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## - ntUNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON D
.C., 20460

VIA FACSIMILE: 201-437-4801 April 19,2012 Attention: Glenn Merritt (201-437-4000) OFFICE OF PREVE NTION, PESTICIDESAND TOXIC SUBSTANCES

Subject: Follow-upto fax transmission of4/13/12 (ePMAremoval ofCBI claimsregarding identified studies) 

### DearMr. Merritt: This is sent to you pursuant ourconversation ofseveral minutes ago. In Larry Robinson's fax of April 13th while there is a waiver ofconfidential business information (CBI) claims in the identified studies there is a an assertion ofcopyrightprotections and some additional text that provides in part ... "Reproduction, reference, summarization, retransmission or otheruse ofthis copyrighted document.
..including electronic transmission, using the internet oremail
....is strictly prohibited
...." While we understand the basis for the copyrightprotections, we have a concern that the language in the fax might be interpreted to mean that ePMA is asserting that the studies submitted perthe TSCA activity are not to bemade available in an Agencye-docket (electronic docket) orbe included in EPA websites among the collection ofTSCAhealth and safety studies. We are thinking that the identified limitations noted in the fax are not intended to apply to EPA's normal procedures for the handling ofnon-CBI materials submitted by entities subject to TSCA regulations (including the posting ofsuch materials ongovernment websites) and submittedby such entities in order to comply with a TSCA requirement, but are rather intended to limit entities outside the government from the unauthorized use or appropriation ofthe studies. Canyou please confirm CPMA's intent? Many thanks.

Scott M. Sherlock, Attorney Advisor Environmental Assistance Division Office ofPollution Prevention and Toxics

# BASF
en-jk 034092-RPT.doc STUDY TITLE REPORT SALMONELLA TYPHIMURIUM /ESCHERICHIA COLI REVERSE MUTATION ASSAY (STANDARD PLATE TEST AND PREINCUBATION TEST) WITH Alkali Blue 

### Dry 61001 Lot 2236308 DATA REQUIREMENT OECD No. 471 EC 2000/32, B. 13 /8.14 AUTHORS Dr. G.Engelhardt (Study Director) Dr. E.Leibold STUDY COMPLETED ON March 23,2004 TESTING FACILITY Experimental Toxicologyand Ecology BASF Aktiengesellschaft 67056 Ludwigshafen, Germany LABORATORY PROJECT IDENTIFICATION Project No.: 40M0437/034093 SPONSOR BASF Akiengeselischaft 67056 Ludwigshafen, Germany 

# IThis
docurnent contains manufacturing andtrade secrets o1 BASF. it as the propertyofBASF and maybe used onlyfor that purpose for which itwas intended by BASF. Every otheror additional use, exploitation, reproduction, publication or submission toother parties require thewritten permission of BASF, with the exception of regulatory agencies acting within the lmits of theiradministrative authority. 

### Page 1of57

### Report; Project No.: 40M0437/034093 THIS PAGE ISINTENTIONALLY LEFT BLANK

Page 2of 57

# BASF Report, Project No.:40M0437/034093 

### GLP COMPLIANCE STATEMENT
This studywas conducted ingeneral accordanceWith 

### the OECD Principles ofGood Laboratory Practiceand the GLP Principles ofthe German "Chemikaliengesete (Chemicals Act) and meetthe United States Environmental Protection Agency Good Laboratory Practice Standards [40 CFR Part 160 (FIFRA) and Part 792 CrSCA)), with the exception that recognized differences exist between the GIP Principles/Standards of OECD and the Principles/Standards of FIFRA andTSCA. However, there was the following deviation from the requirements ofthe above-mentioned principles:

-The 

### stability ofthetest substance inthe vehicleDMVSO and inwater was not

determined analytically.

Dr. rer. nat. G.Engelhardt (Study Director)

Page 3of57

# BASF Report; Project No.:40M04371034093 THIS PAGE ISINTENTIONALLY LEFT BLANK

Page 4 of57Y~~ Aua I&



### Report Project No.: 40M04371034093 

### SIGNATURE PAGE

-Study 

### Director IConduct 

### ofstudy: 22fra 2a'o C 

### Dr. rer. nat. G.Engelhardt

Management 

# Aw.~' zd~

oof Dr. rer. nat E.Leibod

Page 5of 57

### Report; Project No.: 40M0437/034093 

### STATEMENT ofthe QualityAssumnce Unit The QualityAssurance Unit (QAU) inspectedthe study and reported anyinspection resultsto the Study Directorand to Management. Thefinal report reflects the raw data. Phase of study Date of inspection Reported to Study Directoir anid to Management 1[mm-dd-yyyyj ['Fmui-dyyyyj Sdy Plan: 10-20-2003 10-20-2003 Conduct ofStudy: 12-04-2003 12-04-2003 Report: 03-18-2004 03-18-2004

Ludwigshafenl, 

# 22
- ........ ..... . ......

Page6 of57

### Report; Project No.:40M0437/034093 

# LRhdnlandpfaV

ffir Pflaiztnben mad Pflanzenwacbt 

### GLPD-Beschelnlgung /Statement of GLP Compliance (gwnamuI oorfttoI1 lb Abe. I Chwnkcflkflauat) Fine GLP-Inapakdonzur Oboiwachunq derEinhaitung Paissmant ofconfonnit with GLP according der GLP-Grundsilzo geniASChemikalainoet bzw. to Chemikalengesetz and Directive 85J20/EEC at Richilrhe 8'20/EG wurdo durcge~ht i: -jTPrOfelnrlctun Tea flt E) PrOfsandot/Test at BASF Aktiengesellschaft Experimentelle Toxikologie und Okologle 0467058 Ludwigshafen Pr(Ifungen nach Katogorien /Areas of Experli

InMftara~fl Ch"VmVWL Mr. 5.SIOECD riowi)
Datum dorInspoiction /Date of Inspection

......................... .... 5M2001 und vorn 21. bis 28.08.2001......

DIol~or garni ProfenrictuntPr&%tandjort bofindat Theabove mentioned ba llytest m ~ ite Is hIduded aidi Imn natlonalen GL-P-Oberwachungsverfahren urd inthe national GLP-Caiiplance Prograiwno and Is iulrd roglnilfig auf Elnhaltigder GLP-GnmdKsttze Inspected onearegular basi. Auf der Grundiage d83 InapekllonsberliteswWr Mor- Based on the Irapedon report Itcan be cordimd, mit

beatfitigt. doss In dowse Pr~evk~uglemem that W3toutfedWttat ot Isable tW Conductthe Pr~fatandotd ie oben genmnntan Pro*Jf~ngentar Eln- aforementioned studies I comipilance withthe Pikihalfig doe GLP-Grnamtzs durdgelhrt warden ciples ofGLP. I~rnnf.

Untorschiilt Daw/ln ate

LandO&IW2IaOrPtAe,2enbaI WWi WoIWa~b EasW*&~.M 144, 045128MOWT 

### Page 7 of57

# BASF Report; Project No.: 40M0437/034093 CONTENTS TITLE PAGE BLANK PAGE GLP COMPLIANCE STATEMENT BLANK PAGE SIGNATURE PAGE STATEMENT OF THE QUALITY ASSURANCE UNIT STATEMENT OF GLP COMPLIANCE (FROM THE COMPETENT AUTHORITY) CONTENTS 

### 1. SUMMARY 2. INTRODUCTION 2.1. PRINCIPLE 2.2. REGULATORY GUIDELINES 

### 3. MATERIAL AND METHODS 3.1. TEST SUBSTANCE 3.2. TEST SUBSTANCE ANALYSIS 3.2.1. Test substance 3.2.2. Testsubstance preparation 3.3. EXOGENOUS METABOLIC ACTIVATION 3.3.1. S-9 fraction 3.3.2. S-9 mix 3.4. BACTERIA 3.4.1. Salmonella typhimurium 3.4.2. Esoherichia coli 3.4.3. Checking out the tester strains 3.5. EXPERIMENTAL PROCEDURE 3.5-1. Choice ofthe vehicle 3.5.2. Mutagenicitytests 3.5.2.1. Standard plate test 3.5.2.2. Preincubatlon Test 3.5.3. Titer determination 3.5.4. Control articles 3.5.4.1. Negative controls 3.5.4.2. Positive controls 3.5.5. Scope oftests andtest conditions Page 8of57

# BAS. Report; Project No.: 40M0437/034093

3.6. EVALUATION 3.6.1. Mutagenicit 3.6.2. Titer 3.6.3. Toxicity 3.6.4. Solubility 3.6.5. Acceptance criteria 3.6.6. Evaluation criteria 3.7. RETENTION OF RECORDS 4. RESULTS 4.1. MUTAGENICITY 4.1.1. Standard platetest 4.1.1.1. TestswithoutS-9mix 4.1.1.2. TestswithS-9mix 4.1.2. Preincubation test 4.1.2.1. Tests without S-9 mix 4.1.2.2. Tests with S-9 mix 4.2. TOXICITY 

### 4.3. SOLUBILITY 4.4. DISCUSSION 4.5. CONCLUSION 

### 5. REFERENCES
Appendix 1: Tables 1
- 

### 9(Standard plate test) Appendix 2: Tables 10- 14 (Preincubation test) Appendix 3: Sterility control Appendix 4: S-9 fraction characterization Appendix 5: Historical negative control data Appendix 6: Historical positive control data

Page 9of 57

# BASF Report; Project No.: 40M04371034093 

### 1. SUMMARY The substance Alkali Blue Dry 6100 /Lot2236308 was tested for its mutagenic potential based on the abilityto induce point mutations inselected loci ofseveral bacterial strains, i.e. Salmonella typhimurium and Esoherichia col, inareverse mutation assay.
" STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E.coil WP2 uvrA " DOSE RANGE: 0.8 pg - 

### 5750 pg/plate (SPT; Salmonella strains) 23.0 pg -5 750 pg/plate (SPT; E.coliWP2 uvrA)

4.0 pg - 

### 1500 pg/plate (PIT; all testerstrains) *TEST CONDITIONS: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (Aroclor-induced rat liver S-9 mix).I *SOLUBILITY: Precipitation ofthe test substance was found from about 100 pg/plate onward. *TOXICITY: Abacteriotoxic effectwas observed under all test conditions (for details see item 4.2.).

*MUTAGENICITY:

An increase inthe number ofhis* ortrp 4 

### revertants was not observed inthe standard plate test or inthe preincubation test either without S-9 mix orafter the addition ofa metabolizing system. *CONCLUSION: According to the results ofthe presentstudy, the test substance Alkali Blue Dry 6100 / Lot 2236308 is not mutagenic inthe Salmonella typhimurium/Escherichis coli reverse mutation assay under the experimental conditions chosen here.

Page 10 of57

### Report; Project No.: 40M0437/034093
2. INTRODUCTION 2.1. PRINCIPLE Bacteria[ reverse mutation assays using amino-acid requiring strains ofSalmonella typhimuriumn and Escherichia coi are commonly employed as initial screening methods for detecting apoint mutagenic activity ofchemical substances (1,2,5)and are used to screen for possible mammalian mutagens and carcinogens. The principle ofthese assays isthat mutations are detected by the reversion of mutations present inthe bacterial strains.This leads to arestoration of the functional capability of the bacteria to synthesize the essential amino acid and thus to the abilityto grow inthe absence ofthe amino acid required bythe parent strains. Most ofthe substances are not mutagenic or carcinogenicthemselves, but onlyafter metabolic transformation, which invivo iscatalyzed mainly bythe enzyme systems ofthe liver. Therefore, the tests are carried out not only directly, but also inthe presence ofan exogenous metabolic activation system. The most commonly used system is acofactorsupplemented postmitochondrial fraction (S-9) obtained from livers of rats treated with an enzyme-inducing agent, e.g. Aroclor 1254. Experimental starting date: November 6,2003 Experimental completion date December 12, 2003 (evaluation last experiment):

2.2. REGULATORY GUIDELINES The tests are carried out inaccordance with the following guidelines:

e OECD No.471 (July 21, 1997)
e EC Directive 2000/32, B-13 IB.14 (May 19, 2000)

Page 11 of57- - i 

### ~ra~ ~imI IJULIII~u~ui~I~ilL I JLIIIL II L L i IL


# BASF Report; Project No.: 40M0437/034093 

### 3. MATERIAL AND METHODS 3.1. TEST SUBSTANCE Name oftest substance: Alkali Blue 

### Dry 6100/Lot 

### 2236308 Test substance No.: 03/0437-3 Batch No.: Lot 2236308 GAS No.: 1324-76-1 Date offilling: September 18, 2003 Purity/Composition: 88 %main component (See analytical data of October 9,2003) Appearance, consistency: Blue Powder Storage: Room temperature

Page 12 of57

# BASF Report; Project No.: 40M0437/034093

3.2. TEST SUBSTANCE ANALYSIS 3.2.1. Test substance The stability ofthe test substance understorage conditions isguaranteed as indicated by the sponsor, and the sponsor holds this responsibility. The homogeneity ofthe test substance was guaranteed by mixing before preparation of the testsubstance formulations. The analytical investigationswere carried out inthe:

-Analytical 

### Laboratory of BASF Corporation Huntington (characterization)
3.2.2. Test substance preparation The stability ofthe test substance inthe vehicle DMSO and inwater was not determined analytically. More detailed information about thetest substance can befound inthe raw data and may be requested from the sponsor (BASF Aktiengesellschaft).

Page 13 of 57

# BASF Report; Project No.: 40M0437/034093
3.3. EXOGENOUS METABOLIC ACTIVATION 3.3.1. S-9 fraction The S-9 fraction isprepared according toAmes et al. (1,2). At least 5male Sprague-Dawley rats (200
- 300 g,Charles River Deutschland GrnbH) receive a single intraperitoneal injection of500 mg Aroclor 1254 (as asolution incorn oil with a concentration of 20 g/l00 ml) per kg bodyweight 5days before sacrifice. During this time, the animals are housed inMakmolon cages inair-conditioned rooms inwhich central air conditioning guaranteed a range oftemperature of20- 24C and arelative humidity of 30
- 70%. Theday/night rhythm is12 hours (light periodfrom 6.00
- 18.00 hours and dark period from 18.00-6.00 hours). Standardized pelleted feed and tapwater from bottles are available ad libitum. 5days afteradministration, the rats are sacrificed, and the livers are prepared (all preparation steps for obtainingthe liver microsome enzymes are carried out using sterile solvents and glassware at atemperature of+4*C). The livers are weighed 

### andwashed Inan equivalent volume of a150 mM KCI solution, then cut intosmall pieces and homogenized inthreevolumes of KCI solution. Aftercentifugation ofthe homogenate at 9000 xgfor 10 minutes at+40C, 5ml portions ofthe supernatant (so-called S-9 fraction) are stored at -700 to -80*C. The preparation date and protein content ofthe S-9 fraction are given inAppendix 4. 3.3.2. S-9 

### mix The S-9 mix isprepared freshly priorto each experiment (1,2). Forthis purpose, asufficient amount ofS-9 fraction isthawed at room temperature and 1volume of S-9 fraction Ismixedwith 9 volumes of S-9 supplement (cofactors). This preparation, the so-called S-9 mix, iskept on ice until used. The concentrations of the cofactors In the S-9 mix are: MgC 2 8mM KCI 33mM Glucose-6-phosphatase 5mM NADP 4mM Phosphate buffer (pH 7.4) 15 mM The phosphate buffer (6)isprepared by mixing an Na 2HPOA solution with an NaH2PO4 solution in a ratio ofabout 4 :1. To demonstrate the efficacyof the S-9 mix inthis assay, the S-9 batch was characterized with benzo(a)pyrene (seeAppendix 4).

Page 14 of57

### Report; Project No.: 40M04371034093
3.4. BACTERIA Fortesting, deep-frozen (-700 

### Cto -800C) bacterial cultures (Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and E.coi WP2 uvrA) are thawed at room temperature, and 0.1 ml ofthis bacterial suspension isinoculated innutrient broth solution (8gDifco nutrient broth + 

### 5g NaCI/liter) and incubated inthe shaking water bath at37*C for about 10
- 

### 12 hours. As arule, agerm density of> 108 bacteria/ml isreached. These cultures grown overnight are kept iniced water from the beginning ofthe experiment until the end inorderto preventfurther growth. The use ofthe strains mentioned is inaccordancewith the current scientific recommendations forthe conduct ofthis assay. The Salmonella strains were obtained from KNOLL Aktiengesellschaft on October 30, 1989. The Escherichia colistrain was obtained from Merck on September 9, 1991.
3.4.1. Salmonella typhimurium The rate of induced back mutations of several bacteria mutants from histidine auxotrophy (his-) to histidine prototrophy (his*) is determined (2,3,4). The tester strains TA 1535, TA 1537, TA 98 and TA 100 selected byAmes and coworkers are derivatives of Salmonella typhimurium LT2 and have GC base pairs atthe primary reversion site. All strains have adefective excision repair system (uvrB), which prevents the repair of lesions which are induced inthe DNA, and this deficiency results ingreatly enhanced 

### sensitivity of some mutagens. Furthermore, all strains show 

### aconsiderably reduced hydrophilic polysaccharide layer (rfa), which leadsto an increase inpermeability to lipophilic substances. The strains TA 1535 andTA 100 are derived from histidine-prototrophic Salmonella strains bythe substitution mutation his G46 and are used to detect base pair substitutions. TA 1537 and TA 98 are strains for the detection offrameshift mutagens. These strains carry different frameshift markers, i.e. the +1mutant his C 3076 inthe case of TA 1537 and the +2type his D3052 inthe case ofTA 98. The strains TA 98 and TA 100 carry an R factor piasmid pKM 101 (4)and, inaddition to having genes resistant to antibiotics, they have amodified postreplication DNA repair system, which increases the mutation rate by inducing adefective repair inthe DNA; this again leads to aconsiderable increase in sensitivity.

Page 15 

### of 57

# BASV Report; Project No.: 40M04371034093 3.4.2. Escherichia coil Escherichia col WP2 uvrA which has an AT base pair at the primary reversion site isa derivative of E.col WP2 with adeficient excision repair and isused to detect substances which induce base pair substitutions (5). The rate of induced back mutations from tryptophan auxotrophy (tr) totryptophan independence (tVp) is determined.
3.4.3. Checking out the tester strains The Salmonella strains are checked for thefollowing characteristics at regular Intervals: deep rough character (rta); UV sensitivity 

### (,& uvrB); ampicillin resistance (Rfactor plasmid). E.col WP2 uvrA ischecked for UV sensitivity.Iw Histidine and tryptophan auxotrophy isautomatically checked ineach experiment Via the spontaneous rate.

Page 16 of57

### Report; Project No.:40M0437/034093
3.5. EXPERIMENTAL PROCEDURE 3.5.1. Choice ofthe vehicle Due tothe limited solubility ofthe test substance 

### inwater, DMSO was selected as the vehicle, which had been demonstrated to be suitable inbacterial reverse mutation tests and for which historical control data are available.
3.5.2. Mutagenicity tests 3.5.2.1. Standard plate test Salmonella typhimurium The experimental procedure ofthe standard plate test (plate incorporation method) is based on the method ofAmes et al. (1,2). Test tubes containing 2-mi portions of soft agar (overlayagar), which consists of 100 ml agar (0.6% agar +-0.6% NaCI) and 10 ml amino acid solution (minimal amino acidsolution for the determination of mutants: 0.5 mM histidine + 

### 0.5 mM biotin) are kept inawater bath at45*C, and the remaining components are added in thefollowing order: 0.1 ml testsolution or vehicle 0.1 ml fresh bacterial culture 0.5 ml S-9 mix (intests With metabolic activation) or 0.5 ml phosphate buffer (intestswithout metabolic activation) After mixing, the samples are poured onto Vogel-Banner agar plates (minimal glucose agar plates)within approx. 30 seconds. Composition of the minimal glucose agar: 980 ml aqua dest.

20 ml Vogel-Banner Emedium 15g Difcobactoagar 20 g D-glucose, monohydrate. After incubation at 37'C for48
- 

### 72 hours inthe dark, the bacterial colonies (his" revertants) are counted.
Escherichia coli Page 17 of 57

# BASF Report; Project No.: 40M0437/034093 The experimental procedure isbased on the method ofAmes et al. (1,2). Test tubes containing2-mI portions ofsoft agar (overlay agar), which consists of 100 ml agar (0.6% agar +-0.6% NaCl) and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) are kept inawater bath at 45TC, and the remaining components are added Inthe following order 0.1 ml test solution or vehicle 0.1 ml fresh bacterial culture 0.5 ml S-9 mix (in tests with metabolic activation) or 0.5 ml phosphate buffer (intestswithout metabolic activation) After mixing, the samples are poured onto minimal agar plates within approx. 30 seconds. Composition of the minimal agar: The composition ofthe minimal agar (SAl selective agar) isbased on the description ofGreen, M.H.L. and Muriel,W.J. (5), wvith the exception ofsolution E(tryptophan solution), which has previously been added tothe softagar 300 ml solution B(agar) 100 ml solution A (saline solution)

8ml solution C(glucose solution) 10 ml solution D(casein solution) After incubation at 370C for48
- 

### 72 hours inthe dark,the bacterial colonies (trp* revertants) are counted.

Page 18 of 57

# B-RASF Report; Project No.: 40MO437/034093 3.5.2.2. Preincubation Test The experimental procedure is based on the method described by Yahagi etal. (7)and Matsushima et al. (8). 0.

1 ml test solution orvehicle, 0. 1ml bacterial suspension and 0.5 ml S-9 mix or phosphate buffer are incubated at 37*C forthe duration of about20 minutes using a shaker. Subsequently, 2ml ofsoft agar isadded and, after mixing; the samples are poured onto the agar plates within approx. 30 seconds. After incubation at37'C for 48
- 

### 72 hours inthe dark, the bacterial colonies are counted.
3.5.3. Titer determination Inthe standard plate test, 0.1ml ofthe overnight cultures is diluted to 10~in each case. Testtubes containing 2-mI portions ofsoft agar containing maximal amino acid solution (5mM tryptophan or 5mM histidine + 

### 0.5 mM biotin) are kept inawater bath at 450C, and the remaining components are added inthe following order: 0.1 ml vehicle (without and with test substance) 0.1 mlfresh bacterial culture (dilution: 1O0) 0.5 ml S-9 mix Inthe preincubation test, 0. 1ml ofthe overnight cultures isdiluted to 10O ineach case. 0.

1ml vehicle (with and without test substance), 0. 1ml bacterial suspension and 0.5 ml S-9 mix are incubated at 37*C forabout 20 minutes using ashaker. Subsequently, 2ml of softagar containing maximal amino acid solution for titer determination (5mM tryptophan or 5 mM histidine + 

### 0.5 mM biotin) isadded. After mixing, the samples are poured ontothe agar plateswithin approx. 30seconds. After incubation at 37 0

### Cfor48
- 

### 72 hours inthe dark, the bacterial colonies are counted.

Page 19of 57

# BASF Report; Project No.: 40M0437/034093
3.5.4. Control articles 3.5.4.1. Negative controls Parallel with each experiment, negative controls are carried out inorderto checkfor possible contaminants (sterility control) and to determine the spontaneous mutation rate (vehicle control).

*Sterility control

Additional plates are treated with soft agar, S-9 mix, buffer,vehicle or thetest substance butwithout the addition oftester strains (see Appendix 3). *Vehicle control

The vehicle control with and without S-9 mix only contains the vehicle used for-the test substance at the same concentration and volume for alltester strains.
3.5.4.2. Positive controls The following positive controls are used to check the mutability ofthe bacteria and the activity of the S-9 mix: With S-9 mix

*2-aminoanthracene (2-AA) (SIGMA, A-1381)10

- 

### 2.5 pg/plate, dissolved inDM50

- 

### strains: TA 1535, TA 100, TA 1537, TA 98

-60 pg/plate, dissolved inDM50

- 

### strain: Escherichia coli WP2 uvrA

Page 20 of 57

# BASF Report; Project No.: 40M0437/034093 Without S-9 mix

*N-methyl-1'-nitro-N-nitrosoguanidine (MVNNG) (FLUKA, 68051)

-5 pg/plate, dissolved in DMSO

- 

### strains: TA 1535, TA 100 *4-nitro-o-phenylendiamine (NOPID) (SIGMA, N-9504)

- 

### 10 pg/plate, dissolved inDMSO

- 

### strain: TA 98 *9-aminoacridine (AAC) (SIGMA. A-I 135)

- 

### 100 pg/plate, dissolved inDMS0

- 

### strain: TA 1537 *4-nitroquinoline-N-oxide (4-NQO) (SIGMA, N-8141)

- 

### 5 pg/plate, dissolved in DMS0

- 

### strain: E.coli WP2 uvrA The stability ofthe selected positive controls iswell-defined under the selected culture conditions, since theyare well-established reference mutagens.

Page 21 of 57

# BASF Report; Project No.: 40M0437/034093 3.5.5. Scope oftests and test conditions 1st Experiment Strains: TA 1535, TA 100, TA 1537, TA 98, E.col WP2 uvrA Doses: 0; 23; 115; 575:2875 and 5750 pg/plate Vehicle: DMSO Type oftest: Standard plate testwith and without S-9 mix Number of plates: 3test plates per dose or per control 2nd Experiment Strains: TA 1535, TA 100, TA 1537,TA 98, Doses: 0; 0.8; 4; 20; 100 and 500 pg/plate Vehicle: DMS0 Type oftest: Standard platetest with and without S-9 mix Number of plates: 3-testplates per dose or per control 3rd Experiment Strains: TA 1535, TA 100, TA 1537, TA 98, E.coil WP2 uvrA Doses: 0; 4; 20;:100;500 and 1500 pg/plate Vehicle: DMSO Type oftest: Preincubation testwith and without S-9 mix Number of plates: 3test plates per dose or per control

Page 22 of57

# BASF Report; Project No.: 40M0437/034093
3.6. EVALUATION 3.6.1. Mutagenicity Individual plate counts,the mean number of revertant colonies per plate and the standard deviations were given for all dose groups aswell asfor the positive and negative (vehicle) controls inall experiments. Ingeneral, fivedoses ofthe test substance are tested With amaximum of 5mg/plate, and triplicate plating isused forall test groups at least inthe 1st experiment. Dose selection and evaluation as well as the number of plates used inrepeat studies orfurther experiments are based on the findings ofthe 1st experiment.
3.6.2. Titer The titer isgenerally determined only inthe experimental parts with S-9 mix both for the negative controls (vehicle only) and for the two highestdoses in all experiments.,
3.6.3. Toxicity Toxicity detected bya

- 

### decrease inthe number of revertants

- 

### clearing or diminution ofthe background lawn (=reduced his-ortrp background growth)

- 

### reduction inthe titer isrecorded for all testgroups both with and without S-9 mix inall experiments and indicated inthetables.
3.6A4. Solublilty Precipitation ofthe test material isrecorded and indicated inthe tables. As long as precipitation does not interfere with the colony scoring, 5mg/plate is generally selected and analyzed (incasesof nontoxic compounds) asthe maximum dose at least inthe 1st experiment even inthe case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses> 5mg/plate might also be tested inrepeat experiments for further clarification/substantiation.

Page 23 of 57fl~III~inLMKjiLL4IIJ ] At-I I



# BASS: Report; Project No.: 40M0437/034093 3.6.5. Acceptance criteria Generally, the experiment isconsidered valid if the following criteria are met: " The numberof revertant colonies inthe negative controls was within the normal range of the historical control data for each tester strain (see Appendix 5). " The sterility controls revealed no indication ofbacterial contamination. " The positive control articles both with and without S-9 mix induced asignificant increase inthe number ofrevertant colonieswithin the range ofthe historical control data or above (see Appendix 6). *The titer ofviable bacteria was> I081m1.
3.6.6. Evaluation criteria The test chemical isconsidered positive inthis assay ifthe following criteria are met

*A dose-related and reproducible increase inthe number of revertant colonies, i.e.

aboutdoubling ofthe spontaneous mutation rate inat least one tester strain either without S-9 mix orafter adding ametabolizing system. Atest substance isgenerally considered nonmutagenic inthistest If:

*The number of revertants.for all testerstrains were within the historical negative

control range under all experimental conditions intwo experiments caried out independently ofeach other.

Page 24 of 57

### Report; Project No.:40M0437/034093 3.7. RETENTION OF RECORDS GLP-relevarit records and materials are stored at BASF Aktiengesellschaft and at BASF Corporation at leastfor the period oftime specified inthe GLP principles. Details on responsibilities or locations of archiving may be seen from the relevant SONs and from the raw data.

Page 25 of 57

# BASF Report; Project No.: 40M0437/034093 

### 4. RESULTS (Tables 1 - 

### 14) The substanceAlkali Blue Dry 6100 /Lot 2238308 was tested for mutagenicity inthe Salmonella Ityphimurium / Escherichia coi reverse mutation assay both inthe standard plate test and inthe preincubation testwith and withoutthe addition ofametabolizing system (S-9 mix) obtained from rat liver using the Salmonella strains TA 1535, TA 100, TA 1537, TA 98 and Escherichia coil WP2 uvrA.
4.1. MIITAGENICITY 4.1.1. Standard plate test (Appendix 1,Tables I
- 

### 9) 4.1.1.1. Tests without S-9 mix TA 1535: TA 100: TA 1537: No increase inthe number ofhis" ortrp4 

### revertants. TA 98: E.col WP2uvrA:

4.1.1.2. Tests with S-9 mix TA 1535: TA 100: TA 1537: No increase inthe number of his+or trp* revertants. TA 98: E.coi WP2 uvrA:

Page 26 of 57

### Report; Project No.: 40M0437/034093
4.1.2. Preincubation test (Appendix 2,Tables 10
- 

### 14) 4.1.2.1. Tests without S-9 mix TA 1535: TA 100: TA 1537: No increase inthe number of hiseor ti-p' revertants. TA 98: E.coi WP2 uvrA:

4.1.2.2. Tests with S-9 mix TA 1535: TA 100: TA 1537: No increase inthe number of hie 4 

### or trp 4 

### revertants. TA 98: E.

### coi WP2 uvrA:

Page 27 of 57

# RAWF Report; Project No.: 40M0437/034093
4.2. TOXICITY A bacteriotocic effect (reduced background growth, decrease inthe number of revertants, reduction inthe titer) was observed inthe standard plate test using the Salmonella strains at doses >

### 575 pg/plate. With E.coli WP2 uvrA only aslightdecrease inthe number oftrp~revertants was found at doses ! 2875 pg/plate. Inthe preincubation assay bacteriotoxicity (reduced background growth, decrease inthe number of revertants) was occasionally observed at about 1500 pg/plate.
4.3. SOLUBILITY Testsubstance precipitation was found from about 100 pg/plate onward.
4.4. DISCUSSION According to the results ofthe present study, the test substance did not lead to an increase inthe number of revertant colonies either without S-9 mixor after adding a metabolizing system inthree experiments carried out independently of each other (standard platetest and preincubation assay).
4.5. CONCLUSION Thus, underthe experimental conditions chosen here, it isconcluded that Alkali Blue Dry 6100 ILot 2236308 isnot amutagenic agent inabacterial reverse mutation test.

Page 28 of57

### Report; Project No.: 40M0437/034093
&.REFERENCES 1

. Ames, B.N.; McCann, J.; Yamasaki, E.: Methods for detecting carcinogens and mutagenswith the Salmonella/mammalian-microsome mutagenicity test. Mut. Res., 31,.347
- 

### 364 (1975) 2. Maron, D.M.; Ames, B.N.: Revised method for the Salmonella mutagenicity test. Mut. Res., 113,217-233 (1983) 3. Ames, B.N.; Lee, F.D.; Durston, W.E.: An improved bacterial testsystem forthe detection and classification of mutagens and carcinogens. Proc. Nat. Acad. Sci. USA, 7, 782-786 (1973) 4. McCann, J.; Spingam, N.E.; Kobori, J.;Ames, B.N.: Detection of carcinogens as mutagens: Bacterial tester strainswith Rfactor plasmids. Proc. Nat. Acad. Sci. USA, 72, 979
- 

### 983 (1975) 5. Green, M.H.L;,Muriel, W.J.: Mutagen testing using trp* reversions inEscherichia coll. Mut Res., 38, 3
- 32 (1976) 

### 6. DeMarini, D.M.; Dallas, M.M.; Lewias, J.: Cytotoxicity and effect on mutagenicity of buffers Inamicrosuspension assay. Ter. Carc. Mut., 9, 

### 287
- 

### 295 (1989) 7. Yahagi, T.; Nagao, M.; Seino, Y.; Matsushima, T.; Sugimura, T.; Okada, M.: Mutagenicities of N-nitrosoamines in Salmonella. Mut Res.,A8, 121 -130 (1977) 8. Matsushimna, T.; Sugimura, T.; Nagao, M.; Yahagi, T.; Shirai,A.: Factors modulating mutagenicity inmicrobial tests. in: Norpoth, K.H. and R.C. Gamner, Short-Term Test Systems for Detecting Carcinogens. Springer Verlag Berlin, Heidelberg, NewYork (1980).

Page 29 of57

### Report; Project No.: 40M0437/034093



### APPENDIX I TABLES 1-9 (STANDARD PLATE TEST)

Page 30 of 57

# RAWF Report; Project No.: 40M0437/034093

BASF hActteng-eleulanbft 01 Experienta.l Toxicology and Ecology STUDY DIR~ECT~OR: DR. DTGELNAPDT PROJECT NO. 40H0437/034093 SUBSTANcz NAME: Alkali Blue Dry 6100 /Lot 2236309 mSUBTAqCE 140. 03/0437-3

TESTDATB 06.=OV.2003 METHOD :STANDARD PLATE TEST VOLUME 100 ul/plate STRAI TIA 1535 VEHICLE :DM80 WIm :NTHOUT S-9 N= WITH S-9 MIX (1:9) DOSE/PLATE RX" X SD PAC REV X SD EEC TITRE DIw0 16 17 3 1.0 19 16 3 1.0 25 21 14 24 is 16 22 23 ug 17 21 4 1.2 16 14 3 0.8 25 14 20 11 115 ug 16p 21 6 1.2 16P 15 2 0.9 28P 12P 19P) 16P 575 ugI 21P 18 3 1.0 14P 17 5 1.0 15P .22P lop 14P 2 875 ug 33/P 3 2 0.2 03/PI 53/p 03/P 4 2E/p 03/P 3 5 750 ug 03/P

. 0/P 0 03/P 03/P 0 03/P 03/P 0 meb 5.0 ug 936 921 58 53.1 857 970 2-A?. 2.5 ug 170 151 21 9.2 154 128 (BE-revertantf/plate, N=ineai, SD-standard deviation, EEC>-factor) P: PMECIPITALTION B: MRDUCEO BACKGR0UND GROWTH



### Page 31 of 57i

-- 

### It


# BASF Report; Project No.: 40M0437/034093
RM Aktiengesel I dcaft 02 Experimental Toxicology and Ecology STUDY DIPSCTO1R: DR. ENGELHARDT PR0OJ3C7 IM. :40M&0437/034093 SUBSTANCE lMSE: Alkali Blue Dry 62100 /Lot 2236300 suBsTANC no. 03/0437-3

TESTDA22 06.2W.2003 METHOD 87AIIDAD PLATE TEST VOLM : 100 ul/plate STEAME : VA 100 S9-I WITHUT B-9 1= ITo 5-9 MIX1 (1:9) D3EPAI 4V M4 SD 11= P" m SD PAC TIME INO104 110 7 1.0 109 118 8 1.0 32 110 121 28 117 124 28 23 ug 113 107 6 1.0 113 107 5 0.9 106 105 101 103 115 ug 100P 104 5 0.9 103P 103 2 0.9 1031 1051 1091 1011 575 ug 901 95 5 0.9 891 91 3 0.8 991 941 97P 901 2 875 ug 03/1 M1/1 11 4 0.1 1 O3/p 7B/P 0 03/1 143/P 0 5 750 mg 03/P 03/P 0 03/1 OB/p 0 0831 03/p 0 MR=U 5.0 ug 970 969 64 9.8 1031

904 2-AA 2.5 ug 953 917 36 7.8 917 882 (BEV-revertants/plate, Mmmmu, SD-standard daviation, YAC-fantor) 1: PRECIIIWv B: REDUE RXCKG~tOUND GRWTH



### Page 32 of 57-- -~BASF 

### Report; Project No.: 40M04371034093

RAST AktiemlgefllIBChaft 03 Experjmnta. Toxicology and Ecology SUYDPZTR R NEHM PRO.JECT NO. :40M0437/034093 SUBsTA~Cz NMM: Alkali Ithe Dry 6100 /Lot 2236308 ,SUBSTANCE No. :03/0437-3

TBBTDATE 06.U0v.2003 METHOD STANDARD PLATE TEST VOW)UM 100 ul/plate sTRAN : TL 1537 VEHICLE DM80 s9-NIX WITHOUT S-9 M= WITH 8-9 MIX (1:9) DOSX/PLATE REV If SD FAC ?"V M SD PAC T1TER DM80 14 12 2 1.0 10 11 1 1.0 28 12 11 17 10 12 23 23 ug 6 a 3 0.7 7 11 4 1.0 7 15 11 10 =15 ug 12P 11 2 0.9 lop 10 1 0.9 sP lop 12P lip 575 12g 6P 5 2 0.4 13P 9 5 0.8 3P 4P 6?P lip 2 875 ug 03/P 33/P 2 1 0.2 0 03/P 13/P 0 03/P 23/P 0 5 750 ug 03/P 03/P 0 03/P 03/P a 03/P CE/P 0 Axc 100 ug 460 425 32 35.4 419 396 2-A 2.5 ug 147 149 11 13.5 161 139 (pEv-revertants/plate, Mnmn, SD=tandard deviat-ion, yAC-factor) P: PPECIPITATION B: P.EWMME 3P.CK0OtND GROM



### Page 33 of 57

# BASF Report; Project No.: 40M0437/034093

BA81 hkjtjeng~eelahwft 04 zxpermumntal Toioology and Ecology STUDY DIRECTOR: DR. ENGELHMIT PRO.)EC NO. 40W1437/034093 8StXnN kqA): Alka-i E1lie Dry 6100 /Lot 2236308 8U3STAEC No. :03/0437-3

TEBTDAU : 06.2WV.2003 HETBOD : TAiNMRD Pla= E ST VOLUM :100 ul/plate 87PJ13F TA 98 89-m= : VTOUT S-9 3= Tm S-9 xixX (1:9) DOSE/PlATE F"V X4 ED PAC REV K4 SD FAC TITE DM80 38 33 5 1.0 38 38 4 1.0 26 31 41 34 29 34 31 23 

### wg 27 27 3 0.8 28 40 14 1.1 30 36 25 55 115 uq 36P 30 6 0.9 31P 30 2 0.8 25P 27? 28? 311' 575 ug 34P 27 7 0.8 26P 29 6 0.8 28? 24? 20? 36P 2 875 wg 03/P 1OB/P 9 5 0.2 0 03/P 43/P 0 03/P BP 0 5 750 ug 03/P

. 0/P0 03/P oB/D 0 wiow 10 ug 740 740 53 22.7 793 688 2-Ak 2.5 ug 900 877 23 23.3 854 878 (XWV-evertanta/plate, )i-ean, SD-standard deavlation, FAC-factor) P: P3ECIPI2TIMC 3: 3RDMCED BACKGR0UND GROTH



### Page 34 of57

# BASF Report; Project No.: 40M04371034093

EASE AktjeiezuefLschaft 05 Experimental Toxicology anid Ecology SUYDRZ0:D.EGLAD PRO3OCT NO.- 4014437/034093

SM3SMAV MU: Alkali RLue Dry 6100 /Lot 2236308 SMTAMU No. 03/0437-3
TESTI)A22 06.NOV.2003 METHOD STAN~DARD PLATE TEST VOLUME 100 ul/plate STIULIV : .col-t H'2 uvrA VEHICLIE IW 59JLX WTOUT S-9 MIX WITH S-9 k=X (1:9) z)OSE/PLI.Tg REV x SD FAC PXV M SD EAC TITRE

- DM80 30 31. 1 1.0 29 34 5 1.0 37 30 36 35 32 37 44 23 Ug 31 30 6 1.0 25 32 6 0.9 36 33 24 37 1-5 g 35P 32 3 1.0 40P 30 9 0.9 30P 22P 31P? 27P 575 ug' 26? 25 1 0.a 21P 25 4 0.7 25P 26? 24? 28P 29875 U9 22? 19 4 0.6 21? 18 3 0.5 26 14? 27P 22 20? 15? is 5 750 ug lip 15.4 0.5 12P 14 3 0.4 12 19? 17P 17 14P 13P 11 4-N100 5.0 ug 813 754 57 24.6 748 700 2-A7A 60 ug 200 214 12 6.3 217 224 (REVrevetants/plate, Itean, SD-standaxd deviatiori, PAf-factor) P: PEIRITATIK



### Page 35 of 57iwrni WuUJJ'U1 1r1 Un I

# BASF Report; Project No.:40M0437/034093

BASF Aktiengesellseliaft 06 zxperimental. Toxicology and Ecology STUDY DIP2CTOR: DR. E2NQEHA1MT PRD3=C NO.- 40IM437/034093 suB8TA1NE was: Alkali Blue Dry 6100 /Lot 2236308 sDBSTA1vE No. 03/0437-3

TESTDP.TH 04.DEC.2003 14ETHOD STBNAM PLATE TEST VOLtMM 100 ul/plate STRLAN TP. 153 VEHICIS DM8=0 89-wIX :WITHOUT S-9 NI WTH 6-9 NIX (1: 9) DOSE/PlATE REV K SD FAC AMV b SD PAC TITEE DKO16 16 3 1.0 14 16 3 1.0 36 is 20 30 13 i5 30 0.8 u 16 17 1 1.1 16 16 4 1.0 is 20 1s 13 4 ug 11 14 3 0.9 13 15 3 0.9 13 18 17 13 20 ug 22 17 5 1.1 14 13 2 o.e 16 11 12 1.4 100 ug 113? 19 7 1.2 171' 16 2 1.0 22 21P 141' 31 24P' 17P' 27 500 ug 141' 13 .1 0.8 141' 15 2 0.9 16 121' 171' 17 1.33? 13' 21 low 5. 0 ug 1041 1050 17 67.0 1070 1039 2-AIL 2.5 uq 169 163 12 10.0 150 171 (PRvrevertants/plate, M3mam, SDlstardard deviation, FAC-factor) 1': PRECIPITATIN



### Page 36 of57

# BASF Report; Project No.: 40M0471034093

BAsF 3itiengenellschaft 07 xpei~mtai Toxicology and Ecology STUDY DIRECTOR: DR. ZffGMLEABDT R0NC NO. 4010437/034093 StJBSTAM m=N: Alkali Blue Dry 6100 

### /lot 2236308 sUBSTAwcE No. 03/0437-3

TEsTDA : 04.DEC.2003 METHOD :STANDAR PLATE TEST VOLUme 100 12/plate STPam : TAt 100 VEHICZE : DMSO 39-MIX : WITHOUT S-9 B33 WITH 8-9 lax (1:9) DOSE/PLATE REV7 x SD PAC riEV X SD PAC TITAR

- nSO, 140 1-20 18 1.0 114 12.0 4 1.0 24 107 109 27 1.13 107 38 0.8 ug 104 105 3 0.9 107 112 6 1.0 103 110 109 119 4 ug 113 107 7 0.9 102 105 7 1.0 108 100 100 113 20 ug 103 107 4 0.9 113 114 2 1.0 2.11 117 107 113 100 ug 120P 112 9 0.9 102P 105 5 1.0 17 114P ilOP 27 103P 102P 26 500 ug loop 92 7 0.8 uop 104 5 0.9 18 90p 102P 20 87P 100p 17 bRO 5.0 ug 1131 1084 43 9.0 1073 1047 2-hP. 2.5 vq 900 951 45 8.6 983 970 (pzv-revertants/plate, YAmen SD=standaxd devIation, FAC-factor) 1: pRSCIVITEXIO



### Page 37 of 57

# RASP Report; Project No.: 40M0437/034093

3181 hkt~eng~eloshaft 08 Experimental Toxiocogy and Ecology PROJCT N. :4OW47/03093STUDY DIRlECTOR: DR. ENGEL~IEDT SUBESTANCE NAME: Alkali Rlue DrY 6100 /Lot 2236308 SUBS7XM~CZ NO. 03/0437-3

TzsTnagy 04.DZC.2003 METHIW : MT.AD PLATE 7r6ST VOIXWE :100 ul/plate STREAN TA 1537 VEHIcLE DMtw0 S9= :WITHOUT 8-9 WITHfl 8-9 M=X (1:9) DOSE/PlATE MEV K SD PAC RE" x SD NrC TITE DXS0 10 11 2 1.0 15 1.1 4 1.0 31 13 8 29

9 10 28 O.8 ug 14 10 5 0.9 9 7 2 0.6 5 6 10 6 4 uq 7 6 1 0.5 8 12 4 1.1 5 14 5 15 20 ug 10 a 2 0.7 11 n1 0 1.0 6 1 7 1 100 ug 73W 7 1. 0.7 9W 10 2 0.9 24 6P 1"W 27 sW 9p 21. 500 ug 2W 4 .2 0.4 7P 7 4 0.7 26 6P 11P 27 5P 4p 25 AA.C 100 ug 436 411 24 38.6 388 410 2-AlL 2.5 ug 144 134 9 12.2 130 127 (REV-revertants/plate, Mamean, sD-standard deviation, pAC-factor)



### Page 38 of 57FLI 

### ~ 'n~n


# BASF Report; Project No.: 40M0437/034093

BAST )ktiexqfeellaft 09 Expex-imntal Toxicology and Rcology STUDY D3hECTOR: DR~. ENGMHAPDT PROJIECT NO. :40M0437/034093 SS3Txm~ MWd: ALkaUi Blue Dry 6100 /Lot 2236308 sBTSmCR No. :03/0437-3

TESIDAE 04 .DHC. 2003 IdETBODST~MAW PLATE TEST VOLUME 100 ill/plate STPJLF : T 98 VEICLE : Dwso 59-= WI1THOUT S-9 W= WTH B-9 2=X (1:9) DOSE/PZLTE FE" m SD rAc P" SD PAC TITW.E

- DamS 28 29 2 1.0 44 42 3 1.0 24 31 39 28 28 43 34 0. ug 26 30 7 1.0 46 39 6 0.9 38 34 27 37 4 ug 33 28 6 1.0 31 29 2 0.7 30 27 22 30 20 ug 30 27 4 0.9 33 36 3 0.9 22 37 28 39 100 ug 19W 21 4 0.7 42P 35 6 0.8 27 262 30P 24 19P 32W 31 500 ug 16P 20 4 0.7 43P 39 8 0.9 21 23P 44W 28 22Y 30P 24 NOPD 10 ug 602 605 25 20.9 631 582 2-xA 2.5 ug 770 718 47 17.1 681 702 (pEv-revertants/plate, M~mean, SD=standard deviation, FA-factor) P: PiEclPITATWE



### Page 39 of 57

# BASF Report; Project No.: 40M0437/034093



### APPENDIX 2 TABLESI10-14 (PREINCUBATION TEST)

Page 40 of57

# BARF Report; Project No.: 40M0437/034093

BABE Jkkttengeael SCAMat 10 Exper±mntal Toxicology and Ecology STUDY DIPZCCTOR: DR. ERGELHAMT2 P1RO33C N0. 4014437/034093 s1J3STAWIE 2AME: Alkali Blue Dry 6100 /Lot 2236308 sU38TMAc No. 03/0437-3

TESTDAR I0.DEC.2003 dE'THOIC PF23NCUSATION TEST VOLUME 100 ul/plate STRA" : T 1535 VEHICLX DM80 S9-f~ : WITHOUT S-9 MTT WTH 5-9 MIX (1:9) DOSE/PLATE F"V 1 SD XAC REV x SD EA= TITE DM80 18 17 2 1.0 19 17 3 1.0 27 19 13 30 15 1s 23 4 sqg 15 16 2 0.9 12 15 3 0.9 14 17 18 16 20 ug 22 16 5 0.9 11 14 3 0.8 14 16 13 15 100 sq Isp 16 1 0.9 16P 1.5 1 0.9 162 14P ISP 5 500 ug 13P 12 2 0.7 12P 14 2 0.8 17 lop 16P 1s 14P 14P 22 1 500 sq 12BhP 10 2 0.6 83/P 9 1 0.5 20 93/P 103/P 11 83/P 93/P 16 NNWa 5.0 sq 643 644 14 37.1 630 658 2-hA 2.5 sq 100 105 6 6.3 11 103 (EEV.=evertant/plate, Vmuean, SD-.tandard deviation, YAO-factor) P: PRCM7IC2IG 3: P.EcD BAC3PR0UU GROTH



### Page 41 of 57

# BAUSF Report; Project No.: 40M0437/034093

IL"AR.kit-iengesllahaft

Eprimntal Toxicmoog and Ec-ology STUDY DIRECTOR: DR. M!SGfLN)T PROJECT no.- 40W437/034093 suWsAcz NME: A13cali BI. Dry 6100 /Lot 2236308 8tJBSTRNOE NO. 03/0437-3

TESTDA3Z 1O.DZC.2003 METHOD PRZINCMLTX~fI TEST VOLUME 100 ni/plate BSPAXN : 72L 100 VEHICLE DhM0 89-= WXTHOUT 8-9 2= wTH 8-9 MIX (1:9) DOSE/ITE REv x SD FAC REv X SD MC TIuhE DmS0 110 107 4 1.0 103 109 5 1.0 24 102 111 29 108 113 27 4 ug 102 105 a 1. 0 1.02 108 7 1.0 98 1125 114 107 20 ug 103 162 2 1.0 104 105 4 1.0 104 102 100 109 100 ug 120? 103 15 1.0 loop 100 2 0.9 95? 1013? 93? 982, 500 ug 98?, 87 10 0.8 95? 93 3 0.9 22 823P 90P 23 SOP 93P 27 1 500 ug 40R/P 37 .3 0.4 64B/p 63 13 0.6 11 37B/P 50R/P 17 353/P 753/p 16 NIgM 5.0 ug 632 664 29 6.2 672 688 2-uLA 2.5 ug 599 619 21 5.7 641 617 (p3v-=evextants/plate, X-mn SDstaudaxd deviation, Plc-factor) P: PmaEI2W(TIQ 3: PmuXJw BID8ROVUND WRTH2



### Page 42 of57

# BASF Report; Project No.: 40M0437/034093

Ex"Y Aktiengesellschaft 12 Experimental ToxLcology and Ecology STUDY DIhZCTCR: DR. EVGRLSAIRDT POJECT NO. 40M0437/034093 smjsT1Icg iNE: Alkali Blue Dry 6100 

### /Lot 2236308 sUssTAwcz no. 03/0437-3

TEBTDATZ 10.DEC.2003 METHOD : PEIrNCORATXCE TEST VOLUME :100 ul/plate STPA3N :TA 1537 VEHICLE :DM80 S9-m= : WITHOUJT S-9 NE= WITH S-9 M= (1:9) D0SE/PLATE AE" X SD YAC p"V w SD JAC TTE DKSO 12 10 2 1.0 9 9 1 1.0 26 9 9 23 10 a 25 4 ug 14 10 3 1.0 8 7 4 0.8 8 11 9 3 20 ug 10 8 2 0.8 a 8 0 0.9 78
100 ug 4p 4 2 0.4 7P 6 2 0.7 6p 7p 2P 4P 500 ug SP 5 2 0.5 6p 6 1 0.7 24 3P 6p 21 7P 7P 18 1 500 iag 23/p 2 1l 0.2 6B/P 4 2 0.5 15 3B/P 3B/9 1.2 3B/2 3B/P 17 AMc 100 ug 439 446 50 43.2 500 400 2-hit 2.5 ug 103 99 7 1.1.4 91 103 (R-revrtant/plate, ld-mean, D-standard deviation, PAC-factor) P: FRECIpIwkTIOK B: PED~ BACNROUN GROTH



### Page 43 of57

### Report;,Project No.: 40M04371034093
iWAc)tj..nesllschiaft. 13 prxperiueftal. Toxicology and Ecology STUDY DIRZCTOR: DR. ENRIZLUMT pmrOOZC No. 40H0437/034093 STJBBTANC NANE Alkali Blue Dry 6100 /Lot 2236308 SMBTANE Iwo. :03/0437-3

TEMMAS 10.MEC.2003 METBOD : PEDCM~ATIH TAB? VOL= : 100 Wolate ST2lIN TA 98 UWmCLR DWEO 09-3=X WTNOUI' S-9 J= WITHS8-9 baX (1:9) DOSE/PLAE I"V m SD FAC REV X SD PAC TITE UNSO 19 25 6 1.0 33 31 3 1.0 27 28 28 35 29 32 32 4 wg 30 29 5 1.2 32 29 2 0.9 34 28 24 28 20 uO 20 23 3 0.9 30 30 3 1.0 25 33 24 27 100 ug 19P 21 4 0.8 26P 27 2 0.9 26P 29P isp 26P 500 ug 16P 17 2 0.7 30p 29 2 0.9 10 15w 27P 10 19W 30P 15 1500o ug 2S/P 3 .2 0.1 203/P 18 3 0.6 10 23/P 143/P 11 63/P 192/p 15 NOpjD 10 

### wg 870 885 22 34.9 911 875 2-lA 2.5 ug 604 563 46 18.2 571 513 (RMv-revertants/plate, U-ne=x, SD-standard deviatioz, rAC=factor) P: pE3CIpITA2'IK 3: R1DUoE BACPMROD GP.O1T



### Page 44 of57

# BASF Report; Project No.: 40M0437/034093

BASF Aktieng'esellschaft 14 Eaqmei-vental, Toxicology and Ecology, STUDY DIREVCTOR: DR. EWGRJIARr)T WRO17ZC NO. :40M0437/034093 slUSTA=c HAW: Alkali. Blue Dry 6100 /Lot 2236308 sMLSTABcZ No. :03/0437-3

7RSTDA10 I.DEC.2003 METHOD WEIN1CMULTIOT TEST VOLO1ME :100 ni/plate STP~uR : .col± V122 uvxcA VEHICLE : M 89-MIX :W7THOUT S-9 MX WITH S-9 lax (1:9) DOSE/PXATE RE" w SD 3= REV X SD FE5C TITER

- DNBO 30 31 3 1.0 32 32 3 1.0 35 28 29 41 34 34 33 4 ugr 26 31 5 1.0 28 30 5 1.0 34 36 34 27 20 ug 21 27 6 0.9 25 27 2 0.9 28 27 32 29 100 ug 301? 28 2 0.9 302 24 5 0.8 26P 202 28P 22P 500 ug 223? 21 1 0.7 27P 23 5 0.7 26 20P 25P 27 21P lap 22 1 500 ug 24B/P 16 . 7 0.5 123/P 13 2 0.4 15 123/P 14B/P 14 14B/P 15B/P 19 4-NQO 5.0 ug 672 658 31 21.5 622 680 2-AA 60 U9 200 206 6 6.5 207 232 (p~v.revrtants/plate, H-ckan, SD-standard deviationi, rhO-factor) B: PZDtJCED BAOKSROMID GROWTH



### Page 45 of57_____~~- 1 -l II It11~UJu



# BALSF Report; Project No.: 40M0437/034093



### APPENDIX 3 STERILITY CONTROL

Page 46 of 57

# BAS= Report; Project No.: 40M04371034093 

### STERILITY CONTROL 1st Experiment

" 2.0 ml soft agar No growth 0.5 mlS-9 mix No growth 0.5 ml buffer No growth 0.5 ml vehicle No growth 5750jig testsubstance No growth 

### 2nd Experiment

0.5 ml vehicle No growth 500 pgtest substance No growth
3rd Experiment

2.0 ml soft agar No growth 0.5 ml S-9 mix No growth 0.5 ml buffer No growth 0.5 ml vehicle No growth 1500 pg test substance No growth

Page 47 of 57

### ~LL~EI2 UIU 1 I 1~I~1 II~rII:II~~ill'1 L I I L -1i



# BASP Report; Project No.: 40M0437/034093



### APPENDIX 4 S-9 FRACTION CHARACTERIZATION

Page 48 of57

### Report; Project No.: 40M0437/034093
S-9 fraction Preparation ofS-9 fraction: June 17, 2003 Content ofprotein (mean*): 29.0 gI Characterization ofthe S-9 mixwith benzo~ajpyrene: July 14, 

### 2003
METHOD: Standard PlateTest S-9 FRACTIONICOFACTORS: 1:9 STRAIN: TA 100 STRAIN: TA 98 DOSEIPLATE MREV* SD- FAC* DOSEIPLATE MREV* SD* FAG' DMSO 113.7 9.0 1.0 DMSO 34.0 3.0 1.0

0.6 Pg 258.9 32.5 2.3 0.6 pg 87.6 16.8 2.6

- 1.25 pg 368.8 26.8 3.2 1.25 pg 486.8 39.1 

### 5.5

2.5 pg 497.5 69.3 4.4 2.5 pg 352.8 29.1 10.4

5.0 pg 639.4 29.5 5.6 5.0 Pg 492.6 46.4 14.5
(REV = revertants/plate, M=mean.SD standard deviation, FAC =factor)

=pool 1-4

Page 49 

### of 57

# BASF Report; Project No.: 40M0437/034093



### APPENDIX 5 HISTORICAL NEGATIVE CONTROL DATA

Page 50 of57411111111 I No


# BASF Report; Project No.:40M0437/034093 Historical Negative Control Data Strain: TA 1535
Methd S9

mi NoNo. of Mehd SBmx Ngative control 'Period Plates Min*' Mak' Mean* So 

### SiPr Water 03.98
- 09.03 400 11 25 18 2 SPT -DMSO 03.00-09.03 400 11 25 18 2 SPT -Acetone 01.96- 05.03 208 11 25 19 2 SPT 1 :9 Water 03.98
- 09.03 400 12 25 18 2 SPT -1:9 DMSO 03.00-09.03 400 10 24 18 2 SPT -1 :9 Acetone 01.96- 05.03 210 11 25 19 2 No. of Method S-9 mix Negative control Period Plates MIi'* Mae' Mean* SID PI Water 08.97- 06.03 400 13 25 19 2 PIT -DMSO 06-99-07.03 400 11 25 18 2 PIT Acetone 01.96-05.03 168 10 25 ,19 2

_____ 1 :9 Water 04.97-06.03 400 11 25 18 2

PIT 1 :9 DMSO 05.99- 09.03 400 10 23 17 2 PIT 1 :9 

### I Acetone 01.96- 05.03 159 10 -25 18 2 

### Strain: TA 100 No. of

Method S-9 mix Negative control Period plates Ae Max* Mean* So

SPT -Water 05.98-09.03 400 82 '155 117 14 

### SPT DMSO 06.00-09.03 400 91 160 110 10 SPT
- Acetone 01.96- 05.03 258 91 160 118 1s SPT 1:9 Water 06.98--09.03 400 94 156 123 16 SPT 1:9 DMS0 08.00-09.03 400 92 154 112 10 ISPT 1:9 Acetone 01.96- 0503 253 85. 150 -120 16 No. of Method S-9 mix Negative control Period
_ plates Min*' Max* Mean' SID

PIT -Water 06.97- 06.03 400 90 160 117 14 PIT -DMS0 06.99- 09.03 400 64 150 112 12 PIT -Acetone 01.96- 05.03 172 91 160 1117 16 PIT 1:9 Water 05.97-06.03 400 92 159 122 15 PIT 1:9 DMSO 06.99-09.03 400 80 159 112 13 PIT 1:9 Acetone 01.96- 05.03 156 96 160 1119 1
* a revertants/plate PIT = preincubation test SPT =standard plate test SID - standard deviation 

### Page 51 of 57

### Report; Project No.: 40M0437/034093 

### Historical Negative Control Data Strain: TA 1537 No. of

Method S-9 mix Negative control Period plates- Min* Max* Mean*. SID

SPT -Water 03.9 -09.03 400 6 16 10 2 SPT -DMS0 03.00-09.03 400 5 16 10 2 SPT
- Acetone 01.96-05.03 213 5 18 10 12 SPT I :9 Water 03.98 -09.03 400 6 19 11 2 SPT I:9 DMSO 03.00
- 09.03 400 5 19 11 2 I SPT I 1:9 Acetone 01.96-05.03 213 7 20 12 12 No. of Method S-9 mix Negative control Period plates Min' Max* Mean' SID

PIT -Water 05.97 -06.03 400 5 19 10 2 PIT -DM80 06.99
- 09.03 400 5 17 10 2 PT- Acetone 01.96-05.03 168 6 17 10 2 PIT 1 :9 Water 04.97- 06.03 400 6 20 11 2 PIT 1:9 DM80 04.99-09.03 400 5 16 10 2 PIT 1 :9 Acetone 01.96- 05.03 162 15 20 11 2 

### Strain: TA 98 No.of

Method S-9 mix Negative control Period 6lhate. Min' Max* Mean' SID

SPT -Water 06.98
- 09.03 400 17 44 28 5 SPT -DM80 07.00
- 09.03 400 20 45 29 5 SPT -Acetone 01.96-05.03 215 19 50 29 5 SPT 1:9 Water 06.98
- 09.03 400 20 49 36 6 SPT 1 :9 DMSO 08.00
- 09.03 400 20 50 36 6 SPT 1:9 Acetone 01.96
- 05.03 214 26 50_ 38 -5
'No.of Method S-9 mix Negative control Period viates Mm'0* Max* Mean' SID

PIT -Water 05.97
- 06.03 400 18 44 27 4 PIT -DMSO 06.99
- 09.03 1400 17 40 27 4 PIT -Acetone 01.96-05.03 168 19 145 28 5 PIT I:9 Water 04.97
- 06.03 400 17 50 36 6 PIT 1 :9 DMSO 06.99
- 09.03 400 20 49 32 5 PIT I:9 

### 1 Acetone 01.96-05.03 161 20 50 37 6

= revertants/plate PIT = prelncubation test SPT =standard plate test SID = standard deviation 

### Page 52 

### of 57

# BtAS Report; Project No.: 40M0437/034093 Historical Negative Control Data Strain: W132 uvrA No. of

Method S__9_mix Negative control Period plates Min* Max' Mean* SID

SPT -Water 11.97.-09.03 400 20 40 31 5 SPT -DMSO 01.00.-09.03 400 22 50 32 5 SPT -Acetone 01.96-05.03 200 18 150 33 6 SPT 1 :9 Water 01.98-09.03 400 20 49 35 6 SPT 1 :9 DMSO 01.00-09.03 400 23 50. 35 6J SPT 1 :9 Acetone 01.96.05.03 200 115 50 37 71 No. of Method S-9 MIX Negative control Period plates Min* MaX* Mean* SID

PIT -Water 04.97.-06.03 400 21 50 31 5 F PIT -DMS0 07.99 -09.03 400 19 49 32 -5

PIT -Acetone 01.96.-05.03 -157 20 47 31 5 PIT 1 :9 Water 01.97-06.03 400 23 50 37 6 PIT 1:9 DM50 06.99 -09.03 400 19 50 32 6 PIT 1 :9 Acetone 01.96-05.03 157 20 40 33 6

= revertantslplate PIT =preincubation test SPT =standard plate test SD m standard deviation



### Page 53 

### of 57

# RASF Report; Project No.:40M0437/034093



### APPENDIX 6 HISTORICAL POSITIVE CONTROL DATA

Page 54 of57

# BASF Report; Project No.: 40M0437/034093 Historical Positive Control Data Strain: TA 1535 

# IPositivecontrol No. of Meho S- mi -ip Peid pae-i*Mx o*S SPT

- MNNG 5.0 02.01 -09.03 400 500 1649 776 199o SPT 1 :9 2-AA 2.5 02.01 -09.03 400 85 325 146 38J 

# SPos ive control No. of 

# I Method S-9 MIX PglplI Period plates Min* Max* Mean* SD

PIT

- MNNG 5.0 07.00-09.03 400 502 1357 785 150 PIT 1 :9 2-AA 2.59 06.00-09.03 400 so 345 129 31


### Strain: TA 100 

# I 1 Positive control 

# INo.ofII Method S-9 mix Pglpl Period 

### .1plates* #An* Max* 

### ~vrS

SPT 

## I

- 

## I MNNG 5.0 

## I03.01-09.03 

# J400 I501 I1747 871 262j SPT 1:9- _2-AA 2.5- 03.01 -09.03 

# )400 507 1708 859 215 

### Posiive ontrl -N.o Method S-9 mix ipglpi Period plates Min*MaW't* Mean'f SD PIT- NNG 5.0 07.00-09.03 400 502 1583 

## Is 188 PT I :9 2-AA 2.5 06.00- 09.03 400 500 1125 

## I675 135

= revertants/plate SPT =standard platetest PIT =prelncubatlon test SD = standard deviation 2-AA =2-aminoanthracene MNNG N-methyl-N-ntro-N-nltrosoguanidine



### Page 55 of57

# BASF Report; Project No.: 40M0437/034093 Historical Positive Control Data Strain: TA 1537
Positive control No.of



# IMethod
S-9 mix PglplI Period Min* Max* Mean* SD S 

## I AAC 100 

# 301.01-09.031 400 300)1147 443 

### 1041 SPT I :9 2-AA 2.5 

## J01.01

- 09.03 400 71 

### j200 127 2 

## I IPositiveconitrol No. ofI 

## I Method S-9 mix P. 9p Period plates Min* Max* Mean*

### l SDJ PIT 

## I

- AAC 100 07.00-09.03 400_ 278 8851435 

### I9 3 PIT 1:9 2-AA 2.5 06.00-09.03 400__ 50 200 

## I112 I20
Strain: TA98 

# II 1 ~Positive controlN. f



### .- Method' 

### S-59 MIX pa/pl atrio 

### MW Max* M#ean' SD 

## IISPT j

- 

## I NOPD 10 02.109.03 00 34 120 737 132

SPT 1:9 2-AA 2.5 0301-09.03 00 504 138 699 139 Positive control 

## 1No,ofI Method i5-9 mix , Ip Period_ plates Mi*MaX' Mean* SD

PIT
- NOPI1 07.00 -09.03 

## I400 39 1129 

### ~.735 135 PIT 1:9 2-AA 2.5 06.00-09.03 400 1 5621985 90 =revertafltsplnte SPT = standard plate test PIT =proincubatlon test sD wstandard deviation 2- AA =2-aminoanthracene AAC =9-aminaacridine NOPD 4-nitro-o-phenylendiamine



### Page 

### 56 of57

# BASF Report; Project No.:40M0437/034093 Historical Positive Control Data Strain: WP2 uvrA 

# I I IPositive control 

### No.of Method S-9 mix gjgIpl Period plates Min* MWx Mean* SD 

# fiSPT

- 

## I4-NQO 5.0 12-00-09.03 400 500 1620 

## I676 171 

### ISPT I:9. 2-AA 60.0 01.01 -09.03 400 155 476 244 -40 

# I Positive control Method S-9 mix jlig/pl

___________

PIT 4-N00 5.0 02.00
- 09.03 400 500 995 588 76 PIT 1 9 2-AA 60.0 06.00
- 09.03 400 170 384 232 26

arevertanits/plate SPT = standard plate test PIT a preincubation test SD = standard deviation 2-AA =2-aminoanthracene 4-NQO m 4-Nitroquinoline-N-oxide



### Page 57 of57