# FAX

- To: 

## 6'-& NN (VI <':"1 VI \ \ , Fax: 

### '}..O\**ll'?1,LI~ I**  Date: 

## <J 

## 11"1 II <)/ Re: 

## C:\> (VI 

### A.

From:

### '7C::D~ 

## S'"**~,,,,** t '"" 

### fax: 202.564.8251 Phone: ,\--01..'

### Sb~ 

# .**~~** 

## S I Cc: 

### Comments:**\'~** 

## "',,t.v,)\\'" 

# rI OCSPP OPPT EAD 1200 Pennsylvania Ave. NW Washington, DC 20460 202.564.8251

Pages: 

# 'Y

r- Urgent 

## I- For Review 

## I- Please Comme



## - Please Reply



## - Please Recycle



## - 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? Manythanks.

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

# BASF
en-jk 034091 RPT.doc STUDY TITLE 

### REPORT SALMONELLA TYPHIMURIUM / ESCHERICHIA COLI REVERSE MUTATION ASSAY (STANDARD PLATE TEST AND PREINCUBATION TEST) WITH 

### Alkali Blue Dry 6100 1Lot 2219308 DATA REQUIREMENT 

### OECD No. 471 EC 2000132, 

### B. 13 1B.14 AUTHORS 

### Dr. G.Engelhardt (Study Director) Dr. E.Leibold STUDY COMPLETED ON March 15,2004 TESTING FACILITY Experimental Toxicologyand Ecology BASF Aktiengesellschaft 67056 Ludwigshafen, Germany LABORATORY PROJECT IDENTIFICATION Project No.: 40MO437/034091 SPONSOR BASF Aktiengesellschaft 67056 Ludwigshafen, Germany This document contains manufacturing and trade secrets of BASF. Itisthe propert of BASF and may be used onlyforthat purpose for which itwas intended by BASF. Every other or additional use, exploitation, reproduction, publication or submissionto other parties requires thewritten permission ofBASF, with the exception of regulatory agencies acting within the limbit oftheir admninistrative

# BASF
Report; Project No.: 40M0437/034091 2 THIS PAGE ISINTENTIONALLY LEFT BLANK

# BASF Report; Project No.: 40MO437/0340913 

### GIP COMPLIANCE STATEMENT
This study was conducted ingeneral accordance withthe 

### OECD Principles of Good Laboratory Practice and theGLP Principles ofthe German "Chemikaliengesetz" (Chemicals Act) and meet the United States Environmental Protection Agency Good Laboratory Practice Standards [40 CFR Part 160 (FIFRA) and Part792 (TSCA)], with the exception that recognized differences exist between the GLP Principles/Standards of OECD and the Principles/Standards of FIFRA and TSCA. However, there was the following deviation from the requirements of the above-mentioned principles:

-The 

### stabilityofthe test substance inthe vehicle DMSO and inwater was not

determined analytically.

Dr.rer na.

G Enelhrd (Study Director)

# BASF Report; Project No.:40M0437/034091 4 THIS PAGE ISINTENTIONALLY LEFT BLANK

# BASF Report; Project No.: 40M0437/034091 5 

### SIGNATURE PAGE

Study Director/ Conduct ofstudy:A Dr. rer. nat. G. Engelhardt



### Management: 

## . . ........ Dr. rer. nat. E.Leiboid

# BASF
Report; Project No.: 40M0437/034091 6
STATEMENT of the QualityAssurance Unit The Quality Assurance Unit (QAU) performed the inspections given below, and reported any inspection results to the Study Director and to Management. The conduct ofthis short-term study was not inspected; the processes ofthe laboratory and ofthe study involved are inspected inregular intervals. The final report reflects the raw data.

Phase ofstudy Date of inspection Reported to Study Director and to Management (mm-dd-yyyy) (mm-dd-yyyy) Study Plan: 10-20-2003 10-20-2003 

### Report: 03-01-2004 03-01-2004



### Ludwigshafen, ...... ... "tl....... Gartmann_I111 mal I aMUMLs - 

# BIASF Report; Project No.: 40M0437/034091 7



### LandesanstaltS ffirPflazeanund Pfazmschutz 

### GLP-Bescheinigung IStatement ofGLP Compliance COW"A ISccrdingt 1 19 b Abs. I Chslef*INgssslz)
Eins GLP-inspektion zur Oberwachung der E-Inhaltung Assessment of conformity with GLP accong der GLP-Grndals8te gemlilChomlikulengesstz bzw. to Chemikalengeseft and Directive 88420EEC at Riciia 88/30/G wurde durchgsMhr Ina: 

### 0PrGfteanrhbg /Ired tft C]Profutandort /Test sits BASF Aktiengesellschaft Experimentelle Toxdkobogie und Okologie D-67056 Ludwigohaten PKofungen nach Kategoilen)/ Areas of Expert!"e (gsWamoav ChinwV4L.P W.5.SJOSC WdI"=) Datumder Inspoldion IDate of Inspection

................ ............ 1. 5M201 und voni 21. baa; 28.OG0 001 --
Dkier ganenntse P 

### ~eiwchtungfs~tandortbefindet The above mentioned test fadlitylAt shte as Included sicti hI natlonalen GLP-Oberwachamgsvsrtshren und in the national GLP.Comp"acProgramme and Is *Wi regeliuIN9s uf ElniahWV derGLP-Grundattze inspected ona regular basis. Aut derGruncdiage des InspektonbeIchtes wird hier- Bsed on the Inspection report itcan be confirnad. mit besWgt doss Indieser Pr~felnichungldeien that thstestfaclty/lest ot Isable to conductthe prftendot die oben genannten PrOfungen unter Eln- aforementioned studies Incompliance With the Puinhaitajigder GLP-Glundslitze dta'chgaMut warden ciples ofGLP. k~nnen.

unto mSignature, Date 

### ~ ,,Di18 

### ~ M~ (Named= d-avemm-rhn P n t M. W~ ofei neponhlbhPutoo
LneW Ist ft chianeflba vv amw ssdm m 4,D 52 ah

# BASF Report Project No.: 40M0437/034091 8 

### CONTENTS Page TITLE PAGE 1 BLANK PAGE 2 GLP COMPLIANCE STATEMENT 3 BLANK PAGE 4 SIGNATURE PAGE5 STATEMENT OF THE QUALITY ASSURANCE UNIT 6 STATEMENT OF GLP COMPLIANCE (FROM THE COMPETENT AUTHORITY) 7 CONTENTS 8 1. SUMMARY 10 

### 2. INTRODUCTION 11 2.1. PRINCIPLE 11 2.2. REGULATORY GUIDELINES 11 3. MATERIAL AND METHODS 12 3.1. TEST SUBSTANCE 12 3.2. TEST SUBSTANCE ANALYSIS 13 3.2.1. Test substance 13 3.2.2. Test substance preparation 13 3.3. EXOGENOUS METABOLIC ACTIVATION 14 3.3.1. S-9 fraction 14 qw 3.3.2. S-9 MIX 14 3.4. BACTERIA 15 3.4.1. Salmonella typhimurium 15 3.4.2. Escherichia coil 16 3.4.3. Checking out the tester strains 16 3.5. EXPERIMENTAL PROCEDURE 17 3.5.1. Choice ofthe vehicle 17 3.5.2. Mutagenicity tests 17 3.5.2.1. Standard plate test 17 3.5.2.2. Preincubation Test 19 3.5.3. Titer determination 19 3.5.4. Control articles 20 3.5.4.1. Negative controls 20 3.5.4.2. Positivecontrols 20 3.5.5. Scope of tests and test conditions 22

# BASF Report; Project No.: 40M04371034091 9 Page 3.6. EVALUATION 23 3.6.1. Mutagenicity 23 3.6.2. Titer 23 3.6.3. Toxicity 23 3.6.4. Solubility 23 3.6.5. Acceptance criteria 24 3.6.6. Evaluation criteria 24 3.7. RETENTION OF RECORDS 25 4. RESULTS 

### 26 4.1. MUTAGENICITY 26 4.1.1. Standard platetest 26 4.1.1.1. Testswi~thoutS-9 mix 26 4.1.1.2. Tests With S-9 mix 26 4.1.2. Preincubationtest 27 4.1.2.1. Tests without S-9 mbx 27 4.1.2.'2. Tests with S-9 mix 27 4.2. TOXICITY 28 4.3. SOLUBILITY 28 4.4. DISCUSSION 28 4.5. CONCLUSION 28 

### 5. REFERENCES 29
TOTAL PAGES (including cover pages) 

### Report: 29 Appendix 1: Tables 1
- 

### 9(Standard plate test) 10 Appendix 2: Tables 10- 14 (Preincubation test) 6 Appendix3: Sterility control 2 Appendix 4: S-9 fraction characterization 2 Appendix 5: Historical negative control data 4 Appendix 6: Historical positive control data 4IL ___ ____LA



### Report; Project No.: 40MD437/034091 10
1. SUMMARY The substance Alkali Blue Dry 6100/ Lot2219308 was tested for its mutagenic potential based on the abilityto induce point mutations inselected loci ofseveral bacterial strains, i.e. Salmonella typhimuriumn and Escherichia coli, inareverse mutation assay.
" STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E.col WP2 uvrA " DOSE RANGE: 23 pg -5750 pg/plate (SPT) 4pg - 

### 1500 pg/plate (PIT; Salmonella strains) 4jig
- 

### 2500 pg/plate (PIT: E.coli WP2uvrA) " TEST CONDITIONS: Standard platetest (SPT) and preincubation test (PIT) both with and without metabolic activation (Aroclor-induced rat liver S-9 

### mix). " SOLUBILITY: Precipitation ofthe test substance was found from about 100 pg/plate onward. *TOXICITY: A bacteriotoxic effect was observed under alltest conditions (for details see item 4.2.).

*MUTAGENICITY:

An increase inthe number of his* or frp* revertants was not observed inthe standard platetest or inthe preincubation test either without S-9 mix or afterthe addition ofa metabolizing system. *CONCLUSION:

According to the results ofthe present study, the testsubstance Alkali Blue Dry 6100 1Lot 2219308 isnot mutagenic inthe Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental *conditions chosen here.

# BA$F Report; Project No.: 40M0437/034091 11
2. INTRODUCTION 2.1. PRINCIPLE 

### Bacterial reverse mutation assays using amino-acid requiring strains of Salmonella typhimurium and Escherichia call are commonly employed as initial screening methods fordetecting apoint mutagenic activity of chemical substances (1,2,5)and are used to screen for possible mammalian mutagens and carcinogens. The principle ofthese assays isthat mutations are detected bythe reversion of mutations

- 

### present inthe bacterial strains. This leads to arestoration ofthe functional capabilityof the bacteria to synthesize the essential amino acid and thus tothe ability togrow inthe absence ofthe amino acid required bythe parent strains. Most ofthe substances are not mutagenic or carcinogenic themselves, but onlyafter metabolic transformation, which invivo iscatalyzed mainly bythe enzyme systems ofthe liver. Therefore, thetests are carried out notonly directly, but also inthe presence of an exogenous metabolic activation system. The most commonly used system isacofactorsupplemented postmitochondrial fraction (S-9) obtained from livers of rats treated with an enzyme-inducing agent, e.g. Aroclor 1254. Experimental starting date: November 5,2003 Experimental completion date December4, 2003 (evaluation last experiment):
2.2. REGULATORY GUIDELINES The tests are carried 

### out inaccordance with the following guidelines: * OECD No. 471 (July21, 1997) " EC Directive 2000/32, B.13/13B.14 (May 19, 2000)M i ut

m111 ii! tL11liiilfh IllIEIL1I 1 1E



### Report Project No.: 40M0437/034091 12
3. MATERIAL AND METHODS 3.1. TEST SUBSTANCE Name oftest substance: Alkali Blue Dry 61001 Lot 2219308 Test substance No.: 03/0437-1 Batch No.: Lot2219308 CAS 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

# BASF
Report; Project No.: 40M0437/034091 13

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

- 

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

# BASF Report; Project No.: 40M0437/034091 14
3.3. EXOGENOUS METABOLIC ACTIVATION 3.3.1. S-9 fraction The S-9 fraction isprepared according to Ames et al. (1, 2). At least 5male Sprague-Dawley rats') (200 - 300 g)receive asingle intraperitoneal injection of 500 mg Aroclor 1254 (as asolution 

### incornoil with a concentration of 20 g/100 ml) per kg body weight 

### 5days before sacrifice. During this time, the animals are housed inMakrolon cages inair-conditioned rooms inwhich central airconditioning guaranteed arange oftemperature of20- 240C and arelative humidity of 30 - 70%. The day/night rhythm is12 hours (lightperiod from 6.00 - 18.00 hours and dark period from 18.00
- 6.00 hours). Standardized pelleted feed and tap waterfrom bottles are available ad libitum. 5days after administration, the rats are sacrificed, and the livers are prepared (all preparation steps for obtaining the liver microsome enzymes are carried out using sterile solvents and glassware at atemperature of +4 0C). The livers are weighed and washed in an equivalent volume ofa 150 mM KCI solution (1ml :r I gwet liver), then cut into small pieces and homogenized inthree volumes of KCI solution.After centrifugation of the homogenate at 9000x gfor 10 minutes at+4*C, 5ml portions ofthe supernatant (so-called S-9 fraction) are stored at-70*C to 

### -80C. 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 prior to each experiment (1,2). Forthis purpose, a sufficient amount ofS-9 fraction isthawed at room temperature and 1volume of S-9 fraction ismixed with 9volumes of S-9 supplement (cofactors). This preparation, the so-called S-9 mix, iskept on ice until used. The concentrations ofthe cofactors inthe S-9 mix are: MgCI2 

### 8mM KCI 

### 33 mM Glucose-6-phosphatase 5mM NADP 4mM Phosphate buffer (pH 7.4) 15 mM The phosphate buffer (6)isprepared by mixing an Na 2HPO4 solutionwith an NaH 2PO 4 solution in aratio of about4 : 1. To demonstrate the efficacy of the S-9 mix inthis assay, the S-9 batch was characterized with benzo(a)pyrene (see Appendix 

### 4).
~Charles River Deutschland GmbH

# BASF
Report; Project No.:40M0437/034091 15
3.4. BACTIERIA For testing, deep-frozen (-70*C to -80oC) bacterial cultures (Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and E.coil WP2 uvrA) are thawed atroom temperature, and 0.1 ml ofthis bacterial suspension isinoculated innutrient broth solution (8gDifco nutrientbroth +

### 5gNaCI/liter) and incubated inthe shaking water bath at 376C for about 10
- 

### 12 hours. As arule, agerm densityof> 108 bacterialml isreached. These cultures grown overnight are kept Iniced water from the beginning ofthe experiment until the end inorderto 

### prevent further growth. The use of the strains mentioned isinaccordance with the current scientific recommendations for the conduct ofthis assay. The Salmonella strains were obtained from KNOLL Aktiengesellschaft on October 30, 1989. The Escherichia col strain was obtained from Merck on September 9, 1991.
3.4.1. Salmonella typhimurium The rate of induced back mutations ofseveral bacteria mutants from histidine auxotrophy (his-) to histidine prototrophy (his+) isdetermined (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 GO base pairs at the primary reversion site. All strains have adefective excision repair system (uvrB), which prevents the repairof lesionswhich are induced inthe DNA, and this deficiency results ingreatly enhanced sensitivity ofsome mutagens. Furthermore, all strains show aconsiderably reduced hydrophilic polysaccharide layer (fa),which leads to an increase inpermneability to lipophilic substances. The strains TA 1535 and TA 100 are derived from histidine-prototrophic Salmonella strains by the 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 carrydifferent frameshift markers, i.e. the +1mutant his C3076 inthe case of TA 1537 andthe +2type his D3052 inthe case ofTA 98. The strainsTA 98 and TA 100 carry an Rfactor plasmid 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 toaconsiderable increase insensitivity.

### Report; Project No.: 40M0437/034091 16
3.4.2. Escherichia coil Escherichia coil WP2 uvrA which has an AT base pair atthe primary reversion site isa derivative of E.coilWP2 With adeficient excision repair and isused to detect substances which induce base pair substitutions (5). The rate of induced back mutations from tryptophan auxotrophy (trp) to tryptophan independence (trp*) isdetermined.
3.4.3. Checking out the tester strains The Salmonella strains are checked for the following characteristics at regular intervals: deep rough character (rfa); UV sensitivity

### (&uvrB); ampicillin resistance (R factor plasmid). E.col WP2 uvrA ischecked for UV sensitivity. Histidine and tryptophan auxotrophy isautomatically checked ineach experiment viathe spontaneous rate.

# BASF
Report; Project No.: 40M04371034091 17
3.5. EXPERIMENTAL PROCEDURE 3.5.1. Choice ofthe vehicle Due tothe limited solubilityofthe test substance inwater, DMS0 was selected as the vehicle, which had been demonstrated to be suitable inbacterial reverse mutation tests and forwhich 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 at. (1,2). Test tubes containing 2-mi portions ofsoft agar (overlay agar), which consists of 100 ml agar (0.6% agar + 

### 0.6% NaCI) and 10 ml amino acid solution (minimal amino acid solution forthe determination of mutants: 0.5 mM histidine + 

### 0.5 mM biotin) are kept inawater bath at 45*C, and the remaining components are added inthefollowing order: 0.1 ml test solution orvehicle 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-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds. Composition ofthe minimal glucose agar. 980 ml aqua dest.

20 ml Vogel-Bonner Emedium 15g Difcobactoagar 20 g D-glucose, monohydrate. After incubation at 37TC 

### for 48
- 

### 72 hours inthe dark, the bacterial colonies (his' revertants) are counted.

# BASF Report; Project No.: 40M0437/034091 18 Escherichia coli The experimental procedure isbased onthe method ofAmes et al. (1,2). Test tubes containing 2-mI portions of soft agar (overlay agar), which consists of 100 ml agar (0.6% agar + 

### 0.6% NBCI) and 10 ml amino acid solution (minimal amino acid solutionfor the determination of mutants: 0.5 mM tryptophan) are kept Inawater bath at450C, andthe 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 (intests with metabolicactivation) or 0.5 ml phosphate buffer (intests without metabolic activation) Aftermixing, the samples are poured onto minimal agar plates within approx. 30 seconds. Composition ofthe minimal agar The composition ofthe minimal agar (SAl selective agar) isbased on the description of Green, M.H.L. and Muriel, W.J. (5), with the exception ofsolution E(tryptophan solution), which has previously been added to the 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 37*C for 48
- 

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

# BASF Report; Project No.: 40M0437/034091 19
3.5.2.2. Preincubation Test The experimental procedure isbased on the method described by Yahagi et al. (7)and Matsushima et al. (8). 0.1 ml test solution or vehicle, 0.1 ml bacterial suspension and 0.5 ml S-9 mix or phosphate buffer are incubated at 37'C for the duration ofabout20 minutes usinga shaker. Subsequently, 2ml of soft agar isadded and, aftermixing, 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, 0.1 ml of the overnight cultures isdiluted to 10
- 

### ineach Case. Test tubes containing 2-mI portions ofsoft agar containing maximal amino aicid solution (5mM tryptophan or 5mM histidine + 

### 0.5 mM biotin) are kept in awater bath at450 

### C, and the remaining components are added inthe following order 0.1 ml vehicle (without and with testsubstance) 0.1 ml fresh bacterial culture (dilution: 10-6) 0.5 ml S-9 mix Inthe preincubation test, 0.1 ml of the overnight cultures isdiluted to 10-6 

### ineach case. 0.

1ml vehicle (with and without test substance), 0. 1ml bacterial suspension and 0.5 ml S-9 mix are incubated at370C forabout 20 minutes using ashaker. Subsequently, 2ml

- 

### ofsoft agar containing maximal amino acid solution for titer determination (5mM tryptophan or 5mnM histidine +

### 0.5 mnM biotin) isadded. After mixing, the samples are poured onto the agar plates Within approx. 30 seconds. After incubation at 37 0



### Cfor 48
- 

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

# BASF Report; Project No.: 40M0437/034091 20
3.5.4. Control articles 3.54.1. Negative controls Parallel with each experiment, negative controls are carried out inorder to check for possible contaminants (sterility control) and todetermine the spontaneous mutation rate (vehicle control).

*Sterility control

Additional plates are treated with soft agar, S-9 mix, buffer, vehicle orthe test substance butwithout the addition oftesterstrains (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 all tester strains.
3.5.4.2. Positive controls The following positive controls are usedto check the mutability of the bacteria and the activity ofthe S-9 mix: With S-9 mix 2-aminoanthracene (2-AA) (SIGMA, A-1381)

- 

### 2.5 pg/plate, dissolved inDMSO

- 

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

- 

### 60 pg/plate, dissolved inDMSO

- 

### strain: Escherichia coliWP2 uvrA

# BASF Report; Project No.: 40M0437/034091 21 without S-9 mix " N-methyl-N'-flitro-N-flitrosoguaflidifle (MINNG) (FLUKA, 68051)

. 

### 5 Vg/plate, 

### dissolved inDMSO

- 

### strains: TA 1535, TA 100 " 4-nitro-o-pheflylefldiamine (NOPO) (SIGMA, N-9504)

- 

### 10 pg/plate, dissolved InDMS0

- 

### strain: TA 98 " 9-aminoacridine (AAC) (SIGMA, A-I135)

- 

### 100 pg/plate, dissolved inDM50

*- strain: TA 1537 

### * 4-nitroqulnoline-N-oxide (4.NQO) (SIGMA, N-8141)

- 

### 5 pg/plate, dissolved inDMVSO

- 

### strain: E.coli WP2 uvrA The stability ofthe selected positive controls 

### iswell-defined under the selected culture conditions, since they are well-established reference mutagens.

### Report; Project No.: 40M0437/034091 22
3.5.5. Scope oftests and test conditions 1st Expeiriment Strains: TA 1535, TA 100, TA 1537, TA 98, E.col WP2 uvrA Doses: 0; 23; 115; 575; 28756 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, TA98, Doses: 0; 500; 1000; 1500;2 000 and 2500 pg/plate Vehicle: DMSO Type oftest: Standard plate testwith and without S-9 mix Number ofplates: 3test plates per dose or per control *3rd Experiment

Strains: TA 1535, TA 100, TA 1537, TA 98, E.col WP2 uvrA Doses: Salmonella strains: 0; 4; 20; 100; 500 and 1500 pg/plate E.call, 0; 4; 20; 100; 500 and 2500 pg/plate Vehicle: DM80 Type oftest: Preincubation testwith andwithout S-9 mix Number ofplates: 3test plates per dose or percontrol

# BASF Report, Project No.: 40M0437/034091 23
3.6. EVALUATION 3.6.1. Mutagenicity Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were givenfor all dose groups aswell as forthe positive and negative (vehicle) controls inall experiments. Ingeneral, five doses ofthe test substance are tested with a maximum of 5mg/plate, andtriplicate plating isused for all test groups at least inthe 1 st experiment. Dose selection and evaluation as well as the number of plates used inrepeat studies orfurther experiments are based on thefindings 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 highest doses inall experiments.
3.6.3. Toxicity Toxicitydetected by a

- 

### 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 andwithout S-9 mix inall experiments and indicated inthe tables.
3.6.4. Solubility Precipitation ofthe test material isrecorded and indicated inthe tables. As long as precipitation does not interfere with the colonyscoring, 5mg/plate is generally selected and analyzed (incases of nontoxic compounds) as the 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 forfurther clarification/substantiation.

# BASF Report; Project No.: 40M0437/034091 24
3.6.5. Acceptance criteria Generally, the experiment 

### isconsidered valid if the following criteria are met: e The numberof revertant colonies inthe negative controls was within the normal range of the historical control data for each testerstrain (see Appendix 5). * The sterility controls revealed no indication of bacterial contamination. * The positive control articles both with and without S-9 mix induced asignificant increase inthe number of revertant colonies within the range of the historical control data or above (see Appendix 6). a The titer ofviable bacteria was> 10 8 

### /ml.
3.6.6. Evaluation criteria The test chemical isconsidered positive inthis assay ifthe following criteria are met:

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

about doubling ofthe spontaneous mutation rate inat least one tester strain either without S-9 mix or after adding ametabolizing system. Atest substance isgenerally considered nonmutagenic inthis test if:

*The number of revertantsfor all testerstrains were within the historical negative

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

# BASF Report; Project No.: 40M0437/034091 25
3.7. RETENTION OF RECORDS GLP-relevant records and materials are stored at BASFAktiengesellschaft and at BASF Corporation at least for the period of time specified inthe GLP principles. Details on responsibilities or locations of archiving may be seen from the relevant SOPs and from the rawdata.- 

### ~IiwtuuUIU~fl 1~ IIIIILi M,1111 I I I ALLA111i IIiLL.


# BASF Report; Project No.: 40M0437/034091 26


### 4. RESULTS (Tables I
- 

### 14) The substance Alkali Blue Dry6100/Lot 2219308 was tested for mutagenicity inthe Salmonella typhimurium / Escherichia coll reverse mutation assay both inthe standard plate testand inthe preincubation test with andwithout the addition of ametabolizing system (S-9 mix) obtained from rat liver using the Salmonella strains TA 1535, TA 100, TA 1537, TA 98 and Escherichia coilWP2 uvrA.
4.1. MUTAGENICITY4.1.1. Standard plate test (Appendix 1,Tables 1 - 

### 9) 4.1.1.1. Tests without S-9 mix TA 1535: TA 100: TA 1537: No increase inthe numberof his* or trp* revertants. TA98: E.col W132 uvrA:-
4.1.1.2. Tests with S-9 mix TA 1535: TA 100: TA 1537: No increase inthe number of hise or

### trp* revertants. TA 98: E.coi W132 uvrA:

# BASF Report; Project No.: 40M04371034091 27
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 his* ortrp* 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 ofhis* or trp+ revertants. TA 98: E.coilWP2 uvrA:

# I !T 71ASReport; Project No.: 40MD437/034091 28
4.2. TOXICITY A bacteriotoxiceffect (reduced his' background growth, decrease inthe number ofhis+ revertants, reduction inthe titer)was observed inthe standard plate testdepending on the strain andtest conditions from about 575 pg - 

### 1000 pg/plateonward. No bacterlotoxicitywas observed using E.coi WP2 uvrA. Inthe preincubation assay bacteriotoxicity (reduced his' or trp- background growth, decrease inthe number of his+ortrp+ revertants, reduction inthe titer)was observed from about 500 jag/plate onward.
4.3. SOLUBILITY Test substance precipitation was found from about 100 pg/plate onward.
4.4. DISCUSSION According tothe results ofthe present study, thetest substance did not lead to an increase inthe number of revertant colonieseither without S-9 mix or after adding a metabolizing system inthree experiments carried out independently ofeach other (standard plate testand preincubation assay).
4.5. CONCLUSION Thus, under the experimental conditions chosen here, itisconcluded that Alkali Blue Dry 6100 ILot2219308 isnot amutagenic agent inabacterial reverse mutation test.

# BA SF Report; Project No.: 40M0437/034091 29
5.REFERENCES I1. Ames, B.N.; McCann, 

### J.; Yamasaki, E.: Methods fordetecting carcinogens and mutageris with the Salmonellalmammalan-microsome mutagenicitytest. Mut. Res., ;L1, 

### 347
- 

### 364 (1975) 2. Maron, D.M.; Ames, B.N.: Revised method for the Salmonella mutagenicitytest. Mut Res., 113, 217-233 (1983) 3. Ames, B.N.; Lee, F.D.; Durston, W.E.: An improved bacterial test system forthe detection and classification of mutagens and carcinogens. Proc. Nat. Acad. Sci. USA, 70 782
- 

### 786 (1973) 4. McCann, J.; Spingarn, N.E.; Kobori, J.;Ames, B.N.: Detection of carcinogens as mutagens: Bacterial testerstrains with Rfactor plasmids. Proc. Not. Acad. Sci. USA, 72, 979
- 

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

### 6. DeMarini, D.M.; Dallas, M.M.; Lewtas, J.: Cytotoxicity and effecton mutagenicity of buffers inamicrosuspension assay. Ter. Camc. Mut, 9,287 -. 295 (1989) 

### 7. Yahagi, T.; Nagao, M.; Seino, Y.; Matsushima, T.; 

### Sugimura, T.; Okada, W. Mutagenicities ofN-nitrosoamines inSalmonella. Mut. Res.,!48, 121
- 

### 130 (1977) 8. Matsushima, 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. SpringerVerlag Berlin, Heidelberg, New York (1980).

# BASF Report; Project No.: 40M0437/034091

APPENDIX I TABLES 1-9 (STANDARD PLATE TEST)BASF Aktiengesellschaft 01 Experimental Toxicology and Ecology STUDY DIRECTOR: DR. ENGELHARDT PROJECT NO. :40M437/034091 SUBSTANCE NAME: Alkali Blue Dry 6100 

### /Lot 2219308 SUBSTANCE NO. :0310437-1

TESTDATE :05.NOV.2003 METHOD :STANDARD PLATE TEST VOLUME 200 ul/plate STRAIN :TA 1535 VEHICLE :DMS0 89-MIX :WITHOUT S-9 MIX WITH S-9 MIX (1:9) DOSE/PLATE REV M SD FAC REV M SD FAC ?ITRE DMSO 21 17 3 1.0 20 17 4 1.0 24 16 13 19 15 19 29 23 ug 13 15 2 0.9 16 17 1 1.0 16 18 16 16 115 ug 16P 16 2 0.9 15P 18 5 1.0 14P 15P 17P 23P 575 ug 16P 15 4 0.9 16P 13 4 0.8 lip 14P 18P 9P 2 875 ug OB/P 9B/P 8 1 0.5 1 OB /P 73/P 0 OB /P 8B/P 0 5 750 ug 03/P OB/P 0 03/P OB0/P 0 03/P OB/P 0 MNNG 5.0 zig 1217 1176 39 67.9 1139 1173 2-AA 2.5 ug 166 177 12 10.2 189 177 (REV..revertaflts/plate, M~'mean, SD-standard deviation, FAC-factor) P: PRECIPITATION B: REDUCED BACKGROUND GROWTH......... 

### ~~in -Rum LA1-

BASF Aktiengesellschaft 02 Excperimental Toxicology and Ecology STUDY DIRECTOR: DR. ENGELHARDT PROJECT NO. :40HO437/034091 SUBSTANCE NAME: Alkali Blue Dry 6100 ILot 2219308 SUBSTANCE NO. :03/0437-1

TESTDATE :05.NOV.2003 METHOD STANDARD PLATE TEST VOLUME 100 ul/plate STRAIN : TA 100 VEHICLE : DHSO 59-HIX : WITHOUT S-9 MIX WITH S-9 MIX (1:9) DOSE/PLATE REV M SD FAC REV M SD FAC TITE DMSO 113 113 12 1.0 110 117 a 1.0 29 125 114 37 102 120 25 23 ug 104 ill 6 1.0 102 109 6 0.9 113 113 116 ill 115 ug 104P il1 13 1.0 104P 101 a 0.9 103P 107? 126? 92P 575 ug 120P 104 14 0.9 93P 84 8 0.7 95P SOP 97? 79? 2 875 ug 03/P 113/P 15 8 0.1 0 OB/IP 24B/P 1 OB/P 103/P 0 5 750 ug 03/P 03/P 0 OB/IP OB/P 0 03/P 03/P 0 MNNG 5.0 ug 1311 1297 65 11.4 1226 1353 2-MA 2.5 ug 1043 1059 56 9.1 1013 1122 (REV-revertartslplate, M-mean, Sfl-standard deviation, FAC-factor) P: PRECIPITATION B: REDUCED BACKGROUND GROWTHBASF Aktiengesellschaft 03 Experimental Toxicology and Ecology STUDY DIRECTOR: DR. ENGELHARDT PROJECT NO. :40M44371034091 SUBSTANCE NAME: Alkali Blue Dry 6100 

### /Lot 2219308 SUBSTANCE NO. D 3/0437-1

TESTDATE :05.NOV.2003 METHOD :STANDARD PLATE TEST VOLUME : 100 ul/plate STRAIN z TA 1537 VEHICLE : DM50 S9-MIX ;WITHOUT S-9 MIX WITH S-9 MIX (1:9) DOSE/PLATE REV M SD FAC REV M SD FAC TITRE D14SO 8 11 4 1.0 6 8 2 1.0 22 10 9 21 15 9 24 23 ug 7 9 2 0.8 8 9 1 1.2 11 10 10 10 115 ug 7P 10 3 0.9 12P 15 6 1.8 lop 21P 12P lp 575 ug 7P 7 3 0.7 6P 20 4 1.2 lop 13P

5P lop 2 875 ug 03/P 03/P 0 03/P 03/P 0 03/P 03/P 0 5 750 ug 05/P 03/P 0 OB/P OB/P 0 03/P OB/P 0 AAC 100 ug 560 484 70 44.0 421 472 Z-AA 2.5 ug 150 151 18 18.8 169 133 (REV=revertants /plate, 14-mean, SD=standard deviation, PAC=factor) P: PRECIPITATION B: REDUCED BACKGROUND GROWTHI'DIiiM 'i lUm -i

BASF Aktiengesellschaft 04 Experimental Toxicology and Ecology-SUYDRCO:R.EGLAT PROJECT NO. :40M0437/034091 SUBSTANCE NAME: Alkali Blue Dry 6100 ILot 2219308 SUBSTANCE NO. 03/0437-1

TESTDATE :05.NOV.2003 METHOD :STANDARD PLATE TEST VOLUME 100 ul/plate STRAIN :TA 98 VEHICLE DMSO S9-MIX WITHOUT S-9 MIX WITH S-9 MIX (1:9) DOSE/PLATE_ REV M SD FAC REV M SD PAC TITRE DMSO 27 32 6 1.0 41 41 1 1.0 21 29 40 25 39 42 22 23 ug 28 28 0 0.9 30 33 3 0.8__ 28 34 28 35 115 U& 30P 25 5 0.8 33P 30 3 0.7 20? 28P 26P 30P 575 ug 21? 20 3 0.6 39P 37 2 0.9 17P 35P 22P 37P 2 875 ug lOB/P 11 4 0.3 22B1P 18 4 0.4 0 83/P 173/P 0 153/P 14BIP 0 5 750 ug 03/P 03/P 0 03/P 03/P 0 03/P OB/P 0 NOPD 10 ug 993 924 63 29.2 871 907 2-AA 2.5 ug 1117 1120 28 27.3 1149 1093 (REV-revertants/plate, H-mean, SD-standard deviation, FAC-factor) P: PRECIPITATION B: REDUCED BACKGROUND GROWTHBASF Aktiengesellschaft 0 Experimental Toxicology and Ecology STUDY DIRECTOR: DR. ENGELHARDT PROJECT No. :40M404371034091 SUBSTANCE NAME: Alkali Blue Dry 6100 /Lot 2219308 SUBSTANCE NO. :03/0437-1

TESTDATE : 05.NOV.2003 METHOD : STANDARD PLATE TEST VOLUME :100 ul/plate STRAIN zE.coli WP2 uvrA VEHICLE :DMS0 59-MIX : WITHOUT S-9 MIX WITH S-9 MIX (1;9) DOSE/PLATE REV M SD FAC REV 14 SO FAC TITRE DMSO 40 35 5 1.0 38 38 1 1.0 3B 31 37 40 34 38 33 23 ug 32 30 6 0.8 34 35 2 0.9 34 34

- 23 37 115 us 40P 37 3 1.0 37P 31 6 0.8 34P 28P 36P 27P 575 ug 36P 32 3 0.9 33P 34 5 0.9 30P 29? 31P 39P 2 875 ug 30P 30 7 0.8 34P 31 6 0.8 22 23? 36P 35 36P 24P 33 5 750 ug 31P 29 5 0.8 30P 32 2 0.8 17 33P 34P 20 24P 31P 14 4-NQO 5.0 ug 775 770 37 22.0 731 804 2-AA 60 ug 199 200 21 5.3 180 221 (REV-revertants/plate, M-mean, SD=standard deviation, FAC=factor) P: PRECIPITATIONr[a-I,_IALiLiI IiFMTEiHfl 

### A 1

BASF Aktiengesellschaft 06 Eiperimental Toxicology and Ecology SUYDRCO:D.EGLAD PROJECT NO. :40HO4371034091 SUBSTANCE NAME: Alkali Blue Dry 6100 

### ILot 2219308 SUBSTANCE NO. :03/0437-1

TESTDATE :18.NOV.2003 METHOD :STANDARD PLATE TEST VOLUME :100 ul/plate STRAIN : TA 1535 VEHICLE ;DMS0 S9-HIX : WITHOUT S-9 MIX WITH S-9 MIX (1:9) DOSE/PLATE REV M SD FAC REV M SD FAC TITRE DM50 18 17 1 1.0 17 19 2 1.0 29 18 19 22 16 20 27 500 ug 15P 15 2 0.8 14P 14 2 0.8 16P 16P 13P 13P 1 000 ug 16P 15 1 0.8 17P 15 3 0.8 14P 17P 14P lip 1 500 ug 15P 12 3 0.7 lop 11 1 0.6 lop 12P lop lop 2 000 us 43/P 3 1 0.2 7P 7 2 0.4 5 2BIP 8P 8 23/P 5? 1 2 500 118 03/P 63/P 5 1 0.3 5 03/P 63/P 7 03/P 4B/P 2 HNNG 5.0 ug 1039 1063 34 61.3 1102 1047 2-AA 2.5 ug 140 117 20 6.3 107 104 (REV-revertants /plate, M-mean, SD-standard deviation, FAC-factor) P: PRECIPITATION B: REDUCED BACKGROUND GROWTHBASF Aktiengesellschaft 07 Experimental Toxicology and Ecology STUDY DIRECTOR: DR. ENGELHARDT PROJECT NO. 40HO4371034091 SUBSTANCE NAMdE: Alkali Blue Dry 6100 ILot 221.9308 SUBSTANCE NO. 03/0437-1

TESTDATE :18.NOV.2003 METHOD : STANDARD PLATE TEST VOLUME :100 ul/plate STRAIN :TA 100 VEHICLE : DMS0 S9-MIX : WITHOUT S-9 MIX WITH S-9 MIX (1:9) DOSEIPLATE REV M SD FAC REV M SD FAC TITRE DMS0 104 110 5 1.0 119 log 10 1.0 27 112 107 32 114 100 22 500 ug 103P 107 7 1.0 113P 112 3 1.0 104P 11 4P 115F 109P 1 000 ug 95? 103 9 0.9 104P 95 8 0.9 103P 91? 122P 90P 1 50D ug 44P 36 10 0.3 82P 89 7 0.8 40OP 90P 25P 95P 2 000 ug 03/P 75P 71 10 0.7 14 03/P 78P 11 OB/IP 60? 11 2 500 ug 03/P 223/P 18 6 0.2 12 03/P 11BIP 5 03/P 20B/P 4 I4NNG 5.0 ug 1017 1088 64 9.9 1105 1140 2-AA 2.5 ug 811 781 51 7.2 722 811 (REV-revertantslplate, M-mean, SD-standard deviation, FAC=factor) P: PRECIPITATION B: REDUCED BACKGROUNVD GROWTHBASF Aktiengesellschaft 08 Experimental Toxicology and Ecology STUDY DIRECTOR: DR. ENGELE ARDT PROJECT NO. 40140437/034091 SUBSTANCE NAME: Alkali Blue Dry 6100 

### ILot 2219308 SUBSTANCE NO. 0310437-1

TESTUATE 18.NOV.2003 METHOD :STANDARD PLATE TEST VOLUME 100 ul/plate STRAIN TA 1537 VEHICLE :DMSO S9-HIX WITHOUT S-9 MIX WITH S-9 MIX (1:9) DOSE/PLATE REV M SD FAC REV M SD FAC TITRE DMSO 9 9 1 1.0 9 a 1 1.0 28 10 7 24

9 7 25 500 ug 8P 7 2 0.7 7P 7 2 0.9 4P 5P 8P SP 1 000 ug 9P 7 2 0.8 2P 5 3 0.6 5P 8P 7P 4P 1 500 ug 4P 3 1 0.3 7P 5 2 0.6 2P 4P 3P 3P 2 000 ug lB/P 1 0 0.1 3B/P 2 1 0.3 0 1B/P 2BIP 0 1B/P 1BJP 0 2 500 ug 03/P OB/P 0 03/P OB/P 0 03/P 03/P 0 AAC 100 ug 375 398 27 42.7 428 392 2-AA 2.5 ug 141 134 8 17.5 125 136 (REV-revertantsiplate, M-mean, SD=standard deviation, FAC=factor) Pt PRECIPITATION B: REDUCED BACKGROUND GROWTHBASF Aktiengesellschaft 09 Experimental Toxicology and Ecology STUDY DIRECTOR: DR. ENGELHARDT PROJECT NO. :40M0437/034091 SUBSTANCE NAME: Alkali Blue Dry 6100 / Lot 2219308 SUBhSTANCE NO. 03/0437-1

TESTDATE 18.NOV.2003 METHOD :STANDARD PLATE TEST VOLUME 100 ul/plate STRAIN :TA 98 VEHICLE :DMSO S9-MIX WITHOUT S-9 MIX WITH S-9 MIX (1:9) DOSE/PLATE- REV H SD FAC REV M SD PAC TITRE DMS0 19 24 5 1.0 36 31 4 1.0 29 25 29 37 28 29 32 500 ug 18? 22 4 0.9 26P 26 1 0.8 26P 25P 21P 26P 1 000 ug 20? 20 4 0.8 29? 30 3 1.0 24P 34P 17P 28P 1 500 ug 22P 21 4 0.9 30P 26 5 0.8 16P 28P 24P 20P 2 000 ug lOB/P 9 2 0.4 15P 14. 3 0.4 8 63/P 16P 14 lOB/P lop 6 2 500 us 73/P 6 1 0.3 lOB/P 15 5 0.5 8 6B/P 20BIP 10 63/P 143/P 5 NOPD 10 ug 605 615 42 25.6 662 579 2-AA 2.5 ug 609 611 30 19.5 582 642 (REV=revertalts /plate, M-rnean, SD-standard deviation, FAC=factor) P: PRECIPITATION B: REDUCED BACKGROUND GROWTEBASF Aktiengesellschaft 10 Experimental Toxicology and Ecology STUDY DIRECTOR;: DR. ENGELHARDT PROJECT NO. 40M0437/034091 SUBSTANCE NAME: Alkali Blue Dry 6100 ILot 2219308 SUBSTANCE NO. 03/0437-1

TESTDATE 02.DEC.2003. METHOD PREINCUBATION TEST VOLUME :100 ullplate STRAIN :TA 1535 VEHICLE DM80 S9-MIX WITHOUT S-9 MIX WITH S-9 MIX (1:9) DOSEIPLATE REV M SD FAC REV M SD FAC TITRE DMSO 23 16 3 1.0 15 16 1 1.0 24 18 17 38 17 17 2 4 ug 14 15 2 1.0 12 15 3 0.9 15 18 17 14 20 ug 13 16 3 1.0 14 15 2 0.9 18 18 17 14 100 Ug 16P 12 4 0.8 lip 12 3 0.7 9P lop 12P 15P 500 ug 24P 10 3 0.6 81' 9 2 0.6 5 8P 9 8 9P lip .4 1 500 ug OBIP OB/P 1 OB IP OB/P 0 OB/P OB/P 2 MNNG 5.0 ug 705 749 40 46.8 760 783 2-AA 2.5 ug 166 145 19 8.9 139 130 (REV-revertantsjplate, M-mean, SD=staiidard deviation, FAC-factor) P: PRECIPITATIOW B. REDUCED BACKGROUND GROWTHBASF Aktiengesellschaft 1 Experimental Toxicology and Ecology STUDY DIRECTOR: DR. ENGELHARDT PROJECT NO. 40O437/034091 SUBSTANCE NAM'E: Alkali Blue Dry 6100 /Lot 2219308 SUBSTANCE NO. :0310437-1

TESTDATE :02.DEC.2003 MfETHOD :PREINCUBATION TEST VOLUME :100 ul/plate STRAIN : TA 100 VEHICLE :DMSO S9-HIX : WITHOUT S-9 MIX WITH S-9 MIX (1:9) ],OSEIPLATE- REV M SD FAC REV -M SD FAC TITE DMSO 129 120 9 1.0 110 121 10 1.0 36 119 126 34 112 127 32 4 ug 120 121 4 1.0 113 122 9 1.0 117 123 125 131 20 ug 130 118 10 1.0 120 110 9 0.9 112 107 113 202 100 ug 95P 102 20 0.9 102P 98 7 0.8 113P 1 02P 98P 90P 500 ug 48P 44 3 0.4 78P 67 10 0.6 8 42P 6ZP 5 43P 61P 12 2 500. ug 03/P 20B/P 19 2 0.2 5 03/P 21B/P 7 OB /P 173/P 4 MNNG 5.0 ug 753 791 34 6.6 819 801 2-AA 2.5 ug 602 633 35 5.2 671 625 (REV=revertants/plate, M=mean, SD=standard deviation, FACfactar) P: PRECIPITATION B: REDUCED BACKGROUND GROWTH

### ULIL

BASF Aktienigesellschaft 12 Experimental Toxicology and Ecology STUDY DIRECTOR: DR. ENGELHARDT PROJECT NO. :40M0437/034091 SUBSTANCE NAME: Alkali Blue Dry 6100 ILot 2219308 SUBSTANCE NO. :03/0437-1

TESTDATE 202.DEC.2003 METHOD :PREINCUBATION TEST VOLUME : 100 ul/plate STRAIN :TA 1537 VEHICLE DM80 S9-mIX WITHOUT S-9 MIX WITH S-9 MIX (1:9) DOSE/PLATE- REV M SD FAC REV H SD FAC TITRE DMSO 10 9 1 1.0 8 9 1 1.0 25 9 8 25 9 10 21 4 ug 6 6 2 0.7 9 9 2 1.1 8 8 5 11 20 ug 7 6 1 0.7 6 8 3 0.9 6 11 6 7 100 ug 3P 5 2 0.5 B? a 1 0.9 6P SP 6P 7P 500 ug iP 1 1 0.1 lop 6 3 0.7 0 2P 4P 0 iP 5P 2 1 500 ug 03/P OB0/P 2 03/P OBIP 0 03/P 03/P 2 AAC 100 ug 394 377 19 40.4 380 356 2-AA 2.5 ug 112 122 21 14.1 147 108 (REV-revertants/plate, M--mean, SD-standard deviation, FAC-factor) F: PRECIPITATION B: REDUCED BACKGROUND GROWTHBASF Aktiengesellschaft 1 Experimental Toxicology and Ecology STUDY DIRECTOR: DR. ENGELHARDT PROJECT NO. :40M0437/034091 SUBSTANCE NAME: Alkali Blue Dry 6100 

### ILot 2219308 SUBSTANCE NO. :03/0437-1

TESTDATE 02.DEC.2003 METHOD P REINCUBATION TEST VOLUIME : 100 ul/plate STRAIN :TA 98 VEHICLE : DMSO £9-MIX : WITHOUT S-9 MIX WITH S-9 MIX (1:9) DOSE IPLATE- REV M SD FAC REV M SD FAC TITRE DMSO 30 32 7 1.0 37 34 3 1.0 26 40 31 29 26 33 22 4 ug 27 28 4 0.9 29 27 2 0.8 25 25 32 27 20 ug 33 29 4 0.9 36 34 2 1.0 28 33 26 34 100 ug 30P *25 5 0.8 25P 22 3 0.7 24P 22P 21P 19P 500 ug 16P 13 4 0.4 14P is 1 0.4 5 15P 14P 0

9P 16P 2 1 500 ug 03/P 03/P 0 OB/P OB0/P 0 OB/P OB/P 0 NOPD 10 ug 800 766 31 23.9 758 740 2-AA 2.5 ug 528 578 53 17.2 634 572 (REV='revertants/plate, N-mean, SD=standard deviation, FAC-factor) P: PRECIPITATION B: REDUCED BACKGROUND GROWTHNOMMIMNEMLkLLL VA

BASF Aktiengesellschaft 14 Eiperimental Toxicology and Ecology STUDY DIRECTOR: DR. ENGELHARDT PROJECT NO. 40M4437/034092 SUBSTANCE NAME: Alkali Blue Dry 6100 

### /Lot 2219308 SUBSTANCE NO. :03/0437-1

TESTDATE 02.DEC.2003 METHOD : PREINCUBATION TEST VOLUME : 100 ullplate STRAIN : E.coli 1WP2 uvrA VEHICLE :DKSO S9-HIX : WITHOUT S-9 MIX WITH S-9 MIX (1:9) DOSEIPLATE REV M4 SD FAC REV M SD FAC TIR DMO32 31 2 1.0 35 38 3 1.0 31 32 41 34 28 38 25 4 ug 36 31 5 1.0 32 37 6 1.0 29 36 27 44, 20 ug 20 25 5 0.8 28 31 9 0.8 26 41 29 25 100 ug 27P 23 4 0.7 34P 25 11 0.6 20P 12P 21P 28P 500 ug 14P 18 4 0.6 24P 19 5 0r.5 8 2OP 14P 8 21P 19P 2 2 500 ug 8B/P 9 -2 0.3 2B/P 5 3 0.1 3 11B/P 8B/P 7

73/P 4B/P 5 4-NQO 5.0 ug 701 665 33 21.7 658 636 2-AA 60 ug 251 219 28 5.8 207 200 (REV-revertants /plate, 14-mean, SD-standard deviation, FAC..factor) P: PRECIPITATION B: REDUCED BACKGROUND GROWTH

# BASF Report; Project No.: 40M0437/034091



### APPENDIX 2 TABLESI10-14 

### (PREINCUBATION TEST)

### Report; Project No.: 40M0437/034091



### APPENDIX 3 STERILITY CONTROL

# BASF Report; Project No.: 40MO437/034091 

### STERILITY CONTROL 1st Experiment Components 'Results 2.0 ml softagar No growth 0.5 ml S-9 mix No growth 0.5 ml buffer No growth 0.5 ml vehicle No growth 5750 pg test substance No growth 2nd Experiment Components Reisults 2.0 mlsoftagar No growth 0.5 M!S-9 mix No growth 0.5 ml buffer No growth 0.5 ml vehicle No growth 2 500 pg test substance No growth 3rd Experiment Components Resdlts: 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 2500 pg test substance No growth

# BASF Report; Project No.: 40M0437/034091



### APPENDIX 4 S-9 FRACTION CHARACTERIZATION

# BASF Report; Project No.: 40M0437/034091
S-9 fr-action Preparation of S-9 fraction: June 17, 2003 Content of protein (mean*): 29.0 gl Characterization of the S-9 mix with benzofa]pyrene: July 14, 2003
METHOD: Standard Plate Test S-9 FRACTION/COFACTORS: 1:9 STRAIN: TA 100 STRAIN: TA 98 DOSEIPLATE MREW SD- FAC* DOSEIPLATE MREV* SD* FAC* DMSO 113.7 9.0 1.0 DMS0 34.0 3.0 1.0

0.6 jig 258.9 32.5 2.3 0.6 jig 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.A pg 497.5 69.3 4.4 2.5 pg 352.8 29.1 10.4

5.0 pg 639.4 29.5 5.8 5.0 Pg 492.6 46.4 14.5
(REV =revertantsiplate, M =mean,SD =standard deviation, FAC factor)

*=pool 1 -4

# BASF Report; Project No.: 40M0437/034091



### APPENDIX 5 HISTORICAL NEGATIVE CONTROL DATA

### P roject No.: 40M04371034091 

# BASF Historical Negative Control Data Strain: TA 1535
RL SPT -Water 03.98
- 09.03 400 11 25 18 2 PT-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 1 DM50 03.00
- 09.03 400 10 24 18 2 SPT 1:9 Acetone 01.96-05.03 210 11 25 19 2

PIT -Water 08.97
- 06.03 400 13 25 19 2 PI DMSO 06.99 -07.03 400 11 25 18 2 

### PIT Acetone 01.96-05.03 168 10 25 19 2 PIT 1 :9 Water 04.97-0.03 400 11 25 18 2 PIT 1 :9 DMSO 05.99-09.03 400 10 23 17 2 PIT:9 Acetone 01.96-06.03 159 10 25 18 2 

### Strain: TA 100
SpT -Water 05.98
- 09.03 400 82 155 117 14 SPT -DM50 06.00-09.03 400 91 160 110 10

- SPT
- Acetone 01.96-05.03 258 91 160S 118 15 SPT 1:9 Water 06.98-09.03 400 94 156 123 16 SPT 1:9 DMSO 08.00-09.03 400 92 164 112 10 SPT 1 :9 Acetone 01.96-05.03 253 85 160 120 -16

-_et - _ _ _ - PIT -water 06.97 -06.03 400 90- 160 117 14 PIT -DMSO 06.99-09.03 400 84 160 112 12 PIT -Acetone 01.96-05.03 172 91 160 117 115 PIT 1 :9 Water 05.97-06.03 400 92 159 122 15 PIT 1 :9 DM50 06.99-09.03 400 80 159 112 13 PIT 19Acetone 01.96-05.03 156 96 10 119 Is

*= revertants/plate SPT = standard plate test PIT =preincubatloil test SD =standard deviation Page 1 of 3

### Project No.: 40M04371034091 

# BASF Historical Negative Control Data Strain: TA 1537

SPT

____ Water 03.98
- 09.03 400 6 6 10 2 SPT

_____ DM80 03.00
- 09.03 400 5 16 10 2 SPT

_____ Acetone 01.96
- 05.03 213 5 is 10 2 SPT 1:9 Water 03.98 -09.03 400 6 19 11 2 SPT 19DM80 03.00-09.03 400 5 19 11 2 SPT 19 Acetone 01.96
- 05.03 213 _7 120 J12 2

PIT -Water 0".7 -06.03 400 5 19 10 2 PIT -DM80 06.99 -09.03 400 5 17 10 2 PIT -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 a 20 11 2 

### Strain: TA98

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 -05.03 216 Is s0 29 5 SPT 1:9 Water 06.98
- 09.03 400 20 49 36 6 SPT 19 DM80 08.00- 09.03 400 20 so 36 6 ST 19 Acetone 01.96
- 05.03 214 26 50 38 5

PIT -Water 05.97-06.03 400 18 44 27 4 PIT -DMSO 06.99
- 09.03 400 17 40 27 4 PIT -Acetone 01.96-05.03 168 19 45 28 5 -PIT 1: 9 Water 04.97 -06.03 400 17 50 36 6 PIT 1:9 DM80 06.99 -09.03 400 20 49 32 5 PIT 1:9 Acetone 01.96-05.03 161 20 50 -37 6 revertantsiplate SPT standard plate test PIT =preincubation test SD 3standard deviation 

### Page 2of

### 3

### Project No.: 40M0437/034091 

# BASF Historical Negative Control Data Strain: WP2 uvrA

SPT -Water 11.97-09.03 400 20 49 31 6 SPT -DMSO 01.00 - 09.03 400 22 50 32 5 SPT
- Acetone 01.96 -05.03 200 iB 50 33 16 SPT 1:9 

### I Water 01.98-09.03 400 20 49 35 6 SPT 1:9 

# I DMSO 01.00-09.03 400 23 50 35 6 SPT 1:9 Acetone 01.96-05.03 200 15 50 37 -7


### PIT Water 04.97 - 06.03 400 21 50 31 5 if- DMSO 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 so 37 6 PIT 1:9 DMSO 06.99-09.03 400 19 s0 32 6 PIT I= :9 Acetone 01.96 -05.03 157 20 49 33 6

=revertantsiplate SPT = standard plate testPIT = preincubation test SD = standard deviation.



### Page 3of 3

# BASF Report-,Project No.: 40M04371034091



### APPENDIX 6 HISTORICAL POSITIVE CONTROL DATA

### Project No.: 40M0437/034091 

# BASF Historical Positive Control Data Strain: TA 1535

SPT

_____ MNNG 5.0 02.01 -09.03 400 500 149 776 199 SPT 1:9

- 2-AA 2.5 -02.01 -09.03 400 85s 325 -146 -38

PT- MNNG 5.0 07.00
- 09.03 400 502 1357 785 150 

### I PIT 1 :9 2-AA 2.5 06.00-09.03 400 80 345 129 3
Strain: 

### TA 100

SPT

- MNNG 5.0 03.01 -09.03 400 501 1747 871 2 262 SPT 1:9 2-AA 2.5 03.01
-09.03 400 507 1708 859 215

PIT MNNG 5.0 07.00 -09.03 400 502 1583 859 188 PIT 1:9 2-AA 2.5 06.00 -09.03 400 500 1126 7 135

*= revertantsplate SPT =standard plate test PIT = preincubation test SD =standard deviation

MNNG =N-methyl-N'-nitro-N-nftrosoguanldlne 2
- AA =2-aminoanthracene

Page I of3

### Project No.: 40M0437/034091 

# BASF Historical Positive Control Data Strain: TA 1537

SPT

- AAC 100 01.01 -09.03 400 300 1147 443 104 SPT

- 1:9 2-AA 2.6 -01.01 -09.03 400 =71 200 127 25

PIT - AAC 100 07.00 -09.03 400 278 886 435 83 PIT 1:9 2-AA 2.5 06.00 -09.03 400 80 200 112 20


### Strain: TA98

In wa1 ... J-1 4J~ 

# I

P

r] 

### NOPD1 02.01 -09.03 400 347 1200 737 132 SPT 1:9 

# J -AA 2.5 03.01 -09.03 400 

### 1504 I1384 699 139

PIT NOPDI10 07.00-09.03 400 399 1129 735 135 PIT 1: 9 2-AA 2.6 06.00 -09.03 400 600 985 621_ 9_0

wrevertantslplatB SPT =standard platetest PIT = preincubatiofl test SD =standard deviation
2 - AA = 2-amlnoanthracene MAC =9-aminoacridine NOPD =4-nltro-o-phenyiendiamine

Page 

### 2 of 3

### Project No.: 40MD4371034091 

# BASF Historical Positive Control Data Strain: WP2 uvrA

W K

,',-,,.1 SPT
- 4-NQO 5.0 12.00 -09.03 400 500 1620 676 171 SPT 1:9 2-AA 60.0 01.01 -09.03 400 165 478 244 40 P,~ PIT__

____ 4-NOO 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 22 26

= revertants/plate SPT =standard plate test PIT = preincubation test SD =standard deviation

4-NQO =4-Nitroquinoline-N-oxide 2- AA * 2-aminoanthracene



### Page 3of 3