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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 PREVENTION, PESTICIDESAND TOXIC SUBSTANCES



### Subject: Follow-upto fax transmission of4/13/12 (ePMA

### removal 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 orotheruse ofthis copyrighted document.
..including electronic transmission, using the internet oremail
....is strictlyprohibited
...." While we understand the basis for the copyrightprotections, we have a concernthat the language in the fax might be interpreted to mean that ePMA is asserting that the studies submitted perthe TSCA activity are not tobemade 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 orderto complywith 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

# BAS1F 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 2231308 DATA REQUIREMENT OECD No. 471 EC 2000/32, B.13/ B.14 AUTHORS Dr. G.Engelhardt (Study Director) Dr. E.Leibold STUDY COMPLETED ON March 15, 2004 TESTING FACILIITY Experimental Toxicology and Ecology BASF Aktiengesellschaft 67056 Ludwigshafen, Germany LABORATORY PROJECT IDENTIFICATION Project No.: 40M0437/034092 SPONSOR BASF Aktiengesellschaft. 67056 Ludwigshafen, Germany This document contains manufacturing and trade secrets of BASF. Itisthe property ofBASF and may be used only forthat purpose for which itwas intended by BASF. Every other or additional use, exploitation, reproduction, publication or submission to otr parties requires the written permission of BASF, with theexception of regulatory agencies acting within the limits oftheir administrative authoriy

# BASF
Report; Project No.: 40M04371034092 2 THIS PAGE ISINTENTIONALLY LEFT BLANK

# BASF Report Project No.: 40M0437/034092 3 

### GLP COMPLIANCE STATEMENT
This studywas conducted ingeneral accordance with the OECD Principles ofGood Laboratory Practice and the GLP Principles ofthe German "Chemikaliengesetz" (Chemicals Act) and meet the United States Environmental Protection Agency Good Laboratory Practice Standards [40 CFR Part 160 (FIFRA) and Part 792 (TSCA)], with the exception that recognized differences exist between the GLP Principles/Standards of OECD and the Principles/Standards of FIFRA and ISCA. However, there was the following deviationfrom the requirements of the above-mentioned principles:

-The 

### stability ofthe test substance inthevehicle DM50S and inwater was not

determined analytically.

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

# BASF Report, Project No.: 40M0437/034092 4 THIS PAGE ISINTENTIONALLY LEFT BLANK

# BASF Report; Project No.:40M04371034092 5 

### SIGNATURE PAGE



# to Study Director IConduct 

### of study: ~.......... Dr. rer. nat.G.Engelhardt

Management: 

# 4.....Y......... ~~ Dr. rer. nat. E.Leibold

# BASF
Report; Project No.: 40M0437/034092 6
STATEMENT ofthe Quality Assurance Unit The QualityAssurance Unit (QAU) performed the inspections given below, and reported any inspection resultsto the Study Director and to Management The conduct of this short-term study was not inspected; the processes ofthe laboratory and of the 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 10o-20-2003 

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



### Ludwigshafen, 

## 4af A(29 

### ' Gartmann

# BASF Report; Project No.: 40M0437/034092 7 

# .Rheinan* a~ Lmndesusitt8 M~r ?flaiacnb ad Pflancacbulz GL-P-Beschelnlgung 

### IStatement of GLP Compliance WWYI S~ordki to119 b Abe. 1Chamksiiesz

- Elne GLP.Inspeldlon zurOberwachung der Eklia~bng Assessmert ofconformit with GLP accrdlng derGLP-Grundsitte 9"Si ChernkalVIznee bzw. to Chomilkuliengesetzand Directive 8832O/EEC at RictiieBB0201EG wurde dumidgef~hrt 1w O Prftnrtchtung flotsfditty 

### C PfttandortlTeaMite BASF Aktiengesellschat Expenimentelle Toxilkologie und Okolge D-67056 Ludwigshafen Profungen flinch Kategorlen/Areas of Expertise (pnifitJaw" ahmVW4LPHr. S=OEC DmdMv*

.. ,,., .... . ..... ....-... Datum der lnsplitoI Date of inspection

...... ... ........ t5.052001 undvorm 21. bit26.06.2001 ....
Die/Der genannte Pd*cfnrdtun/p~rlstandort befindet The above mentionedtest facllftylkestsite Is Included slcht i natlonaleri GLP-Oberwachugsverfahren und Inthe national GLIP-Compllance Progrmme andIs vild regelmAfllig aizf Eihaftmn der GLP-Grwxdstze Inspected on aregular basis. Obenwwft Aufder Grqwdlge do* lnspektionsbarichteswird hior. eased on the Inspection reportft can be Confirmed, mit besaftigI doss Indlese PrOfislnrlchtungldiesern thatthutestfaclftyflst sMe Isableto conduct the Profstaridolt die oben genannten PrOfungen unter ln,- aforementioned srtudies Incompliance with the PdnhabmugderGLIP-Grundsatte durchgeftrtwsrden ciples of GLP. kdnneii.

Unteradirikt Datum ISignature. at

L&-sndW AVPMUFI2.nbaI umdP2isimmuftiM Eawnihn W, 144. D-55128 AM=ti

# BASFReport; Project No.: 40MO437/034092 8 CONTENTS Page TITLE PAGE 1 BLANK PAGE 2 GLP COMPLIANCE STATEMENT 3 BLANK PAGE 4 SIGNATURE PAGE 5 STATEMENT OF THE QUALITY ASSURANCE UNIT 6 STATEMENT OF GLP COMPLIANCE (FROM THE COMPETENT AUTHORITY) 7 CONTENTS 8 1. SUMMARY 10 11 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-9fraction 14 *4 3.3.2. S-9 mix 14 3.4. BACTERIA 15 3.4.1. Salmonella typhimurium 15 3.4.2. Escherichia call 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. PreincubationTest 19 3.5.3. Titerdetermination 19 3.5.4. Control articles 20 3.5.4.1. Negative controls 20 3.5.4.2. Positive controls 20 3.5.5. Scope oftests and test conditions 22

# BASF Report; Project No.: 40M0437/'0340929 Page 3.6. EVALUATION 23 3.6.1. Mutagenicfty 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 plate test 26 4.1.1.1. Tests without S-9 mix 26 4.1.1.2. Testswith S-9 mix 26 4.1.2. Prelncubation test 27 4.1.2.1. Tests without S-9 mix 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 Appendix 3: Sterility control 2 Appendix 4: S-9 fraction characterization 2 Appendix 5: Historical negative control data 4 Appendix 6: Historical positive control data 4

# BASF Report; Project No.:40M0437/034092 10
1. SUMMARY The substance Alkali Blue 

### Dry 6100/I Lot 2231308 was tested for its mutagenic potential based on the abilityto induce point mutations inselected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia col, inareverse mutation assay.
" STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E.col WP2 uvrA " DOSE RANGE: 23 pg -5 750 pg/plate (SPT) 4 pg - 

### 1500 pg/plate (PIT; Salmonella strains) 4pg -2 500 pg/plate (PIT; E.coliWP2uvrA) " TEST CONDITIONS: Standard plate test (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 all test conditions (fordetails see item 4.2.).

" MUTAGENICITY: An increase inthe number of his' or trp* revertants was not observed inthe standard plate testor inthe preincubation testeither without S-9 mix or afterthe addition of a metabolizing system. *CONCLUSION:

According to the results ofthe present study, the test subst ance Alkali Blue Dry 6100 1Lot 2231308 isnot mutagenic inthe Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen here.

# BASF Report; Project No.: 40M0437/034092 1
2. INTRODUCTION 2.1. PRINCIPLE Bacterial reverse mutation assays using amino-acid requiring strains of Salmonella typhimurium and Escherichia coli are commonly employed as initial screening methods for detecting apoint mutagenic activity of chemical substances (1,2, 5)and are used to screen for possible mammalian mutagens and carcinogens. The principle of these assays isthat mutations are detected bythe 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 ability to grow 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 of the liver. Therefore, thetests are carried out not only 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 (July 21, 1997) * EC Directive 2000/32, B.13 /B.14 (May 19, 2000)El LI1 -- U JI ULW

L lll I LK1A LlI I I I h1RJILiLI JL



### Report; Project No.: 40M0437/034092 12 

### 3. MATERIAL AND METHODS 3.1. TEST SUBSTANCE Name of testsubstance: Alkali Blue Dry 6100/ Lot2231308 Test substance No.: 03/0437-2 Batch No.: Lot 2231308 CAS No.: 1324-76-1 Date offilling: September 19, 2003 Purity/Composition: 88 %main component (See analytical data ofOctober 9,2003) Appearance, consistency: Blue Powder Storage: Room temperature

# BASF Report; Project No.: 40M04371034092 13

3.2. TEST SUBSTANCE ANALYSIS 3.2.1. Test substance The stabilityofthetestsubstance under storage conditions isguaranteed as indicated by the sponsor, and the sponsor holds this responsibility. The homogeneity ofthe test substancewas 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. Test substance preparation The stability ofthetest 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).-~~~~T 1 71 'I i~I~~ 

# BASF
Report; Project No.: 40M0437/034092 14
3.3. EXOGENOUS METABOLIC ACTIVATION 3.3.1. S-9 fraction The S-9 fraction isprepared according toAmes et al. (1,2). At least5male Sprague-Dawley rats') (200 - 300g)receive asingle intraperitoneal injection of 500 mg Aroclor 1254 (as asolution incorn oil with aconcentration of20 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 - 24*0 and arelative humidityof 30- 70%. The day/night rhythm is12 hours (light period from 6.00 - 18.00 hours and dark period__ from 18.00
- 6.00 hours). Standardized pelletedfeed and tap water from bottles are available ad libitum. 5days after administration, the rats are sacrificed, arnd the livers are prepared (all preparation steps for obtaining the liver microsorne enzymes are carried out using sterile solvents and glassware at atemperature of+4C). The livers are weighed and washed inan equivalent volume ofa 150mM KCI solution (1ml A1gwet liver), then cut into small pieces and homogenized inthree volumes of KCI solution.After centrifugation ofthe homogenate at 9000 xgfor 10 minutes at+'40C, 5ml portions ofthe supernatant (so-called S-9 fraction) are stored at-700C to -800C. The preparation date and protein content ofthe S-9 fraction are given inAppendix4. 

### 3.3.2. S-9 mix The S-9 mix isprepared freshly priorto each experiment (1,2). For this purpose, asufficient 4 amount of S-9 fraction isthawed at room temperature and I volume 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 Giucose-6-phosphatase 5mM NADP 4mM Phosphate buffer (pH 7.4) 15 mM The phosphate buffer (6)isprepared by mixing an Na 2HPO4 solutionwith an NaH2PO4 solution In aratio ofabout4:1. To demonstrate the efficacyofthe S-9 mix inthis assay, the S-9 batch was characterized with benzo(a)pyrene (see Appendix

### 4).

ICharles River Deutschland GmbH

### Report; Project No.:40M0437/034092 1
3.4. BACTERIA For testing, deep-frozen (-700C 

### to -80"C) bacterial cultures (Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and E.coli WP2 uvrA) are thawed at room temperature, and 0.1 ml of this bacterial suspension isinoculated innutrient broth solution (8gDifco nutrient broth + 

### 5gNaCI/iter) and incubated inthe shaking water bath at 37*C for about 10
- 

### 12 hours.As arule, agerm density of> 108 bacteria/mi isreached. These cultures grown overnight are kept iniced waterfrom th~e beginning ofthe experiment until the end inorderto prevent further growth. The use ofthe 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 coli 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 by Ames and coworkers are derivatives of Salmonella typhimurium LT2 and have GC base pairs at the 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 sensitivityof some mutagens. Furthermore, all strains show aconsiderably reduced hydrophilic polysaccharide layer (rfa), which leads to an increase inpermeabilityto 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 differentframeshift markers, i.e. the +1mutant his C3076 inthe case ofTA 1537 and the +2type his D3052 inthe case ofTA 98. The strains TA 98 and TA 100 carry an Rfactor plasmid pKM 101 (4) and, inaddition to having genes resistantto 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 insensitivity.

# BASF Report; Project No.: 40MO437/034092 16
3.4.2. Escherichia coil Escherichia Coil WP2 uvrA which has an AT base pair at the primary reversion site isa derivative of E.coli 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 (trp-) to tryptophan independence (trp*) isdetermined.
3.4.3. Checking out the tester strains The Salmonella strains are checkedfor the following characteristics at regular intervals: deep rough character (rfa); UV sensitivity

## (,& uvrB); amnpicillin resistance (Rfactorplasmid). E.coli WP2 uvrA ischecked for UV sensitivity. Histidine and tryptophan auxotrophy isautomatically checked ineach experiment via the spontaneous rate.

# BASF Report; Project No.: 40M0437/034092 17
3.5. EXPERIMENTAL PROCEDURE 

### 3.5.1. Choice ofthe vehicle Due to the limited solubility ofthe test substance inwater, DMSO 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 of Ames et al. (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 for the determination of mutants: 0.5 mM histidine +

### 0.5 mM biotin) are kept inawater bath at 45C, 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 (intests without metabolic activation) After mixing, the samples are poured ontoVogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds. Composition of the minimal glucose agar. 980 ml aqua dest.

20 ml Vogel-Bonner Emedium 15 g Difco bacto agar 20 g D-glucose, monohydrate. After incubation at 37 0



### Cfor 48
- 

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

# BASF Report; Project No.: 40M0437/034092 18

*Escherichia coi

The experimental procedure isbased on the method ofAmes et al. (1,2). Testtubes containing 2-mi portions ofsoft agar (overlay agar), which consists of 100 mi agar (0.6% agar + 

### 0.6% NaCi) and 10 ml amino acid solution (minimal amino acid solution for the determination ofmutants: 0.5 mM tryptophan) are kept inawater bath at45 0



### C,and the remaining components are added inthe following 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 (in tests without metabolic activation) " After mixing, 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 soft agar: 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 379C for 48
- 

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

# BASF Report; Project No.: 40M0437/034092 19
3.5.2.2. Prelnicubation Test The experimental procedure Isbased on the method described byYahagi et al. (7)and Matsushima et al. (8). 0.

1 ml test solution orvehicle, 0.1 ml bacterial suspension and 0.5 ml S-9 mix or phosphate buffer are incubated at 370Cforthe duration of about 20 minutes using a shaker. Subsequently, 2ml of soft agar isadded and, after mixing, the samples are poured onto the agar plates within approx. 30 seconds. After incubation at 37"Cfor 48
- 

### 72 hours inthe dark, the bacterial colonies are counted.
3.5.3. Titerdetermination Inthe standard plate test, 0.1 ml of the overnight cultures isdiluted to 10'6 ineach case. Test tubes containing 2-mi portions of softagar containing maximal amino acid solution (5mM tryptophan or5mM histidine + 

### 0.5 mM biotin) are kept inawater bath at45 0

### C, and the remaining components are added inthe following order 0.1 ml vehicle (withoutand with test substance) 0.1 mlfresh 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. 0.1 ml vehicle (with and without testsubstance), 0.1 ml bacterial suspension and 0.5 ml S-9 mix are incubated at 370C for about 20 minutes using ashaker. Subsequently, 2ml of soft agarcontaining maximal amino acid solution for titerdetermination (5mM tryptophan or 5mM histidine + 

### 0.5 mM biotin) isadded. After mixing, the samples are poured onto the agar plates within approx. 30 seconds. After incubation at37 0



### Cfor 48
- 

### 72 hours inthe dark, the bacterial colonies are counted.- - -~V L I I I 3 1 1 

# BASF Report; Project No.: 40M0437/034092 20
3.5.4. Control articles 3.5... Negative controls Parallel with each experiment, negative controls are carried out inorder to checkfor possible contaminants (sterility control) and to determine the spontaneous mutation rate (vehicle control). " Sterility control Additional plates are treated with softagar, S-9 mix, buffer, vehicle orthe test substance but withoutthe addition oftester strains (see Appendix 3). " Vehicle control The vehicle control with and Without S-9 mix only contains the vehicle usedfor the test substance at the same concentration and volume for all tester strains.
3.5.4.2. Positive controls The following positive controls are used to check the mutability ofthe bacteria and the activity ofthe S-9 mix: WthS-9 mix 41



### *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 in DMSO

- 

### strain: Escherichia coliWP2 uvrA

# BASF Report; Project No.: 40M0437/034092 21 Without S-9 

### mix * N-methyl-N'-nltro-N-nitros oguanidine (MNNG) (FLUKA, 68051)

- 

### 5 pg/plate, dissolved inDMSO

-strains: TA 1535, TA 100 9 4-nitro-o-phenylendiamine (NOPID) (SIGMA, N-9504)

- 

### 10jig/plate, 

### dissolved inDMS0

- 

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

- 

### 100 pg/plate, dissolved inDMSO

- - strain: TA 1537 

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

- 

### 5 pg/plate, dissolved in DM50S

- 

### 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.M111JJ1E N1 ILIILJILI ILL

### Report; Project No.: 40M0437/034092 22
3.5.5. Scope oftests andtest conditions 1st Experiment Strains: TA 1535, TA 100, TA 1537. TA 98, E.coi WP2 uvrA Doses: 0; 23; 115; 575; 2875 and 5750 pg/plate Vehicle: DMS0 Type of test: Standard plate testWith and without S-9 mix Number of plates: 3test plates perdose or per control *2nd Experiment

Strains: TA 1535, TA 100, TA 1537, TA 98, Doses: 0; 500; 1000; 1500;2000 and 2500 pg/plate Vehicle: DMS0 Type oftest: Standard platetestwith 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.coilWP2 uvrA Doses: Salmonella strains: 0; 4; 20; 100;500 and 1500 pg/plate E.coliWP2 uvrA: 0; 4; 20; 100; 500 and 2500 pg/plate Vehice: DMSO Type of test: Preincubation test with and without S-9 mix Number of plates: 3test plates per dose or per control

# BASF Report; Project No.: 40M0437/034092 23
3.6. EVALUATION 3.6.1. Mutageniclty Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups aswell as for the positive and negative (vehicle) controls inall experiments. Ingeneral, five doses ofthe test substance are tested with amaximum of 5mg/plate, and triplicate plating isused for alltest groups at least inthe I1st 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 bothfor the negative controls (vehicle only) and for the two highest doses inall experiments.
3.6.3. Toxicity Toxicitydetected bya

- 

### decrease inthe number of revertants

- 

### clearing or diminution of the background lawn (=reduced his or tr background

- growth)

- 

### reduction inthe titer isrecorded for all test groups both with and without 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 colony scoring, 5mg/plate isgenerallyselected and analyzed (incases ofnontoxic compounds) as the maximum dose at least inthe 1ist experiment even inthe case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses>5 mg/plate might also be tested in repeat experiments for further clarification/substantiation..A4I~liJU1-1 11 J iL


# BASF
Report; Project No.: 40M0437/034092 24
3.6.5. Acceptance criteria Generally, the experiment isconsidered valid ifthe following criteriaare met * The numberof revertant colonies inthe negative controls was within the normal range ofthe historical control data for each tester strain (see Appendix 5). e 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 (seeAppendix 6). * The titer ofViable bacteria was > 10 8

### 1m1. 3.6.6. Evaluation criteria The test chemical isconsidered positive inthis assay If the following criteria are met: *Adose-related and reproducible increase inthe numberof revertant colonies, i.e.

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

*The number of revertants for all tester strainswere within the historical negative

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

# -BASF Report; Project No.: 40M0437/034092 25
3.7. RETENTION OF RECORDS GLP-relevant 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 locationsof archiving may be seen from the relevant SOPs and from the raw data.

# BASF Report; Project No.: 40M0437/034092 26


### 4. RESULTS (Tables I
- 

### 14) The substance Alkali Blue Dry 61001/Lot2231308 was tested for mutagenicity inthe Salmonella typhimurium /Escherichia coi reverse mutation assay both Inthe standard platetest and inthe preincubation testwith and withoutthe 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 coil WP2 uvrA.
4.1. MUTAGENICITY4.1.1. Standard platetest (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* ortrp*' revertants. TA 98: E.coilWP2 uvrA:
4.1.1.2. Tests with S-9 mix TA 1535: TA 100: TA 1537: No increase inthe number ofhis4 or trp* revertants. TA 98: E.coilWP2 uvrA:

# BASF Report; Project No.: 40M0437/034092 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+ortrp4 

### revertants. TA 98: E.col WP2 uvrA:
4.1.2.2. Tests with S-9 mix TA 1535: TA 100: TA 1537: No increase inthe number of his" ortrp+ revertants. TA 98: E.coilWP2 uvrA

# BASF Report; Project No.: 40M0437/034092 28
4.2. TOXICITY A bacteriotoxic effect (reduced background growth, decrease inthe number of revertants, reduction inthe titer) was observed inthe standard plate test depending on the strain and test conditions from about 1500 pg - 

### 2000 pg/plate onward (Salmonella strains) orat doses '. 2875 pg/plate (E.coilWP2 uvrA). Inthe preincubation assay bacteriotoxicity (reduced background growth, decrease Inthe number of revertants, reduction inthe titer) was observed from about 100 pg - 

### 500 pg/plate onward.
4.3. SOLUBILITY Test substance 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 ofrevertant colonieseither without S-9 mix or after adding a metabolizing system inthree experiments carried out independently of each other (standard plate test and preincubation assay).
4.5. CONCLUSION Thus, underthe experimental conditions chosen here, itisconcluded that Alkali Blue Dry6100 /Lot 2231308 isnot amutagenic agent inabacterial reverse mutation test.

# BAUSF Report; Project No.: 40M0437/034092 29 

### 5.REFERENCES 1

. Ames, B.N.; McCann, J.; Yamasaki, E.: Methods for detecting carcinogens and mutagens with the Salmonellalmammalian-microsome mutagenicity test. Mut Res., 3.1, 

### 347
- 

### 364 (1975) 

### 2. Maron, D.M.; Ames, B.N.: Revised method forthe Salmonella mutagenicitytest. Mut. Res., jj13.

### 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. Scl. USA, 7-0. 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 usingtrp' reversions in Escherichia coli. Mut. Res., 38, 

### 3
- 

### 32 (1976) 6. DeMarini, D.M.; Dallas, M.M.; Lewtas, J.: Cytotoxicity and effect on mutagenicity ofbuffers inamicrosuspension assay. Ter. Carc. Mut., 2,

### 287
- 

### 295 (1989) 7. Yahagi, T.; Nagao, M.; Seino, Y.; Matsushima, T.; Sugimura, T.; Okada, M.: 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. Garner, Short-Term Test Systems for Detecting Carcinogens. Springer Verlag Berlin, Heidelberg, New York (1980).

# BASF Report; Project No.: 40M0437/034092



### APPENDIX 1 TABLES 1-9 (STANDARD PLATE TEST)BASF Aktiengesellschaft 01 Experimental Toxicology and Ecology STUDY DIRECTOR: DR. ENGELHARDT PROJECT NO. :40MO437/034092 SUBSTANCE NAMdE: Alkali Blue Dry 6100 ILot 2231308 SUBSTANCE NO. :03/0437-2

TESTDATE 05.NOV.2003 METHOD :STANDARD PLATE TEST VOLUME 100 uliplate STRAIN TA 1535 VEHICLE : DM5 59-MIX WITHOUT S-9 MIX WITH S-9 MIX (1:9) DOSE/PLATE REV H SD FAC REV M SD FAC TITRE DM50 21 17 3 1.0 20 17 4 1.0 24 16 13 19 15 19 29 23 ug 16 19 3 1.1 17 18 7 .1.0 18 12 22 25 115 ug 21P 24 2 1.4 12P 16 4 0.9 25P 16P 25P 19? 575 ug 17P 17 2 1.0 15P 14 1 0.8 19P 13? 15? 15P 2 875 ug lop 8 2 0.5 18P 16 2 0.9 6 7P 16P 0 7P 14P 1 5 750 ug 08/P OB/P 0 OB/P 08/? 0 08/P OB/P 0 HNNG 5.0 ug 1217 2176 39 67.9 1139 1173 2-AA 2.5 ug 166 177 12 10.2 189 177 (REV-revertants /plate, M-mean, SD-standard deviation, FAC=factor) P: PRECIPITATION B: REDUCED BACKGROUND GROWTHiLLS fin z

BASF Aktiengesellschalft 0.2 Experimental Toxicology and Ecology STUDY DIRECTOR: DR. ENGELHARDT PROJECT 9O. 40HO4371034092 SUBSTANCE NAME: Alkali Blue Dry 6100 

### ILot 2231308 SUBSTANCE NO. 0310437-2

TESTDATE :05.NOV.2003 METHOD STANDARD PLATE TEST VOLUME 100 ul/plate STRAIN :TA 100 VEHICLE :DMSO S9-HIX WITHOUT S-9 MIX WITH S-9 MIX (1:9) DOSE/PLATFE REV M SD PAC REV M SD FAC TITRE DMSO 113 113 12 1.0 120 117 8 1.0 29 125 114 37 102 126 25 23 ug 110 111 8 1.0 129 124 6 1.1

_ 104 118 119 124 115 ug 120P 114 5 1.0 124P 109 5 0.9 11OP 10 7P 113P 105P 575 ug loop 100 5 0.9 102P 94 7 0.8 104P 89P

95P 9 OP 2 675 ug 03/P 143/P 23 9 0.2 0 03/P 253/P 0 031P 31E/P 0 5 750 ug OBIP 03/P 0 03/P 03/P 0 03/P 03/P 0 MNNG 5.0 ug 1311 1297 65 11.4 1226 1353 2-AA 2.5 ug 1043 1059 56 9.1 1013 1122 (REV-revertants/plate, M-mean, SD-standard deviation, FAC-factor) P: PRECIPITATION B: REDUCED BACKGROUND GROWTHBASF-Aktiengesellschaft -03 Experimental Toxicology and Ecology STUDY DIRECTOR: DR. ENGELHARDT PROJECT NO. 40M0437/034092 SUBSTANCE NAME: Alkali Blue Dry 6100 /Lot 2231308 SUBSTANCE NO. 03/0437-2

TESTDATE 05.NOV.2003 METHOD STANDARD PLATE TEST VOLUME 100 ul/plate STRAIN TA 1537 VEHICLE :DM50 s9-KIX : WITHOUT S-9 MIX WITS S-9 MIX (1:9) DOSE/PLATE- REV M SD FAC REV M SD FAC TITRE DMSO 8 11 4 1.0 6 8 2 1.0 22 10 9 21 15 9 24 23 ug 8 a 1 0.7 12 12 2 1.5 7 11 9 14 115 ug 7P 8 2 0.8 lop 11 3 1.4 lop 15P

8P 9P 575 ug 12P 11 2 1.0 SP 13 6 1.6 12P 19P

8P 12P 2 875 ug OBIP OB/P 0 OBI/P 03/P 0 03/P 03/P 0 5 750 ug OB/P I 03/P 0 03/P 03/P 0 OB/IP OBIP 0 AAC 100 us 560 484 70 44.0 421 472 2-AA 2.5 ug 150 151 18 18.8 169 133 (REV~revertants /plate, M-mean, SD=standard deviation, FAC-factor) P: PRECIPITATION B: REDUCED BACKGROUND GROWTH- L I L I I I L mI L &zLJ~UIAizjR Iu~ I r

BASF Aktiengesellschaft 04 Expirimental Toxicology and Ecology SUYDRCO:D.EGLAD PROJECT NO. : 40MD437 /034092 SUBSTANCE NAME: Alkali Blue Dry 6100 

### /Lot 2231308 SUBSTANCE NO. D3/0437-2

TESTDA7E :05.NOV.2003 METHOD STANDARD PLATE TEST VOLUME :100 ul/plate STRAIN : TA 98 VEHICLE : DM30 59-MIX : WITHOUT S-9 MIX WITH S-9 MIX (1:9) DOSEIPLATE 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 31 32 4 1.0 36 36 3 0.9 36 39 29 34 115 ug 32P 30 5 0.9 33P 34 1 0.8 34P 33P 24P 35P 575 ug 31P 26 5 0.8 60P 47 12 1.1 24P 36P 22P 45P 2 875 ug OB/P 33P 43 9 1.0 1 OB/IP sip I OB /P 44P 0 5 750 ug aD/P OB/P 0 03/P 03/P 0 OB/IP 03/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, M-mean, SD-standard deviation. FAC-factor) P: PRECIPITATION B. REDUCED BACKGROUND GROWTHBASF-Aktiengesellschaft 05 Experimental Toxicology and Ecology STUDY DIRECTOR: DR. ENGELHARDT PROJECT NO. :40MO437/034092 SUBSTANCE NAME: Alkali Blue Dry 6100 

### /Lot 2231308 SUBSTANCE No. :03/0437-2

TESTDATE 05.NOV.2003 HETHOD STANDARD PLATE TEST VOLUME 100 ul/plate STRAIN : E.coli IWP2 uvrA VEHICLE :DMSO 59-MIX : WITHOUT S-9 MIX WITH S-9 MIX (1:9) DOSE IPLATE REV M SD FAC REV M SD FAC TITE DMSO 40 35 5 1.0 38 38 1 1.0 38 31 37 40 34 38 33 23 ug 34 31 4 0.9 39 35 5 0.9 27 36 31 29 115 ug 34? 34 3 1.0 31P 34 4 0.9 37? 34? 31P 38P 575 ug 34P 35 3 1.0 43P 32 11 0.8 33P 31lP 39P 22P 2 875 ug 34? 34 3 1.0 24P 27 3 0.7 5 31P 28P 7 36? 30? 2 5 750 ug 30P 21 -9 0.6 22? 18 4 0.5 0 22P 18? 0 12P 15? 0 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, Mmzean, SDstandard deviation, FAC-factor) P: PRECIPITATIONBASy Aktiengesellschaft 0 Experimental Toxicology and Ecology STUDY DIRECTOR: DR. ENGELHARDT PROJECT NO. 40M0437/034092 SUBSTANCE NAME: Alkali Blue Dry 6100 ILot 2231308 SUBSTANCE NO. 03/0437-2

TESTDATE 28.NOV.2003 METHOD STANDARD PLATE TEST VOLUME 100 ul/.plate STRAIN TA 1535 VEHICLE : D?4S0 5944IX : WITHOUT S-9 MIX WITH S-9 MIX (1:9) DOSE/PLATE REV M SD PAC REV M SD PAC TITRE DMSO 18 17 1 1.0 17 19 2 1.0 29 18 19 22 16 20 27 500 ug 13P 16 3 0.9 17P 15 3 0.8

_ 17P 12P 18P 17iP 1 000 ug lip 14 3 0.8 iSP 16 3 0.8 17P 19P 13P 13P 1 500 Ug lop 13 4 0.7 14P 12 2 0.7 17P 12P lip lip 2 000 ug 12P 9 3 0.5 8F 6 3 0.3 9 SP 2P 8 7P 7P 5 2 500 ug 6P 6 .2 0.4 6P 6 2 0.3 6 5P 8P 4 BP 5P 1 MNNG 5.0 ug 1039 1063 34 61.3 1102 1047 2-AA 2.5 ug 140 117 20 6.3 107 104 (REVrevertants /plate, M-mean, SD~standard deviation, FAC-factor) P: PRECIPITATIONBASF Aktiengesellschaft 07 Experimental Toxicology and Ecology STUDY DIRECTOR: DR. ENGELHARDT PROJECT NO. :40HO437/034092 SUBSTANCE NAME: Alkali Blue Dry 6100 ILot 2231308 SUBSTANCE NO. 03/0437-2

TESTDATE 18.NOV.2003 METHOD STANDARD PLATE TEST VOLUME 100 uliplate STRAIN TA 100 VEHICLE : DM50 S9-MIX : WITHOUT S-9 MIX WITH S-9 MIX (1:9) DOSE/PLATE REV M SD FAC REV M Sp FAC TITRE DMSO 104 110 5 1.0 119 109 10 1.0 27 112 107 32 114 100 22 500 ug 105P 103 12 0.9 113P 107 7 1.0 113P 10oop

90P 107P 1 000 ug 95P 97 3 0.9 102P 99 4 0.9 95P 99? loop 95P 1 500 ug 96? 94 3 0.9 102P 100 11 0.9 9OP 1lOP 95P 89? 2 000 ug 20P 14 9 0.1 40P 41 11 0.4 0 4P 52P 4 I9P 31P 5 2 500 iig OB/P 1031? 14 5 0.1 1 OB/P 19BIP 0 OB IP 14B/P 2 MNNG 5.0 ug 1017 1088 64 9.9 1108 1140 2-A.A 2.5 ug 811 781 51 7.2 722 811 (REV-revertantslplate, M-mean, SD-standard deviation, FAC-factor) P: PRECIPITATION B: REDUCED BACKGROUND GROWTHBASF Aktiengesellschaft 08 Experimental Toxicology and Ecology STUDY DIRECTOR: DR. ENGELHARDT PROJECT NO. 40H0437/034092 SUBSTANCE NAME: Alkali Blue Dry 6100 1Lot 2231308 SUBSTANCE NO. 0310437-2

TESTDATE :18.NOV.2003 METHOD :STANDARD PLATE TEST VOLUME : 100 ul/plate STRAIN : TA 1537 VEHICLE : DHSO S9-NIX : WITHOUT S-9 MIX WITH S-9 MIX (1:9) DOSEIPLATE REV M SD FAC REV M SD FAC TITRE DMSO 9 9 1 1.0 9 8 1 1.0 28 10 7 24

9 7 25 500 ug 5P 6 1 0.6 7P 6 2 0.8 7P 7P 6P 4P 1 000 ug I? 6 5 0.6 9? 7 4 0.9 5P 9? lip 2P 1 500 ug 2P 2 2 0.3 6P 5 2 0.6 4P 3P 1? 5P 2 000 ug 03/P 2B/P 3 1 0.4 0 OB (P 3B/P 0 OB/P 43/P 0 2 500 ug 03/P OB/P 0 03/P OBIP 0 OB/IP 0BIP 0 AAC 100 ug 375 398 27 42.7 428 392 2-AA 2.5 ug 141 134 8 17.5 125 136 (REV=revertaltsl/plate, M~mean, SD=standard deviation, FAC=factor) P: PRECIPITATION B: REDUCED BACKGROUND GROWTHBASF Aktiengesellschaft 09 Experimental Toxicology and Ecology STUDY DIRECTOR: DR. ENGELHAR.DT PROJECT NO. :40M0437/034092 SUBSTANCE NAME: Alkali Blue Dry 6100 /Lot 2231308 SUBSTANCE NO. 03/0437-2

TESTDATE 18.NOV.2003 METHOD :STANDARD PLATE TEST VOLUME 100 ul/plate STRAIN : TA 98 VEHICLE : DMSO 59-MIX : WITHOUT S-9 MIX WITH S-9 MI (1:9) DOSE/PLATE REV M SD FAC REV M SD FAC TITRE DMSO 19 24 5 1.0 36 31 4 1.0 29 25 29 37 28 29 32 500 ug 18P 20 2 0.8 34P 30 4 1.0 zip 27P 21P 30OP 1000D ug 22P 20 2 0.8 27P 25 4 0.8 20P 2OP I 9P 27P 1 500 ug 12P 10 3 0.4 19P 19 3 0.6 lop 17P

7P 22P 2 000 ug 9B/P 5 4 0.2 20P 16 4 0.5 8 2BIP 17P1 3B/P 12P 5 2 500 ug OR/P 14B/P 11 3 0.3 4 OB/P lOB/P1 OB /P 8B/P 2 NOPD 10 ug 605 615 42 25.6 662 579 2-AA 2.5 ug 609 611 30 19.5 582 642 (REV=revertaratslplate, M--mean, SD=standard deviation, FAC=factor) P: PRECIPITATION B: REDUCED BACKGROUND GROWTH

# BASF Report; Project No.: 40M0437/034092



### APPENDIX 2 TABLES 10-14 (PREINCUBATION TEST)BASF Aktiengesellschaft 10 Experimental Toxicology and Ecology STUDY DIRECTOR: DR. ENGELHARDT PROJECT NO. :40M0437/034092 SUBSTANCE NAME: Alkali Blue Dry 6100 

### ILot 2231308 SUBSTANCE NO. :03/0437-2

TESTDATE 02.DEC.2003 METHOD :PREINCUBATION TEST VOLUME 100 ul/plate STRAIN : TA 1535 VEHICLE : DMS0 59-MIX : WITHOUT S-9 MIX WITH S-9 MIX (1:9) DOSE IPLATE REV M SD FAC REV M SD FAC TITHE DM50 13 16 3 1.0 15 16 1 1.0 24 18 17 58 17 17 23 4 ug 17 17 1 1.1 17 15 2 0.9 18 14 17 14 20 ug 13 16 3 1.0 15 16 1 1.0 16 16 19 17 100 ug 17P 16 1 1.0 16P 14 2 0.9 16P 14P 16P 13P 500 ug 2B/P 5 3 0.3 8H/P 7 2 0.4 1 4H/P 83/P 0 8H IP 43/P 0 1 500 ug OB/P OB/P 0 OBIP OBIP 0 OBIP OH/P 0 MNNG 5.0 ug 705 749 40 46.8 760 7.8

3 2-AA 2.5 ug 166 145 19 8.9 139 130 (REV-revertants /plate, M=mean, SDstandard deviation, FAC-factor) P: PRECIPITATION H: REDUCED BACKGROUND GROWTHEAUY Aktiengesellschaft 1 Experimental Toxicology and Ecology STUDY DIRECTOR: DR. ENGELHARDT PROJECT NO. :40M0437/034092 SUBSTANCE NAME: Alkali Blue Dry 6100 

### ILot 2231308 SUBSTANCE NO. : 03/0437-2

TESTDATE : 02.DEC.2003 METHOD : PREINCUBATION TEST VOLUME : 100 ul/plate STRAIN : TA 100 VEHICLE :DM80 89-MIX : WITHOUT S-9 MIX WITH S-9 MIX (1:9) DOSEIPLATE REV M SD FAC REV M SD- FAC TITRE DMSO 129 120 9 1.0 110 121 10 1.0 36 119 126 34 112 127 32 4 ug 110 113 7 0.9 104 102 6 0.8

_ 108 207 121 95 20 ug 120 113 6 0.9 202 100 2 0.8 110 100 109 99 200 ug 47P 41 7 0.3 9BP 100 2 0.8 34P 102P 4lP loop 500 ug 113/P 6 5 0.0 55B1P 39 14 0.3 11 lB/P 27B/P 2 53/P 36B/P 5 1 500 ug OB/P OB/P 0 OB/P OBIp 0 OB/IP OBI? 0 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, H-mean, SD=standard deviation, FACa-factor) P: PRECIPITATION B.-REUCED BACKGROUND GROWTHBASF Aktiengesellschaft 12 Experimental Toxicology and Ecology STUDY DIRECTOR: DR. ENGELHARDT PROJECT NO. 40140437/034092 SUBSTANCE NAME: Alkali Blue Dry 6100 ILot 2231308 SUBSTANCE NO. :03/0437-2

TESTDATE 02.DEC.2003 METHOD : PREINCUBATION TEST VOLUME :100 ul/plate STRAIN :TA 1537 VEHICLE : DMSO S9-MIX : WITHOUT S-9 MIX WITH S-9 MIX (1:9) DOSEIPLATE REV M SD FAC REV M SD FAC TITRE DMS0 10 9 1 1.0 8 9 1 1.0 25 98 25 9 10 21 4 ug 7 9 2 0.9 5 8 3 0.9 11 8

8 11 20 ug 14 9 4 1.0 6 8 2 0.9 6 8 7 9 100 ug lop 8 3 0.8 5P 5 1 0.6 8? 4P 5P 6P 500 ug 43/P 4 1 0.4 73/P 3 3 0.4 0 5B/P ZBIP 0 3B/P lB/P 0 1 500 ug 03/P 03/P 0 OB/P 03/P 0 OB/IP 03/P 0 MAC 100 ug 394 377 19 40.4 380 356 Z-AA 2.5 ug 112 122 21 14.1 147 108 (REVrevertants/plate, 1-mean, SD-standard deviation, FAC-factor) P: PRECIPITATION B: REDUCED BACKGROUND GROWTHBASF Aktiengesellschaft 1 Experimental Toxicology and Ecology STUDY DIRECTOR: DR. ENGELHARDT PROJECT NO. 40MO4371034092 SUBSTANCE NAME: Alkali Blue Dry 6200 /Lot 2231308 SUBSTANCE NO. 03/0437-2

TESTDATE :02.DEC.2003 METHOD :PREINCUBATION TEST VOLUME :100 ul/plate STRAIN TA 98 VEHICLE : DMS0 S9-MIX :WITHOUT S-9 MIX WITH S-9 MIX (1:9) DOSE/PLATE 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 25 26 5 0.8 34 32 3 0.9 21 28 31 33 20 ug 28 26 5 0.8 25 25 4 0.7 29 28 20 21 100 ug 21P 21 2 0.7 25F 25 4 0.7 23P 22P 20P 29P 50D ug lOB/P 9 1 0.3 14B/P 11 3 0.3 0 SB (P 11BIP 2 83/P 8B/P 2 1 500 ug 03/ O/P 0 03/P 03/P 0 03/P 03/P 0 NOPD 10 ug 800 765 31 23.9 758 740 2-AA 2.5 us 528 578 53 17.2 634 572 (REV-revertants/plate, M-mean, SD-standard deviation, FAC-factor) P: PRECIPITATION B: REDUCED BACKGROUND GROWTHBASF Aktiengesellschaft 14 Experimental Toxicology and Ecology STUDY DIRECTOR: DR. ENGELHARDT PROJECT NO. :401404371034092 SUBSTANCE NAIME: Alkali Blue Dry 6100 ILot 2231308 SUBSTANCE NO. :03/0437-2

TESTDATE :02.DEC.2003 METHOD PREINCUBATION TEST VOLUME 100 ullplate STRAIN : E.coli WP2 uvrA VEHICLE : DMSO S9-MIX : WITHOUT S-9 MIX WITH S-9 MIX (1:9) DOSEIPLATE REV M SD FAC REV M SD FAC TITRE DM50 32 31 2 1.0 35 38 3 1.0 31 32 41 34 28 38 25 4 ug 27 25 3 0.8 32 28 4 0.7 26 25 21 28 20 ug 28 33 7 1.1 30 29 4 0.8 41 25 29 33 100 ug ZIP 23 2 0.7 21P 24 2 0.6 24P 25P 23P 25P 500 ug 18B1P 13 5 0.4 17B/P 11 5 0.3 6 lOB/P 83/P 7 10B/P 9B/P 5 2 500 ug 03/P 03/P 0 OBIP 03/P 0 03I P 03/P 0 4-NQO 5.0 ug 701 665 33 21.7 658 636 2-AA 60 ug 251 219 28' 5.8 207 200 CREV=revertantslplate, M=xean, SDstandard deviation, FACfactor) P: PRECIPITATION B: REDUCED BACKGROUND GROWTH

# BASF Report; 

### Project No.: 40M0437/034092



### APPENDIX 3 STERILITY CONTROL

# BASF Report; Project No.: 40MO437/034092 STERILITY CONTROL 1

st Experiment



# F Components :Resu

### ts

# 2.0ml soft agar No .g.rowth 

### 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 Coniponefts Resilt. 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 3rd Experiment 

### Components 

# *1Results, 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.: 40M0437/034092



## APPENDIX 4 S-9 FRACTION CHARACTERIZATIONS-9 fraction Preparation ofS-9 fraction: June 17, 2003 Content of protein (mean*): 29.0 onl Characterization of the S-9 mix with benzofalpyrene: July 14, 2003
METHOD: Standard Plate Test S-9 FRACTION/COFACTORS: 1:9 STRAIN: TA 100 STRAIN: TA 98 DOSEIPLATE MREV* SD* FAC* DOSE/PLATE M REV* SD* FAC* DMSO 113.7 9.0 1.0 DM80 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) pool11-4

# BASF Report; Project No.: 40M0437/034092



### APPENDIX 5 HISTORICAL NEGATIVE CONTROL DATA

## Historical Negative Control Data Strain: TA 1535

.......... ... .... .... ........ .. iio ....... 

### ~ ~X ia $ SPT

- Water..... 3.9. 9.03 40 1 5 1
SPT 1:9Water 03.98 - 09.03 400 12 25 18 2 SPT 19DM80S 03.00 -09.03 400 11 24 18 2 SPT 19 Acetone 01.96 -05.03 210 11 125 -19 2

PT 1: Water 03.987-0.03 400 13 25 19 2 PT 1 :9 DM50S 05.99 -09.03 400 10 23 17 2 PT 1:9 Acetone 01.96-05.03 15910 25 18 2

PIT -Water. 05.97- 0.03 400 82 155 117 14 PIT -DMVSO 06.00 .0.03 400 91 160 110 10
S PIT -Acetone 01.96 -05.03 258 910 160 116 15 PIT 1:9 Water 04.97B-0.03 400 94 15 123 16 PIT 1 :9 DM80S 08.00 -09.03 400 92 154 112 10 PIT 1 :9- Acetone 01.96-05.03 253 85_ 160 120 16 Strain TA

10

PT 19water 05.97 -0.03 400 92 159 122 15 PT 19DM80S 06.009-09.03 400 80 159 112 13 PT 19Acetone 01.96-05.03 156 96 160 119 16 SPT 1 stndr Waate test.90 40 94 5 13 1

PIT Waeteralo te9st0.3 0 9 6 17 1 SD~~~8 =2tnad eito

## Historical Negative Control Data Strain: TA 1537

-. . . .. . . ..... . . .-..-. SPT -Water 03.98 -09.03 400 6 16 10 2 SPT -DM50 03.00- 09.03 400 5 16 10 2 SPT -Acetone 01-96-05.03 213 5 118 10 12 SPT 1 :9 Water 03.98-09.03 400 6 19 11 2 SPT 1:9 DM50 03.00-09.03 400 8 19 11 2 SPT 1T:9 Acetone 01.96-05.03 213 7 20 12 2

PIT -Water 05.97 -06.03 400 5 19 10 2 PIT -DMSO 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 DM50 04.99-09.03 400 5 16 10 2 PIT 1:9 Acetone 01.96-05.03 162 5 20 11 2 

## Strain: TA98

. 

### ~f

........... . . . . SPT -Water 06.98 -09.03 400 17 44 28 5 SPT -DM50 07.00 -09.03 400 20 45 29 5 SPT -Acetone 01.96-05.03 215 19 50 29 5 SPT 1 :9 Water 06-95 -09.03 400 20 49 36 6 SPT 1:9 DMSO 08.00 -09.03 1400 20 50 36 6- SPT 1 :9 Acetone 01.96-05.03 1214 26 50 38 5

PIT Water 05.97
-0803 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 DM50 06.99 -09.03 400 20 49 32 5 PIT 1:9 Acetone 01.96-.05.03 161 20 50 37 6

*= revertants/platd SPT =standard plate test PIT = preincubation test SD) =standard deviation

## Historical Negative Control Data Strain: WP2 uvrA

............... 

## . .:.:N.o: SPT

- Water 11.97-09.03 400 20 49 31 5 F SPT -DMSO 01.00 - 09.03 400 22 50) 32 5

SPT -Acetone 01.96 - 05.03 200 1s 50 33 6 SPT 1 :9 Water 01.98-09.03 400 20 49 35 6 SPT 1:9 DMVSO 01.00.09.03 400 23 50 35 6 SIFT 1:9 Acetone 01.96-05.03 1200 15 50 37 7

PIT -Water 04.97 -06.03 400 21 50 31 5 Pfl DMSO 07.99-09.03 400 1s 49 32 5 PIT -Acetone 01.96 - 05.03 157 20 147 31 5 PIT 1:9 Water 01.97- 05.03 400 23 50 37 .6 Prr 1 :9 DMVSO- 06.99-09.03 400 19 50 32 6 PIT 1:9 Acetone 01.96-05.03 157 20 49 33 6

* = revertants/plate SIFT =standard platetest PIT =preincubation test SD = standard deviation

# BASF Report; Project No.: 40M0437/034092



### APPENDIX 6 HISTORICAL POSITIVE CONTROL DATA

## Historical Positive Control Data Strain: TA 1535

............. . ......... __ .... 

# 1 1

SPT

- MNNG 5.0 02.01 -09.03 400 500 1649 776 199 SPT

- 1:9

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

. . . . :*:::*-:-:::-::-:-:.........::--... PIT MNNG 5.0 07.00 - 0.03 400 502 1357~785 150 PIT 1:9 2-AAk 2.5 06.00-09.03 400 80 345 129 31 

## Strain: TA 100

SPT 

## J

- MNNG 5.0 03.01 -09.03 400 501 1747 871 262 

# I SPT 

# ) 1:9 2-AA- 2.5 03.01 -09.03 400 507 1708 859 215

M.......... ....... b X.d~ .............. : 

### ~ 

## si I PIT 1:9 2-AA 2.5 06.00 -09.03 400n 500 115 675 135..~4

=revertantsiplate SPT = standard plate test PIT = preincubatlon test SID =standard deviation

MNNG =N-methyl-N-nitro-N-nitrosoguanidine 2- AA *2-aminoanthr-acene

## Historical Positive Control Data Strain: TA 1537

.................. ... ...... 

## 1

.. . .

. . .*.* *
. .. . ........ ....... 

## IF T AAC 100 01.01 -09.03 400 300 1147 443 104



# S

PT 1:9 2-AA 2.5 61.01 -09.03 40 71 200 127 25_

.. . . . . . .......... ._. .. _. ..

... *........: .tjl:: %:. .......... j~ > ?4~e M~ n 

### ~ S PIT

- AC100 07-00 -0903 400 

## I278 885 435 83 PIT 1:9 2-AA 2.5 06-00 -09.03 400 80 200 112 20 

## Strain: TA98

SPT

- 

# J NOPD 10 02.01 -0903 400 34T 1200 737 132 SPT 1:9 

# J 2-AA 2.5 03.01 -09.03 400 504 1384 699 139

_____ - NOPI1 07.00-09.03 400 399 1129 735 135 

## j 

# ILPIT 

# 1 1:9 2-AA 2.5 06.00 -0903 400 500 985 621 90 

## J

= revertantsiplate SPT =standard plate test PIT = preincubation test SID =standard deviation

2 -AA =2-minoanthracene AAC = 9-amino. cridine NOPD =4-nitro-o-phenylendiamine

## Historical Positive Control Data Strain: WP2 uvrA

.................... ......................... SPIT

- 4-NQO 5.0 12.00 -09.03 400 500 1620 676 171 1P 1:9 2-AA 60.0 101.01 -09.03 400 155 478 244 40

._ _ _ _ _ _ _ _ ._ _ _ _ _ _ _ . ._ __ _

. ..

. -

_ _ _..._ _

....................... ........... _ -_. ... _. .. .. PIT

- 4-NQO 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

=revertants/plate SPT =standard plate test PIT *preincubatlon test SD =standard deviation

4-NO 4-Nitroquinoline-N-oxide 2- AA =2-aminoanthracene