## Harcros Chemicals

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Kansa s City, K S 6610 6 Telephone : 913/621-774

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6 E-maif : OrganicsCS@Harcros.co m Dat e 03/07/1 3 Documen t Contro l Offic e (DCO ) (7407M ) Offic e o fPollutio n Contro l an d Toxics (OPPT) U.S

. Environmenta l Protectio n Agenc y(EPA) 120 0 Pennsylvani a Ave
. N Wr- » Washington , D.C

. 2046 0 (202-564-8930 ) g Attn : TSCASectio n 4 40 CFR § 799.5087

(Docket # EPA-HQ-OPPT-2007-0531) ^ %

. ~o<r> 

# i > RE : Micronucleou s Tes t i n th e Mous e CASR N 31138-65- 5 £pj ^ T o Who m I t Ma

yConcern : ^ - I n accordanc e wit h EPA' s fina l rule , Testin g ofCertai n Hig h Productio n Volum e Chemicals ; Secon d Grou p o fChemicals , publishe d i n th e Federa l Registe ro n Januar y7,201 1 at 7 6 fed

. Reg
. 1067 , pleas e fin d enclose d th e fina l repor t fo rth e Micronucleou s Tes t i n th e Mous e o n D-gluco-Heptoni c acid , monosodiu m salt , (2.xi.) - [CAS # 31138-65-5]
. Thi s stud ywa s conducte d t o fulfil l th e requirement s oftestin g categor yE a s se t fort h i n Tabl e 2 of4 0 CF R 799.5087G)

. I fan yfurthe rinformatio n i s required , pleas e d o not hesitat e t o contact m e a t (913 ) 62 1 -780 2 o rat asavini@harcros.com

. Regards ,

An n Mari e Savin i Harcro s Chemical s Inc
. Regulator ySpecialis t cc : Pete rRadford Howard Stevenso n Jame s Grantha m Rand yBuckne r Do n Meuschke Joh n Mile s Fil e 4 0 1 300000 2



## IS O O N SNIV1NO OPAG E 1 O F 2 4 PAGE S 

# harla n

Sodium Glucoheptonate: MICRONUCLEU S TES TI N TH E MOUS E PROJEC TNUMBER : 4110469 0

AUTHOR : LFlander s

STUD YSPONSOR : Harcro s Chemical s Inc
. 345 2 Corporat e Driv e Dalto n G A3072 0 UNITE D STATE S O F AMERIC A TES TFACILITY : Harla n Laboratorie s Lt d Shardlo wBusines s Par k Shar d low Derbyshir e DE7 2 2G D U K Telephone : +4 4 (0 ) 133 2 79289 6

41104690/L F Facsimile : +4 4 (0 ) 133 2 79901 8PROJEC TNUMBER : 4110469 0 PAG E 2 QUALIT YASSURANC E REPOR T Th e Genera ] Stud yP/a n fo rthi s stud ytyp e wa s reviewe d fo rcomplianc e onc e onl yo n initia l production
. Th e conduct o fthi s stud yha s bee n subjec tt o periodi cinspectio n by th e Qualit yAssuranc e Unit
. In addition, general

facilities

are inspected

at least once a year and the results are reported to management Thi s report ha s bee n audite d b yth e Qualit yAssuranc e Unit , an d i s considere d t o b e a n accurat e accoun t o fth e dat a generate d an d o fth e procedure s followed

. I n eac h case , th e outcom e o fQAevaluatio n i s reporte d t o th e Stud yDirecto ran d Managemen t o n th e da yo fevaluation. Audit s o fstud ydocumentatio n an d inspection s appropriat e t o thi s stud ywer e a s follows :

0 1 Jul y201 1 Genera l Stud yPla n Complianc e Audi t § 2 8 Novembe r201 2 Assessmen t o fRespons e § 2 8 Novembe r201 2 Harves t 0 9 January201 3 DraftReportAudi t § Dat e o fQ ASignatur e Fina l Report Audi t § Evaluatio n specifi ct o thi s stud y

Fo rth e Qualit yAssuranc e Unit * DATE : 

## 1 8 JA N 

## 2013

'Authorise d Q ASignatures : Senio rAudi t Staff : J G Rile yBS c(Hons ) MRQA , J M Crowthe rMISc TMRQA , G Wre n ON C MRQA , S Beva n BS c(Hons ) MRQA , LBlane yMRQ AP
. 4
PROJEC TNUMBER : 4110469 0 PAG E 3
GL P COMPLIANC E STATEMEN T Wit h th e exceptio n note d beiow , th e wor k describe d wa s performe d i n complianc e wit h U KGL P standard s (Schedul e 1 , Goo d LaboratoryPractic e Regulation s 199 9 (S I 1999/310 6 a s amende d b yS I 2004/0994))

. Thes e Regulation s ar e i n accordanc e wit h GL P standard s publishe d a s OEC D Principle s o n Goo d LaboratoryPractic e (revise d 1997 , ENV/MC/CHEM(98)17) ; an d are i n accordanc e with , an d implement , th e requirement s o fDirective s 2004/9/E C an d 2004/10/EC

. N o analysi s wa s carrie d ou t t o determin e th e homogeneity , concentratio n o rstabilit yo f th e tes t ite m formulation

. Th e tes t ite m wa s formulate d withi n tw o hours o fi t bein g applie d t o th e tes tsystem ; i t i s assume d tha tth e formulatio n wa s stabl e fo rthi s duration
. Thi s exceptio n i s considere d no tt o affec t th e purpos e o rintegrit yo fth e study
. Thi s reportfull yan d accuratel yreflect s th e procedure s use d an d dat a generated
.

Thi s repor

t ma ybe presente d i n final for m a s a digita l (pdf) document
. Suc h documents ar e prepare d b

yscannin g th e pape roriginal , an d ar e considere d o

fequivalen t integrit

yan d authenticit

yto version s produce d b

yoptica l photocopy
. However , i n al l cases th e hand-signe d pape roriginal , hel d i n secur e archives , i s th e definitive document , DATE : 

## 1 6 JA

N 

## 201 3 LFlander s Stud yDirecto rp . 5
PROJEC TNUMBER : 4110469 0 PAG E 4
CONTENT S QUALIT YASSURANC E REPOR T2 GL P COMPLIANC E STATEMEN T3 CONTENT S 4 SUMMAR Y5 1 . INTRODUCTIO N 7 2 . PRINCIPLE S O F INVESTIGATIO N 7 3 . TES TAN D CONTRO L ITEM S AN D EXPERIMENTA L PREPARATIO N 8 3. 1 Tes t Ite m 8 3. 2 Positiv e Contro l Ite m 9 3. 3 Vehicl e Contro l Ite m 9 4 . METHOD S 9 4. 1 Animal s an d Anima l Husbandr y9 4. 2 Procedur e 1 0 5 . ARCHIVE S 1 3 6 . RESULT S 1 4 6. 1 Range-findin g Toxicit yTes t1 4 6. 2 Micronucleu s Tes t1 5 7
. CONCLUSIO N 1 5 Tabl e 1 Micronucleu s Tes t - Summaryo fGrou p Mea n Dat a 1 6 Tabl e 2 Micronucleu s Tes t - Individua l Dat a an d Grou p Mean s an d Standar d

Deviations : Vehicle Contro l (1 0 ml/kg ) 24-Hou rSamplin g Tim e 1 7 Tabl e 3 Micronucleu s Tes t- Individua l Dat a an d Grou p Mean s an d Standar d

Deviations : Cyclophosphamid e (5 0 mg/kg ) 24-Hou rSamplin g Tim e 1 8 Tabl e 4 Micronucleu s Tes t - Individua l Dat a an d Grou p Mean s an d Standar d

Deviations : Tes tIte m (200 0 mg/kg ) 48-Hou rSamplin g Tim e 1 9 Tabl e 5 Micronucleu s Tes t- Individua l Dat a an d Grou p Mean s an d Standar d

Deviations : Tes tIte m (200 0 mg/kg ) 24-Hou rSamplin g Tim e 2 0 Tabl e 6 Micronucleu s Tes t - Individua l Dat a an d Grou p Mean s an d Standar d

Deviations : Tes t Ite m (100 0 mg/kg ) 24-Hou rSamplin g Tim e 2 1 Tabl e 7 Micronucleu s Tes t - Individua l Dat a an d Grou p Mean s an d Standar d

Deviations : Tes t Ite m (50 0 mg/kg ) 24-Hou rSamplin g Tim e 2 2 Appendi x1 Historica l Contro l Dat a (200 9 an d 2010 ) 2 3 Appendi x2 Statement o fGL P Complianc e i n Accordanc e wit h Directiv e 2004/9/E C 2 4PROJEC TNUMBER : 4110469 0 PAG E 5
Sodiu m Glucoheptonate : MICRONUCLEU S TES TI N TH E MOUS E

SUMMAR Y Introduction
. Th e stud ywa s performe d t o asses s th e potentia l o fth e tes t ite m t o produc e damag e t o chromosome s o raneuploid ywhe n administere d t o mice
. Th e metho d wa s designe d t o b e compatibl e wit h th e 199 7 OEC D Guideline s fo rTestin g o f Chemical s No.47 4 "Mammalia n Erythrocyt e Micronucleu s Test" , Metho d 81 2 o f Commissio n Regulatio n (EC ) No
. 440/200 8 o f3 0 May2008 , th e U S EP A(TSCA ) OPPT S 870.5395 , EP A712-C-98-226 , Augus t 199 8 guidelines , an d b e acceptabl e t o th e Japanes e METI/MHLW/MAF F guideline s fo rtestin g o fne wchemica l substances
. Methods
. Arange-findin g tes t wa s performe d t o fin d suitabl e dos e level s o fth e tes t item , rout e o fadministration , an d t o investigat e i fther e wa s a marke d differenc e i n toxi c respons e betwee n th e sexes . Ther e wa s n o marke d differenc e i n toxicit yo fth e tes t ite m betwee n th e sexes ; therefor e th e mai n tes twa s performe d usin g onl ymal e mice
. Followin g discussion s wit h th e Sponsor , th e micronucleu s tes t wa s conducte d usin g th e intraperitonea l rout e i n group s o fseve n mic e (males ) at th e maximu m recommende d dos e (MRD ) o f200 0 mg/k g an d wit h 100 0 an d 50 0 mg/k g a s th e tw o lowe rdos e levels
. Animal s wer e kille d 2 4 o r4 8 hours later , th e bon e marro wextracted , an d smea r preparation s mad e an d stained
. Polychromati c(PCE ) an d normochromati c(NCE ) erythrocyte s wer e score d fo rth e presenc e o fmicronuclei
. Additiona l group s o fmic e wer e give n a singl e intraperitonea l dos e o fphosphat e buffere d salin e (PBS ) ( 7 mal e mice ) o rdose d orall ywit h cyclophosphamid e ( 5 mal e mice) , t o serv e a s vehicl e an d positiv e control s respectively
. Vehicl e an d positiv e contro l animal s wer e kille d afte r2 4 hours
. Results
. Ther e wer e n o prematur e death s see n i n an yo fth e dos e group s i n th e mai n test
. Clinica l sign s wer e observe d i n animal s dose d wit h th e tes t ite m at an d abov e 100 0 mg/k g i n bot h th e 2 4 an d 48-hou rdos e groups , wher e applicable , an d include d hunche d postur e an d ptosis
.PROJEC TNUMBER : 4110469 0 PAG E 6 Statisticall ysignifican t decrease s i n th e PCE/NC E rati o wer e no t observe d i n an yo fth e tes t ite m dos e group s whe n compare d t o th e vehicl e contro l group . However , th e observatio n o fclinica l sign s wa s take n t o indicat e tha t systemi cabsorptio n ha d occurre d an d exposur e t o th e targe t tissu e ha d bee n achieved
. There was no evidence

of any statistically

significant

increases

in the incidence

of micronucleated

polychromatic

erythrocytes

in animals dosed with the test item when compared

to the vehicle control group. Th e positiv e contro l grou p showe d a marke d increas e i n th e incidenc e o fmicronucleate d polychromati cerythrocyte s henc e confirmin g th e sensitivit yo fth e syste m t o th e know n mutageni cactivit yo fcyclophosphamid e unde rth e condition s o fth e test
. Th e tes t ite m wa s foun d no tt o produc e a toxicologlcall ysignifican tincreas e i n th e frequenc yo fmicronucle i i n polychromati cerythrocyte s o fmic e unde rth e condition s o f th e test
. Conclusion

. Th e tes t ite m wa s considere d t o b e non-mutageni cunde rth e condition s o f th e test
.p . 8
PROJEC TNUMBER : 4110469 0 PAG E 7
Sodiu m Glucoheptonate : MICRONUCLEU S TES TI N TH E MOUS E 1
. INTRODUCTIO N Th e micronucleu s tes ti s a mammalia n i n viv o tes ttha t detect s damag e t o th e chromosome s induce d bychemicals
. I n addition , numerica l change s du e t o chromosom e los s durin g cel l divisio n ca n b e detecte d i n thi s test
. Th e stud ywa s performe d accordin g t o a metho d tha t wa s designe d t o b e compatibl e wit h th e 199 7 OEC D Guideline s fo rTestin g o fChemical s No.47 4 "Mammalia n Erythrocyt e Micronucleu s Test" , Metho d B1 2 o fCommissio n Regulatio n (EC ) No
. 440/200 8 o f3 0 Ma y2008 , th e U S EP A(TSCA ) OPPT S 870.5395 , EP A712-C-98-226 , Augus t199 8 guidelines , an d b e acceptabl e t o th e Japanes e METI/MHLW/MAF F guideline s fo rtestin g o fne wchemica l substances
. Th e result s o fth e tes tar e believe d t o b e o fvalu e i n predictin g th e mutageni cpotentia l o fth e tes t ite m t o man . Th e tes tsyste m wa s chose n becaus e th e mous e ha s bee n show n t o b e a suitabl e mode l fo rthi s typ e o f stud yan d i s th e recommende d specie s i n th e tes t method
. Th e experimenta l phase s o fth e stud ywer e performe d betwee n 2 8 Februar y201 2 an d 1 1 Decembe r2012
. 2 . PRINCIPLE S O F INVESTIGATIO N I n mitoti ccell s i n whic h chromosom e damag e ha s bee n cause d byth e tes t ite m o rit s metabolites , fragment s (centri co racentric ) o rwhol e chromosome s ten d t o la g behin d i n th e anaphas e stag e o fcel l division
. Afte rtelophas e a larg e proportio n o fth e fragment s are no t include d i n th e nucle i o fth e daughte rcell s an d henc e for m a singl e o rmultipl e micronucle i (Howell-Joll ybodies ) i n th e cytoplas m o fthes e cells
. Thes e micronucle i ar e see n i n a wid e variet yo fcel l types , bu terythrocyte s ar e chose n sinc e micronucle i are easil ydetecte d i n thes e cells
. Afe whour s afte rth e las t mitosi s i s completed , erythrocyte s expe l thei rnuclei
. Immatur e erythrocytes , les s tha n 2 4 hours old , stai n blu e wit h May-Grunwald/Giems a du e t o th e presenc e o fminut e fragment s o fnuclea rmateria l i n th e cytoplasm
. Thi s materia l i s mainl yribonuclei caci d (RNA) , whic h graduall ydisappear s s o tha tmor e matur ePROJEC TNUMBER : 4110469 0 PAG E 8 erythrocyte s (normochromati cerythrocytes ) stai n pin k wit h May-Grunwald/Giemsa

. Th e immatur e blu e stainin g cell s ar e know n a s polychromati cerythrocyte s an d mauv e staine d micronucle i ar e easil ydetecte d i n thi s cel l type
. I fscorin g i s restricte d t o polychromati cerythrocytes , al l th e chromosoma l damag e detecte d wil l hav e bee n cause d durin g th e fina l cel l cycl e o fth e nucleate d precurso rcells
. Thu s byexaminin g polychromati ccell s a t variou s period s afte radministration , th e effec to fth e tes tite m ove r th e previou s 3 0 hour s ca n b e monitored
. An ytoxi ceffect s o fth e tes t ite m o n th e immatur e nucleate d cell s maylea d t o a reductio n i n cel l divisio n an d cel l death
. Thi s i n tur n lead s t o a reductio n i n cel l volum e an d t o compensat e fo rthis , periphera l bloo d i s shunte d int o th e bon e marrow . I fth e rati o o f polychromati ct o normochromati cerythrocyte s i s score d an d foun d t o b e significantl y lowe rtha n th e contro l value , thi s i s take n a s bein g indicativ e o fcytotoxicity
. 3 . TES TAN D CONTRO L ITEM S AN D EXPERIMENTA L PREPARATIO N 3. 1 Tes t Ite m Sponsor' s identificatio n : Sodiu m Glucoheptonat e Descriptio n Dar k brow n liqui d Batc h numbe r92100010 0 Purity50.5 % activ e ingredien t (accounte d fo ri n principle ) Dat e receive d 0 9 Februar y201 2 Expir ydat e : 0 9 Februar y201 5 Storag e condition s Roo m temperature , i n th e dar k Th e integrit yo fsupplie d dat a relatin g t o th e identity , purit yan d stabilit yo fth e tes tite m i s th e responsibilit yo fth e Sponsor

. Fo rth e purpos e o fthi s stud yth e tes t ite m wa s freshl yprepare d a s require d a s a solutio n at th e appropriat e concentratio n i n phosphat e buffere d salin e (PBS)

. A n adjustmen t wa s mad e fo rth e wate rconten t o fth e tes tite m (49.5% ) whe n preparin g dos e concentrations
. N o analysi s wa s carrie d ou t t o determin e th e homogeneity , concentratio n o rstabilityo f th e tes t ite m formulation

. Th e tes tite m wa s formulate d withi n tw o hours o fi t bein g applie d t o th e tes t system ; i t i s assume d tha tth e formulatio n wa s stabl e fo rthi s duration
. Thi s i s a n exceptio n wit h regar d t o GL P an d ha s bee n reflecte d i n th e GL P complianc e statement
.p . 1 0
PROJEC TNUMBER : 4110469 0 PAG E 9 

### 3. 2 PositiveControl Ite m Th e positiv e contro l ite m wa s supplie d byAcro s Organics , a s follows : Supplier' s identificatio n Supplier' s lo tnumbe r In-hous e seria l numbe r Dat e receive d Expir ydat e Storag e condition s Cyclophosphamid e A030260 5 R-535 9 2 7 Apri l 201 2 2 7 Apri l 201 4 Approximatel y4° C i n th e dar k Fo rth e purpos e o fthi s stud yth e positiv e contro l ite m wa s freshl yprepare d a s require d a s a solutio n a tth e appropriat e concentratio n i n distille d wate r(Aguettan t Lt d batc h no . 300893 7 01)

. 3. 3 Vehicl e Contro l Ite m Th e vehicl e wa s supplie d a s a 10xconcentrate d solutio n byGibco , a s follows : Supplier' s identificatio n Supplier' s lo t numbe r In-hous e seria l numbe r Dat e receive d Expir ydat e Storag e condition s Dulbecco' s Phosphat e Buffere d Salin e Solutio n 

### 93828 1 V-5241 0 2 Novembe r201 1 0 1 Ma y201 3 Roo m temperatur e Fo rth e purpos e o fthi s stud yth e vehicl e contro l ite m wa s freshl yprepare d a s require d a s a solutio n a tth e appropriat e concentratio n i n distille d wate r(Aguettan t Lt d batc h no . 300893 7 01)

. 4 . METHOD S 4. 1 Animal s an d Anima l Husbandr y Sufficien t albin o Hsd : IC R (CD-1® ) strai n mic e wer e obtaine d fro m Harla n Laboratorie s U KLtd. , Oxon , UK
. A t th e starto fth e mai n tes t th e mic e weighe d 2 1 t o 30 g an d wer e approximatel ysi xt o te n week s old
. Afte ra minimu m acclimatisatio n perio d o ffiv e day sp . 1 1 PROJEC TNUMBER : 4110469 0 PAG E 1 0 th e animal s wer e selecte d a t rando m an d give n a numbe runiqu e withi n th e stud ybytai l markin g an d a numbe rwritte n o n a colou rcode d cag e card
. Th e animal s wer e house d i n group s o fu p t o seve n i n solid-floo rpolypropylen e cage s wit h wood-flak e bedding
. Fre e acces s t o main s drinking wate ran d foo d (Harla n Tekla d 2014 C Globa l Certifie d Roden tDie t supplie d byHarla n Laboratorie s U KLtd. , Oxon , UK) wa s allowe d throughou t th e study
. Th e temperatur e an d relativ e humidit ywer e se tt o achiev e limit s o f1 9 t o 25° C an d 3 0 t o 70% , respectively
. An yoccasiona l deviation s fro m thes e target s wer e considere d not t o hav e affecte d th e purpos e o rintegrit yo fth e study
. Th e rat e o fai rexchang e wa s approximatel yfiftee n change s pe rhou ran d th e lightin g wa s controlle d b ya tim e switc h t o giv e twelv e hour s ligh t an d twelv e hours darkness
. 4. 2 Procedur e 4.2. 1 Range-findin g Toxicit yTes t Arange-findin g toxicit ytes t wa s performe d t o determin e a suitabl e dos e leve l an d rout e o fadministratio n fo rth e micronucleu s test
. Th e dos e leve l selecte d shoul d ideall yb e th e maximu m tolerate d dos e leve l o rtha t whic h produce s som e evidenc e o ftoxicit yu p t o a maximu m recommende d dos e o f200 0 mg/kg

. Th e range-findin g toxicit ytes t wa s als o use d t o determin e i fth e mai n tes t wa s t o b e performe d usin g bot h sexe s o rmale s only
. Wit h n o evidenc e o ftoxicit yvi a th e ora l route , an d followin g discussion s wit h th e Sponsor , i t wa s considere d necessaryt o investigat e th e intraperitonea l rout e of administratio n t o maximis e exposur e o fth e tes t item
.p . 1 2
PROJEC TNUMBER : 4110469 0 PAG E 1 1 Group s o fmic e wer e dose d a s follows : Dos e Leve l Concentratio n Dos e Volum e Numbe ro fMic e (mg/kg ) (mg/ml ) (ml/kg ) Mal e Femal e 200 0 (oral ) 20 0 1 0 1 1 100 0 (ip ) 10 0 1 0 1 1 150 0 (ip ) 15 0 1 0 1 1 200 0 (ip ) 20 0 1 0 1 1 200 0 (ip ) 20 0 1 0 2 0 Al l animal s wer e dose d onc e onl ya tth e appropriat e dos e leve l b ygavag e usin g a meta l cannul a o ra hypodermi cneedl e attache d t o a graduate d syringe
. Th e volum e administere d t o eac h anima l wa s calculate d accordin g t o it s bodyweigh tat th e tim e o f dosing
. Animal s wer e observe d on e hou rafte rdosin g an d subsequentl yonc e dail yfo ru p t o tw o days
. An ydeath s an d evidenc e o fovert toxicit ywer e recorde d at eac h observation
. N o necropsie s wer e performed

. 4.2. 2 Micronucleu s Tes t Groups , eac h o fseve n mal e mice , wer e dose d onc e onl yVi a th e intraperitonea l rout e wit h th e tes t ite m at2000 , 100 0 o r50 0 mg/kg

. On e grou p o fmic e fro m eac h dos e leve l wa s kille d b ycervica l dislocatio n 2 4 hours followin g treatmen t an d a secon d grou p dose d wit h tes tite m at 200 0 mg/k g wa s kille d afte r4 8 hours
. Tw o additiona l group s o fmal e mic e wer e include d i n th e study ; on e grou p (seve n mice ) wa s dose d vi a th e intraperitonea l rout e wit h th e vehicl e alon e (PBS ) an d a secon d grou p (fiv e mice ) wa s dose d orall ywit h cyclophosphamide
. Cyclophosphamid e i s a positiv e contro l ite m know n t o produc e micronucle i unde rth e condition s o fth e test
. Th e vehicl e an d positiv e contro l grou p animal s wer e kille d 2 4 hours followin g dosing
.

i p = Intraperitonea lp . 1 3
PROJEC TNUMBER : 4110469 0 PAG E 1 2
Th e experimenta l design i s summarise d i n th e followin g table :

Dos e Grou p Dos e Leve l (mg/kg ) Concentratio n

(mg/ml ) Dos e

Volum e (ml/kg ) Kil l Tim e

(Hour s Arte r Dosing ) Anima l

Number s 1
. Vehicl e Contro l (PBS ) 0 0 1 0 2 4 1 - 7 2
. Positiv e Contro l (Cyclophosphamide ) 5 0 5 1 0 2 4 8-1 2 3
. Sodiu m Glucoheptonat e 200 0 20 0 1 0 4 8 13-1 9 4
. Sodiu m Glucoheptonat e 200 0 20 0 1 0 2 4 20-2 6 5
. Sodiu m Glucoheptonat e 100 0 10 0 1 0 2 4 27-3 3 6
. Sodiu m Glucoheptonat e 50 0 5 0 1 0 2 4 34-4 0 Animal s wer e observe d fo rsign s o foverttoxicit yan d deat h on e hou rafte rdosin g an d the n onc e dail ya s applicabl e an d immediatel yprio rt o termination
. 4.2. 3 Slid e Preparatio n Immediatel yfollowin g terminatio n (i.e

. 2 4 o r4 8 hour s followin g dosing) , bot h femur s wer e dissecte d fro m eac h survivin g animal , aspirate d wit h foeta l bovin e seru m an d bon e marro wsmear s prepare d followin g centrifugatio n an d re-suspension
. Th e smears were air-dried , fixe d i n absolut e methanol , staine d i n May-Grunwald/Giemsa , allowe d t o air-dr y an d a cove rsli p applie d usin g mountin g medium
. 4.2. 4 Slid e Evaluatio n Staine d bon e marro wsmear s wer e code d an d examine d blin d usin g ligh t microscop yat x100 0 magnification

. Wher e possible , th e incidenc e o fmicronucleate d cell s pe r200 0 polychromati cerythrocyte s (PCE-blu e staine d immatur e cells ) pe ranima l wa s scored
. Micronucle i ar e normall ycircula ri n shape , althoug h occasionall ythe ymayb e ova l o r half-moo n shaped , an d hav e a shar p contou rwit h eve n staining
. I n addition , th e numbe r o fnormochromati cerythrocyte s (NCE-pin k staine d matur e cells ) associate d wit h 100 0 erythrocyte s wa s counted ; thes e cell s wer e als o score d fo rincidenc e o fmicronuclei
.p . 1 4
PROJEC TNUMBER : 4110469 0 PAG E 1 3 Th e rati o o fpolychromati ct o normochromati cerythrocyte s wa s calculate d togethe rwit h appropriat e grou p mea n value s an d standar d deviations
. 4.2. 5 Interpretatio n ofResult s Acompariso n wa s mad e betwee n th e numbe ro fmicronucleate d polychromati c erythrocyte s occurrin g i n eac h o fth e tes t ite m group s an d th e numbe roccurrin g i n th e vehicl e contro l group . Apositiv e mutageni crespons e woul d b e demonstrate d whe n a statisticall ysignificant , dose-responsive , toxicologicall yrelevan tincreas e i n th e numbe ro fmicronucleate d polychromati cerythrocyte s wa s observe d fo reithe rth e 2 4 o r48-hou rkil l time s whe n compare d t o th e vehicl e contro l group . I fthes e criteri a wer e no tfulfilled , the n th e tes t ite m woul d b e considere d non-genotoxi c unde rth e condition s o fth e test
. Apositiv e respons e fo rbon e marro wtoxicit ywoul d b e demonstrate d whe n th e dos e grou p mea n polychromati ct o normochromati crati o wa s show n t o b e statisticall y significantl ylowe rtha n th e vehicl e contro l group . Al l dat a wer e statisticall yanalyse d usin g appropriat e statistica l method s a s recommende d byth e UKEM S Sub-committe e o n Guideline s fo rMutagenicityTestin g Report , Part II I (1989)

. Th e dat a wa s analyse d followin g a TJ(X+1 ) transformatio n usin g Student' s t-tes t (tw o tailed ) an d an ysignifican t result s wer e confirme d usin g th e on e wa y analysi s o fvariance
. 5 . ARCHIVE S Unles s instructe d otherwis e byth e Sponsor , al l origina l dat a an d th e fina l report wil l b e retaine d i n th e Harla n Laboratorie s Ltd , Shardlow , U Karchive s fo rfiv e years , afte rwhic h instruction s wil l b e sough ta s t o furthe rretentio n o rdisposal
.p . 1 5
PROJEC TNUMBER : 4110469 0 PAG E 1 4
6 . RESULT S 6. 1 Range-findin g Toxicit yTes t Th e mortalit ydat a ar e summarise d a s follows : Dos e Leve l (mg/kg ) Se x Numbe ro f Animal s Treate d Rout e Death s o n Da y Tota l Death s Dos e Leve l (mg/kg ) Se x Numbe ro f Animal s Treate d Rout e 0 1 2 Tota l Death s
200 0 Mal e 1 ora l 0 0 0 0/ 2 200 0 Femal e 1 ora l 0 0 0 0/ 2
100 0 Mal e 1 i P 0 0 0 0/ 2 100 0 Femal e 1 i P 0 0 0 0/ 2
150 0 Mal e 1 i p 0 0 0 0/ 2 150 0 Femal e 1 i p 0 0 0 0/ 2
200 0 Mal e 1 i P 0 0 0 0/ 2 200 0 Femal e 1 i P 0 0 0 0/ 2 200 0 Mal e 2 i p 0 0 0 0/ 2 I n animal s dose d wit h th e tes t ite m at200 0 mg/k g vi a th e ora l rout e clinica l sign s wer e no t observe d i n an yo fth e animals
. Therefore , wit h n o evidenc e o ftoxicit yth e intraperitonea l rout e o fadministratio n wa s investigate d t o maximis e exposur e o fth e tes t 

### item
. Usin g th e intraperitonea l route , th e clinica l sign s hunche d posture , ptosis , increase d activity , an d irritatio n an d ove rgroomin g o fth e injectio n sit e wer e observe d i n animal s dose d wit h th e tes t ite m at an d abov e 100 0 mg/kg

. Therefore , wit h evidenc e o fmodes t toxicity , th e maximu m dos e leve l i n th e mai n tes twa s se ta t th e maximu m recommende d dos e o f200 0 mg/k g wit h 100 0 an d 50 0 mg/k g a s th e tw o lowe rdos e levels
. Th e tes t ite m showe d n o marke d differenc e i n it s toxicit yt o mal e o rfemal e mice ; therefor e th e mai n tes t wa s performe d usin g mal e mic e only
.

i p - Intraperitonea lp . 1 6
PROJEC TNUMBER : 4110469 0 PAG E 1 5 6. 2 Micronucleu s Tes t 6.2. 1 MortalityDat a an d Clinica l Observation s Ther e wer e n o prematur e death s see n i n an yo fth e dos e group s i n th e mai n test
. Clinica l sign s wer e observe d i n animal s dose d wit h th e tes tite m a t an d abov e 100 0 mg/k g i n bot h th e 2 4 an d 48-hou rdos e groups , wher e applicable , an d include d hunche d postur e an d ptosis
. 6.2. 2 Evaluatio n ofBon e MarrowSlide s Asummaryo fth e result s o fth e micronucleu s tes ti s give n i n Tabl e 1
. Individua l an d grou p mea n dat a ar e presente d i n Table s 2 t o 7
. Statisticall ysignifican t decrease s i n th e PCE/NC E rati o wer e no tobserve d i n an yo fth e tes t ite m dos e group s whe n compare d t o th e vehicl e contro l group . However , th e observatio n o fclinica l sign s wa s take n t o indicat e tha tsystemi cabsorptio n ha d occurre d an d exposur e t o th e targe t tissu e ha d bee n achieved
. Ther e wa s n o evidenc e o fan ystatisticall ysignifican tincrease s i n th e incidenc e o f micronucleate d polychromati cerythrocyte s i n animal s dose d wit h th e tes tite m whe n compare d t o th e vehicl e contro l group . Th e positiv e contro l grou p showe d a marke d increas e i n th e incidenc e o fmicronucleate d polychromati cerythrocyte s henc e confirmin g th e sensitivit yo fth e syste m t o th e know n mutageni cactivit yo fcyclophosphamid e unde rth e condition s o fth e test
. Th e tes t ite m wa s foun d no t t o produc e a toxicologicall ysignifican t increas e i n th e frequenc yo fmicronucle i i n polychromati cerythrocyte s o fmic e unde rth e condition s o f th e test
. . 7 . CONCLUSIO N Th e tes t ite m wa s considere d t o b e non-mutageni cunde rth e condition s o fth e test
.p . 1 7
PROJEC TNUMBER : 4110469 0 PAG E 1 6 Sodiu m Glucoheptonate : MICRONUCLEU S TES TI N TH E MOUS E Tabl e 1 Micronucleu s Tes t - Summar yo fGrou p Mea n Dat a

Treatmen t Grou p Numbe ro fPC E wit h M/cronucle t pe r200 0 PC E PCE/NC E Rati o Treatmen t Grou p Grou p Mea n S D Grou p Mea n S D 1
. Vehicl e Contro l (PBS ) 1 0 ml/k g 24-hou rSamplin g Tim e 1. 0 1. 2 0.8 4 0.2 7 2 , Positiv e Contro l (Cyclophosphamide ) 5 0 mg/k g 24-hou rSamplin g Tim e 23.0** * 8. 4 0.9 5 0.2 0 3
. Sodiu m Glucoheptonat e 200 0 mg/k g 48-hou rSamplin g Tim e 2. 0 1. 6 1.2 7 0.4 7 4
. Sodiu m Glucoheptonat e 200 0 mg/k g 24-hou rSamplin g Tim e 2. 1 2. 9 1.5 6 0.4 3 5
. Sodiu m Glucoheptonat e 100 0 mg/k g 24-hou rSamplin g Tim e 0. 9 1. 6 1.9 1 0.9 4 6
. Sodiu m Glucoheptonat e 50 0 mg/k g 24-hou rSamplin g Tim e 0. 6 0. 5 1.47 0.3 1

PC E = Polychromati cerythrocyte s NC E = Normochromati cerythrocyte s S D = Standar d deviatio n ** * = p < 0.00 1J* : p . 1 8

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PROJEC TNUMBER : 4110469 0 PAG E 2 3 Sodiu m Glucoheptonate : MICRONUCLEU S TES TI N TH E MOUS E Appendi x1 Historica l Contro l Dat a (200 9 an d 2010 ) 24-Hou rVehicl e Contro l Grou p Mea n Dat a

PC E + M N Pe r200 0 PC E PCE/NC E 100 0 Erythrocyte s Mea n 1.23 0.9 0 S D 0.7 2 0.1 9 Minimu m 0.1 0 0.6 1 Maximu m 3.4 0 1.22

24-Hou rPositiv e Contro l Grou p Mea n Dat a

PC E + M N Pe r200 0 PC E PCE/NC E 100 0 Erythrocyte s Mea n 31.5 9 0.9 2 S D 8.8 1 0.2 4 Minimu m 18.0 0 0.5 5 Maximu m 60.4 0 1.5 3

PC E = Polychromati cerythrocyte s NC E = Normochromati cerythrocyte s S D = Standar d deviatio np . 2 5
PROJEC TNUMBER : 4110469 0 PAG E 2 4 Appendi x2 Statemen t ofGL P Complianc e i n Accordanc e wit h Directive

2004/9/E C

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