1flO((W\O(,I(.tM'\- 1 (.;if,l,.H"4!

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•• l .. _ Fig. 25

1 H-NMR spectrum oftest item -after 1000 **~I** addition, 16h **~"•** Ctu.HHf.l t1 '" 1.4 aCJutae .... .., 1i'01007C10W (Q)61JI'OI:aa.""' , .. **cor.~t61J.'.It** Eu 0 v,.,H: 0 "''
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· Fig. 26

1 H-NMR spectrum of test item •••• after 1000 **~I** addition, 24h page20 of24_. t".ACRlTlP Hi :>311 e•1Jo31, ""' " ' **t1~~tlr o:~K:**  "!t!Aio4i.""C'MC 1.12 .. .,...., C!;(lu:lilf": :'!"JOC.illf **\UI(.(JCJI)"i()~**

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. 27

1 H-NMR spectrum of test item- after 2000 lJI addition, 30 min ·-•CIJ.Utlet,!l.-

.. ,. ..... ._. 1H 1 .. 00'\IM(. .... l291tffttOVI GOD 572'JON.Mt17 ..... **OQCI!«lllr.'M~tl%** w " 031)1-1.· 0 "" _______ L _____ __ _ **XTANV~Ne0H 2ni~**  ... \7 Fig.28

1 H-NMR spectrum of test item **~fter** 2000 IJI addition, 24h •• page 21 of 24•• • tUJ0!1.:6tu **01~11~** .u,.c.a..a:-Es.e: 12i **.:ow~** .,.,_ ..... 100000000- , **~·Ct\.&JMI** f1••••••-.oo Ill 1.:QOw.«. ........ **~7$U~1t1.0W O(il)~-;:r.mr.J.Ifi.·**  **r.~** f«• •A¥.00

. "17 WI C:. IU • O:IOtf: • "'' Study Report Fig.29

1 H-NMR spectrum of test item-after 2600 !JI add., 30 min (pH shift 2.9 ppm!) CHI. .. Hlln •• • •·- "' 1 **~** "'"' UC;f.t. ....... 12 91911610 Ill liOO.J7.11CitaT.Wir r;<>)!l tcn:.oo')1Atii.JIV Ul • il:.JII.; 0 '"" 1-----

- -·- --
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- --- - - ---.---

· --- •• Fig. 30

**~2 tm124t\**  **·~** i \7 page 22 of 24Fig. 31 Fig.32 Study Report -- - -- - **-~**

. **-L ~~~**  **~==~ ---~-** 

-

, ppm . . **~** 1
... **-~** ·· · ; · **~.** .; ... • ' · ; **-~** ; **-~**

- ----t-

. (·-- : · .. .. **~**1
r I 2. 5 2.0 +- -·- , : . **.- ~**  t . ! · -t ' l . =-- - - 1 . f ' "" **~**

.. **~** - r **~** ---......,..=--- 1 .5
1. 0

· !. 0 'T 20 · 60 I ! L : : rl 120 : · 140 :' 1r 60 .. ;l : HMBC spectrum of test item --after 1000 JJI addition, 24h a i .;,--.

j • **~** " ! . , ..... .. . ;. --+-

.... **----~-----** 4.0 3.5 3.0 2.5 2.0 1.5 **~** ' ! .j ! 1------· 1.0

• 80 l ,oo :I ;·c 120 • 140 160 180 .j f 200 \J 0.5 HMBC spectrum of test item-after 2000 JJI addition, 24h **-~·~---** ....,,
.. .. .... _ .. .._.._ "''

, ..... .. ,.,, ...

, , ....... . WoOl

• **.,~** .... **--~ n--**  '"'"
.. ...,. w•- " ··- ""' uoa "LfW t2..11t1H•w POt Mel PU1t1 Mel **-~ o-**  **-·** '""' "" .., 11- - "-2 .... . ••uw .. ,......, ., lfO: llfi::IIC. "» .... **---~HTC: .....,...**  **•- ~~**  IIIC
1 :- **~** .. . . ...,.... .... , . CJI'n ..... ... OI'J2 .. .. **~** .. . .. .... -- - -

, "' "' .01 ,.,.., ""' **~~** tMM mHI ::.0... =- -:,.- . - .. **-~ Mkl**  ..... - ... . ......
... .. . " .... . - ""' ., • 1\l.IDr» ... - - - u ...
. ..,_ .. . page 23of24Study Report 6 SUMMARY The degradation
of strongly pH dependent. At pH values > 12 the hydrolysis product which is relatively stable at hgh pH values

, Degradaton to the corresponding amino compound needs hours up to several days. Lowerirg the pH value enforces
a faster reacton to the amine and inter- resp. ntramolecular formation of urea oligomeric and polymeric compounds. The formation of cyclic moecules could be confirmed by HPLC/MS chromatography

. 7 PERSONNEL Study director: Co-worker: Qually assurance: All are staff members of the test facility

. 8 CONFIRMATON OF THE STUDY REPORT Date: Study director Chemist Chemist Food chemist Biological technical assistant Company stamp: Page 1 of the original report is printed on paper; the report is authorised by original signature and company's stamp. Use or publication
in parts is not authorised on principle and is not allowed to be connected with the company's name or a signature of a staff member. Misuse will be prosecuted.

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