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 


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. It is the property 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 otr parties

requires the written permission of BASF, with the exception of regulatory agencies acting within the limits of their administrative

authoriy
BASF

Report; Project No.: 40M04371034092 2

THIS PAGE IS INTENTIONALLY LEFT BLANK
BASF

Report Project No.: 40M0437/034092 3

GLP COMPLIANCE STATEMENT

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

Laboratory Practice and the GLP Principles of the German "Chem ikaliengesetz"

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

-The stability of the test substance in the vehicle DM50S and in water was not

determined analytically.

... ................ e..........

Dr. rer. nat. G. Engelhardt

(Study Director)
BASF

Report, Project No.: 40M0437/034092 4

THIS PAGE IS INTENTIONALLY LEFT BLANK
BASF

Report; Project No.: 40M04371034092 5

SIGNATURE PAGE

to Study Director IConduct of study: ~..........

Dr. rer. nat. G. Engelhardt

Manageme nt: 4.....Y......... ~~ Dr. rer. nat. E. Leibold
BASF

Report; Project No.: 40M0437/034092 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 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 I Statement of GLP Compliance

WWY I S~ordki to 119 b Abe. 1 Chamksiiesz

- Elne GLP.Inspeldlon zur Oberwachung der Eklia~bng Assessmert of conformit with GLP accrdlng

der GLP-Grundsitte 9"Si ChernkalVIznee bzw. to Chomilkuliengesetz and Directive 8832O/EEC at

Rictiie BB0201EG 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" ahmVW4LP Hr. S=OEC DmdMv*

..,,., .... . ..... ....-...

Datum der lnsplitoI Date of inspection

...... ........... t 5.052001 und vorm 21. bit 26.06.2001 ....

Die/Der genannte Pd*cfnrdtun/p~rlstandort befindet The above mentioned test facllftylkest site Is Included

slcht i natlonaleri GLP-Oberwachugsverfahren und In the national GLIP-Compllance Progrmme and Is

vild regelmAfllig aizf Eihaftmn der GLP-Grwxdstze Inspected on a regular basis.

Obenwwft

Auf der Grqwdlge do* lnspektionsbarichtes wird hior. eased on the Inspection report ft can be Confirmed,

mit besaftigI doss In dlese PrOfislnrlchtungldiesern that thu test faclftyflst sMe Is able to conduct the

Profstaridolt die oben genannten PrOfungen unter ln,- aforementioned srtudies In compliance with the Pdnhabmug der GLIP-Grundsatte durchgeftrt wsrden ciples of GLP.

kdnneii.

Unteradirikt Datum ISignature. at

L&-sndW AVPMUFI2.nbaI umd P2isimmuftiM 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-9 fraction 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 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.: 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. Tests with 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 ability to induce point mutations in selected loci of several

bacterial strains, i.e. Salmonella typhimurium and Escherichia col, in a reverse 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 - 1 500 pg/plate (PIT; Salmonella strains)

4 pg -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 trp* 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 subst ance

Alkali Blue Dry 6100 1 Lot 2231308 is not mutagenic in the 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 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 / B.14 (May 19, 2000)
El JI L L I1 --U lll ULW I LK1A LlI I I I h1RJILiLI JL

Report; Project No.: 40M0437/034092 12

3. MATERIAL AND METHODS

3.1. TEST SUBSTANCE

Name of test substance: Alkali Blue Dry 6100/ Lot 2231308

Test substance No.: 03/0437-2

Batch No.: Lot 2231308

CAS No.: 1324-76-1

Date of filling: September 19, 2003

Purity/Composition: 88 % main component

(See analytical data of October 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 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).
-~~~~T 'I i~I~~ 1 71 

BASF

Report; Project No.: 40M0437/034092 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 - 24*0 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, arnd the livers are prepared (all preparation

steps for obtaining the liver microsorne enzymes are carried out using sterile solvents and

glassware at a temperature of +4C). The livers are weighed and washed in an equivalent

volume of a 150 mM KCI solution (1 ml A 1 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 +'40C, 5 ml portions of the supernatant (so-called S-9 fraction) are

stored at -700C to -800C.

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 4

amount of S-9 fraction is thawed at room temperature and I 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:

MgCI2 8 mM

KCI 33 mM

Giucose-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 NaH2PO4 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).

ICharles River Deutschland GmbH
Report; Project No.: 40M0437/034 092 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 is inoculated in nutrient broth

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

at 37*C for about 10 - 12 hours. As a rule, a germ density of > 108 bacteria/mi is reached.

These cultures grown overnight are kept in iced water from th~e 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 coli 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 GC 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 (rfa), which leads to an increase in permeability 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.
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 is a

derivative of E. coli 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); amnpicillin resistance (R factorplasmid).

E. coli 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.: 40M0437/034092 17

3.5. EXPERIMENTAL PROCEDURE

3.5.1. Choice of the vehicle

Due to the limited solubility of the test substance in water, DMSO 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 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% 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 45C, 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

15 g Difco bacto agar

20 g D-glucose, monohydrate.

After incubation at 370C for 48 - 72 hours in the dark, the bacterial colonies

(his" revertants) are counted.
BASF

Report; Project No.: 40M0437/034092 18

*Escherichia coi

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 mi 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 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 379C for 48 - 72 hours in the dark, the bacterial colonies

(trp* revertants) are counted.
BASF

Report; Project No.: 40M0437/034092 19

3.5.2.2. Prelnicubation 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 370C 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 10'6 in each case.

Test tubes containing 2-mi portions of soft agar containing maximal amino acid 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 mM histidine + 0.5 mM 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.
- I - -~V 3 1 L 1 I I 

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 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:

Wth S-9 mix 41

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

- 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.: 40M0437/034092 21

Without S-9 mix

* N-methyl-N'-nltro-N-nitros oguanidine (MNNG) (FLUKA, 68051) - 5 pg/plate, dissolved in DMSO

-strains: TA 1535, TA 100

9 4-nitro-o-phenylendiamine (NOPID) (SIGMA, N-9504)

- 10 jig/plate, dissolved in DMS0

- strain: TA 98

* 9-aminoacridine (AAC) (SIGMA, A-I 135) - 100 pg/plate, dissolved in DMSO

- - strain: TA 1537

* 4-nitroqui noline-N -oxide (4-NQO) (SIGMA, N-8141) - 5 pg/plate, dissolved in DM50S

- 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.
M111JJ1E N1 ILIILJILI ILLReport; Project No.: 40M0437/034092 22

3.5.5. Scope of tests and test conditions

1st Experiment

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

E. coi WP2 uvrA

Doses: 0; 23; 115; 575; 2 875 and 5 750 pg/plate

Vehicle: DMS0

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: DMS0

Type of test: Standard 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. coil WP2 uvrA

Doses: Salmonella strains:

0; 4; 20; 100; 500 and 1 500 pg/plate

E. coli WP2 uvrA:

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

Vehice: DMSO

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.: 40M0437/034092 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 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 I1st 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 tr 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 1ist

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.
.A4I~liJU1-1 11 iLJ 

BASF

Report; Project No.: 40M0437/034092 24

3.6.5. Acceptance criteria

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

* 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).

e 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).

* The titer of Viable bacteria was > 1 081m1.

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.: 40M0437/034092 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.
BASF

Report; Project No.: 40M0437/034092 26

4. RESULTS

(Tables I - 14)

The substance Alkali Blue Dry 61001/ Lot 2231308 was tested for mutagenicity in the

Salmonella typhimurium / Escherichia coi 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 I - 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. coil WP2 uvrA:

4.1.1.2. Tests with S-9 mix

TA 1535:

TA 100:

TA 1537: No increase in the number of his4 or trp* revertants.

TA 98:

E. coil WP2 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 in the number of his+ or trp4 revertants.

TA 98:

E. col 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
BASF

Report; Project No.: 40M0437/034092 28

4.2. TOXICITY

A bacteriotoxic effect (reduced background growth, decrease in the number of revertants,

reduction in the titer) was observed in the standard plate test depending on the strain and

test conditions from about 1 500 pg - 2 000 pg/plate onward (Salmonella strains) or at

doses '. 2 875 pg/plate (E. coil WP2 uvrA).

In the preincubation assay bacteriotoxicity (reduced background growth, decrease In the

number of revertants, reduction in the 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 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 /Lot 2231308 is not a mutagenic agent in a bacterial 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 for the Salmonella mutagenicity test.

Mut. Res., jj13. 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. 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 strains with R factor plasmids.

Proc. Nat. 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. Carc. Mut., 2, 287 - 295 (1989)

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

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. 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 GROWTH
iLLS 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

11 OP 10 7P

113P 1 05P

575 ug loop 100 5 0.9 102P 94 7 0.8

104P 8 9P

95P 9 OP

2 675 ug 03/P 143/P 23 9 0.2 0

03/P 253/P 0

031 P 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 GROWTH
BASF-Aktiengesellschaft -03

Experimental Toxicology and Ecology

STUDY DIRECTOR: DR. ENGELHARDT

PROJECT NO. 40M0437/034092

SUBSTANCE NAME: Alkali Blue Dry 6100 /Lot 2231308

SUBSTANCE NO. 03/043 7-2

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 1 5P

8P 9P

575 ug 12P 11 2 1.0 SP 13 6 1.6

12P 1 9P

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. D 3/0437-2

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

3 4P 33P

2 4P 35P

575 ug 31P 26 5 0.8 60P 47 12 1.1

24P 3 6P

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 GROWTH
BASF-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: PRECIPITATION
BASy 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 2 8.NOV.2003

METHOD S TANDARD 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 _

1 7P 12P

18P 17iP

1 000 ug lip 14 3 0.8 iSP 16 3 0.8

1 7P 1 9P

13 P 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: PRECIPITATION
BASF 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

11 3P 10oop

90P 10 7P

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

9 OP 1 lOP

95P 89?

2 000 ug 20P 14 9 0.1 40P 41 11 0.4 0

4P 52P 4

I 9P 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 GROWTH
BASF 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)

DOSE IPLATE 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 GROWTH
BASF 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

1 6P 1 4P

1 6P 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

OB IP 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 GROWTH
EAUY 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

3 4P 1 02P

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 GROWTH
BASF 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 GROWTH
BASF 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 GROWTH
BASF 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

2 4P 25P

23P 2 5P

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 ts2.0 ml 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

5 750 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

2 500 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

2 500 pg test substance No growth
BASF

Report; Project No.: 40M0437/034092

APPENDIX 4

S-9 FRACTION CHARACTERIZATION
S-9 fraction

Preparation of S-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 M REV* 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)

pool 11-4
BASF

Report; Project No.: 40M0437/034092

APPENDIX 5

HISTORICAL NEGATIVE CONTROL DATA
Historical Negative Control Data

Strain: TA 1535

.......... ... .... .... ........ ......... i io ~ ~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 10 TA 

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 stndr 1 test.90 Waate 40 94 5 13 1

PIT te9st0.3 Waeteralo 0 9 6 17 1

SD~~~8 eito =2tnad 
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: TA 98

. ~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 01.00.0 9.03 400 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 plate test

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: TA 98

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 IL PIT 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. crid ine

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