Document jgEa7Z222Jea0jwVrQrrNkopN
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1
AR226-3171
TRADE s e c r e t Study TMe
Authors Valentine O. Wagner, m , M.S.
Michelle L.Klug,B.S.
Report CompletionDate
November 16,2000
Performing Laboratory BioReliance
9630 Medical Center Drive Rockville, MD 20850
for
E. I. du Pont de Nemours and Company Stine Haskell Research Center DuPont Haskell Laboratory P.O.Box 50 1090 Elkton Road Newark, DE 19714-0050
Performing Laboratory Study Number
AA34BK.5020Q1 .BTL
`
DuPont Project ID DuPont-4670
W ork
Service Code
Reverse Mutation
Company Sanitized Does not contain TSCA CBI
:
good laboratory practice compliance st a u NT
Study No AA34BK.502001BTL as conducted in compliance wffl, fi* U S EPA n p
Standards 40CFR 160 and 40 CFR 792 thp tit nr
Z U`h' EPA GLP
GLP Stendarf and he OECD wfth the biloteing exceptions:
4,6 ,a,Hnese Laboratay Practice in aU material aspect,
7 " identity, strath, purity and composition or other taractenstics to define die control substances have not been detennraed by the testing facility. The control substances have
as per the Certificates o f Analysis on file with
t Z l Z H * ^ f T 16
Unifom,i^ ( applicable) or
Z T Z Z 1 ^ r C0"trol mktWSS WB" 1161Perfotined
&Clllty' ^ Spoasor *** 1101 Performed these
te sta b ility o f the test or control substances in the test or control
mrxtures has not been determined by the testing facility. The Sponsor has not performed these analyses.
Submitter/Sponsor:
BioReliance Study Director:
BioReliance Study Management:
DuPont Registration Representative:
E. I. du Pont de Nemours and Company Wilmington, DE 9898U.S.A.
O - cXtr~ Valentine O. Wagner, m , M.S. "
ZfeAiW 3,000 Date
Richard H.C. San, PhD.
f&Jey 0(? ~~ Date
Date
Oofflapany Sanitized. Does not eentein TSCA CBI
Authors Valentine O. Wagner, HI, M.S.
Michelle L. Klug, B.S.
Report Completion Date November 16,2000
Performing Laboratory BioReliance
9630 Medical Center Drive Rockville, MD 20850
E. I. du Pont de Nemours and Company Stine Haskell Research Center DuPontHaskell Laboratory P.O.Box 50 1090 Elkton Road Newark, DE 19714-0050
Performing Laboratory Study Number AA34BK.502Q01.BTL DuPont Project ID DuPont-4670
Service Code
i
Company Sanitized. Does not contain TSCACBI
GOOD LABORATORY PRACTICE COMPLIANCE STATEMENT
Study No. AA34BK.502001.BTL was conducted in compliance with the U S EPA gtp 40CFR 160and 40CFR792, the UK GLP
The identity, strength, purity and composition or other characteristics to define the control substances have not been determined by the testing facility. The control substances have been characterized as per the Certificates o f Analysis on file with the testing facility.
Analyses to determine the uniformity (as applicable) or concentration o f the test or control mixtures were not performed by the testing fecility. The Sponsor has not performed these analyses.
The stability o f the test or control substances in the test or control mixtures has not been determined by the testing fecility. The Sponsor has not performed these analyses.
Suhmitter/Sponsor:
BioReliance Study Management:
DuPont Registration Representative:
E. I. du Pont de Nemours and Company Wilmington, DE 19898 U.SA . , / TTT--Valentine 0 . Wagner, l, M.S.
Richard H.C. San, Ph.D.
Hbivov uoao Date
f //P 'y 0 & Date
Date
BioReliance Study No. AA34BK.502001.BTL
Company Sanitized, Bo# 1 contain TSCA SB!
}
Quality Assurance Statement
Study Num ber: AA34BK.5020Q1 .BTL Study Director: Valentine 0 . Wiagner, IH, M.S.
Inspect On 12-Sep-OO - 12-Sep-OO To Study Dir 12-Sep-OO To Mgmt 14-Sep-OQ
Phase
Preparation ofSO mixture
^
Irrapect On 30-0ct-00 - 30-0ct-00 To Study Dir 30-0ct-00 To Mgmt 01-Nov-00
Phase
Draft Report
Inspect On 14-Nov-OO - 14-Nov-OO To Study Dir 14-Nov-00 To Mgmt 14-Nov-00
Phase
Draft to Final Report
IIL u l
Diane Brecha, B.S. QUALITY ASSURANCE
BioReliance Study No. AA34BK.502001.BTL
IL> tiC k f 2 M b
DATE
o * P ^ S, n&K!. 0!ie!fiM n (a|n T sice|
'est with an
acterial Reverse Mutation
CERTIFICATION
DuPont-4670
Z l! iL TM l! ^ igne<i' dedare *1*thia ieport provid<!S m accuia*e evaiuation r>r'"
,
Issued by Study Director:
Valentine 0 . Wagner, it, M.S. BioReliance
IL aX /^OOC Date
Approved by Sponsor Study Monitor:
tsVN -- Maria Donner, PLD. Senior Research Sdentisi
1STfJov 2-000 Date
w BioReliance
Study No. AA34BK.502001.BTL
4 fSompany Sanitized. Does mesntete TSC Cl
I
TABLE OF CONTENTS Good Laboratory Practice Compliance Statement.............
Page ,,
Quality Assurance Statement....... .........................
.
............................................................................... 3
Certification .......................................................................................................... ................... 4
Table o f Contents.........
......................... ........... * ........* ..... .......................................................................
Study Information......................................................................
SUQHU3iy,,,,M,M.... ................................... ..... ............................................................... ................... 8
..............................................
n
Characterization o f Test and Control Substances
,,
Materials and Methods.......................................
tA
....................................................................................10
Results and Discussion ..................................................................................................................
Conclusion................................................. ............................................................................................... ...
References............................................................................................................
lg
Data Tables ................................................. ..................................................................................
Table 1: Preliminary Toxicity Test in Salmonella typhimurium TA98
17
Tabfc 2: M ta in a ry Toxicity Tea m Salmonella typhimurium T A m Z Z Z I Z l IS
Table 3. Preliminary Toxicity Test in Salmonella typhimurium TA1535.....
to
Table 4: Preliminary Toxicity Test in Salmonella typhimurium TA1537
20
Table 5: Preliminary Toxicity Test in Escherichia coli WP2 uvrA............ .Z Z ..Z Z 21
Table 6: Mutagenicity Test in Salmonella typhimurium TA98 without S9.Z .................22
Table 7: Mutagenicity Test in Salmonella typhimurium TA98 with S9.........I Z Z Z 23
Table 8: Mutagenicity Test m Salmonella typhimurium TA100 without S9.............. 24
Table 9: Mutagenicity Test in Salmonella typhimurium TA100 with S9.................... 25
Table 10: Mutagenicity Test in Salmonella typhimurium TA1535 without S9.....................26
Table 11: Mutagenicity Test in Salmonella typhimurium TA1535 with S9............1..Z .. 27
Table 12: Mutagenicity Test in Salmonella typhimurium TA1537 without S9...... ."...""..28
Table 13: Mutagenicity Test in Salmonella typhimurium TA1537 with S9.............Z Z 29
BioReliance Study No. AA34BK.502001JBTL
5
Companjp Sanitized. Does not ontain TSCA CBI
? t | Ie I f ^ uta8enic!t5r Test Escherichia coli WP2 mrA without S9
,n
Tabte 5: Mut^emcity Test in Escherichia c d i WP2 ucrK with S9... ....................??
J ? ; 17- Mm Sut^emS city Tl Test in^Salrmoonneenlla t`yypplh?im,murri"umm TTAA998S w ith"S9 ..........
Tahi6 to w Uta86" Wty TeSt " Salmonella typhimurium TA10O without S9 ................. 34
ItTabe 19: Mutagenicity Test in Salmonella typhimurium TA100 withS9 ^ ............*
M e ^ ^ enic?y TeSt " Salmomlla ^ m u riu m TA1535 without S9.................36
TaJ! 22*
i CStmMmonella typhimurium TA1535 with S9..........
37
S ' Mutagenicity ^ mSalmonella typhimurium TA1537 without S9. ........38
TVh! oa ^ Utageni" ty Test 111Salmonella typhimurium TA1537 with S9
39
Table 24: Mutagenicity Test in Escherichia coli WP2 tivrA withTM* so ................... 5?
P j * * * f f * * * l n . EscherichiacoliWP2 vrA
f r Cli27 l f l Mutagenicity Test " Summary o f Results B l Z ...............
Table 27: Salmonella/E coli Mutagenicity Test - Summary o f Results B2 ......... Z 1 Z .4 3
Appendix A: Historical Control Data..............................................
M M . . . . . . . . .................................................. ...................................... ..
44
Appendix B: Study Protocol................................................... Appendix C: Information for Japanese Regulatory Agencies
.4 6 ..57
BioReliance Study No. AA34BK.502001.BTL
6
C ou p * Sanitized 0@ nol oritaiRi TSCA CBS
STUDY INFORMATION Substance Tested:,
Synonyms/Codes: n 24547
Haskell Number: 24547
Composition: |
Known Impurities: Not supplied by the sponsor
Physical Characteristics: Amber sticky gum
StaMH; The test substance appeared to be stable under the conditions o fthe study; no evidence o f instability was observed.
SE2sr: E. I. du Pont de Nemours and Company Wilmington, Delaware 19898 U.S.A.
Study Initiated/Compieted: August 24,2000 / (see report cover page)
In-LifeInitiated/Completed: August29,2000/October04,2000
BioReliance Study No. AA34BK.50200l.BTL
Company Sanitized. Does not contain TSCA CM
DuPont-4670
SUMMARY
?
The test substance, H-24547, was tested in th# bacterial reverse mutation test usine S typhimunum tester strains TA98, TA1O0, TA1535, and TA1537 and E c n liZ J ^ U '
A^ ? l WaS- f 1!! cted " ^ solvent o f choice based on solubility o f the test substance and I - * .* ^ s ^ - a c L s o iu t io n ^ T *
In the preliminary toxicity test, the maximum dose tested was 5000 pg per plate- this dose was achieved using a concentration o f lOOmg/mL and a 50 pL p la tin g ^ W NeithS
precipitate nor toxicity was observed. Based on the findings o f the toxicity tesMhe maximum dose plated m the mutagenicity test was 5000 pg perplate.
in the mutagenicity test, no positive response was observed. In the initial mutaeenicitv assay d o levels tested were 5000, 3333, 1000, 333 and 100 pg " l T T 2 precipitate nor toxicity was observed, in the independent repeat assavtoedose
were 5000,1800, 600,200 and 75 pg pa- plate. Neitherprecipitate nor toxicity was observed.
A lU r ite ri^ r &valid study were met as described, .the protocol. The results of the
* ... . , ,
fBacterial Reverse Mutation Test
withan Independent Repeat Assay indicate that, under die conditions o f this study, the test
substance did not cause a positive mutagenic response with any o f the tester strains in the
presence and absence o f Aroclor-induced rat liver S9.
BioReliance Study No. AA34BK.502001JBTL
CowBpanf Sauillxed. Does not @ortin TSCA CBt
PURPOSE
^ purpose of this study was to evaluate the m u t r i e potential of the test mMsnrog its ability to inducorevene mutations selected tod ofseven.1 a r i . ,, t
K. J,,
9 ^ 1 ^ . 10018 aSEschaida c o liW P l^ A in t t e pesen end
CHARACTERIZATION OF TEST AND CONTROL SUBSTANCES
a Thl? t f t mlf mce: H"24547' w88 received by BioReKanee on 17 August 2000 and was assigned the code numberAA34BK. The test siihgtaray was rharantmToA kv o ^ ,, ,, _
andC
um "
* " ** -
2 2 ^ t ^ WbSlaaX * * *
yellow solid
^ vehicle used to deliver H-24547 to the test system was acetone fCAS# 67 64. n
obtained from Fisher Scientific.
{ Ab# 6/-64-1),
Strain All Salmonella Strains
WP2 uvrA TA98
TA100, TA1535
TA1537 WP2 uvrA
S9 Activation
+
-
Positive Control
2-aminoanthracene (Sigma Chemical Co.)
2-nitrofluorene (Aldrich Chemical Co., Inc.)
sodium azide (Sigma Chemical Co.)
9-aminoacridine (Sigma Chemical Co.)
methyl meihanesulfonate (Aldrich Chemical Co., Inc.)
Concentration (pg/plate) 1.0 10 1.0
1.0
75
1,000
To determine the sterilily o f the test substance, the highest test substance dose level used in the mutagenicity test was plated on selective agar with an aliquot volume equal to that used in
BioReliance Study No. AA34BK.502001.BTL
9
Company Sanitized. Does not contain TSCA CB!
Test System
MATERIALS AND METHODS
ta t n n f f Stf inS used were ^ Sdmanella typhimurium histidine auxotrophs TA98
stafaW P^153!
^ deSCribed by Ames * a/- (1975), and Escherichia coli tested
1998 T * * S?^ SWereIBCeivd tt 11 Au8ust 1998 and 10 November ?TM ?T Umversity o f California, Berkeley, rf/ was received
L tkm d
*" **
Collection o f Industrial and Marine Bacteria, A berdZ ,
hist^ f f ^ J A 9 8 arid TA1537 are reverted from histidine dependence (auxotrophy) to W ta B in i^ r a d a ie e (prototrophy) by fiameshift mutagens. Tester strain TA1535 isrcvSted
I substitutions. Tester stem TA100 is reverted by mutagens
* * fiameshift andbasepair substitution mutations. Specificity o f the revereion
mectemsm m E colt is sensirive to base-pair substitution mutations, rather than fiameshift
mutations (Green and Muriel, 1976).
iramesmtt
the Cf T SW6re
inoculating fiom tire appropriate master plate or fiom
toe appropriate frozen permanent stock into a vessel containing ~50 mL o f culture medium To
assure theftcultoes were harvested in late log phase, the lengft o fincubation was contelled and
^ ^ r f^ ^ ^ ^ P ^ ^ a r e s tin g sh a k e r /in c u b a to r a tio o m tem perate The shaker/incubator was programmed to begin shaking at approximately 125 ipm at 372 C approxunately 12 hours before the anticipated time o f harvest. Each culm was monitored jiectrophotometricaUy for turbidity and was harvested at a percent transmittance yielding a titer o f greater than or equal to 0.3xl09 cells per milliliter. The actual titers were determined by viable count assays on nutrient agar plates, and the data is on file but not presented in this report The study was conducted to comply with OECD Guideline 471 (Genetic Toxicology: Bacterial Reverse Mutation Test), adopted July 1997 and with the
International Conference on Harmonisation o f Technical Requirements for Registration o f Pharmaceuticals for Human Use (1996 and 1997).
Metabolic Activation System
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was
prepared fiom male Sprague-Dawley rats indue! with a single intaperitoneal injection o f
Aroclor 1254, 500 mg/kg, five days prior to sacrifice. The S9 prepared by BioReliance was
batch prepared on 22 June 2000. The S9 purchased fiom MolTox was batch prepared 17
December 1999. AM S9 batches were stored at <-70C until used. Each bulk preparation o f S9
was assayed for its ability to metabolize 2-aminoanthracene and 7,12-
dimethylbenz(a)anthracene to forms mutagenic to Salmonella typhimurium TA100.
*
BioReliance Study No. AA34BK.502001.BTL
10
paint S a n itise Oses not fafei YSCA Ci?
Reverse Mutation
DuPont-4670
! ,p r e p a y immediately before its use and contained 10% S9 SmM
glucose^-phogihaie, 4 mM Mcotmamide-adenine dimicleotide n S g " 5 J
tL33 mM KC1 in a 100 mM phosphate buffer at ,,w ^ TM 7uP
containing 100 mM phosphate S ffer at pH 7 4 prepared
8 MgCI2 " d
Solubility Test
A to * " '*>TMUde. The test was
nroe ofthe& U om ug: t w t ,Ldimshyl sulfoxide (DMSQ), ethanol and ,- ^ ^ - f h T t a
substance was tested to determine the vehicle, selected in older o f
*u * * ^
prepo^on o f tte highest soluMe or w o d t a h le s t o o k e o n c e n t r a t it M .iS ^ i Pmm'ted
Preliminary Toxicity Test
The preliminary toxicity test was used to establish foe dose-range over which foe test substance irould be ass^ed. Vehiele and ten dose levels of the test X ta n c T ^ T o T f ^ ^ i '.,? ! '..?,!00: 3333^ TM PSPffphte) wereplated. Each dose level w a ite d TM a
s e l e e t i v e n . t o i n ^ a g S b o t h t h e p r e t L e ^ ' a ^ r f M H TM ^ ^ ^ ^ 0,1
Mutagenicity Test
The mutagenicity test initial assay (Experiment B l) and independent repeat assav
S Z t f } T T * ,* ! * " *
P-- * of the test i
i
pTre"sTenbceTMandf Sabs2enc^e o f Tra?t Tliv^er S9 TacAti?v8a'tiTonA.10A'llTdAo1s5e3l5e,vTelAs 1o5f37teasIt^suWbsPta2nceA, vhe.htihclee controls andpositive controls were plated in triplicate.
Plating and Scoring Procedures
2?
T d" 1y'te t " *11,6
test, the test sjstem was exposed
atondmueptdeasttesdubbsytaMncaerovniaanthdeApmlateesi(n1e9o8t3p)o.rationmethodologyas describedby Ames e/al ^fW75)
foe day o f its use, minimal top agar, containing 0.8 % agar (W/V) and 0.5 % NaCl CWV), was melted and supplemented with L-histidine, D-biotin and L-tryptophan solution to a final concentration o f 50 pM each. Top agar not used with S9 or Sham mix Was supplemented
of_water for ^ 100 mL o f minimal top agar. For foe preparation o f media and reagents, all references to water imply sterile, deionized water produced by the Milli-Q Reagent
BioReliance Study No. AA34BK.50200l.BTL
11
Company Sanitized. Does not contain TSCA c a t
DuPont-4670
Water System. Bottom agar was Vogel-Bonner minimal medium E (Vogel and Bonner containing 1 5 % QMV) agar. Nutrient bottom agar * V o g d - f l S l S S I l
o2E52X5 % ^ ^c M m m jl5 ^(W /V) agar and sn sp l< m ^m fl,2 .5 %(w/v)OxoidNii6iaitBi<>tliNo 2
Eat* platewas labeled with a code system that identified the test substance, test phase dose level, tester strain, and activation, as described in detail in BioRelimce's Standard O^ n . . . .
SQ * !5 ! S ii* b S s a use. One-half(0.5) mUliUtet o f 2 0 m T ^ r ^ f ' tW,^ * estersto" " >l 5 (iL ofw M cle or test substance were added to
^ o l e l ^ f S ' T c " ? ? 452C- * * " TM W fc * was overlaid onto the surface o f 25 inL o f mmmal bottom agar. When plating the positive controls, die test
p l f f l e s w e r c and incubated for approximately 48 to 72 hoars f 37f 2 ~ Plates that were not counted immediately following the incubation neriod were stored at 2-8C until colony counting could be conducted.
The condition o f the bacterial background lawn was evaluated for evidence o f test substance
1 tOHCity by using a dissecting microscope. Precipitate was evaluated by visual e * S t i S wiftout magnification. Toxicity and degtee o f precipitation wrae scored teladve t o l T S c k
control plate using the codes shown below.
111016
J BioReliance
Study No. AA34BK.502001.BTL
12
Company Sanfteei. Oo@snoi contain TSCACBI
DuPont-4670
Description
Characteristics
Normal Distinguished by a healthy microcolony lawn.
Slightly Distmguishedby a noticeable thim im ^fthe microcolony lawn and Reduced
Moderately Distinguished by a marked thinning of the microcolony lawn Reduced resulting in a pronounced increase in the size o f the microcolonies compared to the vehicle control pi*
Severely Reduced
Distinguished by an extreme thinning o f the microcolony lawn
resultmg m an mcrease unhe size o f the microcoionies compared to
&e velncte control plate such that the microcolony lawn is visible to tne unaided eye as isolated colonies.
Absent
a CmPlele *** f my microcli' ver
Obscured by % e background bacterial lawn cannot be accurately evaluated due to Precipitate microscopic test substance precipitate.
Non Distinguished by precipitate on the plate that is visible to the naked NP Interfering eye but any precipitate particles detected by the automated colony
Precipitate counter total less than 10% o f the revertant colony count (eg <3 particles on a plate with 30 revertants.)
Distinguished by precipitate on the plate that is visible to the naked IP eye and any precipitate particles detected by the automated colony
" ta y --
^
Revertant colonies for a given tester strain and activation condition, except for nositive
, controls, were counted either entirely by automated colony counter or entirelylby hand*unless
the test was the preliminary toxicity test or the plate exhibited toxicity. Hates
t
test substance precipitate to interfere with automated colony counting were counted manually.
Evaluation of Results
" 1" " " dev" on of " nber 0 , 1
For the test substance to be evaluated positive, it must cause a dose-related, increase in the mean revertants per plate of at least one tester strain with a minimum of two increasing concentrations o f test substance. Data sets for strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than three tunes the mean vehicle control value. Data sets for strains TA98 TA100 and
BioReliance Study No. AA34BK.502001.BTL
13 G o p f SamSfescL Bo not contain TSCA QSi
WP2 uvrA were judged positive if the increase in mean revertanis at the peak of the dose response is equal to or greaterthan two times the mean vehicle control value. Criteria for a Valid Test
entures nras demonstrate the presence of t deq> rough mutation m t o " * *" * G"1" 18 f tester stem s TA98 and TA100 must
te n fo ld mercase m the number of reyertante over the mean value o f the respective v S l e
A mmmum of three non-toxic dose levels arc required to evaluate
Adose
le^isconsider^toM cifoneorfc^ofthefoH ow ingm teriaarem et: (1 )A >50% reduction m the mean number o f reyertante per plate as compared to the mean vehicle control value This
--
1 Archives
^ **"-
The study raw data, protocol and all reoorte will h e m ,*;*! ^
,
Operating Procedure OPQP3040 by theBioReliance I ^ O A ,, n T T f ? J 5 d" d
BioReliance, 14920 Broschart Road, Rockville, MD 20850.
eadquartered at:
o f this study. deVaOnS 600140 pr0toco1 or assay-method SOPs occurred during the conduct
BioReliance Study No. AA34BK.502001.BTL
14
Company Sanitized. Does not contain TSCA CB1
Solubility Test
r esu l ts a n d d isc u ssio n
1f ted 95 016 So5vent o f choice based on solubility o f the test substance and
S S S o . . tar8e' Cd,S'
sotabte" * -- fa
Preliminary Toxicity Test
tBStWS
^ Tables 1 through 5. In the
g j s a a s i s s t r a 1.'; a m 1* ."
Mutagenicity Test
27' These ^ were^gen^eraIt!ed ^in Expfeariments6B1 and BM2. In th"e m"utaigzesndiciity
Z L T t l } m \ m ^ 100 fig per plate. Neither precipitate nor toxicity was 75 puge perpnlattei. Neither precipitate n8oSrS^tox' ?iciftyttwosaes,eobvsde8rtveesdt.ed were 5000,1800,600,200 and
No positive responses were observed with any o f the tester strains in the mTM. na
^ fS9 * --
assay
Theres* o f ( t o
^ B 5 5 B H 5 P H H S P P ( P B P ( i Bacterial Reverse Mutation Test
suh-tenrf*
Repeat Assay indicate that, under tRTconditions o f this study, the test
bstance thd not cause a positive mutagenic response with any o f the tester strains in the presence and absence o f Aroclor-induced rat liver S9.
puny SanlMi6d=Dos not onteSn TSC CIBI
est an
********
R evese Mutation
DuPont-4670
' REFERENCES
Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for Detecting Carcinogens and Mutagens with the SedmonellafMsmrmWm Microsome Mutagenicity Test, Mutation Research, 31:347-364.
Ames, B.N., J. McCann and E. Yamasaki (1975) Methods for Detecting Carcinogens and
Mutagens with the Salmonella/Mstamalim Microsome Mutagenicity Test, Mutation Research, 31:347-364.
Green, M.H.L. and W.J. Muriel (1976) Mutagentesting using tip* reversion in Escherichia coli, Mutation Research 3&3*32
International Conference on Harmonisation (ICH) o f Technical Requirements for Registration o f Pharmaceuticals for Human Use. Guidance on Specific Aspects o f Regulatory Genotoxicity Tests for Pharmaceuticals. S2A document recommended for adoption at step 4 o f die ICH process on July 19,1995. Federal Register 61:18198-18202, April 24,1996.
International Conference on Harmonisation (ICH) o f Technical Requirements for Registration o f Pharmaceuticals for Human Use. Genotoxicity: A Standard Battery for Genotoxicity Testing o f Pharmaceuticals. S2B document recommended for adoption at step 4 o f the ICH process on July 16,1997. Federal Register 62:16026-16030, November 21,1997.
Maron, D.M. and B.N. Ames (1983) Revised Methods for the Salmonella Mutagenicity Test
Mutation Research, 113:173-215.
y^
OECD Gurdeline 471 (Genetic Toxicology: Bacterial Reverse Mutation Test), adopted July
Vogel, H.J. and D.M. Bonner (1956) Acetylomithinase o f coli: Partial Purification and Some Properties, J. Biol. Chem., 218:97-106.
f
BioReliance Study No. AA34BK.502001JBTL
16
Company Sanitized. Does not contain TSCACBS
Bacterial Mutation Test Preliminary Toxicity Assay
Test Substance Id:
Table 1
Study Number Strain
Experiment No. Date Plated Vehicle
A34 .50200l.BTL TA98
1
29 Aug 2000
acetone
Test Substance Concentration pg per plate
Vehicle
With S9 Activation Revertants Background _per plate______ Lawn
21 i
Without S9 Activation
Revertants Background
per plate
Lawn
22 i
6.7 10 33 67 100 333 667 1000 3333 5000
Background Lawn c.nrlo
25 29 24
24 19 17 15 20 20 27
1 1
1 1 1 1 1 1
1 1
26 1
19 1
17 1
19 1 25 1 10 1
17 1
18 1 22 1 21 1
1 Normal; 2-Slightly reduced; 3=Severely reduced
4=Extremely reduced; 5=Absent;
NP=Non-Interfering precipitate C=Contaminated
6=Obscured by precipitate ; IP- Interfering precipitate
i
! !
StudyNo. AA34BK.502001.BTL
CsmapMI Sanitized. Bo oi Gsstfm YiiC?CB!
Bacterial Mutation Test Preliminary Toxicity Assay
Test Substance Ids
Table 2
Study Number Strain
: AA34BK7502001.BTL : TA100
Experiment No. : Al
Date Plated
: 29 Aug 2000
Vehicle
: acetone
Platino Aliquot : 50 uL
*
|
Test Substance
Concentration pg per plate
With S9 Activation
Revertants Background per plate____ Lawn ____
Without S9 Activation
Revertants Background per plate______ Lawn
Vehicle
91 i
129 i~
6.7 10 33 67 100 333 667 1000 3333 5000
Background Lawn Code
108 97
125 117 110 118 132 131 114 121
1 1 1 1 1 1 1 1 1 1
124 96 87
115 89 89
134 128 118 161
l=Nrmal; 2=Slightly reduced; 3=Severely reduced
wpEMtrere*y *educed; 5=Absent; 6=Obscured by precipitate NP=*Non~Interfering precipitate; IP- Interfii^ S ^ i L t e
BioReliance Study No. AA34BK.502001.BTL
18
jDompany Sanitized. Does not contain TSCA CBt
Bacterial Mutation Test Preliminary Toxicity Assay
Test Substance Id:
Table
Study Number Strain Experiment No. Date Plated Vehicle Plating Aliquot
Test Substance Concentration jag per plate
Vehicle
502001.BTL TA1535 A1 29 Aug 2000 acetone 50 p.L
With S9 Activation Revertants Background
Without S9 Activation Revertants Background
6.7 10 33 67 100 333 667 1000 3333 5000
Background Lawn Code
10 13 11 10 15
12 10 16 18 16
1 1
1 1 1 1
1 1 1 1
16 1 15 1 14 1 81 13 1 11 1 13 1 16 1
91
12
JJtexwa# M i f l h t l y reduced; 3=Severely reduced
i f S!ely reduced; 5=Absent; 6=0bscured by precioitate
NP=Non-Interfering precipitate; IB - I n t e r f e ? i r S p i r e t e
BioReliance Study No. AA34BK.502001.BTL
19
Company Sanitized, Does not contain TSCA CBI
Bacterial Reverse Mutation
DuPont-4670
Bacterial Mutation Test Preliminary Toxicity Assay
Test Substance Id:
Table 4
Study Number :
Strain
:
Experiment No. :
Date Plated
:
Vehicle
:
Plating Aliquot :
29 Aug 2000 acetone 50 uL
Test Substance Concentration -Iff per plate
Vehicle
with S9 Activation Revertants Background per Plate_______ Lawn
71
Without S9 Activation Revertants Background per plate______ Lawn
61
6.7 10 33 67 100 333 667 1000 3333 5000
Background Lawn Code
11 8
~
1 1 1 1 1 1 1 1 1 1
~"
10 ""
1 1 1 1 1 1 1
1 1 1
'
l=Normal; 2=Slightly reduced; 3=Severely reduced
m ? i f re!ely reduced; 5=Absent; 6=Obscured by precipitate NP-Non-Interfenng precipitate; IP= Interfering precipitate
BioReliance Study Mo. AA34BK.502001.BTL
20
enspanf SanRIzed. Sees not eortaln TSCA cat
Bacterial Mutation Test Preliminary Toxicity Assay
Test Substance Id:
Table 5
Study Number :
Strain
;
Experiment No. :
Date Plated
: 29 Aug 2000
Vehicle
: acetone
Plating Aliquot : 50 uL
Test Substance
With S9 Activation
Concentration
Revertants Background
g per plate_____ per plate______ Lawn
Vehicle
14 1
Without S9 Activation
Revertants Background
per plate
Lawn
12 1
6.7 10 33 67 100 333 667 1000 3333 5000
Background Lawn Code
16 12 11 14 15 11 10 13 13
15
1 1 1
1 1 1 1 1 1 1
91 15 1
11 1 17 1
12 1 10 1 14 1 10 1 13 1 91
l=Nrmal; 2=Slightly reduced; 3=Severely reduced
Mp-ifref ^ y edVced; 5"Absent; 6=0bscured by precipitate NP Non Interfering precipitate; IP= Interfering precipitate
BioReliance Study No. AA34BK.502001.BTL
21
Company Sanitized. Does not contain TSCA GB?
Test Substance Id
BBtuenai. mutation Test Table 6
100 01 37 1 02 19 1 03 25 1
27
333 01 37 1 02 34 1 03 41 1
37
1000
01 27 1 02 31 1 03 28 1
29
3333
01 28 1 02 31 1 03 25 1
28
5000
01 40 1 02 21 1 03 30 1
30 10
Positive Control 2-nitrofluorene 1.0 pg per plate 01 391 1 02 283 1
__________________ 03
337 1
Background Lawn Code
337
54
1f o r m a i ; 2=S1ight1y reduced; 3=Severely reduced
reduced; 5=Absent; 6=Obscured by precipitate NP=Non-Interferrng precipitate; IP= Interfering precipitate
BioReliance Study No. AA34BK.5Q2001 J3TL
22 Cnpanf Sanitized,, D e not oeontain TSCA CBf
m
Bacterial Mutation Test
Test Substance Id:
Table 7
Study Number Strain
Liver Microsomes Vehicle Plating Aliquot
a a : .502001.BTL TA98
Rat liver S9 acetone 50 uL
Concentration pig per plate
Plate Revertants Number per plate
Vehicle
01 02 03
27 34 27
100
01 02
27 1 29 1
03 26 1
333 01 42 1 02 34 1 03 37 1
1000
01 38 1 02 23 1 03 30 1
3333
01 31 1 02 34 1 03 21 1
5000
01 30 1 02 36 1 03 25 1
Experiment No : B1 Cells Seeded : 1-1 X 10 Date Plated : 12 Sep 2000
Average Standard Revertants Deviation
29
27
38
30
29
30
Positive Control 2-aminoanthracene 1 .0 pg per plate
01 385 1
02 457 1
03 313 1
385
Background Lawn Code
72
l=Nrma1; 2=Slightly reduced; 3=Severely reduced
MDEHtre?efy fed?ced' 5=Absent; 6=0bscured by precipitate NP=Non-Interfering precipitate; IP- Interfering precipitate
BioReliance Study No. AA34BK.5Q2001.BTL
Company Sanitised. Does not contain TSCA Cffl
Bacterial Mutation Test
Test Substance Id:
Table 8
Study Number Strain
Liver Microsomes Vehicle
AA34 .502001.BTL TA100 None
acetone
Experiment No Cells Seeded Date Plated
Concentration pg per plate
Vehicle
95
B1 1.3 X 10fl 12 Sep 2000
1000
01 103 1 02 82 1 03 97 1
94 11
3333
01 83 1
I I
02 91 1 03 93 1
89
5000
01 88 1 02 110 1 03 116 1
105 15
Positive Control sodium azide 1 ,
01 02 __________________03
341 320 426
Background Lawn Code
pg per plate
1 1 1
362
56
l=Normal; 2=Slightly reduced; 3=Severely reduced
MDBMtre?efy ^educed; 5=Absent; 6=Obscured by precipitate NP-Non-Interfering precipitate; IP= Interfering precipitate
BioReliance Study No. AA34BK.502001.BTL
24
Coipamf SanHfacdL D o mi contain TICA C
Bacterial Mutation Test
Test Substance Id:!
Table 9
Study Number Strain
: AA34B; 02001.BTL : TA100
Liver Microsomes : Rat liver S9
Vehicle
: acetone
Plating Aliquot 50 uL
Experiment No Cells Seeded Date Plated
B1
1.3 X 108 12 Sep 2000
Concentration P9 per plate
Vehicle
100
333
1000
Plate Number
01 02 03
01 02 03
01 02 03
01 02 03
Revertants per plate
88 96 107
78 113
96
96 91 81
89 102 105
Background Code
1 1 1
1 1 1
1 1 1
1 1 1
Average Revertants
97
96
89
99
Standard 10 18 8 9
3333
01 79 1 02 81 1 03 61 1
74 11
5000
01 86 1 02 87 1 03 82 1
85 3 .
?ositive Control 2 -aminoanthracene 1 .0 pg per plate
01 321 1
02 971 1
03 445 1
579
345
1-Normal; 2=Slightly reduced; 3=Severely reduced
Mr,Eftremely reduGed; S=Absent; 6=Obscured by precipitate HP-Hon-Interfering precipitate; IP- Interfering P S p i ? , t e
Study No. AA34BK.502001.BTL
Company Sanitized. Does not contain TSCA CBi
al Reverse Mutation
Bacterial Mutation Test
Test Substance Idrj
Table 10
Study Number Strain
Liver Mierosomes Vehicle Plating Aliquot
37ES# 502001. BTL TA1535 None
acetone 50 pL
Experiment No : B1 Cells Seeded : 1.7 X 108 Date Plated : 12 Sep 2000
Concentration jg per plate
Vehicle
Plate Number
01 02 03
Revertants per plate
14 18 13
Background _Code
1 1 1
Average Revertants
15
Standard Deviation
100 01 17 1 02 23 1 03 25 1
22
333 01 15 1 02 20 1 03 15 1
17
1000 3333
01 17 1 02 21 1 03 10 1 01 13 1 02 16 1 03 14 1
16 14
5000
01 19 1 02 15 1 03 16 1
17
Positive Control sodium azide 1.0 pg per plate
01 401 1 02 262 l ________ ___________ 03_______ 281_______1_________
Background Lawn Code
315
75
l=Normal; 2=Slightly reduced; 3=Severely reduced
4-Extremely reduced; 5=Absent; 6=Obscured by precipitata NP-Non-Interfering precipitate; IP= interfering precipitate
BioReliance Study No. AA34BK.502001.BTL
26
igOTspaif Samltad,, D@@snot
TSCA OBI
ucu-Lciiai Mutation Test
Test Substance Id:i
Table 11
Study Number Strain
Liver Microsomes Vehicle
AA34BHI502001.BTL TA1535 Rat liver S9 acetone
Experiment No Cells Seeded Date Plated
B1
1-7 X 108 12 Sep 2000
Concentration
]ig per plate
Vehicle
Plate Number
01 02 03
Revertants per plate
19 8
17
Background Code
1 1 1
Average Reverront
15
100 01 11 1 02 9 1 03 14 1
11
333 01 13 1 02 12 1 03 9 1
11
1000
01 17 1 02 11 1 03 14 1
14
3333
01 15 1 02 13 1 03 11 1
13
5000
01 12 1 02 13 1 03 21 1
15
Positive
Control
2 -aminoanthracene 01 79
1 ..0 pg 1
per
plate
02 69 1
03 71 1
Background Lawn Cr
73
Standard ueviation
6 3 2 3 2 5
5
1"Normal; 2=Slightly reduced; 3=Severely reduced
N?No-Interfer^Cedf 5=^b?entf O b s c u r e d by precipitate NP-Non Interfering precipitate; IP= Interfering precipitate
BioReliance Study No. AA34BK.502001.BTL
27
C om paq Sanitized. Dees not contain TSCA CI
!
DuPont-4670
Bacterial Mutation Test
Table
Test Substance
Study Number Strain
Liver Microsomes Vehicle
AA3 '50200l.BTL TA1537 None
acetone 50 uL
Experiment No Cells Seeded Date Plated
B1
1.1 X 108 12 Sep 2000
Concentration pg per plate
Vehicle
Plate Number
01 02 03
Revertants per plate
5 7 9
Background Code
1 1 1
Average Revertants
7
Standard Deviation
2
100 01 7 1 02 5 1 03 C
61
333 01 7 1 02 4 1 03 6 1
62
1000
01 9 1 02 9 1 03 3 1
73
3333
01 9 1 02 10 1 03 5 1
83
5000
01 9 1 02 6 1 03 2 1
64
Positive Control 9-aminoacridine 75 pg per plate
01
1070
1
02 _________________ 03 ___
Background Lawn Code
978 691
1 1
913 198
1"Normal; 2=Slightly reduced; 3=Severely reduced
4--Extremely reduced; 5=Absent; 6<=Obscured by precipitate
NP=Non-Interfering precipitate; IP= Interfering precipitate ^Contaminated
BioReliance Study No. AA34BK.502001.BTL
28
tom pasjf SanStlixed.!Ooes noticontain TSGA C8
est wita anaindepend
Reverse Mutation
DuPont-4670
Bacterial Mutation Test
Test Substance Id
Table 13
Study Number Strain
Liver Microsornes Vehicle
.502001.BTL TA1537
Rat liver S9 acetone
Experiment No Cells Seeded Date Plated
B1 1.1 X 10s 12 Sep 2000
Concentration tig per plate
Vehicle
Plate Number
01 02 03
Revertants per plate
6 4 5
Background Code
1 1 1
Average Revertants
5
Standard Deviation
1
100 01 4 1 02 3 1 .
03 7 1
52
333 01 5 1 02 5 1
03 8 1
S2
1000
01 6 1 02 3 1
03 8 1
63
3333 01 2 1 02 6 1
03 5 1
42
5000 01 4 1 02 5 1
03 6 1
51
Positive Control 2 -aminoanthracene 1 .0 ug per plate 01 80 1 02 84 1 03 113 1
Background Lawn Code
92
18
1"Normal; 2=Slightly reduced; 3=Severely reduced
4"Extremely reduced; 5 ~Abs0nt! 6=Obscured by precipitate
NP=Non-Interfering precipitate; IP= Interfering precipitate
BioReliance Study No. AA34BK.502001.BTL
29
Company Sanftteed. Does not eontain TSCA cel
J Bacterial Mutation Test
Test Substance Id:
Table 14
Study Number Strain
Liver Microsomes Vehicle Plating Aliquot
S02OO I.BTL WP2 uvrA
None
acetone 50 uL
Experiment No Cells Seeded Date Plated
Concentration pg per plate
Vehicle
Plate Revertants Number 'per plate
01 12 02 16 03 19
Background Code
1 1 1
Average 16
100 01 11 1 02 15 1 03 10 1
12
333 01 12 1 02 16 1 03 22 1
17
1000
01 13 1 02 14 1 03 14 1
14
3333
01 12 1 02 10 1
03 19 1
14
5000
01 13 1 02 14 1
03 8 1
12
B1 3.0 X 108 12 Sep 2000
Standard
4
3
5
1
5
3
Positive Control methyl methanesulfonate 1000 pg per plate
01 73 1
02 69 1
03 72 1
71
2
l=Normal; 2=Slightly reduced? 3=Severely reduced 4=Extremely reduced? 5=Absent? 6=Obscured by precipitate NP=Non-Interfering precipitate? IP-- Interfering precipitate
BioReliance Study N o. AA34BKL5020013TL
" 30
1
G p f Sanitised. 0@es not gate TSCA CBj?
DuPont-4670
oacLsnai Mutation Test
Test Substance Id:
Table 15
Study Number Strain Liver Microsomes Vehicle
502001.BTL WP2 uvrA
Rat liver S9 acetone 50 pL
Experiment No Cells Seeded Date Plated
B1
3^0 X 10 12 Sep 2000
Concentration pg per plate
Vehicle
Plate Number
01 02 03
Revertants per plate
11 16 14
Background Code
1 1 1
Average Revertants
14
Standard 3
100 01 16 1 02 12 1 03 11 1
13 3
333 01 10 1 02 15 1 03 12 1
12 3
1000
01 12 1 02 9 1 03 10 1
10 2
3333
01 11 1 02 13 1 03 10 1
11 2
5000
01 11 1 02 13 1 03 12 1
12 1
Positive Control 2 -aminoanthracene 10 pg per plate U1 94 1 02 70 1 03 171 1
Background Lawn Code
112
53
l=Normal; 2=Slightly reduced; 3=Severely reduced
m Ef rT r y reduced; 5=Absent; GObscured by precipitate NP=Non-Interfenng precipitate; IP Interfering precipitate
BioReliance Study No. AA34BK.502001.BTL
31
*Wnpan* " " w * O w i noi contain TSCA C
Bacterial Mutation Test
Test Substance Id:
Table 16
Study Number Strain
Liver Microsomes Vehicle Plating Aliquot
34Bl 502001.BTL TA98
None
acetone 50 pL
Experiment No Cells Seeded Date Plated
Concentration pg per plate
Vehicle
Plate Number
01 02 03
Revertants per plate
16 17 23
Background Code
1 1 1
Average 19
75 01 17 1 02 12 1 03 15 1
15
200 01 12 1 02 14 1 03 20 1
15
600 01 14 1 02 16 1 03 16 1
15
1800
01 18 1
02 14 1
03 16 1
16
5000
01 17 1 02 16 1 03 18 1
17
Positive Control 2 -nitrofluorene 1 .0 pg per plate 01 191 1 02 190 1 03 170 1
184
B2 2,4 X 10s 20 Sep 2000
'Standard Deviation
12
1-Normal; 2=Slightly reduced; 3=Severely reduced ^Extremely reduced; 5=Absent; 6=Obscured by precipitate NP=Non-Interfering precipitate; if- Interfering p r K p i t a t e
BioReliance Study No. AA34BK.502001JBTL
32
Company Sanitized. Does not contain TSCA CBI
Bacterial Reverse Mutation
Bacterial Mutation Test
Test Substance Id
Table 17
Study Number
Strain
: TA98
02001.BTL
Liver Microsomes : Rat liver S9
Vehicle
: acetone
Plating Aliquot : 50 uL
Experiment No : B2
Cells Seeded ; 2.4 X 108
Date Plated : 29 Sep 2000
Concentration J*9 Per plate
Vehicle
75
200
600
1800
5000
Plate Number
01 02 3
01 02 03
01 02 03
01 02 03
01 02 03
01 02 03
Revertants
19 16 20
18 11 15
14 13 21
14 9 6
13 11 12
13 14 17
Background Code
1 1 1
1 1 1
1 1 1
1 1 1
1 1 1
1 1 1
Average Reyertante
18
15 16 10 12 15
Standard Deviation
Positi*. Control 2-aminoanthracene 1 .0 ,,g p,, plat.
02 --- 03
Background Lawn Code
191 I
293 1
------------- -- ---------------- *
201 88
* S S iy " S d
3s r r 8 ly t8,i,ioed
HP.Hon-Int.rf.ringg prre.coiSpiitSattie-; IP= I?nterfberyipnrgpcriepciitpitt.ate
fesnpanf B anited, Bee
n oi contain TSCACBl
Bacterial Mutation Test
Test Substance Id:j
Table 18
Study Number
Strain
: ^ ^ R k *|[)2001 .BTL : TA100
Liver Microsomes : None
Vehicle
: acetone
.Plating Aliquot
50 pL
Concentration
Jig per plate
Vehicle
Experiment No Cells Seeded Date Plated
B2 1.8 X 108 2? Sep 2000
Background Lawn Code
__ --
4=Extramely~reducedf
3=Severely reduced
NPNon-Interfering precipitai;
Jrfey .preciPltate 'terterrng precipitate
BioReliance Study No. AA34BK.502001.BTL
Company Sanitized. Does not eontain TSCA CB!
Bacterial Mutation Test
Test Substance Id
Table 19
Study Number Strain
: 341 02001.BTL ; TA100
Liver Microsomes ; Rat liver S9
Vehicle
acetone
Platinqr Aliauot ifl riT
Experiment No Cells Seeded Date Plated
B2 1.8 X 10 29 Sep 2000
Positive Control 2-aminoanthracene
01 604
02 571
_______________ 03
520
Background Lawn Code
l^Normai; 2=slightiy reduced; 3=Severel
reduced
NP-NonSTey ^ed?Ced; 5=Absent; 6=Obscured by precipitate NP-Non-Interfering precipitate; IP= Interfering preripiLte
BioReliance Study No. AA34BK.502001.BTL
35
C@napamf Sanitized, Does moteontain rSC C il !
Bacterial Mutation Test
Te.st Substance Id
Table 20
Study Number
Strain
.
--- 02001.BTL TA1535
Liver Microsomes : None
Vehicle
. acetone
.Plating Aliquot - 50 uL
Experiment No Cells Seeded Date Plated
B2 2.5 X 10 29 Sep 2000
Concentration per plate
Vehicle
Plate Number
01 02 03
Revertants Per plate
12 7
10
Background Code
ji. 1
1
Average Standard Revertants iDeviation
10
75 200 600 1800
01 11 1 02 12 1 03 17 1
01 14 1 02 12 1 03 13
01 11 1 02 12 1 03 12 1
01 14 1 02 13 1 03 13 1
13 13
12
13
5000
01 15 1 02 12 1 03 12 1
13
Positive Control sodium azide 1.0 pg per plate
uj. 364 I
D2 245 i
03 268 i Background Lawn Code
292 ____ 63
4=Extremely~reducedf 5 = A b S t 32 ^ alV i ! dUCed . NP-Non-Interfering precipitati; Ii>= Inefering^eiptate
; BioReliance
Study No. AA34BK.502001 .BTL
36
Compemy Sanitized. Does not contain TSCA CBS
i
DuPont-4670
Bacterial Mutation Test
Test Substance Id:
Table 21
Study Number i
502001.BTL
Strain
; TA1535
Liver Microso^es : Rat liver S9
Vehicle
; acetone
_Plating Aliquot t 50 uL
Experiment No Cells Seeded Date Plated
2
2;.5 X 108
29 Sep 2000
_ug per plat^ S t S e r per6p l a ^ S ^ o d e " *1 RAvera9e Standard
Vehicle-------^ --- E~
~10
----- --------- Revertants
1
02 8 1
03 11 1
10
Devl-^
75
01 02
12 1 13 1
03 16 1
14
200 01 8 1 02 9 1 03 8 1
600
01
02
6 8
03 11
1800 5000
01 02
91 71
03 13 1
01 02
13 1 91
03 15 1
10 12
Positive Control 2-aminoanthracene 1.0 pg per plate
02 13933 Xj
03 ---------_____________________ 109
Background Lawn Code
21
p r .c lp lt.ti, ` 5 X .
I
I
I
I i
" " W - O o not M d , , TSC C*l
Reverse Mutation
DuPont-4670
Bacterial Mutation Test
Test Substance Id
Table 22
Study Number Strain
Liver Microsomes Vehicle Plating Aliquot
AAc 02001.BTL
TA1537 None
acetone 50 pL
Experiment No : B2 Cells Seeded : 0.8 X 108 Date Plated * 29 Sep 2000
Concentration pg per plate
Vehicle
Plate Number
01 02
03
Revertants per plate
5
6
Background Code
1 1 1
Average ;Standard Revertants Deviation
75 01
02
03
1 1 1
200 01 02 03
1 1 1
600 01 02
1 1
D 03 1
1800
01 02 03
41 31 41
5000 01 7 1 02 6 1 03 7 1
Positive Control 9-aminoacridine 75 pg per plate ux 215 i
02 196 i
03 167 i Background Lawn Code
_
193
. - 24
SP=Non~Interfering
V S " " 7 r9d"ce'i
erfeiSrprcState
BioReliance StiKfy No. AA34BK.50200J.BTL
38
Company Sanitized. Does not contain TSCA GBl
I
'Bacierai Reverse Mutation
DuPont-4670
Bacterial Mutation Test
Test Substance Id:
Table 23
Study Number Strain
. 37Q 537 02001.BTL
V ehicleCrOSOmeS : Racaettolinvee r S9
Experiment No : B2 Cells Seeded : 0 .8 X 109 Date Plated : 29 Sep 2000
Concentration pg per plate
Vehicle
Plate Number
01 02 03
Revertant Per plate
5 11 7
75 01 6 02 4 03 6
200 01 4 02 7 03 7
600 01 5 02 3 03 6
1800
01 7
02 6
03 6
5000 01 6 02 5 03 2
Positive Control 2 -aminoanthracene 01 89 02 82 03 68
Code
1 1 1
1 1 1
1 1 1
1 1 1
1 1 1
1 1 1
1 1 1
Average Standard ..Ravertants Deviation
80 11
S S ily 'r S c S
r,dU" d
p recip itati, S ^ S f e ^ r S S p t . t .
BioReliance Study No. AA34BK.502001 .BTL
39
Sanitized Does not contain TSCACB!
Test Substance Id
Study Number Strain Liver Microsomes Vehicle
Bacterial Mutation Test Table 24
02001.BTL
Experiment No
Vehicle
01 02 03
12 1
14 1 15 1
14
75 01 15 1 02 13 1 03 11 1
13
200 01 12 1 02 11 1 03 11 1
11
600 01 10 1 02 11 1 03 13 1
11
1800
01 12 1 02 15 1 03 18 1
15
5000
01 12 1 02 16 1 03 16 1
\
15
Positive Control methyl methanesulfonate 1000 pg per plate 01 128 1
02 94 1
03 96 1
1 nc
Background Lawn Code
' *----
19
4=Ejrt-rTni 2 =S1ightly reduced; 3=Severely reduced HDEMtre? efy ^educed; 5=Absent; 6=0bscured by precinitate NP=Non-Interfering precipitate; It- Int.rfSiSg P S p i L t e
Study No. AA34BK.502001.BTL
40
0-**-- "
oT" CB'
__ Test Substance Id
Bacterial Mutation Test Table 25
Study Number
: AA34BiCB02001.BTL
p rain .
: WP2 uwrA
l i m crosMies : Rat liver S9
Vehicle
. acetone
-- J-atinq Aliquot ; 50 uL
Experiment No Cells Seeded Date Plated
B2
3.5 X 10 29 Sep 2000
Vehicle 75 200 500 1800 5000
01 02
03
01 02
03
01 02
03
01 02
03
01 02
03
01 02
03
16 1 12 1 13 1
19 10 12
11 13 11
11 13 11
14 1 14 1 10 1
15 1 12 1 91
Average Revertants
14
14
12
12
13
12
Positive Control 2-aminoanthracene 10 pg per plate
u 145 I
2 153 1
03
r1e8o6
ii
Background Lawn Code
lgl
Standard Deviation
SSl'redSd?
" ely
KP"=Non'Interfering precipite, p-'S f S i ^ ^ J ^
BioRelance
Study No. AA34BK.502001.BTL
41
Company Sanitized. Does not contain TSCA CB
Bacterial Mutation Test Summary of Results
Test Substance Id
Table 26
St-u-d-y-N-u-m-b-e-r--- ---:--*RK33B#5020m .rBtTtL_____ Ex_peri.ment No : B1
Liver M i c r o s o S r ^ r " " 118
PUte *
DeviatiTM
- Dse (pg/plate) TA98_____ TA100
Vehicle 100 333 1000 3333
5000
Positive
31 1 2 96
27 9 99
37
4 91
29 2 94
28 3 89
30 _____ 337
10 105 54 362
15 21 21 11
5 15
56
Liver Microsomes: Rat liver S9
TA1535
15 22 17
16
14 17
315
3 4 3
6
2 2
75
TA1537
7 2 6 1 6 2
7 3 8 3
6 4 913 198
WP2 uvrA
16 12 17 14 14 12 71
4
3 5 1 5 3 2
Dose (pg/plate) TA98______TAIOO
TA1535
TA1537 WP2 uvrA
1
Vehicle 100 333 1000 3333 5000
Positive
29 27 38 30 29 30 385
4 97 10 2 9.6. 18 4 89 8 8 99 9 7 74 11 6 85 3 72 579 345
15 11 11 14
13
15 73
6 3 2 3 2 5 5
5 1 14 3
5 2 13 3
6 2 12 3
6 3 10 2
4 2 11 2
5 1 12 1 92 18 112 53
Vehicle = Vehicle Control
Positive = Positive Control
Plating aliquot: 50 pL
I
f f
BioReliance Study No. AA34BK.502001.BTL
42
@mpas8f Sanitized. Bos not esrtaSn TSC CB
Bacterial Mutation Test Summary of Results
Test Substance Id
Table 27
Studi
Numbe=r7----- ^gAg^TMBggLr.5w0*2000011..BRTtLt.
Tin : B2
Liver Micro3oii" *5B o S VertantS
W e Standard Deviatio^
Dose (
Vehicle 75 200 600 1800 5000 Positive________
Diver Microsoiaes:
Rat liver S9
WP2 uvxA
-Dose (p q /p la te )
Vehicle
in
27 05 0 600 1800 5000
1l g5
12 xg
Positive
Vehicle = Vehicle Control Positive = Positive Control Plating aliquot: 50 pL
BioReiiance Study No. AA34BK.502001.BTL
43
Company Sanitized. Does not contain TSC CSi
APPENDIX A Historical Control Data
BioRfiliance Study No. AA34BK.50201 .BTL
-fflpwy SanftbecL S e e s not contain f $ C A I
Strain
TA98 TA100 TA1535 TA1537 WP2vrA r.
Historical Ngative and Positive Control Values 1997-1999
revertants perplate
Control
Neg Pos Neg Pos Neg Pos Neg Pos Neg Pos ,,.
Activation
Mean 16
397 128 592 11 424
6 819
16 190
N<Mie SD Min
64 216 21 29 53 159 129
51 145 6
30 468 13
53 127 35
Max 59
1536 288 1920 45 1024 30 2786 48 961
Mean 21 649 144
785 12 98 7
102 17 338
Rat liver SD Min
76 33~ 56 30 62 300 106
51 79 8 31 128 2 62 282 16
Max
2454
2871 42
1640
1835
2632
""tijlh. ou, mflA-TUMMmmn vaiue; iNeg=negative control
to feio,,iz8d
** - -* *
BioReliance Study No. AA34BK.5020013TL
"" 1-- 45
Company SanHfeed. Bees hot contain 7SC A c
APPENDIX B Study Protocol
DuPont-4670
BioReliance Study No. AA34BK.502001.BTL
46
1Does not contain TSCA CBi
s* S.-?
1.0 PURPOSE
strains coli W P2uvrA
2.0 SPONSOR
2.1 Name:
22 Address:
2.3 Representative:
EX du Pont de Nemours and Company
Stine Haskell Research Center DuPont Haskell Laboratory P. . B ox 50 1090 BUcton Road Newark, DE 19714-0050
Maria Donner, PhJD.
2.4 Sponsor Project No.:
D uP on t-4670
2.5 W R *
2.6 H askell#:
-
H -24547
2.7 Service Code: Cm 2
3.0 IDENTIFICATION OF TEST AND CONTROL SUBSTANCES
3.1 Test Substance Name:
3.2 Test Substance LD.: 3 3 Controls:
H-24547 (to be used in the report text)
ITotocolSPGT5Q200J 16-Aug-2000
BioRellance Study No. AA34BK.502001 .BTL
o-g-tofO
# B i o MiRcofaeiollogiicjatA.n c f
47
Gwnpanf SanRtaad. 0notain TSCA C il
Sponsor Project Nm nber
DtiPnnt-4 fi7n
BioReliaace Study Nuraber AA34BK<avww p j |
3-4 Test Substance Chanwerization
f
(orvehicle).
3J} T est Substance Rtention Sample
th Sponsor.
4.0 TESTWOFACSJIYANDKBYPERSONNEL
4.1 MiNian mm e. ,:
Toxicoogy Testing Facility BioReliance
4.2 Address:
9630 Medical CenterDrive
R ockville.M D 20850
4 3 Study Directon VaJeatlne O. Wagner ffl, M.S
1 Phone: 301-610-2152
Fax: 301-738-2362 Email: swagW fcbioraliance.a
5.0 PROPOSED STUDY DATES
5.1 Experimental Start D ate
i 30-Asig-2000
5 2 Experimental Termination Date: 17-0ct-2000
5.3 Draft Report Date: 5.4 Final Report Date: 6.0 TEST SYSTEM
|3 1 -0 c t-2 0 0 0 8 -N ov-2000'
K " " A described
E. co li tester strani
ProtocolSPGT502001 l-Aug-2000
BioReliance Study No. AA34BK.502001.BTL
P ^ c flO
48
# BFourinoirMiRcrobiollogiie*a!Unnocaoel.a O tm g m w Samfttad, Bm rn netmataiTS CB1
Sponsor Proj N um ber___ D u P o n t? BioRellance Study Num ber _ A A 34B K *nym i f TT
.Gwotypeofthe StrainU,}for Mi Tettili
Each S . typhinmrium tester strain
..
operon, additional motations that
J ^ P 00 " * * ^ d in e
m wation results in a cell wall d e f i c S w ^ L ^ S t J !
*&*&** The rfa
certain classes o f chem icals such
Permeability o f the ce ll to
otherwise be excluded. H ie
,,j
^ g . sysiem s thai W0^
excision-repair system T e L ^ r S * 5
* & deficient DNA
Plasmid (carrying the R-factor) T U ^ n ^
IT T
P 4101
seassvty t mutagens by m odifying an e a d s t f f S l i ^ ^ p a simd increases conlexinvolvedwithttemtoatt^air^^ *>***1 D N A repair polymerase
T A A O ____ i i n a
TA98 and TA1537 a ie revetted from independence (prototrophy) by
dependence (auxotrophy) to hiBtttnr
'1
fi neshift and base substitution cause base substitutions.
TA 1535 Is revm ed o n l ^ S L ^ s t a
b a secium * a a
duoaKwomeamwinf
k m due
^ ^ 1 b" S- shroge elsewhere ja ^
Tte S. tvphimilrinm tester Cfrsinr
, ..
U niversity>f C a l l f o r a ^ S ^
*** Bn * Ames,
National C o l t e c t i t m T U S ^ d t ^ l S S , ^
" " * * ^ *
Kingdom).
a M anne * " * * * , Aberdeen, Scotland (United
7.0 EXPERIMENTAL DESIGN AND METHODOLOGY
T h e te t ^ t a n c e w iil be tested at a minimum o f fiv e dose levels along with a n rem ri^
WP2 uvrA with
P
controls and positive controls w ill be plated in triplicate. ` f test substance, negative
W1ISPGT502001 16-Aug-2000
BioReliance Study No. AA34BKL5Q2001.BTL
PSgeSrfm
" fBTMl*O'1MR!cE>etiLolDI|icAal*N>iocC7I,7ri
49
Conpaiqr Sanitized, Bag not orniate TSAO il
eject Number
D oP ^ ^ ^ a
BioReUance Study Num ber -- AA34B1T
^
7.1 MubiUtyDetommatjoE
A solubility deienninadoa w ill be conducted to tfetemiin* 1 concentration or workable suspension ud to a
.
72 PreUniinaiy Toxicity Assay to Select Dose Levels
assay. - This preliminaryv assavy a.
" 8 F Bl "ay toxicity
s^tiTsasas^rsssSaSSSr^
J
S Sto lew! will 1, S S S S S S S ^
s ^ s js s s s a s s s
le ss than 5 mg/plate and this dose level is nontoxic.
" **** dose level is
73 Frequency and Route o f Administration
sS B ^ avK sn sssara: a ^ s s s s rjja s is S a 5
% l l t r . g r i S :.1? J ' . ^ . . bee " 1 ^ - - t o ld .y
be 0 b same
i Compaq l a f c r t B e not contain TSCACBt
Sponsor Project Num ber;. DuPont-4A7ft BioRelianceStudy Number: A A 34M r.w nf
s s s s s s x a is s s s s ir ^ *
7.4 Controls 7.4.1 Positive Controls
Strain
WKuvrA TA98
TA100.TA1S35 TA1537
1 WP2 uvrA
7-4-2 Negative Controls
S9 Activation
Sat
Potitiv* Control
None
1
I ..
2-nteeBHKae
S^mmoseridi* methyl raetbtneiulfoiiate
1JO 10 1.0 1.0 75 t,000
7.4.3 Sterility Controls
wiu oc cnecBsa tor sterility.
13 Exogenous Metabolic Activation
* hig y a I " yi B T S R
^ m b io s ^ S S S lm ' r -* * * !* -- * -- - * a
. lQ 0nM pbo^bu)f a ,, H 7 J ^ ^ : l TM
m u w ill b e lOOmM p to sp b a u ta a b -a / b t a '
Protocoi SPGTSQ20Q1
P*85rfJ0
S 5 y J_3i lllM * P "
" " '8 9 s t o m
C p * F i H ind. D@m m i saftsto TSCAm
Sponsor Project Num ber
th rtw .4 6 7 0
BioReliance Study N u n * :___AA34BK Sf)?nm ypy
7.6
A ll cultures w ill be harvested by
^
^SSiAEm ttSr *" * -- * -
7.7 T w t System Identification
Standard Operatmg Ptocedures. 7.8 Test Substance Reparation
^ described in BiuRelnoce's
a i be prcprai Im n e d M , w use. a u test substance dostng w ill be nt room tempenture untteryultow
7.9 Treatm em ofTest System
^ Sm
* -* .
and
vorcex
be t W 6 e inveited n d i S X
7.10 Scoring ^ <pdition o f tbe tMctedalbackgrotiDd lew e w ill b eevaluetedffyw nH .------- -
ProfocolSK3T50200I
warance o f the bacterial backgrmmd lawn.
52
Cnprsf Santtized. Dees not contafn TSCACBi
Reverse Mutation
D uP on t-4670
W
Sponsor Project Number: _ D u iw e g m
BioReliance Study Number: AA34 R g
j pj y
7.11 Tester Strain Verification On the day o f use in the m utagenicity____ .11 - ' . .
.
8 .0 CRITERIA f o r d e t e r m in a t io n o f a v a l id t e s t
8.1 Tester Strain Integrity
strain of
T A rCf t e ^a S So t Si iyS R a S t " *"w"*e'wf sihi,alBm "C*o"han1s0otfoTTMA98*aTMnd
1 8 2 Spontaneous Revertant Background Frequency
Based on historical control data, all tester
characteristic number o f s p tm ta r Z m , . , J S L f ^ f " n T * ejdriWt
(vehicle). The mean
S f f f ^ c o n tr o ls
S S S . S t ,0-st*"* * * * n S H t t S f . 3""
8 3 Tester Strain Titers
8.4 Positive Control Values
a m ? n t e nHISt * * * to # a <hrae fold incn the respective mean negative control value (vehicle) for each tester strain.
over
8.5 Toxicity
a nthtcthn in the
PwMcotsPcrrsoeobt
Page7 ofJO
C w panf Sanitized. D@gn8fffafnTSG m i
\
Sponsor Project Number: D tr tW 4 * fli BioRclianee Study N um ber__ A A 3 4 R r sn?nm p-^
9.0 EVALUATION OF TEST RESULTS
f l ? ! T iDf ted pOSitive' * TM * cause . dose-related t o c ^ in the concentrations o S s u b s ^ i as s p S S T ^ 8 minimUB, f *W 9.1 Strains TA1535 and TA1537
S ^ o s s ^ c i-s g a s :
9.2 Strains TA 98,TA 100 and W P2w A
Data sets w ill be judged positive if the increase in m eant
^ J g e k1 * . M ,,
at d peak o f negative control
10.0 REPORT
1
MayS S S S S K T * * '" " "* " oa,M^e f l **'^
Strains: strains used; numbero f ceiM nL per culture; strain characteristics.
w E S J E S S i^
*wa**`
Discussion o f results. Conclusion.
ProtoeoiSPGT50200I
16-Am-MOO
Re8 ofl0
3 BioReliance
W Study No. AA34BK.502001JBTL
54
W B io R e l ia n c p F arm o tljr M ic r o b io lo g ic a l A s to e ia itt
Company Sanitized. Does not contain TSCA CBt
Sponsor Project Number. DuPont-4670 BioReliance StudyNumber: AA34BK sn?nnf
A ge and >bodyweightsofrats used toprepareS9 11.0 RECORDS ANDARCHIVES
All raw data, die protocol and all reuoits will K
j ,.
12,0 REGULATORY REQUBEMENTS/GOODLABORATORYPRACTICE
S SSS
l
* ' of
disposed of following finalization ofthe 5 p o T *"* *
13.0 r e f e r e n c e s
and mutagen i s S S Z
*T testing carcinogens
Research 31347-364.
" """^HiiiGWiaTO mutagenicity trat Mutation
a S 3 8 ^ 3 2 .MUtagen teSting USiDg tip* reversion in
**i
tegfttoty Genotoxi<% Tests foTpJ^m accuSc
" Spedfic ****** of
aStBS.5^ -- ^ B T S E
. K H , TM , Wj, n , 17. t o t e ,
I I
Protoco!SK3T502001 16-Aug-2000 PageSoflO
i | B R *fotmiarolyMicrobeiollogiiae*!Anssocciatees
BioReliance
i
Study No. AA34BK.502001JBTL
55
CMMpaiif Sarcftfmi. Dos* raowSaln TiG C if
BioReliancg Study Niim ben -- A ^ 8 Ki?Q?J0Ol.BTL.
83(1 A" - B *N- <1976). Detection o f carcmogeas as n im a -n s in
S m S E S " " "* * ,* * * * *.. Z S T lw S lZ r
Sci. U SA 7 2 3 1 3 5 ? ^
* * * 300 cheaucais- * * . Nati. Acad.
M"boi` ** "*
" " ***
14.0 APPROVAI.
-----
r ^ ~~Ty^/^N.
SponsorReprsentative ---------------"
"X.%.
3 .C O O
1
ffla.r.fe. ~Dtm er
ProtocolSPOTSC20Q1
16-Aug-2000
lOoflO
v1 BioReliance
Study No. AA34BK.502001.BTL
56
W B R *toraiiaorlyMicroebiolloij.aealAnttoccistee
eoppanF Santlfeed. Does noi contata T SC A
CBI
APPENDIXC Information for Japanese Regulatory Agencies
CoiMpanf Sanitized Dees not oKtain TSCA CB1
Report o f Results o f Reverse-Mutation Assay in Bacteria
1. General Items
Name o f the new chemical substance (IUPAC nomenclature)
Other name
Structural formula or rational formula (or outline o f manufacturing method, in case both are unknown) --
Purity o f the new chemical substance tested
Name and concentration o f impurities
Fluoroalkyl Urethane
100.0 wt%
Molecular weight
I Appearance at ordinary 2 temperature
Stability g 8 Melting point o > Boiling point
Vapor pressure g
Partition .2 coefficient
1 Solubility
J
Water
l DMSO 'S Degree o f 8 solubility Acetone fIt Other
J _______ )
300 mg/mT. J
extent
P ^ c n e n u c a l properties are reference materials, fffi in spaces to
2 m ^tab1iiiiy for water' other solvents, heat, light, etc. 2- V ^ r ^ e ssu r e -^ i mthe v^K>rpressure of the test s u S ic e S a S X
name o f the solvent
4. ''Solubility,,-Fill in such information as water-soluble, soluble in oil 5. Degree o f solubility"-Fill in the solubility at 25C for each solvent
BioReliance Study No. AA34BK.502001.BTL
58
Company Sanitized. Does not contain TSCA CBI
;
Bacterial Reverse Mutation
2. Tester Strains
(1) Procurement
Strain
TA98 TAI 00 TAI 535 TA1537 TA1538 TA97 TA102 WP2 uvrA WP2 nvrA (pkMIOl) WP2 (pKMIOl)
Obtained from
Date obtained 10 November 1998
* Date inspected the strain lot in storage
Dr. Bruce Ames University o f
California, Berkeley
11 August 1998
13 December 1990 14 November 1990
National Collection o f Industrial and Marine Bacteria
Aberdeen. Scotland
1 July 1987
The genetic markers for each
culture are
confirmed on die day o f use
3.S9MX
BioReliance Study No. AA34BK.502001.BTL
59
ipan^ Sanitized. O net eenteln TSA CB1
DuPont-4670
Species, Strain
Sex Age (in weeks) Weight
Rattus norvegicus, Sprague Dawley Name
Inducing substance Aroclor 1254
Male 9
234 to 252 g
Administration method
'Administration period and amount
intraperitoneal
:5 days, 0.5 gm/kg body weight
4. Positive Control Substance (1) Positive control
3
(2) Preparation and storage ofpositive control solution
PoePare0rSt0re
I Store subdividedsolutions (Storage temp. -5 to - 3QC) ]
BioReliance Study Ho. AA34BK.502001.BTL
Company Sanitized. Does not contain-recACBl
5- Preparation o f Test Substance Solution
Solvent used
"Name Acetone
`Manufacture Fisher Scientific
Stability o f test substance
in the solvent
Unknown
*LotNo. `Grade
`Purity
970721; 943596;
971014
Certified ACS
Reason to choose the solvent
Solubility determination
Method o f suspension when test substance is difficult to dissolve
Not applicable
fiom preparation to use for test
Conversion by purity
BioReliance Study No. AA34BK.502001.BTL
61
WBspiOTfSanitized, Bo* m i eseteSn TSA CBS
6. Conditions o f Pre-culture
(1) Conditions
Nutrient broth
Period o fpre-culture
._
J- from
inoculation to beginning o f
shaking culture
^ ame 1 'Manufacturer
Oxoid Nutrient Broth No. 2
Oxoid Ltd.
121 hours
`Lot No. CH,-B=219916
2 to 5 hours at ambient temperature
Storage time and temp, from end o f culture to use for test
Model and manufacturer o f shaker
Method o f shaking (shaking type, speed, etc.)
hours at 2-8C New Brunswick Scientific, model G-24 Rotary (125rev/min.)
_ Cultore vessel (top e, capacity) | shpe: cylinder. 200 n T
Culture volume Volume o f inoculum
50 mL 1 colony
7. Agar Plate Medium
lB es ofconiate TSCA cm
8. Test Results - Judgement o fthe results
D u P on t-4670
Judgement
I Negative
Reason forjudgement and referential matters:
No positive response was observed with any o fthe tester absence o f A reforinducri m, Uver SQ j
Referential matters
* P ^ e n c . and
VeI^cte ^ Positive control values indicate that nil trcw _ -------- ~ -------------
!are valid.
1
BioReiiance Study No. AA34BK.502001.BTL
63
Bmpaif Sanitized. B n4
TSCACBl
