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 

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OCSPP OPPT EAD 

1200 Pennsylvania Ave. NW 

Washington, DC 20460 

202.564.8251 

Pages: 

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r- Urgent I- For Review I- Please Comme - Please Reply - Please Recycle -

nt 
UNITED 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, PESTICIDES AND 

TOXIC SUBSTANCES 

Subject: Follow-up to fax transmission of 4/13/12 (ePMA removal ofCBI claims regarding 

identified studies) 

Dear Mr. Merritt: 

This is sent to you pursuant our conversation of several minutes ago. In Larry Robinson's fax of 

April 13th while there is a waiver of confidential business information (CBI) claims in the 

identified studies there is a an assertion of copyright protections and some additional text that 

provides in part ... "Reproduction, reference, summarization, retransmission or other use of this 

copyrighted document. .. including electronic transmission, using the internet or email .... is 

strictly prohibited .... " 

While we understand the basis for the copyright protections, we have a concern that the language 

in the fax might be interpreted to mean that ePMA is asserting that the studies submitted per the 

TSCA activity are not to be made available in an Agency e-docket (electronic docket) or be 

included in EPA websites among the collection of TSCA health 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 of non-CBI materials submitted by entities subject to TSCA 

regulations (including the posting of such materials on government websites) and submitted by 

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 of the studies. 

Can you please confirm CPMA's intent? Many thanks. 

Scott M. Sherlock, Attorney Advisor 

Environmental Assistance Division 

Office of Pollution 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 1 Lot 2219308

DATA REQUIREMENT

OECD No. 471

EC 2000132, B. 13 1 B. 14

AUTHORS

Dr. G. Engelhardt (Study Director)

Dr. E. Leibold

STUDY COMPLETED ON

March 15,2004

TESTING FACILITY

Experimental Toxicology and 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. It is the propert of BASF and may be used only for that purpose for

which it was intended by BASF. Every other or additional use, exploitation, reproduction, publication or submission to other parties

requires the written permission of BASF, with the exception of regulatory agencies acting within the limbit of their admninistrative
BASF

Report; Project No.: 40M0437/034091 2

THIS PAGE IS INTENTIONALLY LEFT BLANK
BASF

Report; Project No.: 40MO437/0340913

GIP COMPLIANCE STATEMENT

This study was conducted in general accordance with the OECD Principles of Good

Laboratory Practice and the GLP Principles of the 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 TSCA. However, there was the

following deviation from the requirements of the above-mentioned principles:

-The stability of the test substance in the vehicle DMSO and in water was not

determined analytically.

Dr.rer G Enelhrd na. 

(Study Director)
BASF

Report; Project No.: 40M0437/034091 4

THIS PAGE IS INTENTIONALLY LEFT BLANK
BASF

Report; Project No.: 40M0437/034091 5

SIGNATURE PAGE

Study Director/ Conduct of study:A

Dr. rer. nat. G. Engelhardt

Management: .. ........ Dr. rer. nat. E. Leiboid
BASF

Report; Project No.: 40M0437/034091 6

STATEMENT

of the Quality Assurance 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

of this short-term study was not inspected; the processes of the laboratory and of the

study involved are inspected in regular intervals.

The final report reflects the raw data.

Phase of study 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

ffir Pflazeanund Pfazmschutz

GLP-Bescheinigung I Statement of GLP Compliance

COW"A I Sccrdingt 1 19 b Abs. I Chslef*INgssslz)

Eins GLP-inspektion zur Oberwachung der E-Inhaltung Assessment of conformity with GLP accong

der GLP-Grndals8te gemlil Chomlikulengesstz 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"=)

Datum der Inspoldion I Date of Inspection

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

Dkier ganenntse P ~eiwchtungfs~tandort befindet The above mentioned test fadlitylAt shte as Included

sicti hI natlonalen GLP-Oberwachamgsvsrtshren und in the national GLP.Comp"ac Programme and Is

*Wi regeliuIN9s uf ElniahWV der GLP-Grundattze inspected ona regular basis.

Aut der Gruncdiage des InspektonbeIchtes wird hier- Bsed on the Inspection report it can be confirnad.

mit besWgt doss In dieser Pr~felnichungldeien that ths test faclty/lest ot Is able to conduct the

prftendot die oben genannten PrOfungen unter Eln- aforementioned studies In compliance With the Puinhaitajig der GLP-Glundslitze dta'chgaMut warden ciples of GLP.

k~nnen.

unto m Signature, Date ~ ,,Di18 ~ M~

(Named= d-avemm-rhn P n t M. W~ ofei neponhlbh Putoo

LneW Ist ft chianeflba vv am w 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 of the 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. Positive controls 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 plate test 26

4.1.1.1. Testswi~thout S-9 mix 26

4.1.1.2. Tests With S-9 mix 26

4.1.2. Preincubation test 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

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
IL ___ ____LA

Report; Project No.: 40MD437/034091 10

1. SUMMARY

The substance Alkali Blue Dry 6100 / Lot 2219308 was tested for its mutagenic

potential based on the ability to induce point mutations in selected loci of several

bacterial strains, i.e. Salmonella typhimuriumn and Escherichia coli, in a reverse mutation

assay.

" STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E. col WP2 uvrA

" DOSE RANGE: 23 pg -5750 pg/plate (SPT)

4 pg - 1 500 pg/plate (PIT; Salmonella strains) 4 jig - 2 500 pg/plate (PIT: E. coli WP2uvrA)

" 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 of the test substance was found from about

100 pg/plate onward.

*TOXICITY: A bacteriotoxic effect was observed under all test conditions

(for details see item 4.2.).

*MUTAGENICITY:

An increase in the number of his* or frp* revertants was not observed in the standard

plate test or in the preincubation test either without S-9 mix or after the addition of a

metabolizing system.

*CONCLUSION:

According to the results of the present study, the test substance

Alkali Blue Dry 6100 1 Lot 2219308 is not mutagenic in the 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

for detecting a point mutagenic activity of chemical substances (1, 2, 5) and are used to

screen for possible mammalian mutagens and carcinogens.

The principle of these assays is that mutations are detected by the reversion of mutations

- present in the bacterial strains. This leads to a restoration of the functional capability of

the bacteria to synthesize the essential amino acid and thus to the ability to grow in the

absence of the amino acid required by the parent strains.

Most of the substances are not mutagenic or carcinogenic themselves, but only after

metabolic transformation, which in vivo is catalyzed mainly by the enzyme systems of the

liver. Therefore, the tests are carried out not only directly, but also in the presence of an

exogenous metabolic activation system. The most commonly used system is a cofactorsupplemented 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 December 4, 2003

(evaluation last experiment):

2.2. REGULATORY GUIDELINES

The tests are carried out in accordance with the following guidelines:

* OECD No. 471 (July 21, 1997)

" EC Directive 2000/32, B.13/13B.14 (May 19, 2000)
i m111 M ut ii! tL11liiilfh IllIEIL1I 1 1E

Report Project No.: 40M0437/034091 12

3. MATERIAL AND METHODS

3.1. TEST SUBSTANCE

Name of test substance: Alkali Blue Dry 6 1001 Lot 2219308

Test substance No.: 03/0437-1

Batch No.: Lot 2219308

CAS No.: 1324-76-1

Date of filling: 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.: 40M 0437/034091 13

3.2. TEST SUBSTANCE ANALYSIS

3.2.1. Test substance

The stability of the test substance under storage conditions is guaranteed as indicated by

the sponsor, and the sponsor holds this responsibility.

The homogeneity of the test substance was guaranteed by mixing before preparation of

the test substance formulations.

The analytical investigations were carried out in the:

- Analytical Laboratory of BASF Corporation Huntington (characterization)

3.2.2. Test substance preparation

The stability of the test substance in the vehicle DMSO and in water was not determined

analytically.

More detailed information about the test substance can be found in the 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 is prepared according to Ames et al. (1, 2).

At least 5 male Sprague-Dawley rats') (200 - 300 g) receive a single intraperitoneal injection of

500 mg Aroclor 1254 (as a solution in corn oil with a concentration of 20 g/1 00 ml) per kg body

weight 5 days before sacrifice.

During this time, the animals are housed in Makrolon cages in air-conditioned rooms in which

central air conditioning guaranteed a range of temperature of 20 - 240C and a relative humidity of

30 - 70%. The day/night rhythm is 12 hours (light period from 6.00 - 18.00 hours and dark period

from 18.00 - 6.00 hours).

Standardized pelleted feed and tap water from bottles are available ad libitum.

5 days 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 a temperature of +40C). The livers are weighed and washed in an equivalent

volume of a 150 mM KCI solution (1 ml :r I g wet liver), then cut into small pieces and

homogenized in three volumes of KCI solution. After centrifugation of the homogenate at

9 000 x g for 10 minutes at +4*C, 5 ml portions of the supernatant (so-called S-9 fraction) are

stored at -70*C to -80C.

The preparation date and protein content of the S-9 fraction are given in Appendix 4.

3.3.2. S-9 mix

The S-9 mix is prepared freshly prior to each experiment (1, 2). For this purpose, a sufficient

amount of S-9 fraction is thawed at room temperature and 1 volume of S-9 fraction is mixed with

9 volumes of S-9 supplement (cofactors). This preparation, the so-called S-9 mix, is kept on ice

until used. The concentrations of the cofactors in the S-9 mix are:

MgCI 2 8 mM

KCI 33 mM

Glucose-6-phosphatase 5 mM

NADP 4 mM

Phosphate buffer (pH 7.4) 15 mM

The phosphate buffer (6) is prepared by mixing an Na2HPO4 solution with an NaH 2PO4 solution in

a ratio of about 4 : 1.

To demonstrate the efficacy of the S-9 mix in this 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 at room

temperature, and 0.1 ml of this bacterial suspension is inoculated in nutrient broth

solution (8 g Difco nutrient broth + 5 g NaCI/liter) and incubated in the shaking water bath

at 376C for about 10 - 12 hours. As a rule, a germ density of > 108 bacterialml is reached.

These cultures grown overnight are kept In iced water from the beginning of the

experiment until the end in order to prevent further growth.

The use of the strains mentioned is in accordance with the current scientific

recommendations for the conduct of this 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 of several bacteria mutants from histidine auxotrophy

(his-) to histidine prototrophy (his+) is determined (2, 3, 4). The tester strains TA 1535,

TA 1537, TA 98 and TA 100 selected by Ames and coworkers are derivatives of

Salmonella typhimurium LT2 and have GO base pairs at the primary reversion site. All

strains have a defective excision repair system (uvrB), which prevents the repair of

lesions which are induced in the DNA, and this deficiency results in greatly enhanced

sensitivity of some mutagens. Furthermore, all strains show a considerably reduced

hydrophilic polysaccharide layer (fa), which leads to an increase in permneability to

lipophilic substances.

The strains TA 1535 and TA 100 are derived from histidine-prototrophic Salmonella

strains by the substitution mutation his G 46 and are used to detect base pair

substitutions. TA 1537 and TA 98 are strains for the detection of frameshift mutagens.

These strains carry different frameshift markers, i.e. the +1 mutant his C 3076 in the case

of TA 1537 and the +2 type his D 3052 in the case of TA 98.

The strains TA 98 and TA 100 carry an R factor plasmid pKM 101 (4) and, in addition to

having genes resistant to antibiotics, they have a modified postreplication DNA repair

system, which increases the mutation rate by inducing a defective repair in the DNA; this

again leads to a considerable increase in sensitivity.
Report; Project No.: 40M0437/034091 16

3.4.2. Escherichia coil

Escherichia coil WP2 uvrA which has an AT base pair at the primary reversion site is a

derivative of E. coil WP2 With a deficient excision repair and is used to detect substances which induce base pair substitutions (5). The rate of induced back mutations

from tryptophan auxotrophy (trp) to tryptophan independence (trp*) is determined.

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 is checked for UV sensitivity.

Histidine and tryptophan auxotrophy is automatically checked in each experiment via the

spontaneous rate.
BASF

Report; Project No.: 40M04371034091 17

3.5. EXPERIMENTAL PROCEDURE

3.5.1. Choice of the vehicle

Due to the limited solubility of the test substance in water, DMS0 was selected as the

vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests

and for which historical control data are available.

3.5.2. Mutagenicity tests

3.5.2.1. Standard plate test

*Salmonella typhimurium

The experimental procedure of the standard plate test (plate incorporation method) is

based on the method of Ames et at. (1, 2).

Test tubes containing 2-mi portions of soft 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 in a water bath at 45*C, and the remaining components are added in the following

order:

0.1 ml test solution or vehicle

0.1 ml fresh bacterial culture

0.5 ml S-9 mix (in tests with metabolic activation)

or

0.5 ml phosphate buffer (in tests without metabolic activation)

After mixing, the samples are poured onto Vogel-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 E medium

15g Difco bacto agar

20 g D-glucose, monohydrate.

After incubation at 37TC for 48 - 72 hours in the dark, the bacterial colonies

(his' revertants) are counted.
BASF

Report; Project No.: 40M0437/034091 18

Escherichia coli

The experimental procedure is based on the method of Ames 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 solution for the determination of mutants: 0.5 mM tryptophan) are kept In a water

bath at 450C, and the remaining components are added in the following order

0.1 ml test solution or vehicle

0.1 ml fresh bacterial culture

0.5 ml S-9 mix (in tests with metabolic activation)

or

0.5 ml phosphate buffer (in tests without metabolic activation)

After mixing, the samples are poured onto minimal agar plates within approx.

30 seconds.

Composition of the minimal agar

The composition of the minimal agar (SAl selective agar) is based on the description

of Green, M.H.L. and Muriel, W.J. (5), with the exception of solution E (tryptophan

solution), which has previously been added to the soft agar

300 ml solution B (agar)

100 ml solution A (saline solution)

8 ml solution C (glucose solution)

10 ml solution D (casein solution)

After incubation at 37*C for 48 - 72 hours in the dark, the bacterial colonies

(trp* revertants) are counted.
BASF

Report; Project No.: 40M0437/034091 19

3.5.2.2. Preincubation Test

The experimental procedure is based 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 of about 20 minutes using a

shaker. Subsequently, 2 ml of soft agar is added and, after mixing, the samples are

poured onto the agar plates within approx. 30 seconds.

After incubation at 37*C for 48 - 72 hours in the dark, the bacterial colonies are counted.

3.5.3. Titer determination

In the standard plate test, 0.1 ml of the overnight cultures is diluted to 10 - in each Case.

Test tubes containing 2-mI portions of soft agar containing maximal amino aicid solution

(5 mM tryptophan or 5 mM histidine + 0.5 mM biotin) are kept in a water bath at 450C,

and the remaining components are added in the following order

0.1 ml vehicle (without and with test substance)

0.1 ml fresh bacterial culture (dilution: 10-6)

0.5 ml S-9 mix

In the preincubation test, 0.1 ml of the overnight cultures is diluted to 10-6 in each case.

0. 1 ml vehicle (with and without test substance), 0. 1 ml bacterial suspension and 0.5 ml

S-9 mix are incubated at 370C for about 20 minutes using a shaker. Subsequently, 2 ml

- of soft agar containing maximal amino acid solution for titer determination (5 mM

tryptophan or 5 mnM histidine + 0.5 mnM biotin) is added.

After mixing, the samples are poured onto the agar plates Within approx. 30 seconds.

After incubation at 370C for 48 - 72 hours in the 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 in order to check for

possible contaminants (sterility control) and to determine the spontaneous mutation rate

(vehicle control).

*Sterility control

Additional plates are treated with soft agar, S-9 mix, buffer, vehicle or the test

substance but without the addition of tester strains (see Appendix 3).

*Vehicle control

The vehicle control with and without S-9 mix only contains the vehicle used for the test

substance at the same concentration and volume for all tester strains.

3.5.4.2. Positive controls

The following positive controls are used to check the mutability of the bacteria and the

activity of the S-9 mix:

With S-9 mix

2-aminoanthracene (2-AA) (SIGMA, A-1 381) - 2.5 pg/plate, dissolved in DMSO - strains: TA 1535, TA 100, TA 1537, TA 98

- 60 pg/plate, dissolved in DMSO

- strain: Escherichia coli WP2 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 in DMSO

- strains: TA 1535, TA 100

" 4-nitro-o-pheflylefldiamine (NOPO) (SIGMA, N-9504)

- 10 pg/plate, dissolved In DMS0

- strain: TA 98

" 9-aminoacridine (AAC) (SIGMA, A-I 135)

- 100 pg/plate, dissolved in DM50

*- strain: TA 1537

* 4-nitroqu lnoline-N -oxide (4.NQO) (SIGMA, N-8141) - 5 pg/plate, dissolved in DMVSO

- strain: E. coli WP2 uvrA

The stability of the selected positive controls is well-defined under the selected culture

conditions, since they are well-established reference mutagens.
Report; Project No.: 40M0437/034091 22

3.5.5. Scope of tests and test conditions

1st Expeiriment

Strains: TA 1535, TA 100, TA 1537, TA 98,

E. col WP2 uvrA

Doses: 0; 23; 115; 575; 2 8756 and 5 750 pg/plate

Vehicle: DMSO

Type of test: Standard plate test with and without S-9 mix

Number of plates: 3 test plates per dose or per control

*2nd Experiment

Strains: TA 1535, TA 100, TA 1537, TA 98,

Doses: 0; 500; 1 000; 1 500; 2 000 and 2 500 pg/plate

Vehicle: DMSO

Type of test: Stan dard plate test with and without S-9 mix

Number of plates: 3 test 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 1 500 pg/plate

E. call,

0; 4; 20; 100; 500 and 2 500 pg/plate

Vehicle: DM80

Type of test: Preincubation test with and without S-9 mix

Number of plates: 3 test plates per dose or per control
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 given for all dose groups as well as for the positive and negative

(vehicle) controls in all experiments. In general, five doses of the test substance are

tested with a maximum of 5 mg/plate, and triplicate plating is used for all test groups at

least in the 1 st experiment. Dose selection and evaluation as well as the number of

plates used in repeat studies or further experiments are based on the findings of the 1st

- experiment.

3.6.2. Titer

The titer is generally determined only in the experimental parts with S-9 mix both for the

negative controls (vehicle only) and for the two highest doses in all experiments.

3.6.3. Toxicity

Toxicity detected by a

- decrease in the number of revertants

- clearing or diminution of the background lawn (= reduced his' or trp' background

- growth)

- reduction in the titer

is recorded for all test groups both with and without S-9 mix in all experiments and

indicated in the tables.

3.6.4. Solubility

Precipitation of the test material is recorded and indicated in the tables. As long as

precipitation does not interfere with the colony scoring, 5 mg/plate is generally selected

and analyzed (in cases of nontoxic compounds) as the maximum dose at least in the 1st

experiment even in the 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.
BASF

Report; Project No.: 40M0437/034091 24

3.6.5. Acceptance criteria

Generally, the experiment is considered valid if the following criteria are met:

e The number of revertant colonies in the negative controls was within the normal range

of the historical control data for each tester strain (see Appendix 5).

* The sterility controls revealed no indication of bacterial contamination.

* The positive control articles both with and without S-9 mix induced a significant

increase in the number of revertant colonies within the range of the historical control

data or above (see Appendix 6).

a The titer of viable bacteria was > 108/ml.

3.6.6. Evaluation criteria

The test chemical is considered positive in this assay if the following criteria are met:

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

about doubling of the spontaneous mutation rate in at least one tester strain either

without S-9 mix or after adding a metabolizing system.

A test substance is generally considered nonmutagenic in this test if:

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

control range under all experimental conditions in two experiments carried out

independently of each other.
BASF

Report; Project No.: 40M0437/034091 25

3.7. RETENTION OF RECORDS

GLP-relevant records and materials are stored at BASF Aktiengesellschaft and at

BASF Corporation at least for the period of time specified in the GLP principles. Details

on responsibilities or locations of archiving may be seen from the relevant SOPs and

from the raw data.
- ~IiwtuuUIU~fl 1~ IIIIILi M,1 111 I I I ALLA111i IIiLL.

BASF

Report; Project No.: 40M0437/034091 26

4. RESULTS

(Tables I - 14)

The substance Alkali Blue Dry 6100 / Lot 2219308 was tested for mutagenicity in the

Salmonella typhimurium / Escherichia coll reverse mutation assay both in the standard

plate test and in the preincubation test with and without the addition of a metabolizing

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 plate test

(Appendix 1, Tables 1 - 9)

4.1.1.1. Tests without S-9 mix

TA 1535:

TA 100:

TA 1537: No increase in the number of his* or trp* revertants.

TA 98:

E. col W132 uvrA:-

4.1.1.2. Tests with S-9 mix

TA 1535:

TA 100:

TA 1537: No increase in the 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 in the number of his* or trp* revertants.

TA 98:

E. coi WP2 uvrA:

4.1.2.2. Tests with S-9 mix

TA 1535:

TA 100:

TA 1537: No increase in the number of his* or trp+ revertants.

TA 98:

E. coil WP2 uvrA:
I !T 71ASReport; Project No.: 40MD437/034091 28

4.2. TOXICITY

A bacteriotoxic effect (reduced his' background growth, decrease in the number of his+

revertants, reduction in the titer) was observed in the standard plate test depending on

the strain and test conditions from about 575 pg - 1 000 pg/plate onward. No bacterlotoxicity was observed using E. coi WP2 uvrA.

In the preincubation assay bacteriotoxicity (reduced his' or trp- background growth,

decrease in the number of his+ or trp+ revertants, reduction in the 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 to the results of the present study, the test substance did not lead to an

increase in the number of revertant colonies either without S-9 mix or after adding a

metabolizing system in three experiments carried out independently of each other

(standard plate test and preincubation assay).

4.5. CONCLUSION

Thus, under the experimental conditions chosen here, it is concluded that

Alkali Blue Dry 6100 ILot 2219308 is not a mutagenic agent in a bacterial reverse

mutation test.
BA SF

Report; Project No.: 40M0437/034091 29

5.REFERENCES

I1. Ames, B.N.; McCann, J.; Yamasaki, E.:

Methods for detecting carcinogens and mutageris with the

Salmonellalmammalan-microsome mutagenicity test.

Mut. Res., ;L1, 347 - 364 (1975)

2. Maron, D.M.; Ames, B.N.:

Revised method for the Salmonella mutagenicity test.

Mut Res., 113, 217 -233 (1983)

3. Ames, B.N.; Lee, F.D.; Durston, W.E.:

An improved bacterial test system for the 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 tester strains with R factor plasmids.

Proc. Not. Acad. Sci. USA, 72, 979 - 983 (1975)

5. Green, M.H.L.; Muriel, W.J.:

Mutagen testing using trp" reversions in Escherichia coli.

Mut. Res., 38, 3 - 32 (1976)

6. DeMarini, D.M.; Dallas, M.M.; Lewtas, J.:

Cytotoxicity and effect on mutagenicity of buffers in a microsuspension assay.

Ter. Camc. Mut, 9, 287 -.295 (1989)

7. Yahagi, T.; Nagao, M.; Seino, Y.; Matsushima, T.; Sugimura, T.; Okada, W.

Mutagenicities of N-nitrosoamines in Salmonella.

Mut. Res.,!48, 121 - 130 (1977)

8. Matsushima, T.; Sugimura, T.; Nagao, M.; Yahagi, T.; Shirai, A.:

Factors modulating mutagenicity in microbial tests.

In: Norpoth, K.H. and R.C. Gamner, Short-Term Test Systems for Detecting

Carcinogens.

Springer Verlag 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 2 00 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

1 4P 15P

1 7P 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 Aktiengesellschaf t 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

103 P 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 GROWTH
BASF 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 GROWTH
I'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 GROWTH
BASF 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: PRECIPITATION
r[ a-I, _IALiLiI IiFMTEiHfl A 1

BASF Aktiengesellschaft 06

Eiperimental Toxicology and E cology 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

1 6P 1 6P

13P 13P

1 000 ug 16P 15 1 0.8 17P 15 3 0.8

14P 1 7P

1 4P 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

2B IP 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 GROWTH
BASF 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 GROWTH
BASF Aktiengesellschaft 08

Experimental Toxicology and Ecology

STUDY DIRECTOR: DR. ENGELE ARD T

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 GROWTH
BASF 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 GROWTE
BASF 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 GROWTH
BA SF 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

11 3P 1 02P

98P 90P

500 ug 48P 44 3 0.4 78P 67 10 0.6 8

4 2P 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 GROWTH
BASF 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 1 9P

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 GROWTH
NOMMIMNEMLkLLL 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 soft agar No growth

0.5 ml S-9 mix No growth

0.5 ml buffer No growth

0.5 ml vehicle No growth

5 750 pg test substance No growth

2nd Experiment

Components Reisults

2.0 ml soft agar 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

2 500 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 M REW SD- FAC* DOSEIPLATE M REV* 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: TA 98

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

reverta ntsip late

SPT standard plate test

PIT =preincubation test

SD 3standard deviation

Page 2 of 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 3 of 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 of 3
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: TA 98

In wa1 ... 4J~ J-1 I P r] NOPD 1 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

w revertantslplatB

SPT = standard plate test

PIT = preincubatiofl test

SD = standard deviation

2 - AA = 2-a mlnoa nthracene

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 3 of 3