### 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

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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

21

22

22
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:

1 51 

### 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 10 6 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 10 6 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 cm 2 

### plastic flasks (GREINER, 72632 Frickenhausen, Germany). About 5· 10 5 

### 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 NaHC0 3 

### 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· 10 6 

### (single culture) and 5· 10 2 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 Na 2 HP04· 7H20 290 mg KH 2 P04 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 0 6 

### cells per experimental point were subcultivated in 175 cm 2 

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

cm 2 

### cell culture flasks were seeded with about 3

- 5· 10 5 

### cells each in medium containing 6- TG. Two additional 25 cm 2 

### 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 % C0 2 

### 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 

## 10 6 

### 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 10 6 

### 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 0 6 

### 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 · 10 6 

### divided by the number of cells survived

mutant colonies per 1 0 6 

### cells 

## I mutant colonies per 10 6 

### 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 0 6 

### 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 0 6 

### 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 10 6 

### cells; the range of the groups treated with the test item was from 11.9 up to 38.1 mutants per 1 0 6 

### 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 originalStudyNumber1234903LeucoSulfurBlack113ANNEX1:TABLESOFRESULTS13.1Pre-testTable3:Toxicitydatacone.S9DurationofcellsmixtreatmentseededTestgroup(h)1111Column12 34Solventcontrol.4509Testitem3.9.4509Testitem7.8.4509Testitem15.6.4509Testitem31.3.4509Testitem62.5.4509Testitem125.0.4509Testitem250.0.4509Testitem500.0.4509Solventcontrol+4509Testitem3.9+4509Testitem7.8+4509Testitem15.6+4509Testitem31.3+4509Testitem62.5+4509Testitem125.0+4509Testitem250.0+4509Testitem500.0+4509Solventcontrol-24509Testitem3.9-24509Testitem7.8-24509Testitem15.6-24509Testitem31.3-24509Testitem62.5-24509Testitem125.0-24509Testitem250.0-24509Testitem500.0-24509----ReportPage23of58numberofcoloniesperflaskCE%CE%foundabsoluterelativeIprecipitationIIImean567891046138942583.5100.051148649997.9117.332340636571.685.845442844186.6103.832827930459.671.436326931662.174.425926526251.561.6precipitation10322516432.238.6Iprecipitation9681.51.8Iprecil)itation49949149597.2100.050844947994.096.747750849396.899.544044744487.189.643838641280.983.236831834367.469.338040139176.778.9precipitation49042946090.392.8Iprecipitation38233535970.472.4'precipitation48250049196.5100.050644647693.596.945942744387.090.242239340880.183.042338140279.081.939736538174.977.635127131161.163.314218916632.533.7!precipjtation5L__661.11.1,precipitationStudyNumber1234903LeucoSulfurBlack113.2MainExperiment13.2.1ExperimentICultureITable4:CloningEfficiencyI(Survival),cultureIcellscone.S9seededJ.Jg/mlmix1111column1 2 3Solventcontrolwithwater-509PositivecontrolwithEMS150.0-509Testitem15.6-509Testitem31.3-509Testitem62.5-509Testitem125.0-509Testitem250.0-509Testitem500.0-509Solventcontrolwithwater+512PositivecontrolwithDMBA1.1+512Testitem15.6+512Testitem31.3+512Testitem62.5+512Testitem125.0+512Testitem250.0+512Testitem500.0+512----ReportnumberofcoloniesperflaskCEIfoundabsoluteIJlmean45 6 73513123320.654403323860.762823623220.632492242370.462051881970.392071871970.391491441470.2912371970.192783042910.571411261340.261732171950.382122002060.401491451470.291311251280.2581110960.1912211QL__________jj§0.23-#culturewasnot continuedsinceaminimumofonlyfouranalysableconcentrationsisrequiredPage24of58CEIcells/mlcelldensityrelativeat1st%%subcultivationofcontrol8910100.03818000100.0116.43861000101.197.1culturewasnotcontinued#I71.34325000113.359.3270000070.759.4230000060.2!44.2229700060.229.3343700090.0100.03980000100.045.9265600066.767.0culturewasnotcontinued#70.84770000119.850.54070000102.344.04112000103.332.84015000100.939.9380100095.5StudyNumber1234903LeucoSulfurBlack1ReportTable5:CloningEfficiencyII(Viability),experimentI,cultureIcellsnumberofcoloniesperflaskcone.S9seededfoundIJQ/mlmix1111IIImeancolumn1234 56Solventcontrolwithwater-616446461453.5PositivecontrolwithEMS150.0-616398378388.0Testitem15.6-culturewasnotcontinued#Testitem31.3-576440454447.0Testitem62.5-506401384392.5Testitem125.0-500397363380.0Testitem250.0-502381374377.5Testitem500.0-502297304300.5Solventcontrolwithwater+505327343335.0PositivecontrolwithDMBA1.1+503284334309.0Testitem15.6+culturewasnotcontinued#Testitem31.3+506276295285.5Testitem62.5+504298262280.0Testitem125.0+508330311320.5Testitem250.0+504288269278.5Testitem500.0+510356371363.5CEIIabsolute70.740.630.780.780.760.750.600.660.610.560.560.630.550.71#culturewasnotcontinuedsinceaminimumofonlyfouranalysableconcentrationsisrequiredPage25of58CEIIrelativecellscells%seededsurvived889100.045270033327885.6412200259632105.4461400358066105.4385500299029103.2443100336756102.133930025515181.3489000292718100.036240024040492.641040025211585.140065022605883.735070019483395.139800025110083.3400500221308107.4373800266424StudyNumber1234903LeucoSulfurBlack1ReportTable6:Mutagenicitydata(Mutationrates),experimentI,cultureInumberofmutantcoloniesperflaskcone.S9foundafterplatinginTGmediumug/mlmixIIIIllIVvcolumn1 2 345 67Solventcontrolwithwater-715894PositivecontrolwithEMS150.0-2740303739Testitem15.6-culturewasnotcontinued#Testitem31.3-1135 3 4Testitem62.5-43 2 5 4Testitem125.0-3 4 643Testitem250.0-2 7353Testitem500.0-367 56Solventcontrolwithwater+97 7 75PositivecontrolwithDMBA1.1+197226227250224Testitem15.6+culturewasnotcontinued#Testitem31.3+4 3 125Testitem62.5+235 5 4Testitem125.0+4 4 4 23Testitem250.0+2 6 647Testitem500.0+6 2 5126mean88.634.65.23.64.04.05.47.0224.83.03.83.45.06.2#culturewasnotcontinuedsinceaminimumofonlyfouranalysableconcentrationsisrequiredPage26of58mutantIstandardcoloniesdeviation_per106cells9104.025.815.8133.33.314.511.112.011.211.92.015.71.518.41.429.1'18.8891.71.613.31.319.50.913.52.022.63.623.3StudyNumber1234903LeucoSulfurBlack113.2.2ExperimentICultureIITable7:CloningEfficiencyI(Survival),cultureIIcellscone.S9seededI.JQ/mlmix1111column12 3Solventcontrol'Nithwater-509PositivecontrolwithEMS150.0-509Testitem15.6-509Testitem31.3-509Testitem62.5-509Testitem125.0-509Testitem250.0-509Testitem500.0-509Solventcontrol'Nithwater+512PositivecontrolwithDMBA1.1+512Testitem15.6+512Testitem31.3+512Testitem62.5+512Testitem125.0+512Testitem250.0+512Testitem500.0+512ReportnumberofcoloniesperflaskCEIfoundabsoluteIIImean45674053223640.713923523720.732513643080.603053503280.643873273570.702882272580.512272402340.461231461350.262953353150.621341221280.252162652410.472142122130.421231231230.24134941140.229062760.155898780.15#culturewasnotcontinuedsinceaminimumofonlyfouranalysableconcentrationsisrequiredPage27of58CEIcells/mlcelldensityrelativeat1st%%subcultivationofcontrol8910100.02925000100.0102.3224900076.984.6culturewasnotcontinued"90.12935000100.398.23111000106.470.8290200099.264.23205000109.637.03075000105.1100.04842000100.040.6326500067.476.3culturewasnotcontinued"67.6334500069.139.0345000071.336.2451900093.324.1410000084.724.8355600073.4StudyNumber1234903LeucoSulfurBlack1ReportTable8:CloningEfficiencyII(Viability),experimentI,cultureIIcellsnumberofcoloniesperflaskcone.S9seededfoundIJQ/mlmix1/11IIImeancolumn12345 6Solventcontrolwithwater-506468439453.5PositivecontrolwithEMS150.0-504364358361.0Testitem15.6-culturewasnotcontinuecfTestitem31.3-500369374371.5Testitem62.5-503383391387.0Testitem125.0-500326359342.5Testitem250.0-500265313289.0Testitem500.0-504291254272.5Solventcontrolwithwater+503327378352.5PositivecontrolwithDMBA1.1+631309321315.0Testitem15.6+culturewasnotcontinuecfTestitem31.3+501334302318.0Testitem62.5+500336297316.5Testitem125.0+501293331312.0Testitem250.0+507345322333.5500.0+638311337324.0CEllCEllrelativeabsolute%780.90100.00.7279.90.7482.90.7785.80.6976.40.5864.50.5460.30.70100.00.5071.20.6390.60.6390.30.6288.90.6693.90.5172.5#culturewasnotcontinuedsinceaminimumofonlyfouranalysableconcentrationsisrequiredPage28of58cellscellsseededsurvived89427500383145474600339942409500304259440700339067426900292427429000247962465000251414426300298749411450205399I4422002806784093502591194555502836964309502834751447450227232jStudyNumber1234903LeucoSulfurBlack1ReportTable9:Mutagenicitydata(Mutationrates),experimentI,cultureIInumberofmutantcoloniesperflaskcone.S9foundafterplatinginTGmediumIJQ/mlmixIIIIllIVvcolumn1234 567Solventcontrolwithwater-44334PositivecontrolwithEMS150.0-3841423531Testitem15.6-culturewasnotcontinued#Testitem31.3-663 54Testitem62.5-12136 7 9Testitem125.0-8 510910Testitem250.0-64 794Testitem500.0-37 4106Solventcontrolwithwater+54585PositivecontrolwithDMBA1.1+279337304284256Testitem15.6+culturewasnotcontinued#Testitem31.3+53 5 6 6Testitem62.5+ 9 2 3 4 6Testitem125.0+31515Testitem250.0+39964Testitem500.0+4 2 474mean83.637.44.89.48.46.06.05.4292.05.04.83.06.24.2#culturewasnotcontinuedsinceaminimumofonlyfouranalysableconcentrationsisrequiredPage29of58mutantstandardcoloniesdeviationper106cells9100.59.44.5110.01.315.83.027.72.128.72.124.22.723.91.518.1.30.41421.61.217.82.818.52.010.62.821.91.818.5StudyNumber1234903LeucoSulfurBlack113.2.3ExperimentII,CultureITable10:CloningEfficiencyI(Survival),cultureIcellscone.S9seededmix1/11column123Solventcontrolwithwater-512PositivecontrolwithEMS75.0-512Testitem15.6-512Testitem31.3-512Testitem62.5-512Testitem125.0-512Testitem250.0-512Testitem500.0-512Solventcontrolwithwater+512PositivecontrolwithDMBA1.1+512Testitem15.6+512Testitem31.3+512Testitem62.5+512Testitem125.0+512Testitem250.0+512Testitem500.0+512ReportnumberofcoloniesperflaskCEIfoundabsoluteIIImean45 6 74024284150.814234684460.874464634550.894624464540.893764123940.774424074250.832572592580.506674700.143614013810.741401381390.273974134050.793643563600.703063203130.612873112990.582913183050.592772902840.55#culturewasnotcontinuedsinceaminimumofonlyfouranalysableconcentrationsisrequiredPage30of58CEIcells/mlcelldensityrelativeat1st%%subcultivationofcontrol8910100.05480000100.0107.3435500079.5109.5culturewasnotcontinued"109.4523900095.694.95683000103.7102.3537400098.162.25597000102.116.9540900098.7100.04444000100.036.5301400067.8106.3culturewasnotcontinued#94.55005000112.682.25054000113.778.54982000112.179.95170000116.374.45426000122.1StudyNumber1234903LeucoSulfurBlack1ReportTable11:CloningEfficiencyII(Viability),experimentII,cultureIcellsnumberofcoloniesperflaskcone.S9seededfoundIJQ/mlmix1/11IIImeancolumn12345 6Solventcontrolwithwater-503412402407.0PositivecontrolwithEMS75.0-504318330324.0Testitem15.6-culturewasnotcontinuecfTestitem31.3-502332344338.0Testitem62.5-501237273255.0Testitem125.0-638383379381.0Testitem250.0-505329305317.0Testitem500.0-505321359340.0Solventcontrolwithwater+501489453471.0PositivecontrolwithDMBA1.1+500394434414.0Testitem15.6+culturewasnotcontinuecfTestitem31.3+502403447425.0Testitem62.5+508382431406.5Testitem125.0+503445433439.0Testitem250.0+514438430434.0Testitem500.0+508456451453.5CEllabsolute70.810.640.670.510.600.630.670.940.830.850.800.870.840.89#culturewasnotcontinuedsinceaminimumofonlyfouranalysableconcentrationsisrequiredPage31of58CEllrelativecellscells%seededsurvived889100.045840037091279.445390029179383.236270024420862.939090019896173.844910026819377.645000028247583.2454200305798100.039165036819888.136990030627790.136600030986185.132730026190492.840050034954289.830780025989395.0387000345481StudyNumber1234903LeucoSulfurBlack1ReportTable12:Mutagenicitydata(Mutationrates),experimentII,cultureInumberofmutantcoloniesperflaskcone.S9foundafterplatinginTGmediumIJg/mLmixIIIIllIVvcolumn12 3 456 7Solventcontrolwithwater.9 546 9PositivecontrolwithEMS75.0.6871798570Testitem15.6.culturewasnotcontinued#Testitem31.3.678 52Testitem62.5.242 5 6Testitem125.0.7 95 5 5Testitem250.0.57 3 47Testitem500.0.4 5 5 32Solventcontrolwithwater+7 564 6PositivecontrolwithDMBA1.1+229231226219235Testitem15.6+culturewasnotcontinued#Testitem31.3+4 6 7510Testitem62.5+36614Testitem125.0+4 6 45 3Testitem250.0+345 37Testitem500.0+976 6 9#culturewasnotcontinuedsinceaminimumofonlyfouranalysableconcentrationsisrequiredPage32of58mutantstandardcoloniesmeandeviationper106cells89106.62.317.874.67.2255.75.62.322.93.81.819.16.21.823.15.21.818.43.81.312.45.61.115.2228.06.0744.46.42.320.74.02.115.34.41.112.64.41.716.97.41.521.4StudyNumber1234903LeucoSulfurBlack113.2.4ExperimentII,CultureIITable13:CloningEfficiencyI(Survival),cultureIIcellscone.S9seededmix1/11column12 3Solventcontrolwithwater-512PositivecontrolwithEMS75.0-512Testitem15.6-512Testitem31.3-512Testitem62.5-512Testitem125.0-512Testitem250.0-512Testitem500.0-512Solventcontrolwithwater+512PositivecontrolwithDMBA1.1+512Testitem15.6+512Testitem31.3+512Testitem62.5+512Testitem125.0+512Testitem250.0+512Testitem500.0+512ReportnumberofcoloniesperflaskCEIfoundabsoluteIIImean45 674473864170.813674494080.804434354390.864324984650.914093934010.784164214190.822923483200.633543390.084204044120.80105941000.193543283410.673293213250.633133223180.622752872810.552042432240.442142382260.44#culturewasnotcontinuedsinceaminimumofonlyfouranalysableconcentrationsisrequiredPage33of58CEIcells/mlcelldensityrelativeat1st%%subcultivationofcontrol8910100.04562000100.098.0451800099.0105.4culturewasnotcontinued#111.65663000124.196.35530000121.2100.55415000118.776.84747000104.19.45166000113.2100.04898000100.024.2285600058.382.8culturewasnotcontinued#78.9483700098.877.1467800095.568.2487200099.554.24931000100.754.94942000100.9StudyNumber1234903LeucoSulfurBlack1ReportTable14:CloningEfficiencyII(Viability),experimentII,cultureIIcellsnumberofcoloniesperflaskcone.S9seededfound(.Jg/mlmix1111IIImeancolumn1 2 3 4 5 6Solventcontrolwithwater-502268240254.0PositivecontrolwithEMS75.0-504229213221.0Testitem15.6-culturewasnotcontinuecfTestitem31.3-505221218219.5Testitem62.5-506167199183.0Testitem125.0-501236228232.0Testitem250.0-509219236227.5Testitem500.0-505187171179.0Solventcontrolwithwater+505317286301.5PositivecontrolwithDMBA1.1+509270283276.5Testitem15.6+culturewasnotcontinuecfTestitem31.3+505349372360.5Testitem62.5+505304301302.5Testitem125.0+500214262238.0Testitem250.0+500.197172184.5Testitem500.0+505277256266.5CEllabsolute70.510.440.430.360.460.450.350.600.540.710.600.480.370.53#culturewasnotcontinuedsinceaminimumofonlyfouranalysableconcentrationsisrequiredPage34of58CEllrelativecellscells%seededsurvived889100.042450021478786.742300018548285.941220017916471.537140013432191.540800018893488.341250018436970.1415500147276100.0421500251648191.04812002613981!119.6436500311600100.342750025607779.741520019763561.8462300170589188.44083002154691StudyNumber1234903LeucoSulfurBlack1ReportTable15:Mutagenicitydata(Mutationrates),experimentII,cultureIInumberofmutantcoloniesperflaskcone.S9foundafterplatinginTGmediumIJglrnLmixIIIIllIVvcolumn12 3 4 5 6 7Solventcontrolwithwater.5 34 23PositivecontrolwithEMS75.0.6972797165Testitem15.6.culturewasnotcontinued#Testitem31.3.52 2 5 4Testitem62.5.3 6 6 54Testitem125.0.6115 8 6Testitem250.0.5 5 3 611Testitem500.0.3 62 2 1Solventcontrolwithwater+3 2 3 4 2PositivecontrolwithDMBA1.1+219 235231 201214Testitem15.6+culturewasnotcontinued#Testitem31.3+3 5 52 4Testitem62.5+ 45 6 5 3Testitem125.0+5 37 3 5Testitem250.0+5433 4Testitem500.0+43231#culturewasnotcontinuedsinceaminimumofonlyfouranalysableconcentrationsisrequiredPage35of58mutantstandardcoloniesmeandeviationper106cells89103.41.115.871.25.1383.93.61.520.14.81.335.77.22.438.16.03.032.52.81.919.02.80.811.1220.013.6841.63.81.312.24.61.118.04.61.723.33.80.822.32.61.112.1

### Study Number 1234903 Leuco Sulfur Black 1 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 10 6 

### 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

### Study Number 1234903 Leuco Sulfur Black 1 Report



# 15 ANNEX Ill: COPY OF STUDY PLAN

Annex Ill contains 15 pages. Page 37 of 58

### Study Number 1234903 Leuco Sulfur Black 1 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 Leuco Sulfur Black 1

Study Number 1234903 Leuco Sulfur Black 1



## 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 Leuco Sulfur Black 1

Study Number 1234903 Leuco Sulfur Black 1



# 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

### Study Number 1234903 Leuco Sulfur Black 1

Study Number 

### 1234903 Leuco Sulfur Black 1



## 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

### Study Number 1234903 Leuco Sulfur Black 1

Study Number 1234903 Leuco Sulfur Black 1 

### 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.

### Study Number 1234903 Leuco Sulfur Black 1

Study Number 1234903 Leuco Sulfur Black 1



## 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 Leuco Sulfur Black 1

Study Number 1234903 Leuco Sulfur Black 1 

### 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.

### Study Number 1234903 Leuco Sulfur Black 1

Study Number 1234903 Leuco Sulfur Black 1



## 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.

### Study Number 1234903 Leuco Sulfur Black 1

Study Number 1234903 Leuco Sulfur Black 1



## 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. 10 5 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 Leuco Sulfur Black 1

Study Number 1234903 Leuco Sulfur Black 1



### 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.

### Study Number 1234903 Leuco Sulfur Black 1

Study Number 1234903 Leuco Sulfur Black 1 

### Report

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 NaHC0 3 

### 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 

### 1.5-10 6
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 Na 2 HP04 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 

### 10 6 cells per experimental point are subcultivated in 175 

## em> flasks containing 30 ml medium.

### Study Number 1234903 Leuco Sulfur Black 1

Study Number 1234903 Leuco Sulfur Black 1 

### Report

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 

### 10 5 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 

### 10 8 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 

### %.

### Study Number 1234903 Leuco Sulfur Black 1

Study Number 1234903 Leuco Sulfur Black 1 

### Report

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 Leuco Sulfur Black 1

Study Number 1234903 Leuco Sulfur Black 1



# 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

### Study Number 1234903 Leuco Sulfur Black 1

Study Number 1234903 Leuco Sulfur Black 1 

### Report

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

### Study Number 1234903 Leuco Sulfur Black 1 Report Page 53 of 58



# 16 ANNEX 

# IV: COPY OF FIRST AMENDMENT TO STUDY PLAN

Annex IV contains 5 pages.

### Study Number 1234903 Leuco Sulfur Black 1 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

1 51 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