p. 1



### Harcro s Chemical s Inc . 520 0 Speake rRoa d Kansa s C'rty , K S 6610 6 Telephone : 913/621-780 2 Fax : 913/621-787 6 E-mail : asavini@harcros.co m Dat e 05/22/1 3 Documen t Contro l Offic e (DCO ) (7407M ) Offic e ofPollutio n Contro l an d Toxic s (OPPT) U.S
. Environmenta l Protectio n Agency(EPA) 120 0 Pennsylvani a Ave . N W Washington , D.C
. 2046 0 (202-564-8930 ) Attn : TSC ASectio n 4 4 0 CF R § 799.508 7 (Docke t # EPA-HQ-OPPT-2007-0531 ) 

# 354347

RE : Ame s Tes t Amendmen t fo rCASR N 31138-65- 5 T o Who m I t Ma

yConcern : I n accordanc e wit h EPA' s fina l rule , Testin g o fCertai 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 , Harcro s Chemical s Inc . submitte d a fina l repor t fo rth e Ame s tes t fo rD-gluco-Heptoni cacid , monosodiu m salt , (2.xi.) - [CAS # 31138-65-5 ] o n Decembe r18 , 2012 . I n respons e t o a reques t fro m EPAt o provid e informatio n o n th e dilutio n o fth e sampl e use d i n th e study , pleas e fin d attache d a n amendmen t t o th e Ame s tes t containin g th e require d information . Thi s stud ywa s conducte d t o fulfil l th e requirement s o ftestin g categor yE a s se t fort h i n Tabl e 2 o f4 0 CF R 799.50870) . I fan yfurthe rinformatio n i s required , pleas e d o no t hesitat e t o contact m e at (913 ) 621-780 2 o rat asavini@harcros.com
.

An n Mari e Savin i Harcro s Chemical s Inc . Regulator ySpecialis t cc : Pete rRadford Joh n Mile s Regards ,

Jame s Grantha m Rand yBuckma n Fil e 

# iiiiiiiiiigii)
4 0 1 3000003 9 

# II

I

CONTAIN S N O CB I

# harla n HARLA N LABORATORIE S LT D AUTHENTICATIO N O F AMENDMEN TT O FINA L REPOR T

Sodiu m Glucoheptonate : REVERS E MUTATIO N ASSA Y"AME S TEST " USIN G SALMONELL ATYPHIMURIU M AN D ESCHERICHI ACO U

PROJEC TNUMBER : 4110468 8 REASO N FO R AMENDMEN T Ates t ite m purit ycorrectio n fo r49.5 % wate rpresen t i n th e sampl e wa s mad e i n Experiment s 1 an d 2 . However , thi s informatio n wa s no t recorde d i n th e appropriat e sectio n o fth e fina l report . DETAIL S O F AMENDMEN T Sectio n 3. 2 Preparatio n o fTes t an d Referenc e Items : Th e secon d paragrap h ha s bee n amende d wit h a n additiona l sentenc e t o confir m tha t a n appropriat e purityallowanc e wa s mad e i n Experiment s 1 an d 2 . Th e additiona l sentenc e force d th e fina l paragrap h o n pag e 9 ont o pag e 10 ; therefor e 2 page s hav e bee n amended . Page s 9 an d 1 0 ar e hereb yamended . Thi s amendmen t doe s no t affec t th e validit yo rinterpretatio n o fth e data .

P WThompso n HN C Stud yDirecto r Thi s amendmen t ha s bee n audite d byHarla n Laboratorie s Ltd , Shardlow , U KQualit y Assuranc e Uni t an d i s considere d t o b e a n accurat e accoun t o fth e project . DATE :

DATE : 

### 1 6 MA

Y201 3 Fo rth e Qualit yAssuranc e Unit *

"Authorise d Q

ASignatures : SeniorAudit Staff ; J G Rile yBS c(Hons ) MRQA , J M Crowthe rMISc

TMRQA , G Wren ON

C MRQA , S Beva n BS c(Hons ) MRQA , LBlane yMRQ APROJEC TNUMBER : 4110468 8 PAG E 9 (Amendment ) cause s a n inactivatio n o fth e excisio n repai rsyste m an d a dependence o n exogenou s biotin . I n th e strain s TA9 8 an d TA100 , th e R-facto rplasmi d pKM10 1 enhance s chemica l an d UV-induce d mutagenesi s vi a a n increas e i n th e error-pron e repai rpathway . Th e plasmi d als o confer s ampicilli n resistanc e whic h act s a s a convenien t marke r (Mortleman s an d Zeige r(2000)) . I n additio n t o a mutatio n i n th e tryptopha n operon , th e E . col i teste rstrai n contain s a uvr ADN Arepai rdeficienc ywhic h enhance s its sensitivity t o som e mutageni ccompounds . Thi s deficiencyallow s th e strai n t o sho wenhance d mutabilit ya s th e uvrArepai rsyste m woul d normall yact t o remov e an d repai rth e damage d sectio n o fth e DN Amolecul e (Gree n an d Murie l (1976)) . Th e strain s o fbacteri a use d i n th e tes t wer e obtaine d fro m th e Universit yo fCalifornia , Berkeley , o n cultur e discs , o n 0 4 Augus t 199 5 o rfro m th e Britis h Industria l Biologica l Researc h Association , o n nutrien t aga rplates , o n 1 7 Augus t 1987 . Al l o fth e strain s wer e store d a t approximatel y-196° C i n a Statebourn e liqui d nitroge n freezer , mode l SX R 34 . I n thi s assay , overnigh t sub-culture s ofth e appropriat e code d stoc k culture s wer e prepare d i n nutrien t brot h (Oxoi d Limited; lot number s 107836 4 08/16 , 111937 6 10/1 7 an d 101457 0 03/16 ) an d incubate d a t 37° C fo rapproximatel y1 0 hours . Eac h cultur e wa s monitore d spectrophotometricall yfo rturbidit ywit h titre s determine d byviabl e coun t analysi s o n nutrien t aga rplates . 3. 2 Preparatio n ofTes t an d Referenc e Item s Th e tes t ite m wa s full ymiscibl e i n steril e distille d wate ra t 5 0 mg/m l i n solubilit ychecks performe d in-house . Steril e distille d wate rwa s therefor e selecte d a s th e vehicle . Th e tes t ite m wa s accuratel yweighe d an d approximat e half-lo g dilution s prepare d i n steril e distille d wate rbymixin g o n a vorte xmixe ro n th e da yo feac h experiment . Formulate d concentration s wer e adjuste d i n Experiment s 1 an d 2 t o allo wfo rth e state d wate rconten t (49.5% ) o fth e tes t item . Al l formulation s wer e use d withi n fou rhours o f preparatio n an d wer e assume d t o b e stabl e fo rthi s period . Analysi s fo rconcentration , homogeneit yan d stabilit yo fth e tes t ite m formulation s i s no t a requiremen t o fth e tes t guideline s an d was , therefore , no t determined . Thi s i s a n exceptio n wit h regard t o GL P an d ha s bee n reflecte d i n th e GL P complianc e statement .PROJEC TNUMBER : 4110468 8 PAG E 1 0 (Amendment ) Asolven t treatmen t grou p wa s use d a s th e vehicl e contro l an d th e positiv e control s use d i n th e serie s o fplate s withou t S9-mi xwer e a s follows : N-ethyl-N'-nitro-N-nitrosoguanidin e N-ethyl-N'-nitro-N-nitrosoguanidin e N-ethyl-N'-nitro-N-nitrosoguanidin e 9-Aminoacridin e (9AA) : 4-Nitroquinoline- 1 -oxid e (4NQO) : (ENNG) : 2 ug/plat e fo r\NP2uvrA (ENNG) : 3 ug/plat e fo rTA10 0 (ENNG) : 5 ug/plat e fo rTA153 5

- 8 0 ug/plat e fo rTA153 7 0. 2 ug/plat e fo rTA9 8 I n addition , 2-Aminoanthracen e (2AA ) an d Benzo(a)pyren e (BP) , whic h ar e non-mutageni ci n th e absenc e o fmetabolisin g enzymes , wer e use d i n th e serie s of plate s wit h S9-mi xa t th e followin g concentrations : 2-Aminoanthracen e (2AA ) 2-Aminoanthracen e (2AA ) 2-Aminoanthracen e (2AA ) Benzo(a)pyren e (BP) : 1 ug/plat e forTA10 0 2 ug/plate for TA1535 and TA1537 10 ug/plate for \NP2uvrA 5 ug/plat e fo rTA9 8 3. 3 Microsoma l Enzym e Fractio n Th e S 9 Microsoma l fractio n wa s prepare d in-hous e (1 8 Septembe r201 1 (preliminar y toxicit ytest) , 0 1 Jul y201 2 (Experimen t 1 ) an d 1 6 Septembe r201 2 (Experimen t 2) ) fro m rat s induce d wit h Phenobarbitone/p-Naphthoflavon e a t 80/10 0 mg/kg/day , orally , fo r3 day s prio rt o preparatio n o n da y4 . Th e S 9 homogenat e wa s produce d byhomogenisin g th e live ri n a 0.15 M KC I solutio n (1 g live rt o 3 m l KCI ) followe d bycentrifugatio n a t 900 0 g . Th e protei n conten t o fth e resultan t supernatan t wa s adjuste d t o 2 0 mg/ml
. Aliquot s o fth e supernatan t wer e froze n an d store d a t approximatel y-196°C
. Prio rt o use , eac h batc h o fS 9 wa s teste d fo rit s capabilit yt o activat e know n mutagen s i n th e Ame s test . 3. 4 S9-Mi xan d Aga r Th e S9-mi xwa s prepare d immediatel ybefor e us e usin g sterilise d co-factor s an d maintaine d o n ic e fo rth e duratio n o fth e test .p . 5

ORIGI NID : KCK

A<913 ) 621-773 3 JAK E BQORE M HARCRO S CHEMICAL

S IN C 520 0 SPEAKE

R R D KANSA S CITY . K

S 6610 6 UNITE D 8TATE S U S SHI P DATE : 22MAY1 3 ACTWBT : 0. 6 L

B MA N CAD : 017980t/CAFE260 8 BIL L THIR

D PART Y TO 

## (DCO)(7407M)(OPPT

) U S EP ATSC ASECTIO N 4 120 0 PENNSYLVANI

A AV E N W 

# WASHINGTO

N D C 2046 0 (202)564-893 0 REF: POSTAG

E 

# i

FedEx , Express f 8

TRKf f 10201 , 

# 565 4 323 9 795 8 

# X C RDV A TH U - 2 3 MA Y10:30 A PRIORIT YOVERNIGH T 2046 0 DC-U S DC A