DISCLAIMER 

This report is the property of Clariant. 

Its content is confidential and not for general 

release. It is not allowed to cite and reveal the report 

or its content to a third party without prior written 

approval of Clariant. 

STUDY NUMBER 1234903 

GENE MUTATION ASSAY 

IN CHINESE HAMSTER V79 CELLS 

IN VITRO 

( V79/ HPRT) 

WITH 

Leuco Sulfur Black 1 

REPORT 

STUDY COMPLETION DATE: 

August07,2009 

harlanTM .... 

CONTAINS NO C~~ 

- l ~ -~ r;;n 7'--;::L 

-
Study Number 1234903 

Leuco Sulfur Black 1 

Report 

1 COPY OF GLP CERTIFICATE 

Gute Laborpraxis/Good Laboratory Practice 

GLP-Bescheinigung/Statement of GLP Compliance 

(gemlilliaccording to § l9b Abs. l Chemikaliengeset7.) 

Page 2 of 58 

HESS EN 

Eine GLP-lnspektion 7.ur Oberwachung der Einhaltung 

der GLP-Grundslltze gemlill Chemikaliengesetz bzw. 

Richtlinie 2004/9/EG wurde durchgeftlhrt in 

Assessment of conformity with GLP according to 

Chemikaliengeset7. and Directive 200419/EEC at: 

[83 PrUfeinrichtung/Test facility 0 PrUfstandort/Test site 

Harlan Cytotest Cell Research GmbH 

Harlan Cytotest Cell Research GmbH 

In den Leppsteinswiesen 19 

64380 RoBdorf 

(Unvcrwcchselbare Bezeichnung und AdresseiUncquivocal name and adress) 

PrOfungco oach KatL-gorien/Areas of Expertise 

(geml!Biaccording chemVwV-GLP Nr. 5.3/0ECD guidance) 

2 Pnlfungen zur llestimmung der toxikologischen 

Eigenschafte n 

3 Pnlfungen zur Bestimmung rler erbgutveriin 

dernden Eigenschaft<"n (in vitro und in vivo) 

6 PrQfungen zur llestimmung von RQckstanden 

I Analytisrhe Prufungen an biologischen Materialien 

2 Toxicity studies 

3 Mutagenicity studies 

6 Residues 

8 Analytical studies on biological materials 

15.08. und 27. - 29.10.2008 

Datum der lnspektion/Date of Inspection 

(Tag Monat Jahriday month year) 

Die genannte PrUfeinrichtung befmdet sich im nationalen GLP-Oberwachungsverfahren und wird regel· 

mllllig auf Einhaltung der GLP-Grundslltze Uberwacht 

Auf der Grundlage des lnspektionsberichtes wird hiermit 

bestlltigt, dass in dieser PrUfeinrichtung die oben ge· 

nannten PrUfungen unter Einhaltung der GLP· Grundsatze durchgetllhrt werden kl!nnen. 

The above mentioned test facility is included 

in the national GLP Compliance Programme and is 

inspected on a regular basis. 

Based on the inspection report it can be confirmed. 

that this test facility is able to conduct the 

aforementioned studies in compliance with the 

Principles of GLP. 

Energie, l..llndwir!Khafl uud Verbraucher.ochutz, 

80 065189 Wiesbaden &<11.-i~J ... ,P and address of the GLP Monitoring Authority 
Study Number 1234903 

Leuco Sulfur Black 1 

Report 

2 CONTENTS 

1 COPY OF GLP CERTIFICATE 

2 CONTENTS 

3 PREFACE 

3.1 General 

3.2 Responsibilities 

3.3 Schedule 

3.4 Project Staff Signatures 

3.5 Good Laboratory Practice 

3.6 Guidelines 

3.7 Archiving 

3.8 Deviations to the study plan 

4 STATEMENT OF COMPLIANCE 

5 STATEMENT OF QUALITY ASSURANCE UNIT 

6 SUMMARY 

7 OBJECTIVE 

7.1 Aims of the Study 

7.2 Relevance of the Test System 

8 MATERIALS AND METHODS 

8.1 Test Item 

8.2 Controls 

8.2.1 Solvent Controls 

8.2.2 Positive Control Substances 

8.3 Test System 

8.3.1 Reasons for the Choice of the Cell Line V79 

8.3.2 Cell Cultures 

8.4 Mammalian Microsomal Fraction S9 Mix 

8.4.1 S9 (Preparation by Harlan CCR) 

8.4.2 S9 Mix 

8.5 Pre-Test on Toxicity 

8.6 Dose Selection 

8.7 Experimental Performance 

8.7.1 Seeding 

8.7.2 Treatment 

8.8 Data Recording 

8.9 Acceptability of the Assay 

8.10 Evaluation of Results 

8.11 Statistical Analysis 

Page 3 of 58 

2 

3 

5 

5 

5 

5 

6 

6 

6 

6 

6 

7 

8 

9 

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11 

11 

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14 

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15 

15 

15 

17 

17 

17 

18 

18 

18 

19 
Study Number 1234903 

Leuco Sulfur Black 1 

CONTENTS CONTINUED 

Report 

9 CALCULATION AND PROCESSING OF THE DATA 

10 RESULTS AND DISCUSSION 

11 REFERENCES 

12 DISTRIBUTION OF THE REPORT 

13 ANNEX 1: TABLES OF RESULTS 

13.1 Pre-test 

13.2 Main Experiment 

13.2.1 Experiment I Culture I 

13.2.2 Experiment I Culture II 

13.2.3 Experiment II, Culture I 

13.2.4 Experiment II, Culture II 

14 ANNEX II: HISTORICAL DATA 

15 ANNEX Ill: COPY OF STUDY PLAN 

16 ANNEX IV: COPY OF FIRST AMENDMENT TO STUDY PLAN 

Page 4 of 58 

20 

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23 

23 

24 

24 

27 

30 

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36 

37 

53 
Study Number 1234903 

Leuco Sulfur Black 1 

3 PREFACE 

3.1 General 

Report Page 5 of 58 

Title: Gene Mutation Assay 

Sponsor: 

Study Monitor 

Test Facility: 

Contract Institute: 

Reference Number: 

3.2 Responsibilities 

Study Director: 

Deputy Study Director: 

Management: 

Head of Quality Assurance Unit: 

3.3 Schedule 

Experimental Starting Date: 

Experimental Completion Date: 

in Chinese Hamster V79 Cells 

in vitro (V79/HPRT) 

with Leuco Sulfur Black 1 

Clariant Products (Switzerland) Ltd. 

Rothausstrasse 61 

4132 Muttenz 1 

Switzerland 

Contact Person: Dr. H. W. Schneider 

Dr. Thomas Roth 

sec GmbH 

Mikroforum Ring 1 

55234 Wendelsheim 

Germany 

Harlan Cytotest Cell Research GmbH (Harlan CCR) 

In den Leppsteinswiesen 19 

64380 Rossdorf 

Germany 

Harlan Laboratories Ltd. 

4452 ltingen 

Switzerland 

C31133 

Dr. Hans-Eric Wollny 

Dipl. Bioi. Andrea Sokolowski 

Dr. Wolfgang Volkner 

Frauke Hermann 

May 12, 2009 

June 23, 2009 
Study Number 1234903 

Leuco Sulfur Black 1 

Report 

3.4 Project Staff Signatures 

Study Director 

Management Dr. Wolfgang Volknerr---

Page 6 of 58 

... _\~,J .. \. :, \, ..•........... [ .... · f, F'oth 

Date: August 07, 2009 

3.5 Good Laboratory Practice 

The study was performed in compliance with: 

"Chemikaliengesetz" (Chemicals Act) of the Federal Republic of Germany, "Anhang 1" (Annex 1 ), version dated July 02, 2008 ("BGBI.", p. 1146)." 

"OECD Principles of Good Laboratory Practice", as revised in 1997 [C(97)186/Final]. 

3.6 Guidelines 

This study was conducted according to the procedures indicated by the following internationally accepted guidelines and recommendations: 

Ninth Addendum to the OECD Guidelines for the Testing of Chemicals, February 1998, 

adopted July 21, 1997, Guideline No. 476 "In vitro Mammalian Cell Gene Mutation Test". 

Commission Regulation (EC) No. 440/2008 8.17, dated May 30, 2008. 

3. 7 Archiving 

Harlan CCR will archive the following data for 15 years: 

Raw data, study plan, report, and a sample of the test item. 

No data will be discarded without the sponsor's prior written consent. 

3.8 Deviations to the study plan 

There were no deviations to the study plan. 
Study Number 1234903 

Leuco Sulfur Black 1 

Report 

4 STATEMENT OF COMPLIANCE 

Study Number: 

Test Item: 

Study Director: 

Title: 

1234903 

Leuco Sulfur Black 1 

Dr. Hans-Eric Wollny 

Gene Mutation Assay 

in Chinese Hamster V79 Cells 

in vitro (V79/HPRT) 

with Leuco Sulfur Black 1 

Page 7 of 58 

This study performed in the test facility of Harlan CCR was conducted in compliance with 

Good Laboratory Practice Regulations: 

"Chemikaliengesetz" (Chemicals Act) of the Federal Republic of Germany, "Anhang 1" (Annex 1), dated July 25, 1994 ("BGBI. 11994", pp. 1703), last revision June 27,2002. 

"OECD Principles of Good Laboratory Practice", as revised in 1997 [C(97)186/Final). 

There were no circumstances that may have affected the quality or integrity of the study. 

Study Director Harlan CCR 

Dr. Hans-Eric Wollny 

l

' ,,j(. '-1 

[, L L~- I 

Date: August 07, 2009 
Study Number 1234903 

Leuco Sulfur Black 1 

Report Page 8 of 58 

5 STATEMENT OF QUALITY ASSURANCE UNIT 

Study Number: 1234903 

Test Item: Leuco Sulfur Black 1 

Study Director: Dr. Hans-Eric Wollny 

Title: Gene Mutation Assay 

in Chinese Hamster V79 Cells 

in vitro (V79/HPRT) with Leuco Sulfur Black 1 

The general facilities and activities of Harlan CCR are inspected periodically and the results are reported to the responsible person and the Management. 

Study procedures were inspected periodically. The study plan and this report were audited 

by the Quality Assurance Unit. The dates are given below. 

I i Phases and Dates of QAU Inspections/ Audits 

II; ·-·--·-.... -... Study Plan: 

151 Amendment to Study Plan: 

i 

March 05, 2009 

Dates of Reports to the Study 

Director and to Management 

March 05, 2009 

[ Pro~~~~ ~~~~ection 

June 19, 2009 

June 24, 2009 

June 19, 2009 

June 24, 2009 .. ·-~--~--- -~j 

Test Performance: 

Report: 

April 28, 2009 

July 23, 2009 

April 28, 2009 

July 23, 2009 

This statement is to confirm that the present report reflects the raw data. 

Head of Quality Assurance Unit . Frauke Hermann 

... Man.uella. I ho.m.s.~.o .................................... . 

Date: August 07, 2009 

I 
Study Number 1234903 

Leuco Sulfur Black 1 

6 SUMMARY 

Report Page 9 of 58 

The study was performed to investigate the potential of Leuco Sulfur Black 1 to induce 

gene mutations at the HPRT locus in V79 cells of the Chinese hamster. 

The assay was performed in two independent experiments, using two parallel cultures 

each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a 

treatment time of 4 hours with and 24 hours without metabolic activation. 

The highest applied concentration (500 j.lg/ml) was limited by the solubility properties of 

the test item in aqueous medium. 

The tested concentrations are described in Table 2 (page 16). The evaluated experimental points and the results are summarised in Table 1 (page 10). 

No substantial and reproducible dose dependent increase of the mutation frequency was 

observed in both main experiments. 

Appropriate reference mutagens, used as positive controls, induced a distinct increase in 

mutant colonies and thus, showed the sensitivity of the test item and the activity of the 

metabolic activation system. 

Conclusion 

In conclusion it can be stated that under the experimental conditions reported the test 

item did not induce gene mutations at the HPRT locus in V79 cells. 

Therefore, Leuco Sulfur Black 1 is considered to be non-mutagenic in this HPRT assay. 
Study Number 1234903 

Leuco Sulfur Black 1 

Table 1: Summary of results 

cone. 11g S9 

per mL mix 

column 1 2 

Experiment I, 4 h treatment 

Solvent control with water - Positive control with EMS 150.0 -

Test item 15.6 -

Test item 31.3 - Test item 62.5 -

Test item 125.0 -

Test item 250 (p) -

Test item 500 (p) 

Solvent control with water + 

Positive control with DMBA 1.1 + 

Test item 15.6 + 

Test item 31.3 + 

Test item 62.5 + 

Test item 125 (p) + 

Test item 250 (p) + 

Test item 500 (p) + 

Experiment II 24 h treatment 

Solvent control with water 

Positive control with EMS 75.0 

Test item 15.6 

Test item 31.3 

Test item 62.5 

Test item 125.0 

Test item 250 (p) 

Test item 500 (p) 

Experiment II, 4 h treatment 

Solvent control with water + 

Positive control with DMBA 1.1 + 

Test item 15.6 + 

Test item 31.3 + 

Test item 62.5 + 

Test item 125 (p) + 

Test item 250 (P) + 

Test item 500 (p) + 

relative 

cloning 

efficiency I 

% 

3 

100.0 

116.4 

97.1 

71.3 

59.3 

59.4 

44.2 

29.3 

100.0 

45.9 

67.0 

70.8 

50.5 

44.0 

32.8 

39.9 

100.0 

107.3 

109.5 

109.4 

94.9 

102.3 

62.2 

16.9 

100.0 

36.5 

106.3 

94.5 

82.2 

78.5 

79.9 

74.4 

Report 

relative mutant 

cloning colonies/ induction 

efficiency II 106 cells factor 

% 

4 5 6 

culture I 

100.0 25.8 1.0 

65.6 133.3 5.2 

culture was not continued' 

105.4 14.5 0.6 

105.4 12.0 0.5 

103.2 11.9 0.5 

102.1 15.7 0.6 

81.3 18.4 0.7 

100.0 29.1 1.0 

92.6 891.7 30.6 

culture was not continued' 

65.1 13.3 0.5 

63.7 19.5 0.7 

95.1 13.5 0.5 

63.3 22.6 0.8 

107.4 23.3 0.8 

culture I 

100.0 17.8 1.0 

79.4 255.7 14.4 

culture was not continued' 

63.2 22.9 1.3 

62.9 19.1 1.1 

73.8 23.1 1.3 

77.6 18.4 1.0 

63.2 12.4 0.7 

100.0 I 15.2 I 1.0 

68.1 744.4 48.9 

culture was not continued' 

90.1 20.7 1.4 

65.1 15.3 1.0 

92.8 12.6 0.8 

89.8 16.9 1.1 

95.0 21.4 1.4 

Page 10 of 58 

relative relative mutant 

cloning cloning colonies/ induction 

efficiency I efficiency II 106 cells factor 

% % 

7 8 9 10 

culture II 

100.0 100.0 I 9.4 I 1.0 

102.3 79.9 110.0 11.7 

64.6 culture was not continued' 

90.1 82.9 15.8 1.7 

98.2 85.8 27.7 3.0 

70.8 76.4 28.7 3.1 

64.2 64.5 24.2 2.6 

37.0 60.3 23.9 2.5 

100.0 100.0 18.1 1.0 

40.6 71.2 1421.6 78.6 

76.3 culture was not continued' 

67.6 90.6 17.8 1.0 

39.0 90.3 18.5 1.0 

36.2 68.9 10.6 0.6 

24.1 93.9 21.9 1.2 

24.8 72.5 18.5 1.0 

culture II 

100.0 100.0 15.8 1.0 

98.0 86.7 383.9 24.2 

105.4 culture was not continued' 

111.6 85.9 20.1 1.3 

96.3 71.5 35.7 2.3 

100.5 91.5 36.1 2.4 

76.8 68.3 32.5 2.1 

9.4 70.1 19.0 1.2 

100.0 100.0 11.1 I 1.0 

24.2 91.0 841.6 75.6 

82.8 culture was not continued' 

78.9 119.6 12.2 1.1 

77.1 100.3 18.0 1.6 

68.2 79.7 23.3 2.1 

54.2 61.8 22.3 2.0 

54.9 68.4 12.1 1.1 

# culture was not continued since a minimum of only four analysable concentrations is required 

P = precipitation 
Study Number 1234903 

Leuco Sulfur Black 1 

7 OBJECTIVE 

7.1 Aims of the Study 

Report Page 11 of 58 

This in vitro assay was performed to assess the potential of the test item to induce gene 

mutations by means of two independent HPRT experiments using the Chinese hamster 

cell line V79. The treatment time was 4 hours in the first experiment with and without 

metabolic activation. The experimental part with metabolic activation was terminated prior 

to the generation of any data on mutagenicity due to microbial contamination of the cultures. This experimental part was repeated in experiment lA. The data of the repeat experiment are reported under experiment I with metabolic activation. In the second 

experiment the cells were exposed to the test item for 4 hours with and 24 hours without 

metabolic activation. 

7.2 Relevance of the Test System 

In vitro methods are valuable when it is desired to accurately control the concentration and 

exposure time of cells to the test item under study. However, the limited capacity of metabolic activation of potential mutagens requires an exogenous metabolic activation system. 

This in vitro test is an assay for the detection of forward gene mutations in mammalian 

cells. Gene mutations are discussed as an initial step in the carcinogenic process (2). 

The V79 cells are exposed to the test item both with and without exogenous metabolic 

activation. At a defined time interval after treatment the descendants of the treated original 

population are monitored for the loss of functional HPRT enzyme. 

HPRT (hypoxanthine-guanine phosphoribosyl transferase) catalyzes the conversion of the 

nontoxic 6TG (6-thioguanine) to its toxic ribophosphorylated derivative. Therefore, cells 

deficient in HPRT due to a forward mutation are resistant to 6TG. These cells are able to 

proliferate in the presence of 6TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The 

phenotypic expression is achieved by allowing exponential growth of the cells for 7 - 9 

days. The expression period is terminated by adding 6TG to the culture medium (3). 

Mutant frequency is determined by seeding known numbers of cells in medium containing 

the selective agent to detect mutant cells, and in medium without selective agent to determine the surviving cells. After a suitable period the colonies are counted. Mutant frequencies are calculated from the number of mutant colonies corrected for cell survival. 

In order to establish a concentration response effect of the test item at least four concentration levels are tested. These concentration levels should yield a concentration related 

toxic effect. The highest concentration level should induce a reduced level of survival. 

To demonstrate the sensitivity of the test system reference mutagens are tested in parallel 

to the test item. 
Study Number 1234903 

Leuco Sulfur Black 1 

Report Page 12 of 58 

8 MATERIALS AND METHODS 

8.1 Testltem 

Internal Test Item Number: S 973811 

The test item and the information concerning the test item were provided by the sponsor. 

Identification: 

CAS No.: 

Description: 

Batch No.: 

Composition: 

Molecular Weight: 

Stability of Test Item: 

Storage: 

Expiry Date: 

Leuco Sulfur Black 1 

66241-11-0 

Thixotrophic, black, wet paste 

ESA0007595 

Leuco Sulfur Black 1: 

2,4-Dinitrochlorobenzene: 

2,4-Dinitrophenole: 

Sulfate: 

Chloride: 

Sodium: 

Water: 

52.75% (calculated by amount of 

by-compounds) 

< 20 ppm 

< 20 ppm 

970 ppm 

750 ppm 

1.3% (w/w) 

45.75% (w/w) by Karl-Fischer 

titration 

Not applicable, because test item is an UVCB substance 

Stable under storage conditions 

At room temperature (range 20 +/- 5o C), light protected 

July 11, 2011 

On the day of the experiment (immediately before treatment), the test item was suspended in 

deionised water. The final concentration of deionised water in the culture medium was 10 % 

v/v. 

The osmolarity and pH-value were determined in the solvent control and the maximum concentration of the pre-experiment without metabolic activation: 

solvent control Leuco Sulfur Black 1 

500 IJQ/ml 

Osmolarity mOsm 282 284 

pH-value 7.41 7.37 
Study Number 1234903 

Leuco Sulfur Black 1 

8.2 Controls 

8.2.1 Solvent Controls 

Report Page 13 of 58 

Concurrent solvent control (water) were performed. 

8.2.2 Positive Control Substances 

8.2.2.1 Without metabolic activation 

Name: 

Supplier: 

Purity: 

Lot no.: 

Expiry date: 

Dissolved in: 

Final concentration: 

EMS; ethylmethane sulfonate 

ACROS ORGANICS, 2440 Geel, Belgium 

~98% 

A0259466 

March 2010 

Nutrient medium 

0.150 mg/mL = 1.2 mM (experiment I) 

0.075 mg/mL = 0.6 mM (experiment II) 

8.2.2.2 With metabolic activation 

Name: 

Supplier: 

Lot no.: 

Expiry date: 

Dissolved in 

Supplier: 

Final concentration: 

DMBA; 7,12-dimethylbenz(a)anthracene 

SIGMA CHEMIE GMBH, 82041 Deisenhofen, Germany 

096K1881 

July 2012 

DMSO, Dimethylsulfoxide; 

(E. MERCK, 64293 Darmstadt; Germany, Purity 99.5 %) 

final concentration in nutrient medium 0,5 % 

1.1 IJg/mL = 4.3 1-1M 

The dilutions of the stock solutions were prepared on the day of the experiment and used 

immediately. 

The stability of both positive control substances in solution is unknown, but a mutagenic 

response in the expected mutation range is sufficient evidence of biological stability. The 

dilutions of the stock solutions were prepared on the day of the experiment and used immediately. 
Study Number 1234903 

Leuco Sulfur Black 1 

8.3 Test System 

Report 

8.3.1 Reasons for the Choice of the Cell Line V79 

Page 14 of 58 

The V79 cell line has been used successfully in in vitro experiments for many years. 

Especially the high proliferation rate (doubling time 12- 16 h in stock cultures) and a good 

cloning efficiency of untreated cells (as a rule more than 50 %) both necessary for the 

appropriate performance of the study, recommend the use of this cell line. The cells have 

a stable karyotype with a modal chromosome number of 22 (3). 

8.3.2 Cell Cultures 

Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing; Technical University; 64287 Darmstadt, Germany) are stored in liquid nitrogen in the cell bank of 

Harlan CCR allowing the repeated use of the same cell culture batch in experiments. 

Before freezing, the level of spontaneous mutants was depressed by treatment with HATmedium as described in [3]. Each batch is screened for mycoplasma contamination and 

checked for karyotype stability and spontaneous mutant frequency. Consequently, the 

parameters of the experiments remain similar because of the reproducible characteristics 

of the cells. 

Thawed stock cultures are propagated at 37 oc in 80 cm2 plastic flasks (GREINER, 

72632 Frickenhausen, Germany). About 5· 105 cells are seeded into each flask with 

15 mL of MEM (minimal essential medium; SEROMED, 12247 Berlin, Germany) supplemented with 10% fetal calf serum (FCS; PAA Laboratories GmbH, 35091 Colbe, Germany) and 1 % neomycin. The cells are subcultured twice weekly. The cell cultures are 

incubated at 37 oc in a 4.5% carbon dioxide atmosphere (95.5% air). 

For the selection of mutant cells the medium is supplemented with 11 IJg/mL thioguanine 

(6TG, SIGMA GmbH, 82041 Deisenhofen, Germany). 

8.4 Mammalian Microsomal Fraction 59 Mix 

Lacking metabolic activities of cells under in vitro conditions are a disadvantage of assays 

with cell cultures as many chemicals only develop a mutagenic potential when they are 

metabolized by the mammalian organism. However, metabolic activation of chemicals can 

be achieved at least partially by supplementing the cell cultures with mammalian liver 

microsome preparations (S9 mix). 
Study Number 1234903 

Leuco Sulfur Black 1 

Report 

8.4.1 S9 (Preparation by Harlan CCR) 

Page 15 of 58 

Phenobarbital/j3-Naphthoflavone induced rat liver S9 was used as the metabolic activation 

system. The S9 is prepared from 8- 12 weeks old male Wistar HsdCpb:WU rats(Harlan 

Laboratories GmbH, 33178 Borchen, Germ<;~ny), weight approx. 220- 320 g induced by 

applications of 80 mg/kg b.w. Phenobarbital i.p. (Desitin; 22335 Hamburg, Germany) and 

f3-Naphthoflavone p.o. (Aldrich, 89555 Steinheim, Germany) each on three consecutive 

days. The livers were prepared 24 hours after the last treatment. The S9 fractions were 

produced by dilution of the liver homogenate with a KCI solution (1+3) followed by centrifugation at 9000 g. Aliquots of the supernatant were frozen and stored in ampoules at80° C. Small numbers of the ampoules were kept at -20°C for up to one week. 

The protein concentration in the S9 preparation was 35.6 mg/mL (Lot No.: 270309) in the 

pre-experiment and both main experiments. 

8.4.2 S9 Mix 

An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution 

to result in a final protein concentration of 0. 75 mg/mL in the cultures. Cofactors were added to the S9 supernatant to reach following concentrations in the S9 mix: 

8 mM MgCI2 

33 mM KCI 

5 mM glucose-6-phosphate 

4 mM NADP 

in 100 mM sodium-ortho-phosphate-buffer, pH 7.4. 

During the experiment, the S9 mix was stored in an ice bath. The S9 mix preparation was 

performed according to Ames et al. (1). 

8.5 Pre-Test on Toxicity 

A pre-test was performed in order to determine the concentration range for the mutagenicity experiments. The general culture conditions and experimental conditions in this pre-test 

were the same as described for the mutagenicity experiment below. In this pre-test the 

colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item was observed and compared to the controls. 

Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE). 

8.6 Dose Selection 

According to the recommendations of the guidelines (see page 6), several concentrations 

(usually at least four) of the test item should be used. These should yield a concentrationrelated toxic effect. The highest concentration should produce a low level of survival and 

the survival in the lowest concentration should approximate the solvent control. Relatively 

insoluble substances should be tested up to their limit of solubility under culture conditions. 

For freely-soluble nontoxic substances the maximum concentration should be 5 mg/mL or 

1 0 mM. If the maximal concentration is based on cytotoxicity the cloning efficiency should 

be reduced to less than 50 % and/or culture growth at subcultivation should be at least 

20 % of the corresponding solvent control. 
Study Number 1234903 

Leuco Sulfur Black 1 

Report Page 16 of 58 

The concentration range of the pre-test was limited by the solubility of the test item in 

water and aqueous media. Test item concentrations between 3.9 and 500 IJg/mL were 

used to evaluate toxicity in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Relevant cytotoxic effects were noted at 250 and 500 IJg/mL 

without metabolic activation. In the presence of metabolic activation no cytotoxicity occurred up to the maximum concentration. 

The test medium of the pre-experiment was checked for precipitation at the end of each 

treatment period (4 or 24 hours) just prior to removal of the test item. Precipitation of the 

test item visible to the unaided eye occurred in at 125 IJg/mL and above following 4 hour 

treatment with and without metabolic activation. Following continuous treatment for 24 

hours without metabolic activation precipitation occurred at 250 IJg/mL and above. 

The osmolarity and pH-value of the medium was measured at the maximal concentration 

and the solvent control and showed no relevant deviation. 

Based on the results of the pre-experiment, the concentration range of the main experiments was selected. In both main experiments the individual concentrations were spaced 

by a factor of 2 and covered soluble and precipitating concentrations. 

Table 2: Doses applied in the gene mutation assay with Leuco Sulfur Black 1: 

concentrations in IJglmL 

Experiment I 

without S9 mix 15.6 31.3 62.5 125.0 250.0 500.0 

with S9 mix 15.6 31.3 62.5 125.0 250.0 500.0 

Experiment II 

without S9 mix 15.6 31.3 62.5 125.0 250.0 500.0 

with S9 mix 15.6 31.3 62.5 125.0 250.0 500.0 

The cultures at the lowest concentration (15.6 IJg/mL) were not continued since a minimum of only four analysable concentrations is required by the guidelines. 
Study Number 1234903 

Leuco Sulfur Black 1 

Report 

8. 7 Experimental Performance 

8.7.1 Seeding 

Page 17 of 58 

Two days old (experiment I with S9 mix and experiment II with and without S9 mix) and 

three days old (experiment I without S9 mix) exponentially growing stock cultures (more 

than 50 % confluent) were trypsinized at 37 oc for 5 minutes. Then the enzymatic digestion was stopped by adding complete culture medium and a single cell suspension was 

prepared. The trypsin concentration for all subculturing steps was 0.2% in Ca-Mg-free 

salt solution (Trypsin: Difco Laboratories, Detroit, USA). 

The Ca-Mg-free salt solution had the following constituents (per litre): 

NaCI 8000 mg 

KCI 400 mg 

Glucose 1000 mg 

NaHC03 350 mg 

Prior to the trypsin treatment the cells were rinsed with Ca-Mg-free salt solution containing 

200 mg/1 EDTA (ethylenediamine tetraacetic acid). 

The cell suspension was seeded into plastic culture flasks (Greiner, 

72632 Frickenhausen, Germany). Approximately 1.5· 106 (single culture) and 5· 102 cells 

(in duplicate) were seeded in MEM with 10% FCS (complete medium) for the determination of mutation rate and toxicity, respectively. 

8. 7.2 Treatment 

After 24 h the medium was replaced with serum-free medium containing the test item, 

either without S9 mix or with 50 IJI/ml S9 mix. Concurrent solvent and positive controls 

were treated in parallel. After 4 h this medium was replaced with complete medium following two washing steps with "saline G". In the second experiment the cells were exposed to 

the test item for 24 h in complete medium in the absence of metabolic activation. 

The "saline G" solution had the following constituents (per litre): 

NaCI 8000 mg 

KCI 400 mg 

Glucose 11 00 mg 

Na2HP04· 7H20 290 mg 

KH2P04 150 mg 

The pH was adjusted to 7.2 

The colonies used to determine the cloning efficiency (survival) were fixed and stained 

approx. 7 days after treatment as described below. 

Three days after treatment 1. 5· 1 06 cells per experimental point were subcultivated in 

175 cm2 flasks containing 30 ml medium. Following the expression time of 6 days five 80 

cm2 cell culture flasks were seeded with about 3 - 5· 105 cells each in medium containing 

6-TG. Two additional 25 cm2 flasks were seeded with approx. 500 cells each in nonselective medium to determine the viability. 
Study Number 1234903 

Leuco Sulfur Black 1 

Report Page 18 of 58 

The cultures were incubated at 37 oc in a humidified atmosphere with 4.5 % C02 for about 

8 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution (E. 

MERCK, 64293 Darmstadt, Germany). 

The stained colonies with more than 50 cells were counted. In doubt the colony size was 

checked with a preparation microscope (Nikon, 40407 Dusseldorf, Germany). 

8.8 Data Recording 

The data generated were recorded in the raw data. The results are presented in tabular 

form, including experimental groups with the test item, solvent, and positive controls. 

8.9 Acceptability of the Assay 

The gene mutation assay is considered acceptable if it meets the following criteria: 

the numbers of mutant colonies per 106 cells found in the solvent controls fall within 

the laboratory historical control data range (see Annex II). 

the positive control substances must produce a significant increase in mutant colony 

frequencies (see Annex II, Historical data). 

the cloning efficiency II (absolute value) of the solvent controls must exceed 50%. 

The data of this study comply with the above mentioned criteria (see Annex I (Tables of 

Results, mutation rate and factor calculated referring to the C.E. of the untreated cultures) 

and Annex II (Historical data)]. 

8.10 Evaluation of Results 

A test item is classified as positive if it induces either a concentration-related increase of 

the mutant frequency or a reproducible and positive response at one of the test points. 

A test item producing neither a concentration- related increase of the mutant frequency 

nor a reproducible positive response at any of the test points is considered non-mutagenic 

in this system. 

A positive response is described as follows: 

A test item is classified as mutagenic if it reproducibly induces a mutation frequency that 

is at least three times above the spontaneous mutation frequency at least at one of the 

concentrations in the experiment. 

The test item is classified as mutagenic if there is a reproducible concentration-related 

increase of the mutation frequency. Such evaluation may be considered also in the case 

that a threefold increase of the mutant frequency is not observed. 
Study Number 1234903 

Leuco Sulfur Black 1 

Report Page 19 of 58 

However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate in 

the range normally found (0.6- 31.7 mutants per 106 cells) a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into 

consideration. 

8.11 Statistical Analysis 

A linear regression (least squares) was performed to assess a possible dose dependent 

increase of mutant frequencies using SYSTA~11 (SYSTAT Software, Inc., 501, Canal 

Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant 

colonies obtained for the groups treated with the test item were compared to the solvent 

control groups. A trend is judged as significant whenever the p-value (probability value) is 

below 0.05. However, both, biological and statistical significance were considered together. 

experimental group p-value 

experiment I, culture I without S9 mix 0.983 

experiment I, culture II without S9 mix 0.424 

experiment I, culture I with S9 mix 0.733 

experiment I, culture II with S9 mix 0.686 

experiment II, culture I without S9 mix 0.088 

experiment II, culture II without S9 mix 0.871 

experiment II, culture I with S9 mix 0.309 

experiment II, culture II with S9 mix 0.983 
Study Number 1234903 

Leuco Sulfur Black 1 

Report Page 20 of 58 

9 CALCULATION AND PROCESSING OF THE DATA 

The data listed in the tables (Annex I) are calculated and processed as described below . 

. Pre-test 

cloning efficiency, absolute 

cloning efficiency, relative 

Main test 

Cloning efficiency I (survival): 

Cloning efficiency II (viability): 

cloning efficiency I 

(survival, absolute): 

cloning efficiency I 

(survival, relative): 

cell density % of control 

cloning efficiency II 

(viability, absolute): 

cloning efficiency II 

(viability, relative): 

cells survived (after plating in TG 

containing medium) 

mutant colonies /1 06 cells: 

induction factor: 

mean number of colonies per flask divided by the 

number of cells seeded· 100 

(mean number of colonies per flask divided by the 

mean number of colonies per flask of the corresponding control) · 100 

cloning efficiency determined immediately after 

treatment to measure toxicity. 

cloning efficiency determined after the expression 

period to measure viability of the cells without 

selective agent. 

mean number of colonies per flask divided by the 

number of cells seeded 

(mean number of colonies per flask divided by the 

mean number of colonies per flask of the corresponding control) · 100 

(cell density at 1st subcultivation divided by the cell 

density at 1st subcultivation of the corresponding 

control) · 1 00 

mean number of colonies per flask divided by the 

number of cells seeded 

cloning efficiency II absolute divided by the cloning 

efficiency II absolute of the corresponding control · 

100 

number of cells seeded · cloning efficiency II absolute 

mean number of mutant colonies per flask found 

after plating in TG medium · 106 divided by the 

number of cells survived 

mutant colonies per 1 06 cells I mutant colonies per 

106 cells of the corresponding solvent control 
Study Number 1234903 

Leuco Sulfur Black 1 

Report 

10 RESULTS AND DISCUSSION 

Page 21 of 58 

The test item Leuco Sulfur Black 1 was assessed for its potential to induce gene mutations 

at the HPRT locus using V79 cells of the Chinese hamster. 

The study was performed in two independent experiments, using identical experimental 

procedures. In the first experiment the treatment period was 4 hours with and without 

metabolic activation. The second experiment was performed with a treatment time of 4 

hours with and 24 hours without metabolic activation. 

The cell cultures were evaluated at the following concentrations: 

Experiment 1: 

without S9 mix: 

with S9 mix: 

Experiment II: 

without S9 mix: 

with S9 mix: 

31.3; 62.5; 125.0; 250.0; and 500.0 IJg/mL 

31.3; 62.5; 125.0; 250.0; and 500.0 IJg/mL 

31.3; 62.5; 125.0; 250.0; and 500.0 IJg/mL 

31.3; 62.5; 125.0; 250.0; and 500.0 IJg/mL 

Precipitation of the test item was observed at 125 j.Jg/ml and above with and at 

250 IJg/mL and above without metabolic activation. 

Relevant cytotoxic effects defined as a relative cloning efficiency I below 50% in both 

parallel cultures occurred at 500 IJglmL in both experiments without metabolic activation. 

In the presence of metabolic activation cytotoxic effects as described above were noted at 

125 IJg/mL and above in the first experiment. 

No relevant and reproducible increase in mutant colony numbers/1 06 cells was observed in 

the main experiments up to the maximum concentration. All mutant frequencies remained 

well within the historical range of solvent controls. The induction factor was equal to or 

exceeded slightly the threshold of three times the corresponding solvent control in the 

second culture of experiment I without metabolic activation at 62.5 and 125.0 j.Jg/ml (induction factor of 3.0 and 3.1, respectively). This marginal effect was judged as biologically 

irrelevant since it was based upon the rather low solvent control of 9.4 mutant colonies/1 06 

cells. Furthermore, the increase was not reproduced in the parallel culture under identical 

conditions and not dose dependent as indicated by a lacking statistical significance 

A linear regression analysis (least squares) was performed to assess a possible dose 

dependent increase of mutant frequencies using SYSTAT® statistics software. No significant dose dependent trend of the mutation frequency indicated by a probability value of 

<0.05 was determined in any of the experimental groups. 

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 9.4 up to 29.1 mutant colonies per 106 cells; the range of the groups treated 

with the test item was from 11.9 up to 38.1 mutants per 1 06 cells. 

EMS (0.15 mg/ml in experiment I and 0.075 mg/ml in experiment II) and DMBA 

(1.1 IJg/mL) were used as positive controls and showed a distinct increase in induced 

mutant colonies. 
Study Number 1234903 

Leuco Sulfur Black 1 

Conclusion 

Report Page 22 of 58 

In conclusion it can be stated that under the experimental conditions reported the test 

item did not induce gene mutations at the HPRT locus in V79 cells. 

Therefore, Leuco Sulfur Black 1 is considered to be non-mutagenic in this HPRT assay. 

11 REFERENCES 

1. B.N. Ames, J. McCann, and E. Yamasaki 

Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test 

In: B.J. Kilbey et al. (Eds.) "Handbook of Mutagenicity Test Procedures", Elsevier, 

Amsterdam, 1-17, 1977 

2. P. Howard-Flanders 

Mutagenesis in mammalian cells 

Mutation Research 86, 307-327, 1981 

3. M.O. Bradley, B. Bhuyan, M.C. Francis, R. Langenbach,.A. Peterson and E. Huberman 

Mutagenesis by chemical agents in V79 Chinese hamster cells: a review and analysis 

of the literature. A report of the gene-tax program 

Mutation Research 87, 81-142, 1981 

12 DISTRIBUTION OF THE REPORT 

Study Monitor 

Study Director 

2x copy 

1x original 
Study Number 1234903 

Leuco Sulfur Black 1 

13 ANNEX 1: TABLES OF RESULTS 

13.1 Pre-test 

Table 3: Toxicity data 

cone. S9 Duration of cells 

mix treatment seeded 

Test group (h) 1111 

Column 1 2 3 4 

Solvent control . 4 509 

Test item 3.9 . 4 509 Test item 7.8 . 4 509 

Test item 15.6 . 4 509 Test item 31.3 . 4 509 

Test item 62.5 . 4 509 

Test item 125.0 . 4 509 Test item 250.0 . 4 509 

Test item 500.0 . 4 509 

Solvent control + 4 509 

Test item 3.9 + 4 509 

Test item 7.8 + 4 509 

Test item 15.6 + 4 509 

Test item 31.3 + 4 509 

Test item 62.5 + 4 509 

Test item 125.0 + 4 509 

Test item 250.0 + 4 509 

Test item 500.0 + 4 509 

Solvent control - 24 509 

Test item 3.9 - 24 509 

Test item 7.8 - 24 509 

Test item 15.6 - 24 509 

Test item 31.3 - 24 509 

Test item 62.5 - 24 509 

Test item 125.0 - 24 509 

Test item 250.0 - 24 509 Test item 500.0 - 24 509 

- -- -

Report 

number of colonies per flask CE% CE% 

found absolute relative I pr

I II mean 

5 6 7 8 9 

461 389 425 83.5 100.0 

511 486 499 97.9 117.3 

323 406 365 71.6 85.8 

454 428 441 86.6 103.8 

328 279 304 59.6 71.4 

363 269 316 62.1 74.4 

259 265 262 51.5 61.6 pr

103 225 164 32.2 38.6 I pr

9 6 8 1.5 1.8 I pr

499 491 495 97.2 100.0 

508 449 479 94.0 96.7 

477 508 493 96.8 99.5 

440 447 444 87.1 89.6 

438 386 412 80.9 83.2 

368 318 343 67.4 69.3 

380 401 391 76.7 78.9 pr

490 429 460 90.3 92.8 I pr

382 335 359 70.4 72.4 'pr

482 500 491 96.5 100.0 

506 446 476 93.5 96.9 

459 427 443 87.0 90.2 

422 393 408 80.1 83.0 423 381 402 79.0 81.9 

397 365 381 74.9 77.6 

351 271 311 61.1 63.3 

142 189 166 32.5 33.7 ! pr

5 L__ 6 6 1 .1 1 .1 , pr
Study Number 1234903 

Leuco Sulfur Black 1 

13.2 Main Experiment 

13.2.1 Experiment I Culture I 

Table 4: Cloning Efficiency I (Survival), culture I 

cells 

cone. S9 seeded 

J.Jg/ml mix 1111 

column 1 2 3 

Solvent control with water - 509 

Positive control with EMS 150.0 - 509 

Test item 15.6 - 509 Test item 31.3 - 509 Test item 62.5 - 509 Test item 125.0 - 509 Test item 250.0 - 509 

Test item 500.0 - 509 

Solvent control with water + 512 

Positive control with DMBA 1.1 + 512 

Test item 15.6 + 512 

Test item 31.3 + 512 

Test item 62.5 + 512 

Test item 125.0 + 512 

Test item 250.0 + 512 

Test item 500.0 + 512 

- ---

Report 

number of colonies per flask CEI 

found absolute 

I Jl mean 

4 5 6 7 

351 312 332 0.65 

440 332 386 0.76 

282 362 322 0.63 

249 224 237 0.46 205 188 197 0.39 207 187 197 0.39 

149 144 147 0.29 123 71 97 0.19 

278 304 291 0.57 

141 126 134 0.26 

173 217 195 0.38 

212 200 206 0.40 

149 145 147 0.29 

131 125 128 0.25 

81 110 96 0.19 

122 11Q L__________jj§ 0.23 -

# culture was not continued since a minimum of only four analysable concentrations is required 

CEI cells/ml c

relative at 1st 

% subcultivation o

8 9 

100.0 3818000 116.4 3861000 

97.1 culture was not co

71.3 4325000 

59.3 2700000 

59.4 2300000 

44.2 2297000 

29.3 3437000 

100.0 3980000 

45.9 2656000 

67.0 culture was not co

70.8 4770000 

50.5 4070000 

44.0 4112000 

32.8 4015000 

39.9 3801000 
Study Number 1234903 

Leuco Sulfur Black 1 

Report 

Table 5: Cloning Efficiency II (Viability), experiment I, culture I 

cells number of colonies per flask 

cone. S9 seeded found 

IJQ/ml mix 1111 I II mean 

column 1 2 3 4 5 6 

Solvent control with water - 616 446 461 453.5 

Positive control with EMS 150.0 - 616 398 378 388.0 

Test item 15.6 - culture was not continued# 

Test item 31.3 - 576 440 454 447.0 

Test item 62.5 - 506 401 384 392.5 

Test item 125.0 - 500 397 363 380.0 

Test item 250.0 - 502 381 374 377.5 

Test item 500.0 - 502 297 304 300.5 

Solvent control with water + 505 327 343 335.0 

Positive control with DMBA 1 .1 + 503 284 334 309.0 

Test item 15.6 + culture was not continued# 

Test item 31.3 + 506 276 295 285.5 

Test item 62.5 + 504 298 262 280.0 

Test item 125.0 + 508 330 311 320.5 

Test item 250.0 + 504 288 269 278.5 

Test item 500.0 + 510 356 371 363.5 

CE II 

absolute 

7 

0.74 0.63 

0.78 0.78 

0.76 0.75 

0.60 0.66 

0.61 

0.56 0.56 

0.63 0.55 

0.71 

# culture was not continued since a minimum of only four analysable concentrations is required 

CE II 

relative cells ce

% seeded surv

8 8 9 

100.0 452700 

85.6 412200 

105.4 461400 105.4 385500 103.2 443100 102.1 339300 

81.3 489000 

100.0 362400 

92.6 410400 

85.1 400650 

83.7 350700 

95.1 398000 

83.3 400500 

107.4 373800 
Study Number 1234903 

Leuco Sulfur Black 1 

Report 

Table 6: Mutagenicity data (Mutation rates), experiment I, culture I 

number of mutant colonies per flask 

cone. S9 found after plating in TG medium 

ug/ml mix I II Ill IV v 

column 1 2 3 4 5 6 7 

Solvent control with water - 7 15 8 9 4 

Positive control with EMS 150.0 - 27 40 30 37 39 

Test item 15.6 - culture was not continued# 

Test item 31.3 - 11 3 5 3 4 

Test item 62.5 - 4 3 2 5 4 

Test item 125.0 - 3 4 6 4 3 

Test item 250.0 - 2 7 3 5 3 

Test item 500.0 - 3 6 7 5 6 

Solvent control with water + 9 7 7 7 5 

Positive control with DMBA 1.1 + 197 226 227 250 224 

Test item 15.6 + culture was not continued# 

Test item 31.3 + 4 3 1 2 5 

Test item 62.5 + 2 3 5 5 4 

Test item 125.0 + 4 4 4 2 3 

Test item 250.0 + 2 6 6 4 7 

Test item 500.0 + 6 2 5 12 6 

mean 

8 

8.6 

34.6 

5.2 3.6 

4.0 4.0 

5.4 

7.0 

224.8 

3.0 3.8 

3.4 

5.0 

6.2 

# culture was not continued since a minimum of only four analysable concentrations is required 

mutant I 

standard colonies 

deviation _per 1 06 cells 

9 10 

4.0 25.81 

5.8 133.3 3.3 14.51 

1.1 12.01 

1.2 11.9 

2.0 15.7 

1.5 18.4 1.4 29.1' 

18.8 891.7 

1.6 13.3 

1.3 19.5 

0.9 13.5 

2.0 22.6 

3.6 23.3 
Study Number 1234903 

Leuco Sulfur Black 1 

13.2.2 Experiment I Culture II 

Table 7: Cloning Efficiency I (Survival), culture II 

cells 

cone. S9 seeded 

I.JQ/ml mix 1111 

column 1 2 3 

Solvent control 'Nith water - 509 

Positive control with EMS 150.0 - 509 

Test item 15.6 - 509 

Test item 31.3 - 509 

Test item 62.5 - 509 Test item 125.0 - 509 

Test item 250.0 - 509 Test item 500.0 - 509 

Solvent control 'Nith water + 512 

Positive control with DMBA 1.1 + 512 

Test item 15.6 + 512 

Test item 31.3 + 512 

Test item 62.5 + 512 

Test item 125.0 + 512 

Test item 250.0 + 512 

Test item 500.0 + 512 

Report 

number of colonies per flask CEI 

found absolute 

I II mean 

4 5 6 7 

405 322 364 0.71 

392 352 372 0.73 

251 364 308 0.60 

305 350 328 0.64 

387 327 357 0.70 

288 227 258 0.51 

227 240 234 0.46 

123 146 135 0.26 

295 335 315 0.62 

134 122 128 0.25 

216 265 241 0.47 214 212 213 0.42 

123 123 123 0.24 134 94 114 0.22 

90 62 76 0.15 58 98 78 0.15 

# culture was not continued since a minimum of only four analysable concentrations is required 

CEI cells/ml c

relative at 1st 

% subcultivation 

8 9 

100.0 2925000 102.3 2249000 

84.6 culture was not co

90.1 2935000 98.2 3111000 

70.8 2902000 

64.2 3205000 

37.0 3075000 

100.0 4842000 

40.6 3265000 

76.3 culture was not co

67.6 3345000 

39.0 3450000 36.2 4519000 

24.1 4100000 

24.8 3556000 
Study Number 1234903 

Leuco Sulfur Black 1 

Report 

Table 8: Cloning Efficiency II (Viability), experiment I, culture II 

cells number of colonies per flask 

cone. S9 seeded found 

IJQ/ml mix 1/11 I II mean 

column 1 2 3 4 5 6 

Solvent control with water - 506 468 439 453.5 

Positive control with EMS 150.0 - 504 364 358 361.0 

Test item 15.6 - culture was not continuecf 

Test item 31.3 - 500 369 374 371.5 

Test item 62.5 - 503 383 391 387.0 

Test item 125.0 - 500 326 359 342.5 

Test item 250.0 - 500 265 313 289.0 

Test item 500.0 - 504 291 254 272.5 

Solvent control with water + 503 327 378 352.5 

Positive control with DMBA 1.1 + 631 309 321 315.0 

Test item 15.6 + culture was not continuecf 

Test item 31.3 + 501 334 302 318.0 

Test item 62.5 + 500 336 297 316.5 

Test item 125.0 + 501 293 331 312.0 

Test item 250.0 + 507 345 322 333.5 

500.0 + 638 311 337 324.0 

CEll 

CEll relative 

absolute % 

7 8 

0.90 100.0 

0.72 79.9 

0.74 82.9 

0.77 85.8 0.69 76.4 

0.58 64.5 0.54 60.3 0.70 100.0 0.50 71.2 

0.63 90.6 

0.63 90.3 0.62 88.9 

0.66 93.9 0.51 72.5 

# culture was not continued since a minimum of only four analysable concentrations is required 

cells cells 

seeded surviv

8 9 

427500 38

474600 33

409500 30

440700 33426900 29429000 24

465000 25

426300 29

411450 20

442200 28409350 25455550 28430950 28447450 22
Study Number 1234903 

Leuco Sulfur Black 1 

Report 

Table 9: Mutagenicity data (Mutation rates), experiment I, culture II 

number of mutant colonies per flask 

cone. S9 found after plating in TG medium 

IJQ/ml mix I II Ill IV v 

column 1 2 3 4 5 6 7 

Solvent control with water - 4 4 3 3 4 

Positive control with EMS 150.0 - 38 41 42 35 31 

Test item 15.6 - culture was not continued# 

Test item 31.3 - 6 6 3 5 4 

Test item 62.5 - 12 13 6 7 9 

Test item 125.0 - 8 5 10 9 10 

Test item 250.0 - 6 4 7 9 4 

Test item 500.0 - 3 7 4 10 6 

Solvent control with water + 5 4 5 8 5 

Positive control with DMBA 1.1 + 279 337 304 284 256 

Test item 15.6 + culture was not continued# 

Test item 31.3 + 5 3 5 6 6 

Test item 62.5 + 9 2 3 4 6 

Test item 125.0 + 3 1 5 1 5 

Test item 250.0 + 3 9 9 6 4 

Test item 500.0 + 4 2 4 7 4 

mean 

8 

3.6 

37.4 

4.8 

9.4 

8.4 

6.0 6.0 

5.4 

292.0 

5.0 

4.8 

3.0 

6.2 

4.2 

# culture was not continued since a minimum of only four analysable concentrations is required 

mutant 

standard colonies 

deviation per 106 cells 

9 10 

0.5 9.4 

4.5 110.0 

1.3 15.8 

3.0 27.7 

2.1 28.7 2.1 24.2 

2.7 23.9 

1.5 18.1. 

30.4 1421.6 

1.2 17.8 

2.8 18.5 

2.0 10.6 

2.8 21.9 

1.8 18.5 
Study Number 1234903 

Leuco Sulfur Black 1 

13.2.3 Experiment II, Culture I 

Table 1 0: Cloning Efficiency I (Survival), culture I 

cells 

cone. S9 seeded 

mix 1/11 

column 1 2 3 

Solvent control with water - 512 

Positive control with EMS 75.0 - 512 

Test item 15.6 - 512 

Test item 31.3 - 512 

Test item 62.5 - 512 

Test item 125.0 - 512 

Test item 250.0 - 512 Test item 500.0 - 512 

Solvent control with water + 512 

Positive control with DMBA 1.1 + 512 

Test item 15.6 + 512 Test item 31.3 + 512 

Test item 62.5 + 512 

Test item 125.0 + 512 

Test item 250.0 + 512 

Test item 500.0 + 512 

Report 

number of colonies per flask CEI 

found absolute 

I II mean 

4 5 6 7 

402 428 415 0.81 

423 468 446 0.87 

446 463 455 0.89 

462 446 454 0.89 

376 412 394 0.77 

442 407 425 0.83 

257 259 258 0.50 

66 74 70 0.14 

361 401 381 0.74 

140 138 139 0.27 

397 413 405 0.79 

364 356 360 0.70 

306 320 313 0.61 

287 311 299 0.58 

291 318 305 0.59 

277 290 284 0.55 

# culture was not continued since a minimum of only four analysable concentrations is required 

CEI cells/ml 

relative at 1st 

% subcultivatio

8 9 

100.0 5480

107.3 4355

109.5 culture was 109.4 5239

94.9 5683

102.3 5374

62.2 5597

16.9 5409

100.0 4444

36.5 3014

106.3 culture was 

94.5 5005

82.2 5054

78.5 4982

79.9 517074.4 5426
Study Number 1234903 

Leuco Sulfur Black 1 

Report 

Table 11: Cloning Efficiency II (Viability), experiment II, culture I 

cells number of colonies per flask 

cone. S9 seeded found 

IJQ/ml mix 1/11 I II mean 

column 1 2 3 4 5 6 

Solvent control with water - 503 412 402 407.0 

Positive control with EMS 75.0 - 504 318 330 324.0 

Test item 15.6 - culture was not continuecf 

Test item 31.3 - 502 332 344 338.0 

Test item 62.5 - 501 237 273 255.0 

Test item 125.0 - 638 383 379 381.0 

Test item 250.0 - 505 329 305 317.0 

Test item 500.0 - 505 321 359 340.0 

Solvent control with water + 501 489 453 471.0 

Positive control with DMBA 1.1 + 500 394 434 414.0 

Test item 15.6 + culture was not continuecf 

Test item 31.3 + 502 403 447 425.0 

Test item 62.5 + 508 382 431 406.5 

Test item 125.0 + 503 445 433 439.0 

Test item 250.0 + 514 438 430 434.0 

Test item 500.0 + 508 456 451 453.5 

CEll 

absolute 

7 

0.81 0.64 0.67 

0.51 

0.60 0.63 0.67 0.94 0.83 0.85 0.80 

0.87 0.84 

0.89 

# culture was not continued since a minimum of only four analysable concentrations is required 

CEll 

relative cells 

% seeded 

8 8 

100.0 458400 

79.4 453900 

83.2 362700 

62.9 390900 

73.8 449100 77.6 450000 

83.2 454200 

100.0 391650 

88.1 369900 90.1 366000 85.1 327300 

92.8 400500 

89.8 307800 

95.0 387000 
Study Number 1234903 

Leuco Sulfur Black 1 

Report 

Table 12: Mutagenicity data (Mutation rates), experiment II, culture I 

number of mutant colonies per flask 

cone. S9 found after plating in TG medium 

IJg/mL mix I II Ill IV v 

column 1 2 3 4 5 6 7 

Solvent control with water . 9 5 4 6 9 

Positive control with EMS 75.0 . 68 71 79 85 70 

Test item 15.6 . culture was not continued# 

Test item 31.3 . 6 7 8 5 2 

Test item 62.5 . 2 4 2 5 6 

Test item 125.0 . 7 9 5 5 5 

Test item 250.0 . 5 7 3 4 7 

Test item 500.0 . 4 5 5 3 2 

Solvent control with water + 7 5 6 4 6 

Positive control with DMBA 1.1 + 229 231 226 219 235 

Test item 15.6 + culture was not continued# 

Test item 31.3 + 4 6 7 5 10 

Test item 62.5 + 3 6 6 1 4 

Test item 125.0 + 4 6 4 5 3 

Test item 250.0 + 3 4 5 3 7 

Test item 500.0 + 9 7 6 6 9 

# culture was not continued since a minimum of only four analysable concentrations is required 

mutant 

standard colonies 

mean deviation per 106 cel

8 9 10 

6.6 2.3 17

74.6 7.2 255

5.6 2.3 22

3.8 1.8 19

6.2 1.8 23

5.2 1.8 183.8 1.3 125.6 1.1 15

228.0 6.0 744

6.4 2.3 20

4.0 2.1 15

4.4 1.1 12

4.4 1.7 16

7.4 1.5 21
Study Number 1234903 

Leuco Sulfur Black 1 

13.2.4 Experiment II, Culture II 

Table 13: Cloning Efficiency I (Survival), culture II 

cells 

cone. S9 seeded 

mix 1/11 

column 1 2 3 

Solvent control with water - 512 

Positive control with EMS 75.0 - 512 

Test item 15.6 - 512 Test item 31.3 - 512 Test item 62.5 - 512 Test item 125.0 - 512 

Test item 250.0 - 512 Test item 500.0 - 512 

Solvent control with water + 512 

Positive control with DMBA 1.1 + 512 

Test item 15.6 + 512 

Test item 31.3 + 512 Test item 62.5 + 512 Test item 125.0 + 512 Test item 250.0 + 512 

Test item 500.0 + 512 

Report 

number of colonies per flask CEI 

found absolute 

I II mean 

4 5 6 7 

447 386 417 0.81 

367 449 408 0.80 

443 435 439 0.86 432 498 465 0.91 

409 393 401 0.78 416 421 419 0.82 

292 348 320 0.63 

35 43 39 0.08 

420 404 412 0.80 

105 94 100 0.19 

354 328 341 0.67 

329 321 325 0.63 

313 322 318 0.62 

275 287 281 0.55 

204 243 224 0.44 214 238 226 0.44 

# culture was not continued since a minimum of only four analysable concentrations is required 

CEI cells/ml 

relative at 1st 

% su bcultivatio

8 9 

100.0 4562

98.0 4518

105.4 culture was 

111.6 5663

96.3 5530

100.5 5415

76.8 4747

9.4 5166

100.0 4898

24.2 2856

82.8 culture was 

78.9 4837

77.1 4678

68.2 4872

54.2 4931

54.9 4942
Study Number 1234903 

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Report 

Table 14: Cloning Efficiency II (Viability), experiment II, culture II 

cells number of colonies per flask 

cone. S9 seeded found 

(.Jg/ml mix 1111 I II mean 

column 1 2 3 4 5 6 

Solvent control with water - 502 268 240 254.0 

Positive control with EMS 75.0 - 504 229 213 221.0 

Test item 15.6 - culture was not continuecf 

Test item 31.3 - 505 221 218 219.5 

Test item 62.5 - 506 167 199 183.0 

Test item 125.0 - 501 236 228 232.0 

Test item 250.0 - 509 219 236 227.5 

Test item 500.0 - 505 187 171 179.0 

Solvent control with water + 505 317 286 301.5 

Positive control with DMBA 1.1 + 509 270 283 276.5 

Test item 15.6 + culture was not continuecf 

Test item 31.3 + 505 349 372 360.5 

Test item 62.5 + 505 304 301 302.5 

Test item 125.0 + 500 214 262 238.0 

Test item 250.0 + 500. 197 172 184.5 

Test item 500.0 + 505 277 256 266.5 

CEll 

absolute 

7 

0.51 0.44 0.43 

0.36 

0.46 0.45 0.35 0.60 

0.54 

0.71 0.60 0.48 

0.37 0.53 

# culture was not continued since a minimum of only four analysable concentrations is required 

CEll 

relative cells ce

% seeded surv

8 8 9 

100.0 424500 

86.7 423000 85.9 412200 

71.5 371400 

91.5 408000 

88.3 412500 

70.1 415500 

100.0 421500 

91.0 481200 2

119.6 436500 3

100.3 427500 2

79.7 415200 

61.8 462300 

88.4 408300 2
Study Number 1234903 

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Report 

Table 15: Mutagenicity data (Mutation rates), experiment II, culture II 

number of mutant colonies per flask 

cone. S9 found after plating in TG medium 

IJglrnL mix I II Ill IV v 

column 1 2 3 4 5 6 7 

Solvent control with water . 5 3 4 2 3 

Positive control with EMS 75.0 . 69 72 79 71 65 

Test item 15.6 . culture was not continued# 

Test item 31.3 . 5 2 2 5 4 

Test item 62.5 . 3 6 6 5 4 

Test item 125.0 . 6 11 5 8 6 

Test item 250.0 . 5 5 3 6 11 

Test item 500.0 . 3 6 2 2 1 

Solvent control with water + 3 2 3 4 2 

Positive control with DMBA 1.1 + 219 235 231 201 214 

Test item 15.6 + culture was not continued# 

Test item 31.3 + 3 5 5 2 4 

Test item 62.5 + 4 5 6 5 3 

Test item 125.0 + 5 3 7 3 5 

Test item 250.0 + 5 4 3 3 4 

Test item 500.0 + 4 3 2 3 1 

# culture was not continued since a minimum of only four analysable concentrations is required 

mutant 

standard colonies 

mean deviation per 106 cel

8 9 10 

3.4 1.1 15

71.2 5.1 383

3.6 1.5 20

4.8 1.3 35

7.2 2.4 38

6.0 3.0 32

2.8 1.9 192.8 0.8 11

220.0 13.6 841

3.8 1.3 12

4.6 1.1 18

4.6 1.7 23

3.8 0.8 22

2.6 1.1 12
Study Number 1234903 

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Report Page 36 of 58 

14 ANNEX II: HISTORICAL DATA 

These values represent the historical control data from 2006 - 2008 

Number of mutant colonies per 106 cells 

4 hours treatment without metabolic activation 

Positive control Solvent control 

EMS (medium, water, DMSO, 

acetone, ethanol, THF) 

range: 58.3-394.0 0.6-31.7 

Mean value: 137.0 9.9 

Standard deviation: 63.0 6.1 

Number of studies: 38 38 

4 hours treatment with metabolic activation 

Positive control Solvent control 

DMBA (medium, water, DMSO, 

acetone, ethanol, THF) 

range: 133.1 - 2873.9 0.8-31.3 

Mean value: 1007.4 10.9 

Standard deviation: 561.0 7.1 

Number of studies: 38 38 

24 hours treatment without metabolic activation 

Positive control Solvent control 

EMS (medium, water, DMSO, 

acetone, ethanol, THF) 

range: 54.3-980.6 2.2-31.5 

Mean value: 270.5 12.0 

Standard deviation: 155.2 6.8 

Number of studies: 38 38 
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Report 

15 ANNEX Ill: COPY OF STUDY PLAN 

Annex Ill contains 15 pages. 

Page 37 of 58 
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Report 

STUDY NUMBER 1234903 

GENE MUTATION ASSAY 

IN CHINESE HAMSTER V79 CELLS 

IN VITRO 

(V79/ HPRT) 

WITH 

Leu co Sulfur Black 1 

STUDY PLAN 

Page 38 of 58 
Study Number 1234903 

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Study Number 1234903 

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1 SIGNATURES 

Study Director 

Management 

Head of Quality Assurance Unit 

Study Monitor 

Report Page 39 of 58 

Study Plan Page 2 of 15 

Dr. Hans-Eric Wollny 

Date March 06, 2009 

Dr. Wolfgang VOikner 

Date March 06, 2009 

Frauke Hermann 

Date .... ~.~.& .. ~.\r.~) ..... 

dr. Thomas Roth 

Date 
Study Number 1234903 

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Study Number 1234903 

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2 CONTENTS 

1 SIGNATURES 

2 CONTENTS 

3 PREFACE 

3.1 General 

3.2 Responsibilities 

3.3 Schedule 

3.4 Good Laboratory Practice 

3.5 Guidelines 

Report 

Study Plan 

3.6 Amendment and Deviation Procedures 

3.7 Archiving 

4 OBJECTIVE 

4.1 Aims of the Study 

4.2 Relevance of the Test System 

5 MATERIALS AND METHODS 

5.1 Test Item 

5.2 Safety precautions 

5.3 Controls 

5.4 Test System 

5.5 Mammalian Microsomal Fraction S9 Mix 

5.6 Pre-Test on Toxicity 

5.7 Dose Selection 

5.8 Experimental Performance 

5.9 Data Recording 

5.10 Acceptability of the Assay 

5.11 Evaluation of Results 

5.12 Statistical Analysis 

6 REPORTING 

7 DISTRIBUTION 

8 REFERENCES 

9 ANNEX: HISTORICAL DATA 

Page 40 of 58 

Page 3 of 15 

2 

3 

4 

4 

4 

4 

5 

5 

5 

5 

6 

6 

6 

7 

7 

7 

8 

9 

9 

10 

10 

11 

12 

12 

13 

13 

13 

13 

14 

15 
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3 PREFACE 

3.1 General 

Report 

Study Plan 

Title: Gene Mutation Assay 

in Chinese Hamster V79 Cells 

in vitro (V79/HPRT) 

'Nith Leuco Sulfur Black 1 

Sponsor: Clariant Products (S'Nitzerland) Ltd. 

Study Monitor 

Rothausstrasse 61 

4132 Muttenz 1 

S'Nitzerland 

Contact Person: Dr. H. W. Schneider 

Dr. Thomas Roth 

SCCGmbH 

Mikroforum Ring 1 

55234 Wendelsheim 

Germany 

Page 41 of 58 

Page 4 of 15 

Test Facility: Harlan Cytotest Cell Research GmbH (Harlan CCR) 

(former RCC Cytotest Cell Research GmbH) 

Contract Institute: 

Reference Number: 

3.2 Responsibilities 

In den Leppsteins'Niesen 19 

64380 Rossdorf 

Germany 

Harlan Laboratories Ltd. 

4452 ltingen 

Switzerland 

C31133 

Study Director: Dr. Hans-Eric Wollny 

Deputy Study Director: Dipl. Bioi. Andrea Sokolo'NSki 

Management: Dr. Wolfgang Volkner 

Head of Quality Assurance Unit: Frauke Hermann 

3.3 Schedule 

Study Initiation Date: 

Proposed Experimental Starting Date: 

Proposed Experimental Co111>letion Date: 

Proposed Date of the Draft Report: 

March 06, 2009 

March 2009 

June 2009 

June 2009 
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Report 

Study Plan 

3.4 Good Laboratory Practice 

The study will be performed in compliance with: 

Page 42 of 58 

Page 5 of 15 

"Chemikaliengesetz" (Chemicals Act) of the Federal Republic of Germany, "Anlage 1" 

(Annexa 1) dated July 25, 1994 ("BGBI. I 1994", pp. 1703), last revision dated 

June 27, 2002. 

"OECD Principles of Good Laboratory Practice", as revised in 1997 r C(97)186/Final 

3.5 Guidelines 

This study will be conducted according to the procedures indicated by the following internationally accepted guidelines and recommendations: 

Ninth Addendum to the OECD Guidelines for the Testing of Chemicals, February 1998, 

adopted July 21, 1997, Guideline No. 476 "In vitro Mammalian Cell Gene Mutation Test". 

Commission Regulation (EC) No. 440/2008 8.17, dated May 30,2008. 

3.6 Amendment and Deviation Procedures 

Amendments (planned changes) to the study plan will be issued and signed by the Study 

Director. The sponsor will receive the original and a copy of the amendment. The original is 

to be countersigned upon agreement and returned to Harlan CCR. The amendment will be 

distributed (see Distribution) and added to all copies of the study plan. 

Deviations (unplanned changes) to the study plan will be documented and maintained 

with the raw data. The report will reflect any deviations. The sponsor will be promptly 

informed of any relevant deviations from the study plan. 

3.7 Archiving 

Harlan CCR will archive the following data for 15 years: 

Raw data, study plan, report, and a sample of the test item. 

No data will be discarded without the sponsor's consent. 
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4 OBJECTIVE 

4.1 Aims of the Study 

Report Page 43 of 58 

Study Plan Page 6 of 15 

This in vitro experiment will be performed to assess the potential of the test item to induce 

gene mutations using the Chinese hamster cell line V79. Two parallel cultures will be used 

throughout the assay. The first experiment will be performed with a treatment time of 

4 hours with and without metabolic activation. · 

If the result of the first experiment is negative or inconclusive, a second experiment will be 

performed with a treatment time of 24 hours without and 4 hours treatment with metabolic 

activation. 

If the first experiment shows a positive (mutagen) result, a second experiment is not necessary. 

4.2 Relevance of the Test System 

In vitro methods are valuable to accurately control the concentration and exposure time of 

cells to the test item under study. However, due to the limited capacity for metabolic activation of potential mutagens an exogenous metabolic activation system is necessary. 

This in vitro test is an as~y for the detection of forward gene mutations in mammalian 

cells. Gene mutations are discussed as an initial step in the carcinogenic process (2). 

The V79 cells are exposed to the test item both with and without exogenous metabolic 

activation. At. a defined time interval after treatment the descendants of the treated original 

population are monitored for the loss of functional H PRT enzyme. 

HPRT (hypoxanthine-guanine phosphoribosyl transferase) catalyses the conversion of the 

nontoxic 6TG (6-thioguanine) to its toxic ribophosphorylated derivative. Therefore, cells 

deficient in HPRT due to a forward mutation are resistant to 6TG. These cells are able to 

proliferate in the presence of 6TG whereas the non-mutated cells die. However. the mutant 

phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 - 9 days. The 

expression period is terminated by adding 6TG to the culture medium (3). 

Mutant frequency is determined by seeding known numbers of cells in medium containing 

the selective agent to detect mutant cells, and in medium without selective agent to determine the surviving cells. After a suitable period the colonies are counted. Mutant frequencies are calculated from the number of mutant colonies corrected for cell survival. 

In order to establish a concentration response effect of the test item at least four concentration levels are tested. These concentration levels should yield a concentration related 

toxic effect. The highest concentration should induce a reduced level of survival. 

Reference mutagens are tested in parallel to the test item in order to demonstrate the 

sensitivity of the test system. 
Study Number 1234903 

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Report 

Study Plan 

5 MATERIALS AND METHODS 

5.1 Test Item 

Internal Test Item Number: S 973811 

Page 44 of 58 

Page 7 of 15 

The test item and the information concerning the test item were provided by the spon· 

sor. 

Identity: 

Batch No.: 

Composition: 

Molecular Weight: 

Purity: 

Stability in Water: 

Leuco Sulfur Black 1 

ESA0007595 

Leuco Sulfur Black 

2.4-Dinitrochlorobenzene 

2,4-Dinitrophenol 

Sulfate 

Chloride 

Sodium 

Water 

Not available 

Not indicated by the sponsor 

> 4 hours 

45.5% (wlw) 

< 20ppm 

< 20 ppm 

970 ppm 

750 ppm 

1.3% (w/w) 

53.0% (w/w) 

Storage: M room temperature, light protected 

Expiration Date: July 11 . 2011 

On the day of the experiment (immediately before treatment). the test item will be dissolved in deionised water. The solvent was chosen according to its solubility properties and 

its relative nontoxicity to the cells. The final concentration of deionised water in the culture 

medium will not exceed 10% v/v. 

The pH will be adjusted to physiological values if required. 

5.2 Safety precautions 

Routine hygienic procedures will be sufficient to ensure personnel health and safety. 
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5.3 Controls 

Report 

Study Plan 

5.3.1 Solvent Controls 

Concurrent solvent controls (deionised water) will be performed. 

5.3.2 Positive Control Substances 

Without metabolic activation 

Name: 

Supplier: 

Purity: 

Lot no.: 

Expiry date: 

Dissolved in: 

Final concentration: 

EMS; ethylmethane sulfonate 

ACROS ORGANICS, 2440 Geel. Belgium 

~98% 

VVill be added in the report 

VVill be added in the report 

Nutrient medium 

0.075-0.3 mg/mL = 0.6- 2.4 mM 

With metabolic activation 

Name: DMBA; 7, 12-dimethylbenz(a)anthracene 

Page 45 of 58 

Page 8 of 15 

Supplier: 

Lot no.: 

SIGMA CHEMIE GMBH. 82041 Deisenhofen, Germany 

Will be added in the report 

Expiry date: 

Dissolved in: 

Final concentration: 

Will be added in the report 

DMSO, Dimethylsulfoxide; 

(E. MERCK. 64293 Darmstadt; Germany 

final concentration in nutrient medium 0.5 %) 

11-2.5~~mL=43-98~M 

The dilutions of the stock solutions will be prepared on the day of the experiment and used 

immediately. 

The stability of both positive control substances in solution is proven by the mutagenic response in the expected range. 
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5.4 Test System 

Report 

Study Plan 

5.4.1 Reasons for the Choice of the Cell Line V79 

Page 46 of 58 

Page 9 of 15 

The V79 cell line has successfully been used in in vitro experiments for many years. Especially the high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good 

cloning efficiency of untreated cells (as a rule more than 50 %) both necessary for the 

appropriate performance of the study, recorrvnend the use of this cell line. The cells have a 

stable karyotype with a modal chromosome number of 22 (3). 

5.4.2 Cell Cultures 

Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing; Technical University; 64287 Darmstadt, Germany) are stored in liquid nitrogen in the cell bank of 

Harlan CCR allowing the repeated use of the same cell culture batch. Before freezing, the 

level of spontaneous mutants is depressed by treatment with HAT-medium as described in 

(3]. Each batch is screened for mycoplasma contamination and checked for karyotype 

stability and spontaneous mutant frequency. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells. 

Thawed stock cultures are propagated at 37 •c in 80 cnt plastic flasks (GREINER, 72632 

Frickenhausen, Germany). About 5. 105 cells are seeded into each flask with 15 mL of 

MEM (minimal essential medium; SEROMED, 12247 Berlin, Germany) supplemented with 

10% foetal calf serum (FCS, PAA Laboratories GmbH, 35091 COibe, Germany) and 1 % 

neomycin. The cells are subcultured twice weekly. The cell cultures are incubated at 37 ·c 

in a 4.5% carbon dioxide atmosphere (95.5% air). 

For the selection of mutants the medium is supplemented with 11 JJg/mL thioguanine (6TG, 

SIGMA GmbH, 82041 Deisenhofen, Germany). 

5.5 Mammalian Microsomal Fraction 59 Mix 

Lacking metabolic activities of cells under in vitro conditions are a disadvantage of assays 

with cell cultures as many chemicals only develop mutagenic potential when they are 

metabolised by the mammalian organism. Hovvever, metabolic activation of chemicals can 

be achieved at least partially by supplementing the cell cultures with rat liver microsome 

preparations (S9 mix). 

5.5.1 S9 (Preparation by Harlan CCR) 

Phenobarbital/p-Naphthoflavone induced rat liver S9 is used as the metabolic activation 

system. The S9 is prepared from 8- 12 vveeks old male Wistar HsdCpb:WIJ rats (Harlan 

Laboratories GmbH. 33178 Borchen, Germany) .. vveight approx. 220- 320 g induced by 

applications of 80 mglkg b.w. Phenobarbital i.p. (Desitin; 22335 Hamburg, Germany) and 

(3-Naphthoflavone p.o. (Aldrich, 89555 Steinheim, Germany) each on three consecutive 

days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with a KCI solution (1+3) follovved by centrifugation at 9000 g. Aliquotes of the supernatant are frozen and stored in ampoules at -so• C. 

Small numbers of the ampoules can be kept at -20"C for up to one vveek. The protein 

concentration in the S9 preparation is usually between 20 and 45 mglml. 
Study Number 1234903 

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5.5.2 89 Mix 

Report Page 47 of 58 

Study Plan Page 10of15 

An appropriate quantity of 59 supernatant is thawed and mixed with S9 cofactor solution to 

result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors are added to 

the 59 supernatant to reach the following concentrations in the 59 mix: 

8 mM MgCI2 

33mM KCI 

5 mM glucose-6-phosphate 

4 mM NADP 

in 100 mM sodium -phosphate-buffer, pH 7.4. 

During the experiment, the 59 mix is stored in an ice bath. The 59 mix preparation is performed according to Ames et al. (1 ). 

5.6 Pre-Test on Toxicity 

If necessary, a pre-test will be performed in order to determine the toxicity of the test item. 

In addition the pH-value and the osmolarity will be measured. The general culturing and 

experimental conditions in this pre-test will be the same as described below for the 

mutagenicity experiment. 

In this pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item will be observed and compared to the controls. Toxicity of the test item is evident as a reduction of the cloning 

efficiency (CE). 

5. 7 Dose Selection 

According to the appropriate guidelines (see page 5), several concentrations (usually at 

least four) of the test item should be used in two parallel cultures. These should yield a 

concentration-related toxic effect. The highest concentration should produce a low level of 

survival (10- 20 %) compared to the corresponding solvent control and the survival at the 

lowest concentration should approximate the solvent control. Freely-soluble nontoxic items 

should be tested up to 5 mg/mL or 10 mM whatever occurs first. Non-toxic substances of 

limited solubility in aqueous medium should be tested into the precipitating concentration 

range. Precipitation should be evaluated at the end of treatment by the naked eye. 

Changes of the pH-value and the osmolarity must be taken into consideration. 
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Study Plan 

5.8 Experimental Performance 

5.8.1 Seeding 

Page 48 of 58 

Page 11 of 15 

TIIYO or three days old exponentially growing stock cultures (more than 50 % confluent) 

are trypsinized at 37 ·c for 5 minutes. Then the enzymatic digestion is stopped by adding 

complete culture medium and a single cell suspension is prepared. The trypsin concentration for all subculturing steps is 0.2 % in Ca-Mg-free salt solution (Trypsin: Difco Laboratories, Detroit, USA). 

The Ca-Mg-free salt solution is composed as follows (per litre): 

NaCI 8000mg 

KCI 400mg 

Glucose 1000 mg 

NaHC03 350 mg 

Prior to the trypsin treatment the cells are rinsed with Ca-Mg-free salt solution containing 

200 mgll EDT A (Ethylene diamine tetraacetic acid). The cell suspension is seeded into 

plastic culture flasks (Greiner, 72632 Frickenhausen, Germany). Approximately 1.5-106 

and 5 x 1<¥ cells are seeded in each flask, respectively. The medium is MEM (minimal 

essential medium) with 10% FCS (complete medium). 

5.8.2 Treabnent 

After 24 h the medium of each culture in parallel is replaced with serum-free medium containing the test item, either without S9 mix or with 50 IJI/ml S9 mix. Concurrent solvent and 

positive controls are treated in parallel. After 4 h this medium is replaced with complete 

medium following two washing steps with "saline G". If no mutagenicity is observed in the 

first experiment, the treatment time may be extended to 24 h in the second experiment 

without metabolic activation. In that case the medium containing the test item will be MEM 

with 10% FCS. 

The "saline G" solution is composed as follows (per litre): 

NaCI 

KCI 

Glucose 

Na2HP04 7Hz0 

KH2P04 

pH is adjusted to 7.2 

8000mg 

400mg 

1100 mg 

290mg 

150mg 

The colonies used to determine the cloning efficiency (survival) are fixed and stained 6 to 8 

days after treatment as described below. 

Three days after treatment 1.5 106 cells per experimental point are subcultivated in 

175 em> flasks containing 30 ml medium. 
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Study Plan 

Page 49 of 58 

Page 12 of 15 

Following the expression time of approximately 6 days five 80 cl'lT cell culture flasks are 

seeded with about 3 - 5 105 cells each in medium containing 6-TG (11 JJg/mL). Two additional 25 ci1T flasks are seeded with approx. 500 cells each in non-selective medium to 

determine the viability. The cultures are incubated at 37 •c in a humidified atmosphere 

with 1.5 % C02. 

After 7- 10 days the colonies are stained with 10% methylene blue in 0.01 % KOH solution (E. MERCK, 64293 Darmstadt, Germany). 

Colonies with more than 50 cells are counted. In doubt the colony size will be checked with 

a preparation microscope (Nikon, 40407 DUsseldorf, Germany). 

5.9 Data Recording 

The data generated will be recorded in the raw data. The results will be presented in tabular form, including experimental groups with the test item, solvent, and positive controls. 

5.10 Acceptability of the Assay 

The gene mutation assay is considered acceptable if it meets the following criteria: 

a) the numbers of mutant colonies per 108 cells found in the solvent controls falls within 

the laboratory historical control data from 2006 - 2008 (see Annex). 

b) the positive control substances should produce a significant increase in mutant colony 

frequencies. 

c) the cloning efficiency II (absolute value) of the solvent controls should exceed 50 %. 
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Study Plan 

5.11 Evaluation of Results 

Page 50 of 58 

Page 13 of 15 

A test item is classified as positive if it induces either a concentration-related increase of 

the mutant frequency or a reproducible positive response for one of the test points. 

A test item producing neither a concentration-related increase of the rnrtant frequency nor 

a reproducible positive response at any of the test points is considered to be nonmutagenic in this system. 

A mutagenic response is described as follows: 

The test item is classified as mutagenic if it induces reproducibly with one of the concentrations a mutation frequency that is three times higher than the spontaneous mutation frequency in the experiment. 

The test item is classified as mutagenic if there is a reproducible concentration-related 

increase of the rnrtation frequency. Such evaluation may be considered also in the case 

that a threefold increase of the rnrtant frequency is not observed. 

Ho\Never, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. 

5.12 Statistical Analysis 

A linear regression (least squares) will be performed to assess a possible dose dependent 

increase of mutant frequencies using SYSTAT®11 (SYSTAT Software, Inc., 501, Canal 

Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant 

colonies obtained for the groups treated with the test item will be compared to the solvent 

control groups. A trend is judged as significant whenever the p-value (probability value) is 

below 0.05. However, both, biological and statistical significance will be considered together. 

6 REPORTING 

A GLP-compliant draft report will be submitted to the sponsor for scientific review. Following receipt of the sponsor's comments, a QA-audited report as well as a robust summary of 

the study will be issued as paper and electronic version. 

7 DISTRIBUTION 

Study Monitor 

Study Director 

QAU 

2x (1x original to be returned to Harlan CCR, 1x copy) 

1x (copy) 

1x (copy) 
Study Number 1234903 

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8 REFERENCES 

Report 

Study Plan 

1. B.N. Ames, J. McCann, and E. Yamasaki 

Page 51 of 58 

Page 14 of 15 

Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test 

In: B.J. Kilbey et al. (Eds.) "Handbook of Mutagenicity Test Procedures". Elsevier, Amsterdam, 1-17, 1977 

2. P. Howard-Flanders 

Mutagenesis in mammalian cells 

Mutation Research 86. 307-327, 1981 

3. M.O. Bradley, B. Bhuyan, M.C. Francis. R. Langenbach,.A. Peterson and E. Huberman 

Mutagenesis by chemical agents in V79 Chinese hamster cells: a review and analysis of 

the literature. A report of the gene-tox program 

Mutation Research 87. 81-142. 1981 
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Study Plan 

9 ANNEX: HISTORICAL DATA 

Page 52 of 58 

Page 15 of 15 

These values represent the historical control data from 2006- 2008 

Number of mutant colonies per 1o'cells 

4 h treatment 

Solvent control 

Positive control (medium, water. DMSO, acetone. 

ethanol, THF) 

without S9 mix withS9 mix 

Without S9 m1x with S9 m1x EMS DMBA 

range· 58.3-394.0 133.1-28739 0 6-31.7 0.6-31.3 

Mean value· 137.0 1007.4 99 10.9 

Standard deviat1on 630 561.0 61 7 1 

Number of studies 38 38 38 38 

24 h treatment 

Solvent control 

Positive control (medium. water. DMSO. acetone. 

ethanol, THF) 

Without S9 m1x 

wrthout S9 m1x EMS 

range 543-960.6 2.2-31.5 

Mean value 270 5 12.0 

Standard deviation 155 2 66 

Number of studies 36 38 
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Report Page 53 of 58 

16 ANNEX IV: COPY OF FIRST AMENDMENT TO STUDY 

PLAN 

Annex IV contains 5 pages. 
Study Number 1234903 

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Report 

STUDY NUMBER 1234903 

GENE MUTATION ASSAY 

IN CHINESE HAMSTER V79 CELLS 

IN VITRO 

(V79/ HPRT) 

WITH 

Leuco Sulfur Black 1 

1st AMENDMENT TO STUDY PLAN 

(5 pages) 

Page 54 of 58 

Sponsor: Clariant Products (Switzerland) Ltd. 

Rothausstrasse 61 

Study Monitor 

Date of 

4132 Muttenz 1 

Switzerland 

Contact Person: Or. H. W. Schneider 

Or. Thomas Roth 

SCCGmbH 

Mikroforum Ring 1 

55234 \Nendelsheim 

Germany 

151 Amendment to Study Plan: June 26, 2009 
Study Number 1234903 

Leuco Sulfur Black 1 

Study Number 1234903 

Leuco Sulfur Black 1 

Report 

1" Amendment to Study Plan 

A) CONCERNING: 3.4 Good Laboratory Practice 

-page 5-

PRESENT: 

Page 55 of 58 

Page 2 of5 

"Chemikaliengesetz" (Chemicals Act) of the Federal Republic of Germany. "Anhang 1" 

(Annex 1). dated July 25. 1994 ("BGBI. 11994". pp. 1703). last revision dated June 27. 

2002. 

NEW: 

"Chemikaliengesetz" (Chemicals Act) of the Federal Republic of Germany. "An hang 1" 

(Annex 1). version dated July 02, 2008 ("BGBI.", p. 1146)." 

REASON FOR THE ALTERATION: 

Updating. 

B) CONCERNING: 3.7 Archiving 

-page 5-

PRESENT: 

No data will be discarded without the sponsor's consent. 

NEW: 

No data will be discarded without the sponsor's prior written consent. 

REASON FOR THE ALTERATION: 

Updating. 
Study Number 1234903 

Leuco Sulfur Black 1 

Study Number 1234903 

Leuco Sulfur Black 1 

C) CONCERNING: 5.1 Test Item 

-page 7-

PRESENT: 

Report 

1" Amendment to Study Plan 

Internal Test Item Number: S 973811 

Page 56 of 58 

Page 3 of5 

The test item and the information concerning the test item were provided by the 

sponsor. 

Identity: 

Batch No.: 

Composition 

Molecular \Neight: 

Purity: 

Storage: 

Expiration Date: 

Leuco Sulfur Black 1 

ESA0007595 

Leuco Sulfur Black 

2,4-Dinitrochlorobenzene 

2.4-Dinitrophenol 

Sulfate 

Chloride 

Sodium 

water 

Not available 

45.5% (w/w) 

< 20 ppm 

< 20 ppm 

970 ppm 

750 ppm 

1.3% (w/w) 

53.0% (w/w) 

Not indicated by the sponsor 

At room temperature, light protected 

July 11. 2011 
Study Number 1234903 

Leuco Sulfur Black 1 

Report Page 57 of 58 

Study Number 1234903 

Leuco Sulfur Black 1 

1st Amendment to Study Plan Page 4 of 5 

NEW: 

Internal Test Item Number: S 973811 

The test item and the information concerning the test item were provided by the 

sponsor. 

Identification: Leuco Sulfur Black 1 

CAS No.: 66241-11-Q 

Description: Thixotrophic, black, \Net paste 

Batch No.: 

Composition: 

ESA0007595 

Leuco Sulfur Black 1: 

2,4-Dinitrochlorobenzene: 

2.4-Dinitrophenole: 

Sulfate: 

Chloride: 

Sodium: 

Water: 

Molecular Weight: Not available 

52.75% (calculated by amount of 

by-compounds) 

< 20 ppm 

< 20 ppm 

970 ppm 

750ppm 

1.3% (w/w) 

45.76% (w/w) by Kart-Fischer 

titration 

Stability of Test Item: Stable under storage conditions 

Storage: At room temperature (range 20 +1- 5° C), light protected 

Expiry Date: July 11, 2011 

REASON FOR THE ALTERATION: 

Updating as requested by the sponsor. 

These alterations have no detrimental impact on the outcome of the study. 
Study Number 1234903 

Leuco Sulfur Black 1 

Study Number 1234903 

Leuco Sulfur Black 1 

SIGNATURES 

Study Director 

Management 

Head of Quality 

Assurance Unit 

Study Monitor 

DISTRIBUTION 

Study Monitor 

Study Director 

QAU 

Report Page 58 of 58. 

1" Amendment to Study Plan Page 5 of 5 

Dr. Hans-Eric Wollny 

....... :/···························· ································· Date: June 26, ,2009 

Dr. Wolfgang Volkner 

Date: June 26, 2009 

Frauke Hermann 

Lyuld r<udoiph 

Date: ................................................. . 

, .. r 

Date: 

2x (1x original to be returned to Harlan CCR. 1x copy) 

1x (copy) 

1x (copy) 

188 ON SNI\tlN08