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# STUDY NUMBER 1234902

IN VITRO CHROMOSOME ABERRATION TEST IN CHINESE HAMSTER V79 

# CELLS WITH LEUCO SULFUR BLACK 1

REPORT
StudyCompletion Date: July07, 2009

CONTAINS NOCBI

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# r2..Study Number 1234902 Leuco Sulfur Black 1 Report


# 1 COPYOF THE GLP CERTIFICATE - page 2 of36 -



## HESSEN GuteLaborpraxis/GoodLaboratoryPractice GLP-Bescheinigung!StatementofGLPCompliance (gemJll.llaccordingto § 19b Abs. IChemikaliengesetz)

EineGLP-Inspektion T.UrOberwachungder Einhaltung derGli'-Grundsl!t:regemlillChemikaliengesetzbzw. Richtlinie 2004/'1/EG wurdedurchgefilhnin i2li'!Ufeinrichtung'l'estfacility As.o;essmentofconfonnitywith GLI'accordingto ChemikaliengesetzandDirecti•·e 2004.'9/EEC at: 

### 0 l'rtlfSiandort!J'est site HarlanCytotestCell ResearchGmbH Harlan C}1otestCell Research GmbH Indenl.cppsteinswicsen 19 643811 RoJldorf (IJnverwechselbare Bezeichnungund Adresse/Unequivocalname andadress) PrOfugenauchKatcgorien/Arcuof**Expcrti~e** (gemat.liaccordingchemVwV-GLPNr.5.3/0ECDguidance) 2 PnHungenzurBt'Slirnmungdcr-**tox1kologischt~n** Etgf'nschaftf'n 3 PrU.fungen zur Hestimmung**cl~r**erbgutveriinderndenEigenschaften {in vitro und invivo) 6 PrUfungen zurBestimmungvon**RilckMAnct~n** 8 Ana.lytischr PrUfungcn an biolog.ischcn**Mat~nabcn** 2**1'ox:icit~·** studtes 3**Mutag~nicity** studies 6**R~9tdues** I Ana.lyllcal studit-s onbwlogtcal matenals

l!i.08. und27.-29.10.20011 Datum derInspektion!Date oflnspeclion

(Tag MonatJahr-'day month year)
Die genannle l'rilfeinrichtungbefindet sich im nationalen GI.P-OberwachungsverfallfCnund wird rcgcl· malligaufEinhaltungder GLP-Grundsatz.eUberwacht. AufderGrundlagedeslnspektionsbcrichteswird hicnnit besliltib'l. dass in dieserPrOteinrichtungdie obengenannten l'rUfungenunterEinhaltungderGLP- Grundsat7.edurchgefilhrt werdenkOnncn. Theabovementionedtestfucilit} is included in the national GLPCompliancel'rogrammeand is inspected on aregularbasis. Basedontheinspectionreponitcan beconfinned. that thistest facilil) is abletoconductthe aforementioned studiesincompliancewiththe l'rinciplesofGLP.

Hess. Mini•tcriumfiir Lmwelt,Encrgic, Landwirt.<ehaftundVerbrauellcnc:llutz, MainzerStralk: liO I>Mt89Winbaden (1\ameund Adressc d•'l'GLI'-Ltberwachungsbehorde/Name andaddressoftheGLPMonitoringAuthority 

### JAJ1**A::'l:]!~p()'"'** 

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### Study Number 1234902 

### Report Leuco Sulfur Black 1 

# 2 CONTENTS 1 COPY OF THE GLP CERTIFICATE 2 CONTENTS 3 PREFACE

3.1 General 3.2 Responsibilities 3.3 Schedule 3.4 ProjectStaffSignatures 3.5 Good Laboratory Practice 3.6 Guidelines 3.7 Archiving 3.8 Deviations from the Study Plan 4 STATEMENT OF COMPLIANCE 5 STATEMENTOF QUALITYASSURANCE UNIT 6 SUMMARY OF RESULTS

6.1 Conclusion 7 INTRODUCTION

7.1 Aims ofthe Study 8 MATERIALSAND METHODS

8.1 Test Item 8.2 Controls 8.3 Test System 8.4 Mammalian Microsomal Fraction S9 Mix 8.5 Pre-experiment 8.6 Dose Selection 8.7 Experimental Performance 8.8 Acceptability ofthe Test 8.9 Evaluation of Results 9 RESULTS AND DISCUSSION 10 DISTRIBUTION OF THE REPORT 11 REFERENCES 12 ANNEX 

### 1: TABLES OF RESULTS

12.1 Summary ofResults 12.2 Experiments Iand II: Determination ofToxicity 12.3 Experiment I 12.4 Experiment II 12.5 Biometry 13 ANNEX II

13.1 

### ChromosomeAberrations: Classification and Criteria 14 ANNEX Ill

14.1 Historical laboratory control data - page 3of36 -



### 2 3 4 4 4 

### 5 

### 5 5 5 6 6 7 

### 8 9 10 11 11 12 12 13 14 15 16 16 17 19 20 21 

### 22 23 24 24 25 27 30 

### 33 34 34 35 35Study Number 1234902 Leuco Sulfur Black 1 

# 3 PREFACE 3.1 General
Title:
Sponsor:

Study Monitor:

Test Facility:

Contracting Institute:

Reference Number: 

## 3.2 Responsibilities Study Director: Deputy Study Director: Management: Head of QualityAssurance Unit: Report



### In vitro 

### ChromosomeAberration Test in Chinese Hamster V79

### Cells with Leuco Sulfur Black 1 Clariant Products (Switzerland) Ltd. Rothausstrasse61 4132 Muttenz 1 Switzerland Contact Person: Dr. H. W. Schneider Dr. Thomas Roth 

# sec GmbH Mikroforum Ring 1 55234 Wendelsheim Germany - page4 of36 -



### Harlan Cytotest Cell Research GmbH (Harlan CCR) In den Leppsteinswiesen 19 64380 Rossdorf Germany Harlan Laboratories Ltd. 4452 ltingen Switzerland C31122

Dr. Caroline Hall Dr. Susanne Bohnenberger Dr. Wolfgang Volkner Frauke Hermann

### Study Number 1234902 Leuco Sulfur Black 1 

## 3.3 Schedule Experimental Starting Date: Report

Experimental Completion Date: March 11 ,2009 April 20, 2009 

## 3.4 Project StaffSignatures Study Director Dr. Caroline Hall

' •••••••••• t •••• ' •**~** ••••••••••••••••• ' ••••••••••••••• 

### Date: July 07, 2009 Management Dr. Wolfgang Volkner

Date: July 07, 2009 

## 3.5 Good Laboratory Practice The studywas performed in compliance with: - page 5of36 -

"Chemikaliengesetz" (Chemicals Act) of the Federal Republic of Germany, "Anhang 1" (Annex 1), version dated July 02, 2008 ("BGBI.", p. 1146). "OECD Principles ofGood Laboratory Practice", as revised in 1997 [C(97)186/Final]. 

## 3.6 Guidelines This study followed the procedures indicated by the following internationally accepted guidelines and recommendations: Ninth Addendum to the OECD Guidelines for Testing of Chemicals, February 1998, adopted July 21, 1997, Guideline No. 473 "In vitro Mammalian Chromosome Aberration Test". Commission Regulation (EC) No. 440/2008, 810: "Mutagenicity - In vitro Mammalian ChromosomeAberration Test", dated May 30, 2008.Study Number 1234902 Leuco Sulfur Black 1 

## 3.7 Archiving Report



### Harlan CCR will archivethe following data for 15 years: Rawdata, study plan, report, and a sample ofthetest item. Microscopicslideswill be archived forat least 12 years. No datawill be discarded withoutthe sponsor's priorwritten consent. 

## 3.8 Deviationsfrom the Study Plan
Therewere no deviationsfrom the study plan. - page 6 of36 -Study Number 1234902 Leuco Sulfur Black 1 Report


# 4 STATEMENT OF COMPLIANCE Study Number: Test Item: Study Director: Title: 1234902 Leuco Sulfur Black 1 Dr. Caroline Hall In vitro 

### Chromosome Aberration Test in Chinese Hamster V79

### Cells with Leuco Sulfur Black 1 - page 7of36 -



### 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), version dated July 02, 2008 ("BGBI.", p. 1146). "OECD Principles ofGood Laboratory Practice", as revised in 1997 [C(97)186/Final]. Therewere no circumstances that may have affectedthe quality or integrity ofthe study.

Study Director Harlan CCR Dr. Caroline Hall

• j


### Date: July 07, 2009Study Number 1234902 Leuco Sulfur Black 1 Report


# 5 STATEMENT OF QUALITYASSURANCE UNIT Study Number: Test Item: Study Director: Title: 1234902 Leuco Sulfur Black 1 Dr. Caroline Hall In vitro 

### ChromosomeAberration Test in Chinese Hamster V79 

### Cells with Leuco Sulfur Black 1 - page 8of36 -



### 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 QualityAssurance Unit. The datesare given below. Phases and Dates ofQAU Inspections/Audits Dates ofReports to the Study Director and to Management r----------·- 

### Study Plan: March 02, 2009 March 02, 2009 1st

### Amendmentto Study Plan: June 05, 2009 June 05, 2009 Study: lns12ection Evaluation: April 15, 2009 April 15, 2009

--------------- --------.------------------· 

### Report: June 05, 2009 June 05, 2009 This statement is to confirm thatthe present report reflectsthe rawdata. Head ofQualityAssurance Unit Frauke Hermann 

## Lyc1ia F<udolph

Date: July07, 2009Study Number 1234902 Leuco Sulfur Black 1 

# 6 SUMMARY OF RESULTS Report - page 9 of36 -



### The test item Leuco Sulfur Black 1, suspended in deionised water, was assessed for its potential to induce structural chromosome aberrations in V79 cells ofthe Chinese hamster in vitro in two independent experiments. The following study designwas performed: WithoutS9 mix With S9 mix Exp.l Exp. II Exp.l Exp. II Exposure period 4 hrs 18 hrs 4 hrs 4 hrs Recovery 14 hrs 

## - 14 hrs 14 hrs Preparation 18 hrs 18 hrs 18 hrs 18 hrs

interval 

### In each experimental group two parallel cultures were set up. 100 metaphases per culture were evaluated forstructural chromosome aberrations. The highest applied concentration in the pre-test on toxicity (5000 IJg/mL) was chosen with respectto the current OECD Guideline 473. 

### Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation. The chosen treatment concentrations are described in chapter 8.6 (page 17). 

### The evaluated experimental points and the results are summarised in Table 1 (page 24). In the absence and presence of S9 mix no cytotoxicity was observed up to the highest evaluable concentration. Due to strong test item precipitation concentrations higher than those examined could not be evaluated for cytogenetic damage. No clastogenicity was observed at the concentrations evaluated either with or without metabolic activation. However, in Experiment II in the presence of S9 mix a single statistically significant increase in the number of aberrant cells excluding gaps (3.0 

### %) 

### was observed at the highest evaluable concentration (150 IJg/mL). This value was clearly in the laboratory's historical solvent control data range (0.0- 4.0% aberrant cells excluding gaps) and is therefore regarded as biologically irrelevant. No evidence ofan increase in polyploid metaphaseswas noticed aftertreatmentwith the test item as compared to the control cultures. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 

### 0.05) in cellswith structural chromosome aberrations.Study Number 1234902 Leuco Sulfur Black 1 

## 6.1 Conclusion Report
- page 1 0of36 -



### In conclusion, it can be stated that underthe experimental conditions reported, the test item did not induce structural chromosome aberrations in V79 

### cells (Chinese hamster cell line) in vitro. 

### Therefore, Leuco Sulfur Black 1 is considered to be non-clastogenic in this chromosome aberration test in the absence and presence of metabolic activation, when tested up to the highest evaluable concentrations.Study Number 1234902 Leuco Sulfur Black 1 

# 7 INTRODUCTION Report - page 11 of36 -



### According to national and international acts chemicals have to be tested before introduction to the market for a possible hazard to humans and the environment. Genotoxicity studies provide important information for the assessment of the mutagenic potential of these substances (1, 4). The in vitro Chromosome Aberration Test performed in this study is an essential partofgenotoxicity test batteries ofsubstances. This in vitro test is a test for the detection of structural chromosomal aberrations. Such aberrations are frequently lethal to the damaged cells (8, 1 0). However, cytogenetic damage in somatic cells is an indicator ofa potential to induce subtle chromosomal damagethat may be compatible with cell division. Similar damage induced in germ cells may lead to heritable cytogenetic abnormalities. Heritable cytogenetic abnormalities are known to have deleterious effects in man, e.g. induction of neoplastic events or birth defects. Also, chromosome abnormalities in somatic cells may become one of the reasons why a transformed cell population may develop into cancer. Chromosome aberrations are generally evaluated in first post treatment mitoses. The majority of chemical mutagens induced structural aberrations are ofthe chromatid type, but chromosome type aberrations also occur. For treatment, cell populations should be in exponential growth to guarantee that there are cells in all stages of the cell cycle (i.e. an asynchronous population). Since the normal cell cycle time is 14 hours (see page 14) and the guidelines require fixation times ofabout 1.5- fold of the normal cell cycle, a fixation time of around 18 hours is appropriate. Due to the limited capacity ofthe V79 cells for metabolic activation ofpotential mutagens an exogenous metabolicactivation system is included (7). To validate the test reference mutagenswere tested in parallel to the test item. 

## 7.1 Aims ofthe Study
This in vitro test was performed to assess the potential of Leuco Sulfur Black 1 to induce structural chromosome aberrations. Evaluation of cytogenetic damage induced in V79 cells (cell line from the lung of the Chinese Hamster) in the absence and the presence of metabolic activation was performed in two independent experiments at one preparation interval (18 hours).Study Number 1234902 Leuco Sulfur Black 1 Report
- page 12 of36 - 

# 8 MATERIALSAND METHODS
8.1 Test Item
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:

MolecularWeight: Stability ofTest 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:
Notavailable 52.75% (calculated by amountof by-compounds) < 

### 20 ppm <

### 20 ppm 970 ppm 750 ppm 1.3% (w/w) 45.75 % (w/w) by Karl-Fischertitration

Stable under storage conditions At room temperature (range 20 

### +/- 5° C), light protected July 11, 2011
On the day ofthe 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 solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.Study Number 1234902 Leuco Sulfur Black 1 

## 8.2 Controls
8.2.1 Solvent Controls Report



### Concurrent solventcontrols (deionised water) were performed.


### 8.2.2 Positive Control Substances
Without metabolicactivation 

### Name: Supplier: Purity: Lot no.: Expiration Date: Dissolved in: Final Concentration: EMS; Ethylmethane sulfonate ACROS ORGANICS, 2440 Geel, Belgium 2:98% A0246840 April2009 Nutrient medium 400 and 1000 IJg/mL (4.0 and 8.0 mM) - page 13 of36 -



### Solutions were prepared on the day of experiment. The stability of the positive control substance in solution was proven by the mutagenic response in the expected range. With metabolicactivation 

### Name: Supplier: Purity: Lot no.: Expiration Date: Dissolved in: Final Concentration: CPA; Cyclophosphamide Sigma-Aldrich Chemie GmbH, 82024 Taufkirchen, Germany 2:98% 097K1311 March 2011 Saline (0.9% [w/v]) 1.4 IJg/mL (5.0 IJM) The dilutions ofthe stock solutions were prepared on the day ofexperiment. The stability of CPA in solution at room temperature is good. At 25

# oc only 3.5% of its potency is lost after 24 hours (6).Study Number 1234902 Leuco Sulfur Black 1 

## 8.3 Test System Report



### 8.3.1 Reasons forthe Choice ofthe Cell Line V79 - page 14of36 -



### The V79 

### cell line has been used successfully for many years in in vitro 

### experiments. The high proliferation rate (doubling time ofclone V79/D3 in stock cultures: 14 hours, determined on April 18, 2008) and a reasonable plating efficiency ofuntreated cells (as a rule more than 70 %) both necessary for the appropriate performance ofthe study, support the use ofthis cell line. The cells have a stable karyotype with a modal chromosome numberof22. Lacking metabolic activities ofcells under in vitro 

### conditions are a disadvantage oftestswith 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 liver microsome preparations (S9 mix). 

### 8.3.2 Cell Cultures Large stocks of the V79 

### cell line (supplied by Laboratory for Mutagenicity Testing, LMP, Technical University Darmstadt, 64287 Darmstadt, Germany) were stored in liquid nitrogen in the cell bank of Harlan CCR allowing the repeated use ofthe same cell culture batch in experiments. Before freezing each batch was screened for mycoplasma contamination and checked for karyotype stability. Consequently, the parameters of the experiments remain similar because ofstandardized characteristics ofthe cells. Thawed stock cultures were propagated at 37 

# oc in 80 cm2 

### plastic flasks (Greiner, 72632 Frickenhausen, Germany). About 5x 105 

### cells per flask were seeded into 15 mL of MEM (Minimal Essential Medium; Invitrogen Gibco, 76131 Karlsruhe, Germany) supplemented with 10% fetal calf serum (FCS; Invitrogen Gibco, 76131 Karlsruhe, Germany). Additionally, the medium was supplemented with 1% 100x Penicillin/ Streptomycin solution (10.000 Units/mL Penicillin, 10 mg/mL Streptomycin; PAA Laboratories GmbH, 35091 Colbe, Germany) and 1% Amphotericin 8-solution (250 IJg/mL, PAA Laboratories GmbH, 35091 Colbe, Germany). The cells were sub-cultured twice weekly. The cell cultures were incubated at 37 

# oc in a humidified atmosphere with 1.5 % carbon dioxide (98.5 %air).

### Study Number 1234902 Leuco Sulfur Black 1 Report


## 8.4 Mammalian Microsomal Fraction 59 Mix

8.4.1 

### 89 (Preparation 

### by Harlan CCR) - page 15of36 -**Phenobarbitai/~-Naphthoflavone** 

### induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from 8- 12 weeks old male Wistar rats (HsdCpb:WU, Harlan Laboratories GmbH, 33178 Borchen, Germany) weight approx. 220 - 320 g induced by intraperitoneal applications of 80 mg/kg b.w. Phenobarbital (Desitin; 22335 Hamburg, Germany) and by peroral administrations of80 mg/kg b.w.**~-Naphthoflavone** 

### (Sigma-Aldrich Chemie GmbH, 82024 Taufkirchen, Germany) each, on three consecutive days. The livers were prepared 24 hours after the last treatment. The S9 fractions were produced by dilution ofthe liver homogenate with a KCI solution (1 :3 parts) followed by centrifugation at 9000 g. Aliquots ofthe supernatantwere frozen and stored in ampoules at -80 °C. Small numbers of the ampouleswere kept at-20

# oc for up to oneweek. The protein concentration was 35.0 mg/mL (Lot no. 211108).


### 8.4.2 

### 89 Mix
The S9 mix preparation was performed according to Ames et al. (1). An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of0.75 mg/mL in the cultures. Cofactorswere added to the S9 mixto reach the following concentrations: 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 experimentthe S9 mixwas stored in an ice bath.Study Number 1234902 Leuco Sulfur Black 1 

## 8.5 Pre-experiment Report
- page 16of36-



### A pre-test on cell growth inhibition was performed in order to determine the toxicity of the test item (2), the solubility during exposure and changes in osmolarity and pH value at experimental conditions. In agreement with the OECD guideline no. 473 the test item was applied up to a maximum concentration of 5000 IJg/mL. This dose selection was not influenced by solubility, pH value, orosmolarity. The experimental conditions in this pre-experiment were identical to those required and described belowforthe mutagenicity assay. 

## 8.6 Dose Selection
The highest concentration used in the cytogenetic experiments were chosen considering the current OECD Guideline for in vitro 

### mammalian cytogenetic tests requesting for the top concentration clear toxicity with reduced cell numbers or mitotic indices below 50% of control, whichever is the lowest concentration, and/orthe occurrence ofprecipitation. In case of non-toxicity the maximum concentration should be 5 mg/ml, 5 IJLimL or 10 mM, whichever is the lowest, ifformulation in an appropriate solvent is possible. 5000 IJg/mL of Leuco Sulfur Black 1was applied as top concentration for treatment of the cultures. Cytotoxicity was determined using the same slides as for the evaluation of cytogenetic damage. Precipitation of the test item was observed at 78.1 IJg/mL and above after 4 hours treatment time in the absence and presence of S9 mix. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I. Dose selection of Experiment II was influenced by the occurrence of the test item precipitation obtained in Experiment I, since no relevant cytotoxicity indicated by reduced cell numbers or reduced mitotic indices below 50 % 

### of control was observed. Therefore, 300 IJg/mL was chosen as top treatment concentration in the absence and presence of S9 mix.Study Number 1234902 Leuco Sulfur Black 1 Report - page 17 of36 -



### Doses applied in the Chromosome aberration testwith Leuco Sulfur Black 1: Preparation Exposure Exp. Concentration

interval period in**~g/mL** Without59 mix 18 hrs 4 hrs I 19.5 39.1 78.1p 156.3p 312.5p 625.0p 1250.0p 2500.0p 5000.0p 18 hrs 18 hrs II 2.3 4.7 9.4 18.8 37.5 75.0 150.0p 300.0p With 59 mix 18 hrs 4 hrs I 19.5 39.1 78.1p 156.3p 312.5p 625.0p 1250.0p 2500.0p 5000.0p 18 hrs 4 hrs II 2.3 4.7 9.4 18.8 37.5 75.0 150.0p 300.0p

Evaluated experimental points are shown in bold characters p Precipitation occurred 4 hours afterstartoftreatment 

### The cytogenetic evaluation of higher concentrations in the respective intervals (with and without S9 mix) was impossible dueto strong test item precipitation. 

## 8.7 Experimental Performance

8.7.1 Seeding ofthe Cultures Exponentially growing stock cultures more than 50% confluent were treated with trypsinEDTA-solution at 37

# oc for approx. 5 minutes. Then the enzymatictreatmentwas stopped by adding complete culture medium and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.5% (w/v) in Ca-Mg-free salt solution (Invitrogen Gibco, 76131 Karlsruhe, Germany). Prior to the trypsin treatment the cells were rinsed with Ca-Mg-free salt solution, which was composed as follows (per litre): NaCI KCI KH2P04 Na2HP04 8000 mg 200 mg 200 mg 150 mg The cells were seeded into Quadriperm dishes (Greiner, 72636 Frickenhausen, Germany) that contained microscopic slides (at least 2chambers per dish and test group). In each chamber 1 x 104

- 6 x 104cells were seeded with regard to the preparation time. The medium was MEM with 10% FCS (complete medium), 1% 100x Penicillin/ Streptomycin and 1% Amphotericin B.Study Number 1234902 Leuco Sulfur Black 1


### 8.7.2 Treatment Exposure period 4 hours Report
- page 18of36 -

The culture medium of exponentially growing cell cultures was replaced with serum-free medium with 1 % 1OOx Penicillin/-Streptomycin-solution and 1% Amphotericin B-solution containing the test item. For the treatment with metabolic activation 50 IJL S9 mix per ml medium were used. Concurrent solvent and positive controls were performed. After 4 hours the cultures were washed twice with "Saline G" and then the cells were cultured in complete medium forthe remaining culture time. The "Saline G" solution was composed asfollows (per litre):

pH was adjusted to 7.2. NaCI 8000 mg KCI 400 mg Glucose x H20 1100 mg NazHP04 x 7 HzO 290 mg KHzP04 150 mg


### Exposure period 18 hours The culture medium of exponentially growing cell cultures was replaced with complete medium (with 10 % FCS, 1 % 1OOx Penicillin/-Streptomycin-solution, 1 % Amphotericin Bsolution) containing different concentrations of the test item without S9 mix. The medium was notchanged until preparation ofthe cells. All cultureswere incubated at37

# oc in a humidified atmospherewith 1.5 % C02 (98.5 % air).


### 8.7.3 Preparation ofthe Cultures
Colcemid was added (0.2 j.Jg/ml culture medium) to the cultures 15.5 hours afterthe start of the treatment. The cells on the slides were treated 2.5 hours later, in the chambers with hypotonic solution (0.4 % KCI) for 20 min at 37

# oc. After incubation in the hypotonic solution the cells were fixed with a mixture of methanol and glacial acetic acid (3:1 parts, respectively). Perexperimenttwo slides per groupwere prepared. After preparation the cells were stained with Giemsa (E. MERCK, 64293 Darmstadt, Germany).Study Number 1234902 Leuco Sulfur Black 1


### 8.7.4 Evaluation ofCell Numbers Report - page 19 of36 -



### The evaluation of cytotoxicity indicated by reduced cell numbers was made after the preparation of the cultures on spread slides. The cell numbers were determined microscopically by counting 10 defined fields per coded slide. The cell number of the treatmentgroups isgiven in percentage compared to the respective solvent control. 

### 8.7.5 Analysis of Metaphase Cells
Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der lndustrie, Cytogenetik" [5]) using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 100well spread metaphases per culture were evaluated for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To describe a cytotoxiceffectthe mitotic index(% cells in mitosis)was determined. 

## 8.8 Data Recording
The data generated were recorded in the raw data file. The results are presented in tabular form, including experimental groupswith the test item, solvent, and positive controls. 

## 8.9 Acceptabilityofthe Test The chromosome aberration test performed in our laboratory is considered acceptable, if it meetsthe following criteria: a) The numberofstructural aberrationsfound in the solvent controls falls within the range of the laboratory's historical control data (see chapter 14.1.1, page 35). b) The positive control substances produce significant increases in the number of cells with structural chromosome aberrations, which are within the range of the laboratory's historical control data (see chapter 14.1.1, page 35).Study Number 1234902 Leuco Sulfur Black 1 

## 8.10 Evaluation ofResults Atest item is classified as non-clastogenic if: Report
- page 20 of36 -



### - the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of the laboratory's historical control data range (see chapter 14.1.1, page 35). and/or

- no significant increase ofthe numberofstructural chromosome aberrations is observed. Atest item is classified as clastogenic if:

- the number of induced structural chromosome aberrations is not in the range of the laboratory's historical control data range (see chapter 14.1.1, page 35). and

- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed. Statistical significance was confirmed by means of the Fisher's exact test (9) (p < 

### 0.05). However, both biological and statistical significance should be considered together. If the criteria mentioned above forthe test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed. Although the inclusion ofthe structural chromosome aberrations is the purpose ofthis study, it is important to include the polyploids and endoreduplications. The following criterion is valid: Atest item can be classified as aneugenic if:

- the number of induced numerical aberrations is not in the range of the laboratory's historical control data range (see chapter 14.1.2, page 36).Study Number 1234902 Leuco Sulfur Black 1 Report


# 9 RESULTS AND DISCUSSION - page 21 of36 -



### The test item Leuco Sulfur Black 1, suspended in deionised water, was assessed for its potential to induce structural chromosome aberrations in 

### V79 cells ofthe Chinese hamster in 

### vitro 

### in the absence and presence of metabolic activation by S9 mix. Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 4 hours with S9 mix and 18 hours without S9 mix. The chromosomes were prepared 18 hours afterstartoftreatmentwith the test item. In each experimental group two parallel cultures were set up. 100 metaphases per culture were evaluated for structural chromosome aberrations. In both experiments no relevant influence of the test item on pH value or osmolarity was observed (Exp. 

### 1: solvent control 286 mOsm, pH 7.4 versus 285 mOsm and pH 7.4 at 5000 IJg/mL; Exp. II: solvent control 284 mOsm, pH 7.4 versus 290 mOsm and pH 7.4 at 300 IJg/mL). Precipitation of the test item in culture medium was observed after treatment with 78.1 IJg/mL and above in the absence and presence of S9 mix in Experiment I and with 150 IJg/mL and above in the absence and presence of S9 mix in Experiment II. No clear toxic effects indicated by reduced mitotic indices or reduced cell numbers were observed after treatment with the test item (see Table 2, 3 and 6, pages 25 to 27 and 30). Due to strong test item precipitation concentrations higherthan those examined could not be evaluated for cytogenetic damage. In both experiments in the absence and presence of S9 mix no biologically relevant increase in the number ofcells carrying structural chromosome aberrations was observed (see Table 4, 5, 7 and 8, pages 28, 29, 31 and 32). The aberration rates ofthe cells aftertreatmentwith the test item (0.0- 3.0 % aberrant cells, excluding gaps) were close to the range of the solventcontrol values (0.5
- 2.0 % aberrantcells, excluding gaps) and within the range ofthe laboratory's historical solvent control data (0.0- 4.0% aberrant cells, excluding gaps). A single significant increase was observed in Experiment II in the presence of S9 mix after treatmentwith 150 IJg/mL. Although this increase of3.0% aberrant cells excluding gapswas statistically significant compared to the low response (0.5% aberrant cells) in the solvent control data, the response is within the historical control data range (0.0
- 4.0 % aberrant cells, excluding gaps). Therefore, this observation has to be regarded as biologically irrelevant. No evidence ofan increase in polyploid metaphaseswas noticed aftertreatmentwith the test item as compared to the controls.Study Number 1234902 Leuco Sulfur Black 1 Report
- page 22 of36 -



### In both experiments, either EMS (400 or 1000 IJg/mL) or CPA (1.41Jg/ml) were used as positive controls and showed distinct increases in the number of cells with structural chromosome aberrations. In conclusion, it can be stated that underthe experimental conditions reported, the test item Leuco Sulfur Black 1did not induce structural chromosome aberrations in V79

### cells (Chinese hamstercell line) when tested up to the highestevaluable concentrations. 

# 10 DISTRIBUTION OF THE REPORT Sponsor Study Director 2x (1x copy, 1xelectronic copy as PDF-file) 1x (original)Study Number 1234902 Leuco Sulfur Black 1 

## 11 REFERENCES Report

1. Ames, B.N., McCann, J. and Yamasaki, E. (1975) - page 23 of36 -

Methodsfor detecting carcinogens and mutagenswith the Salmonella/mammalian microsome mutagenicitytest. Mutation Res., 31, 347-363 2. Armstrong, M.J., Bean, C.L. and Galloway, S.M. (1992) A quantitativeassessmentofthe cytotoxicityassociatedwith chromosome aberration detection in Chinese hamsterovarycells. Mutation Res., 265, 45-60 3. Bean, C.L., Armstrong, M.J. and Galloway, S.M. (1992) Effectofsampling time on chromosome aberration yield for 7chemicals in Chinese hamsterovary cells. Mutation Res., 265, 31-44 4. Bradley, M.O., Bhuyan, B., Francis, M.C., Langenbach, R., Peterson, A. and Huberman, E. (1981) Mutagenesis bychemical agents in V79Chinese hamstercells: a review and analysis ofthe literature. A report ofthe gene-tax program Mutation Res., 87, 81-142 5. Engelhardt, G. (1987) "Arbeitsgruppe der lndustrie, Cytogenetik". Standard-Protokollzurcytogenetischen Auswertung von Mitose- und Meiosechromosomen bei der Routineuntersuchung. 6. Gallelli, J.F. (1967) Stabilitystudies ofdrugs used in intraveneous solutions. Am. J. Hasp. Pharm., 24, 425-433 7. Glatt, H.R. (1993) Fremdstoffmetabolismus und Mutagenitatsprufung, in: Fahrig, R. (ed.): Mutationsforschung und genetischeToxikologie, Wiss. Buchgesellschaft, Darmstadt, 169-185 8. Preston, R.J.J., Au, W., Bender, M.A., Brewen, J.G., Carrano, A.V., Heddle, J.H., McFee, A.F., WolffS. and Wassom, J.S. (1981) Mammalian in vivo and in vitro cytogenetictests. A report ofthe U.S. EPA'sgene-tax program. Mutation Res., 87, 143-188 9. Richardson, C., Williams, D.A., Allen, J.A., Amphlett, G., Chanter, D.O., Phillips, B. (1989) Analysis ofdata from in vitro cytogenetictests Kirkland, D.J. (ed.). "Statistical evaluation ofmutagenicitytest data", Cambridge University Press, Cambridge, 141-154 10. Swierenga, S.H.H., Heddle, J.A., Sigal, E.A., Gilman, J.P.W., Brillinger, R.L., Douglas G.R. and Nestmann, E.R. (1991) Recommended protocols based on the surveyofcurrent practice in genotoxicitytesting laboratories, IV: Chromosome aberration and sisterchromatid exchange in Chinese hamster ovary, V79 Chinese hamster lung and human lymphocytecultures. Mutation Res., 246, 301-322 Supplementary References: Kirkland, D.J. (1992) Chromosome aberration tests in vitro: problemswith protocol design and interpretation ofresults. Mutagenesis, 2, 95-106Study Number 1234902 Report
- page24of36 - Leuco Sulfur Black 1 

## 12ANNEX 

# 1: TABLES OF RESULTS 12.1 SummaryofResults Table 1: Summary ofresults ofthe chromosome aberration studywith Leuco Sulfur Black 1

Exp. Preparation Test item Cell numbers Mitotic indices Aberrant cells interval concentration in% in% in% in !Jg/mL ofcontrol ofcontrol incl. gaps* excl. gaps* Exposure period4 hrswithout59mix 18 hrs Solvent control 1 100.0 100.0 1.5 1.0 Positive control2 n.t. 89.5 14.0 14.05 19.5 85.0 87.5 1.5 1.5 39.1 110.8 95.0 2.5 2.5 78.1p 100.2 107.0 2.0 2.0 Exposure period 18 hrswithout59 mix II 18 hrs Solvent control 1 100.0 100.0 1.0 1.0 Positive control3 n.t. 70.5 13.0 11.55 37.5 97.5 96.9 0.0 0.0 75.0 82.2 90.3 2.5 2.0 150.0p 77.0 112.8 1.5 1.5 Exposure period4 hrswith 59 mix 18 hrs Solvent control1 100.0 100.0 2.0 2.0 Positive control4 n.t. 89.2 10.5 10.05 19.5 79.4 113.5 2.0 1.0 39.1 113.8 120.0 0.0 0.0 78.1p 90.3 93.0 2.5 2.0 II 18 hrs Solvent control 1 100.0 100.0 0.5 0.5 Positive control4 n.t. 87.0 15.5 13.55 37.5 96.4 104.0 2.5 2.5 75.0 93.1 106.0 0.5 0.5 150.0p 84.1 113.0 3.5 3.05 * Including cells carrying exchanges n.t. Nottested p Precipitation occurred 5 Aberrationfrequencystatisticallysignificant higherthan corresponding control values

2 3 4 Deionisedwater 10.0% (v/v) EMS 1000.0j.Jg/ml EMS 400.0 j.Jg/ml CPA 1.4 j.Jg/ml with exchanges

0.0 4.0 1.0 0.5 0.0

0.0 3.0 0.0 0.0 0.5

0.0 2.5 0.0 0.0 0.0 0.0 2.5 1.0 0.0 0.5Study Number 1234902 Leuco Sulfur Black 1 Report - page25of36 -


## 12.2 Experiments Iand II: Determination ofToxicity
The toxicity ofthe test item was examined using the determination of the cell number. Cell numbers oftwo cultures (10 coordinate defined fields per culture) were determined for each experimental group, exceptthe positive control. Table 2: Numberofcells in% ofsolventcontrol
WithoutS9 mix Experiment 1: 4 hrs exposure Experiment II: continuous exposure Preparation Concentration Numberof Cells in% Preparation Concentration Numberof Cells in%

interval in j.Jg/ml cells ofsolvent interval in j.Jg/ml cells ofsolvent control control 18 hrs Solventcontrol 711 100.0 18 hrs Solventcontrol 740 100.0

" 19.5 604 85.0 " 2.3 637 86.0 " 39.1 787 110.8 " 4.7 731 98.8 " 78.1p 712 100.2 " 9.4 595 80.4 " 156.3p n.e. n.e. " 18.8 773 104.4 " 312.5p n.e. n.e. " 37.5 722 97.5 " 625.0p n.e. n.e. " 75.0 609 82.2 " 1250.0p n.e. n.e. " 15D.DP 570 77.0 " 2500.0p n.e. n.e. " 300.0p 599 80.9 " 5000.0p n.e. n.e. Experimental groups evaluatedfor cytogenetic damage are shown in bold characters p Precipitation occurred 4 hrs afterstart oftreatment. n.e. Notevaluable duetotest item precipitation.Study Number 1234902 Leuco Sulfur Black 1 Report



### Table 2 (cont.): Numberofcells in% ofsolventcontrol
With S9 mix Experiment 1: 4 hrsexposure Preparation Concentration Numberof Cells in% Preparation

interval in IJg/mL cells ofsolvent interval control 18 hrs Solventcontrol 807 100.0 18 hrs

" 19.5 640 79.4 " " 39.1 918 113.8 " " 78.1p 728 90.3 " " 156.3p n.e. n.e. " " 312.5p n.e. n.e. " " 625.0p n.e. n.e. " " 1250.0p n.e. n.e. " " 2500.0p n.e. n.e. " " 5000.0p n.e. n.e. - page 26 of36 -

Experiment II: 4 hrs exposure Concentration Numberof Cells in% in IJg/mL cells ofsolvent control Solventcontrol 558 100.0 2.3 520 93.1 4.7 411 73.6 9.4 488 87.4 18.8 666 119.3 37.5 538 96.4 75.0 520 93.1 150.0p 469 84.1 300.0p 508 91.0
Experimental groups evaluated for cytogenetic damage are shown in bold characters p Precipitation occurred4 hours afterstartoftreatment. n.e. Notevaluableduetotest item precipitation.Study Number 1234902 Leuco Sulfur Black 1 

## 12.3 Experiment I
Table 3: Mitotic index; Report



### preparation interval 18 hrs with and without S9 mix
Treatment Cone. 59 Exposure Mitotic indices*

group per ml mix period/ Absolute Mean Recovery 1 2 Solv. control# 10.0 % 

## - 4/14 hrs 9.1 10.9 10.0 Pos. control## 1000.01-Jg 

## - 4/14 hrs 9.2 8.7 9.0 Test item 19.5 IJ9 

## - 4/14hrs 9.2 8.3 8.8 " 39.1 IJ9 

## - 4/14hrs 10.7 8.3 9.5 " 78.1 IJ9 

## - 4/14hrs 10.3 11.1 10.7 Solv. control# 10.0 % + 4/14hrs 10.0 8.5 9.3 Pos. control### 1.4 IJ9 + 4/14 hrs 6.9 9.6 8.3 Test item 19.5 IJ9 + 4/14hrs 11.1 9.9 10.5 " 39.1 IJ9 + 4/14hrs 12.8 9.4 11.1 " 78.1 IJ9 + 4/14 hrs 6.5 10.7 8.6 - page 27 of36 -

%**
100.0 89.5 87.5 95.0 107.0 100.0 89.2 113.5 120.0 93.0 * ** The mitoticindexwas determined in a sample of 1000cells perculture ofeachtestgroup in% Forthe positive control groups and thetest item groups, the relativevalues ofthe mitotic index are related tothe solventcontrols # Deionisedwater ## EMS

- CPAStudy Number 1234902 Leuco Sulfur Black 1 Report



### Table4: Structural chromosome aberrations Experiment I; preparation interval18 hrswithout S9 mix: exposure period 4 hrs Slide Cells %Aberrant cells Aberrations no. scored incl. excl. with ex- - page 28 of36 -

gaps* gaps* changes Gaps 

# I g ig Chromatid type 

# I Chromosometype

# I Other b f d ex ib if id ex ma cd Without59 mix Solventcontrol: Deionisedwater 10.0% 1 100 0 0 2 0 0

# ~I 0 0 0

# ~I 0 0 2 100 2 0 3 0 0 0 0 0 0 0 1+ 2 200 1.5 1.0 0.0 2 0 5 0 0 0 0 0 0 0 Positive control: EMS 1000.0 !Jg I mL 1 100 0 0 13 0 0 3 0 0 0 0 0 0 2 100 1 0 6 2 0 5 0 1 0 0 1 0 1+ 2 200 14.0 14.0 4.0 1 0 19 2 0 8 0 1 0 0 1 0 Test item: 19.51Jg I mL

# ~I 1 100 0 0 0 0 0 2 1 0 0 0 0 2 100 0 0 2 0 0 0 0 0 0 0 0 1+ 2 200 1.5 1.5 1.0 0 0 2 0 0 2 1 0 0 0 0 Test item: 39.1 !Jg ImL 1 100 0 0 2 0 0 0 0 0 0 0 0 0 2 100 0 0 1 1 0 1 0 0 0 0 0 0 1+ 2 200 2.5 2.5 0.5 0 0 3 1 0 1 0 0 0 0 0 0 Test item: 78.1 !Jg ImL 1 100 0 0 1 0 0 0 0 0 0 0 1 0 2 100 0 0 2 0 0 0 0 0 0 0 0 0 1+ 2 200 2.0 2.0 0.0 0 0 3 0 0 0 0 0 0 0 1 0 

### • Inclusive cells carrying exchanges Abbreviations g = gap, ig = iso-gap (gaps are achromatic lesions ofchromatid orchromosometype where no or only a minimal misalignmentofchromosomal material is visible), b=break, ib = iso-break, f =fragment, if= iso-fragment, d =deletion, id = iso-deletion, rna= multiple aberration (=morethan 4 events in one cell [excluding gaps]), ex=chromatid type exchange, ex=chromosometype exchange, cd =chromosomal disintegration (= pulverization)Study Number 1234902 Leuco Sulfur Black 1 Report



### Table 5: Structural chromosome aberrations Experiment I; preparation interval18 hrswith S9 mix: exposure period 4 hrs Slide Cells %Aberrant cells Aberrations no. scored incl. excl. with ex- - page 29 of36 -

gaps* gaps* changes Gaps 

# I g ig Chromatid type 

# I Chromosometype

# I Other b f d ex ib if id ex rna cd With 59 mix Solventcontrol: Deionised water 10.0 % 

# u H H 1 100 0 1 0 0 0 0 0 0 0 2 100 0 2 0 0 1 0 0 0 0 1+ 2 200 2.0 2.0 0.0 0 3 0 0 1 0 0 0 0 Positive control: CPA 1.4**~g** I ml 1 100 1 0 8 1 0 3 2 0 0 1 0 0 2 100 0 0 8 0 0 2 0 0 0 0 0 0 1 + 2 200 10.5 10.0 2.5 1 0 16 1 0 5 2 0 0 1 0 0 Test item: 19.5**~g** I ml

# ~I 1 100 1 0 2 0 0 0 0 0 0 0 0 2 100 1 1 0 0 0 0 0 0 0 0 0 1+ 2 200 2.0 1.0 0.0 2 1 2 0 0 0 0 0 0 0 0 Test item: 39.1**~g** I ml

# ~1 1 100 0 0 0 0 0 0 0 0 0 0 0 2 100 0 0 0 0 0 0 0 0 0 0 0 1+ 2 200 0.0 0.0 0.0 0 0 0 0 0 0 0 0 0 0 0 Test item: 78.1**~g** I ml 1 100 1 0 4 0 0 0 0 0 0 0 0 0 2 100 1 0 1 0 0 0 0 0 0 0 0 0 1+ 2 200 2.5 2.0 0.0 2 0 5 0 0 0 0 0 0 0 0 0 * Inclusive cells carrying exchanges Abbreviations g = gap, ig = iso-gap (gaps are achromatic lesions ofchromatid or chromosome type where no or only a minimal misalignmentofchromosomal material is visible), b= break, ib

# = iso-break, f =fragment, if= iso-fragment, d = deletion, id = iso-deletion, rna = multiple aberration (= more than 4 events in one cell [excluding gaps]), ex=chromatid type exchange, ex=chromosometype exchange, cd =chromosomal disintegration (= pulverization)Study Number 1234902 Leuco Sulfur Black 1 

## 12.4 Experiment II
Table 6: Mitotic index; Report



### preparation interval 18 hrswith and without S9 mix
Treatment Cone. S9 Exposure Mitotic indices*

group per ml mix period/ Absolute Mean Recovery 1 2 Solv. control' 10.0 % 

## - 18/- hrs 12.2 10.5 11.4 Pos. control" 400.0 

### IJ9 

## - 18/-hrs 7.3 8.7 8.0 Test item 37.5 

### IJ9 

## - 18/-hrs 12.3 9.7 11.0 " 75.0 

### IJ9 

## - 18/-hrs 10.3 10.2 10.3 " 150.0 

### IJ9 

# - 18/- hrs 13.1 12.5 12.8 Solv. control# 10.0 % + 4/14hrs 8.8 11.2 10.0 Pos. control- 1.4 

### IJ9 + 4/14hrs 8.6 8.8 8.7 Test item 37.5 

### IJ9 + 4/14hrs 9.6 11.2 10.4 " 75.0 

### IJ9 + 4/14 hrs 10.2 11.0 10.6 " 150.0 

### IJ9 + 4/14 hrs 12.4 10.2 11.3 - page30of36 -

%**
100.0 70.5 96.9 90.3 112.8 100.0 87.0 104.0 106.0 113.0 * ** The mitotic indexwas determined in a sample of 1000cells perculture ofeach testgroup in % Forthe positive control groups and thetest item groups, the relativevalues ofthe mitotic index are related tothe solventcontrols # Deionised water 

# " EMS ### CPAStudy Number 1234902 Leuco Sulfur Black 1 Report



### Table 7: Structural chromosome aberrations Experiment II; preparation interval18 hrswithout S9 mix: exposure period 18 hrs Slide Cells %Aberrantcells Aberrations no. scored incl. excl. with ex- - page 31 of36 -

gaps* gaps* changes

## Gap~

# l g tg Chromatid type 

# I Chromosometype

# I Other b f d ex ib if id ex rna cd Without S9 mix Solventcontrol: Deionised water 10.0%

# ~I**~I ~I**  1 100 0 1 0 0 0 0 0 0 0 2 100 0 0 0 0 0 1 0 0 0 1+2 200 1.0 1.0 0.0 0 1 0 0 0 1 0 0 0 0 Positive control: EMS400.0 j.Jg/mL 1 100 1 0 9 0 0 4 0 0 0 0 0 0 2 100 3 0 11 1 0 1 1 0 0 1 0 0 1 +2 200 13.0 11.5 3.0 4 0 20 1 0 5 1 0 0 1 0 0 Test item: 37.5 iJg/mL

# ~I**~I** 1 100 0 0 0 0 0 0 0 0 0 0 2 100 0 0 0 0 0 0 0 0 0 0 1+2 200 0.0 0.0 0.0 0 0 0 0 0 0 0 0 0 0 Test item: 75.0 IJg/mL 1 100 0 0 1 0 0 0 0 0 0 0 0 0 2 100 1 0 3 0 0 0 0 0 0 0 0 0 1+2 200 2.5 2.0 0.0 1 0 4 0 0 0 0 0 0 0 0 0 Test item: 150.0 IJg/mL 1 100 1 0 1 0 0 0 0 0 0 1 0 0 2 100 0 0 1 0 0 0 0 0 0 0 0 0 1 +2 200 1.5 1.5 0.5 1 0 2 0 0 0 0 0 0 1 0 0 Inclusive cells carrying exchanges Abbreviations g = gap, ig = iso-gap (gaps are achromatic lesions ofchromatid or chromosome type where no or only a minimal misalignmentofchromosomal material is visible), b= break, ib = iso-break, f =fragment, if= iso-fragment, d = deletion, id = iso-deletion, rna = multiple aberration (= more than 4 events in one cell [excluding gaps]), ex =chromatid type exchange, ex =chromosometype exchange, cd = chromosomal disintegration (= pulverization)Study Number 1234902 Leuco Sulfur Black 1 Report



### Table 8: Structural chromosome aberrations Experiment II; preparation interval18 hrswith S9 mix: exposure period 4 hrs Slide Cells %Aberrant cells Aberrations no. scored incl. excl. with ex- - page 32 of36 -

gaps* gaps* changes Gaps 

# I Chromatid type 

# I Chromosometype

# I Other g ig b f d ex ib if id ex ma cd With 59 mix Solventcontrol: Deionised water 10.0%

# ~I 1 100 0 0 0 0 0 0 0 0 0 0 0 2 100 0 1 0 0 0 0 0 0 0 0 0 1 + 2 200 0.5 0.5 0.0 0 1 0 0 0 0 0 0 0 0 0 Positive control: CPA 1.4 IJg/ml 1 100 4 0 15 0 0 4 0 1 0 0 0 0 2 100 2 0 4 2 0 2 2 2 0 0 0 0 1+ 2 200 15.5 13.5 2.5 6 0 19 2 0 6 2 3 0 0 0 0 Test item: 37.5 IJg/ml

# ~I 1 100 0 3 0 0 2 2 0 0 0 0 0 2 100 0 0 0 0 0 0 0 0 0 0 0 1+ 2 200 2.5 2.5 1.0 0 3 0 0 2 2 0 0 0 0 0 Test item: 75.0 IJg/ml 1 100 0 0 0 0 0 0 0 0 0 0 0 0 2 100 0 0 1 0 0 0 0 0 0 0 0 0 1+ 2 200 0.5 0.5 0.0 0 0 1 0 0 0 0 0 0 0 0 0 Test item: 150.0 IJg/ml 1 100 2 0 3 0 1 1 1 0 0 0 0 0 2 100 0 0 2 0 0 0 0 0 0 0 0 0 1+ 2 200 3.5 3.0 0.5 2 0 5 0 1 1 1 0 0 0 0 0 

### • Inclusive cells carrying exchanges Abbreviations g = gap, ig = iso-gap (gaps are achromatic lesions ofchromatid orchromosome type where no or only a minimal misalignment ofchromosomal material is visible), b=break, ib = iso-break, f =fragment, if= iso-fragment, d = deletion, id = iso-deletion, rna = multiple aberration (= more than 4 events in one cell [excluding gaps]), ex=chromatid type exchange, ex=chromosometype exchange, cd =chromosomal disintegration (= pulverization)Study Number 1234902 Leuco Sulfur Black 1 

## 12.5 Biometry Report - page 33 of36 -



### Statistical significance at the five per cent level (p < 

### 0.05) was evaluated by means of the Fisher's exacttest. Evaluation was performed only for cells carrying aberrations excluding gaps. Table 9: Biometry of Experiment I
Testgroupversus Preparation Exposure S9 mix p-value

solventcontrol interval period Testgroup 19.5 IJg/mL 18 hrs 4 hrs 

## - 0.343 " 39.1 IJg/mL 18 hrs 4 hrs 

## - 0.143 " 78.1 IJg/mL 18 hrs 4 hrs 

## - 0.225

......... .-·--**~**··- ........ ··-- --· . ---- . ········-- ··----- " 19.51Jg/ml 18 hrs 4 hrs + n.c. " 39.1 IJg/mL 18 hrs 4 hrs + n.c. " 78.1 IJg/mL 18 hrs 4 hrs + n.c. Positive control versus solventcontrol EMS 1000.0 IJg/mL 18 hrs 4 hrs 

## - < 0.001 5 CPA 1.4 IJg/mL 18 hrs 4 hrs + < 0.001 5
n.c. Notcalculated asthe aberration rate is equal orlowerthanthe corresponding control rate s Aberration rate is statisticallysignificant higherthan the control rate 

### Table 10: Biometry of Experiment II
Testgroupversus Preparation Exposure S9 mix p-value

solventcontrol interval period Testgroup 37.5 IJg/mL 18 hrs 18 hrs 

## - n.c. " 75.0 IJg/mL 18 hrs 18 hrs 

## - 0.225 " 150.0 IJg/mL 18 hrs 18 hrs
- 0.343**-~------··------·-··--·--·-·--·-··**-·-·····.. ....**----~----··--** -----·- ···----- " 37.5 IJg/mL 18 hrs 4 hrs + 0.061 " 75.0 IJg/mL 18 hrs 4 hrs + n.c. " 150.0 IJg/mL 18 hrs 4 hrs + 0.0345 Positive control versus solventcontrol EMS 400.0 IJg/mL 18 hrs 18 hrs
- < 0.001 5 CPA 1.4 IJg/mL 18 hrs 4 hrs + < 0.001 5
n.c. Notcalculated asthe aberration rate is equal orlowerthan the corresponding control rate 5 Aberration rate is statisticallysignificant higherthan the control rateStudy Number 1234902 Leuco Sulfur Black 1 

# 13 ANNEX II Report



### 13.1 ChromosomeAberrations: Classification and Criteria
1. Gaps - page 34 of36 -

Gaps are small areas of the chromosome, which are unstained. The chromatids remain aligned as normal and the gap does not extend alongthe chromatid for a distance greaterthan thewidth ofa chromatid. Ifthe gapoccurs on one chromatid only it is a chromatid gap (g). 2. Chromatid Breaks Chromatid breaks (b) vary in appearance. The chromatid may remain aligned but show a gap which is too large to classify as a gap. Alternatively, the chromatid may be broken so that the broken fragment is displaced. In some cases, the fragment is not seen at all. A chromatid fragment (f) should be evaluated ifthe chromosome oforigin cannot be identified. In addition, deletions can occur as a result of a break. The missing terminal end of a chromatid in the assessed metaphase is classified as deletion (d). 3. Chromosome Breaks Chromosome breaks (ib) are breaks in both chromatids ofthe chromosome. A fragment with two chromatids is formed and this may be displaced by varying degrees. Breaks are distinguishedfrom gaps bythe size ofthe unstained region. A chromosome break is evaluated ifthefragment is associated with a chromosomefrom which itwas probablyderived. However, fragments are often seen in isolation and are then evaluated as chromatid fragments (if). In addition, isodeletions can occur as a result of an isobreak. The missing terminal end of a chromosome in theassessed metaphase is classified as isodeletion (id). 4. Exchanges Exchanges are formed by faulty reJOining of broken chromosomes and may be of the chromosome or chromatid type. Chromatid exchanges (ex) have numerous differentforms but are generally not further classified. Where multiple exchanges have occurred each exchange point is counted as one chromatid exchange. Chromosome exchanges (ex) generally appear as either a dicentric or a ring form, either ofwhich can be associated with a fragment, which if possible should be evaluated as partofthe exchange. 5. MultipleAberrations If many aberrations are present in one metaphase, the exact details may not be evaluable. This is particularly the case when chromosome pulverisation (cd) occurs. If the number of aberrations is greaterthan 4then the cell is classified as multiple aberrant (rna). 6. Chromosome Number If the chromosome (centromere) number is 22 

### ± 1 then it is classified as a diploid cell and evaluated for aberrations. Iflessthan 22

### ± 1chromosomes are counted then the cell is ignored under the assumption, that some chromosomes may have been lost for technical reasons. If greater than 22 

### ± 1 chromosomes are evaluated then the count is recorded and the cell classified as an aneuploid cell. If multiple copies of the haploid chromosome number (other than diploid) are evaluated then the count is recorded and the cell classified as polyploid. Ifthe chromosomes are arranged in closely apposed pairs, i.e. 4 chromatids instead of2, the cell is evaluated asendoreduplicated (e).Study Number 1234902 Leuco Sulfur Black 1 

# 14 ANNEX Ill Report - page 35 of36 -



## 14.1 Historical laboratorycontrol data 14.1.1 Percentage ofaberrantcells in Chinese hamster 

### V79 cell cultures (January2006toJanuary2008)

TreatmenU preparation interval (hours)

4/18 18/18 28/28

4/18 (800 j.Jg/ml) 4/18 (900 j.Jg/ml) 18/18 (400 j.Jg/ml) 18/18 (500 j.Jg/ml) 28/28 (400 j.Jg/ml) 28/28 (500 j.Jg/ml)

TreatmenU preparation interval

4/18 4/28

4/18 (1.0 j.Jg/ml) 4/18 (1.4 j.Jg/ml) 4/28 (1.4 IJg/ml) 4/28 (2.0 IJg/mL) Without59 mix Aberrantcells excluding gaps (%) No. ofperformed Range Mean Standard studies (Min.- Max.) deviation Negative control 103 0.0-4.0 1.6 0.9 72 0.0-3.5 1.4 0.8 70 0.0-4.0 1.4 1.0 Positive control Ethyl methanesulfonate (EMS) 8 9.0-17.0 12.8 2.3 53 8.0-38.0 13.9 6.0 10 7.0-34.0 14.0 8.1 45 8.5-41.0 15.5 6.5 11 5.0-35.0 12.7 8.1 43 8.0-51.0 26.3 10.7

With 59 mix Aberrant cells excluding gaps (%) No. ofperformed Range Mean studies Negative control 110 0.0-4.0 2.0 66 0.0-4.0 1.6 Positive control Cyclophosphamide (CPA)

20 115 12 74 5.0-21.0 7.5-38.0 4.5-23.5 7.5-44.0 10.7 13.2 11.0 12.1 

### ;)Qe;:)**-·~.rr** Standard deviation

0.9 0.9

4.1 4.9 6.0 6.2Study Number 1234902 Leuco Sulfur Black 1 Report
- page 

### 36of

### 36

-



### 14.1.2 Percentage ofpolyploid cells in Chinese hamster V79 

### cell cultures (January2006toJanuary2008)
Negative control Polyploidcells (%) TreatmenUpreparation No. ofperformed Range Mean Standard interval (hours) studies (Min.- Max.) deviation WithoutS9 mix 4/18 102 1.0-4.6 2.3 0.8 18/18 72 0.0-5.2 2.6 0.9 28/28 70 1.0-4.4 2.4 0.8 With S9 mix 4/18 110 0.6-4.3 2.3 0.8 4/28 66 0.7-5.3 2.5 1.0



# CONTAINS NO CBI 

# /