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 1234902 

IN VITRO 

CHROMOSOME ABERRATION TEST 

IN CHINESE HAMSTER V79 CELLS 

WITH 

LEUCO SULFUR BLACK 1 

REPORT 

Study Completion Date: 

July 07, 2009 

CONTAINS NO CBI 

p~ 

3 y0 J55 

harlan·M .¥ 

fa I-;).. l/ r2.. 
Study Number 1234902 

Leuco Sulfur Black 1 

Report 

1 COPY OF THE GLP CERTIFICATE 

- page 2 of 36 -

HESS EN 

Gute Laborpraxis/Good Laboratory Practice 

GLP-Bescheinigung!Statement of GLP Compliance 

(gemJll.llaccording to § 19b Abs. I Chemikaliengesetz) 

Eine GLP-Inspektion T.Ur Oberwachung der Einhaltung 

der Gli'-Grundsl!t:re gemlill Chemikaliengesetz bzw. 

Richtlinie 2004/'1/EG wurde durchgefilhn in 

i2li'!Ufeinrichtung'l'est facility 

As.o;essment of confonnity with GLI' according to 

Chemikaliengesetz and Directi•·e 2004.'9/EEC at: 

0 l'rtlfSiandort!J'est site 

Harlan Cytotest Cell Research GmbH 

Harlan C}1otest Cell Research GmbH 

In den l.cppsteinswicsen 19 

643811 RoJldorf 

(IJnverwechselbare Bezeichnung und Adresse/Unequivocal name and adress) 

PrOfugen auch Katcgorien/Arcu of Expcrti~e 

(gemat.liaccording chemVwV-GLP Nr. 5.3/0ECD guidance) 

2 PnHungen zur Bt'Slirnmung dcr- tox1kologischt~n 

Etgf'nschaftf'n 

3 PrU.fungen zur Hestimmung cl~r erbgutveriindernden Eigenschaften {in vitro und in vivo) 

6 PrUfungen zur Bestimmung von 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 on bwlogtcal matenals 

l!i.08. und 27.-29.10.20011 

Datum der Inspektion!Date oflnspeclion 

(Tag Monat Jahr-'day month year) 

Die genannle l'rilfeinrichtung befindet sich im nationalen GI.P-OberwachungsverfallfCn und wird rcgcl· 

mallig auf Einhaltung der GLP-Grundsatz.e Uberwacht. 

Auf der Grundlage des lnspektionsbcrichtes wird hicnnit 

besliltib'l. dass in dieser PrOteinrichtung die oben genannten l'rUfungen unter Einhaltung der GLP- Grundsat7.e durchgefilhrt werden kOnncn. 

The above mentioned test fucilit} is included 

in the national GLP Compliance l'rogramme and is 

inspected on a regular basis. 

Based on the inspection repon it can be con finned. 

that this test facilil) is able to conduct the 

aforementioned studies in compliance with the 

l'rinciples of GLP. 

Hess. Mini•tcrium fiir Lmwelt, Encrgic, Landwirt.<ehaft und Verbrauellcnc:llutz, 

Mainzer Stralk: liO I>Mt89 Winbaden 

(1\ame und Adressc d•'l' GLI'-Ltberwachungsbehorde/Name and address ofthe GLP Monitoring Authority 

JAJ1 A::'l:]!~p()'"' .i,:)2T 

v1()i' t.: .~~ : .i/'. 

j/)/ 

(aa<J~~; (t J,, ; 
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 Project Staff Signatures 

3.5 Good Laboratory Practice 

3.6 Guidelines 

3.7 Archiving 

3.8 Deviations from the Study Plan 

4 STATEMENT OF COMPLIANCE 

5 STATEMENT OF QUALITY ASSURANCE UNIT 

6 SUMMARY OF RESULTS 

6.1 Conclusion 

7 INTRODUCTION 

7.1 Aims of the Study 

8 MATERIALS AND 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 of the 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 of Results 

12.2 Experiments I and II: Determination of Toxicity 

12.3 Experiment I 

12.4 Experiment II 

12.5 Biometry 

13 ANNEX II 

13.1 Chromosome Aberrations: Classification and Criteria 

14 ANNEX Ill 

14.1 Historical laboratory control data 

- page 3 of 36 -

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 

35 
Study 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 

Quality Assurance Unit: 

Report 

In vitro Chromosome Aberration Test 

in Chinese Hamster V79 Cells 

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 

- page 4 of 36 -

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 Staff Signatures 

Study Director Dr. Caroline Hall 

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

Date: July 07, 2009 

Management Dr. Wolfgang Volkner 

Date: July 07, 2009 

3.5 Good Laboratory Practice 

The study was performed in compliance with: 

- page 5 of 36 -

"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 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 

Chromosome Aberration Test", dated May 30, 2008. 
Study Number 1234902 

Leuco Sulfur Black 1 

3. 7 Archiving 

Report 

Harlan CCR will archive the following data for 15 years: 

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

Microscopic slides will be archived for at least 12 years. 

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

3.8 Deviations from the Study Plan 

There were no deviations from the study plan. 

- page 6 of 36 -
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 7 of 36 -

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 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. Caroline Hall 

• j 

Date: July 07, 2009 
Study Number 1234902 

Leuco Sulfur Black 1 

Report 

5 STATEMENT OF QUALITY ASSURANCE UNIT 

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 8 of 36 -

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. 

Phases and Dates of QAU Inspections/ Audits Dates of Reports to the Study Director 

and to Management r----------·-

Study Plan: March 02, 2009 March 02, 2009 

1st Amendment to 

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 that the present report reflects the raw data. 

Head of Quality Assurance Unit Frauke Hermann 

Lyc1ia F<udolph 

Date: July 07, 2009 
Study Number 1234902 

Leuco Sulfur Black 1 

6 SUMMARY OF RESULTS 

Report - page 9 of 36 -

The test item Leuco Sulfur Black 1, suspended in deionised water, was assessed for its 

potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in 

vitro in two independent experiments. The following study design was performed: 

Without S9 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 for structural chromosome aberrations. 

The highest applied concentration in the pre-test on toxicity (5000 IJg/mL) was chosen with 

respect to 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 of an increase in polyploid metaphases was noticed after treatment with 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 cells with structural chromosome aberrations. 
Study Number 1234902 

Leuco Sulfur Black 1 

6.1 Conclusion 

Report - page 1 0 of 36 -

In conclusion, it can be stated that under the 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 of 36 -

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 part of genotoxicity test batteries of substances. 

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 of a potential to induce subtle chromosomal damage that 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 of the 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 of about 1.5-

fold of the normal cell cycle, a fixation time of around 18 hours is appropriate. Due to the 

limited capacity of the V79 cells for metabolic activation of potential mutagens an exogenous 

metabolic activation system is included (7). 

To validate the test reference mutagens were tested in parallel to the test item. 

7.1 Aims of the 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 of 36 -

8 MATERIALS AND 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: 

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: 

Not available 

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 

Stable under storage conditions 

At room temperature (range 20 +/- 5° 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 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 solvent controls (deionised water) were performed. 

8.2.2 Positive Control Substances 

Without metabolic activation 

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 of 36 -

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 metabolic activation 

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 of the stock solutions were prepared on the day of experiment. 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 for the Choice of the Cell Line V79 

- page 14 of 36 -

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

high proliferation rate (doubling time of clone V79/D3 in stock cultures: 14 hours, determined 

on April 18, 2008) and a reasonable plating efficiency of untreated cells (as a rule more than 

70 %) both necessary for the appropriate performance of the study, support the use of this 

cell line. The cells have a stable karyotype with a modal chromosome number of 22. 

Lacking metabolic activities of cells under in vitro conditions are a disadvantage of tests 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 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 of the 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 of standardized characteristics of the cells. 

Thawed stock cultures were propagated at 37 oc in 80 cm2 plastic flasks (Greiner, 

72632 Frickenhausen, Germany). About 5 x 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 15 of 36 -

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 of 80 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 

of the liver homogenate with a KCI solution (1 :3 parts) followed by centrifugation at 9000 g. 

Aliquots of the supernatant were frozen and stored in ampoules at -80 °C. Small numbers of 

the ampoules were kept at -20 oc for up to one week. 

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 of 0. 75 mg/mL in the cultures. Cofactors were added 

to the S9 mix to 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 experiment the S9 mix was stored in an ice bath. 
Study Number 1234902 

Leuco Sulfur Black 1 

8.5 Pre-experiment 

Report - page 16 of 36-

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, or osmolarity. 

The experimental conditions in this pre-experiment were identical to those required and 

described below for the 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/or the occurrence of precipitation. In case 

of non-toxicity the maximum concentration should be 5 mg/ml, 5 IJLimL or 10 mM, 

whichever is the lowest, if formulation in an appropriate solvent is possible. 

5000 IJg/mL of Leuco Sulfur Black 1 was 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 of 36 -

Doses applied in the Chromosome aberration test with Leuco Sulfur Black 1: 

Preparation Exposure Exp. Concentration 

interval period in ~g/mL 

Without 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 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 after start of treatment 

The cytogenetic evaluation of higher concentrations in the respective intervals (with and 

without S9 mix) was impossible due to 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 trypsinEDT A-solution at 37 oc for approx. 5 minutes. Then the enzymatic treatment was 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 2 chambers per dish and test group). In each 

chamber 1 x 104 - 6 x 104 cells 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 18 of 36 -

The culture medium of exponentially growing cell cultures was replaced with serum-free 

medium with 1 % 1 OOx 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 for the remaining culture time. 

The "Saline G" solution was composed as follows (per litre): 

pH was adjusted to 7.2. 

NaCI 8000 mg 

KCI 400 mg 

Glucose x H20 11 00 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 % 1 OOx Penicillin/-Streptomycin-solution, 1 % Amphotericin Bsolution) containing different concentrations of the test item without S9 mix. The medium 

was not changed until preparation of the cells. 

All cultures were incubated at 37 oc in a humidified atmosphere with 1.5 % C02 (98.5 % air). 

8. 7.3 Preparation of the Cultures 

Colcemid was added (0.2 j.Jg/ml culture medium) to the cultures 15.5 hours after the 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). Per experiment two slides per group were 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 of Cell Numbers 

Report - page 19 of 36 -

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 

treatment groups is given 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. 100 well spread meta phases 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 

cytotoxic effect the 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 groups with the test item, solvent, and positive controls. 

8.9 Acceptability of the Test 

The chromosome aberration test performed in our laboratory is considered acceptable, if it 

meets the following criteria: 

a) The number of structural aberrations found 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 of Results 

A test item is classified as non-clastogenic if: 

Report - page 20 of 36 -

- 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 of the number of structural chromosome aberrations is observed. 

A test 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 for the 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 of the structural chromosome aberrations is the purpose of this study, 

it is important to include the polyploids and endoreduplications. The following criterion is 

valid: 

A test 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 of 36 -

The test item Leuco Sulfur Black 1, suspended in deionised water, was assessed for its 

potential to induce structural chromosome aberrations in V79 cells of the 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 after start of treatment with 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 higher than 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 of cells carrying structural chromosome aberrations was observed (see Table 

4, 5, 7 and 8, pages 28, 29, 31 and 32). The aberration rates of the cells after treatment with 

the test item (0.0- 3.0 % aberrant cells, excluding gaps) were close to the range of the 

solvent control values (0.5 - 2.0 % aberrant cells, excluding gaps) and within the range of the 

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 

treatment with 150 IJg/mL. Although this increase of 3.0% aberrant cells excluding gaps was 

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 of an increase in polyploid metaphases was noticed after treatment with the test 

item as compared to the controls. 
Study Number 1234902 

Leuco Sulfur Black 1 

Report - page 22 of 36 -

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 under the experimental conditions reported, the test item 

Leuco Sulfur Black 1 did not induce structural chromosome aberrations in V79 cells (Chinese 

hamster cell line) when tested up to the highest evaluable concentrations. 

10 DISTRIBUTION OF THE REPORT 

Sponsor 

Study Director 

2x (1x copy, 1x electronic 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 of 36 -

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

mutagenicity test. 

Mutation Res., 31, 347-363 

2. Armstrong, M.J., Bean, C.L. and Galloway, S.M. (1992) 

A quantitative assessment of the cytotoxicity associated with chromosome aberration detection in 

Chinese hamster ovary cells. 

Mutation Res., 265, 45-60 

3. Bean, C.L., Armstrong, M.J. and Galloway, S.M. (1992) 

Effect of sampling time on chromosome aberration yield for 7 chemicals in Chinese hamster ovary 

cells. 

Mutation Res., 265, 31-44 

4. Bradley, M.O., Bhuyan, B., Francis, M.C., Langenbach, R., Peterson, A. and Huberman, E. (1981) 

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

literature. A report of the gene-tax program 

Mutation Res., 87, 81-142 

5. Engelhardt, G. (1987) 

"Arbeitsgruppe der lndustrie, Cytogenetik". Standard-Protokoll zur cytogenetischen Auswertung 

von Mitose- und Meiosechromosomen bei der Routineuntersuchung. 

6. Gallelli, J.F. (1967) 

Stability studies of drugs 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 

genetische Toxikologie, 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., 

Wolff S. and Wassom, J.S. (1981) 

Mammalian in vivo and in vitro cytogenetic tests. A report of the U.S. EPA's gene-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 of data from in vitro cytogenetic tests 

Kirkland, D.J. (ed.). "Statistical evaluation of mutagenicity test 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 survey of current practice in genotoxicity testing 

laboratories, IV: Chromosome aberration and sister chromatid exchange in Chinese hamster 

ovary, V79 Chinese hamster lung and human lymphocyte cultures. 

Mutation Res., 246, 301-322 

Supplementary References: 

Kirkland, D.J. (1992) 

Chromosome aberration tests in vitro: problems with protocol design and interpretation of results. 

Mutagenesis, 2, 95-106 
Study Number 1234902 Report - page 24 of 36 -

Leuco Sulfur Black 1 

12 ANNEX 1: TABLES OF RESULTS 

12.1 Summary of Results 

Table 1: Summary of results of the chromosome aberration study with Leuco Sulfur Black 1 

Exp. Preparation Test item Cell numbers Mitotic indices Aberrant cells 

interval concentration in% in% in% 

in !Jg/mL of control of control incl. gaps* excl. gaps* 

Exposure period 4 hrs without 59 mix 

18 hrs Solvent control1 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 hrs without 59 mix 

II 18 hrs Solvent control1 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 period 4 hrs with 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 control1 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. Not tested p Precipitation occurred 

5 Aberration frequency statistically significant higher than corresponding control values 

2 

3 

4 

Deionised water 10.0% (v/v) 

EMS 1000.0 j.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 

0.5 
Study Number 1234902 

Leuco Sulfur Black 1 

Report - page 25 of 36 -

12.2 Experiments I and II: Determination of Toxicity 

The toxicity of the test item was examined using the determination of the cell number. Cell 

numbers of two cultures (10 coordinate defined fields per culture) were determined for each 

experimental group, except the positive control. 

Table 2: Number of cells in% of solvent control 

Without S9 mix 

Experiment 1: 4 hrs exposure Experiment II: continuous exposure 

Preparation Concentration Number of Cells in% Preparation Concentration Number of Cells in% 

interval in j.Jg/ml cells of solvent interval in j.Jg/ml cells of solvent 

control control 

18 hrs Solvent control 711 100.0 18 hrs Solvent control 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 evaluated for cytogenetic damage are shown in bold characters p Precipitation occurred 4 hrs after start of treatment. 

n.e. Not evaluable due to test item precipitation. 
Study Number 1234902 

Leuco Sulfur Black 1 

Report 

Table 2 (cont.): Number of cells in% of solvent control 

With S9 mix 

Experiment 1: 4 hrs exposure 

Preparation Concentration Number of Cells in% Preparation 

interval in IJg/mL cells of solvent interval 

control 

18 hrs Solvent control 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 of 36 -

Experiment II: 4 hrs exposure 

Concentration Number of Cells in% 

in IJg/mL cells of solvent 

control 

Solvent control 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 occurred 4 hours after start of treatment. 

n.e. Not evaluable due to test 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 of 36 -

%** 

100.0 

89.5 

87.5 

95.0 

107.0 

100.0 

89.2 

113.5 

120.0 

93.0 

* 

** 

The mitotic index was determined in a sample of 1000 cells per culture of each test group in% 

For the positive control groups and the test item groups, the relative values of the mitotic index are 

related to the solvent controls 

# Deionised water 

## EMS 

- CPA 
Study Number 1234902 

Leuco Sulfur Black 1 

Report 

Table 4: Structural chromosome aberrations Experiment I; 

preparation interval18 hrs without S9 mix: exposure period 4 hrs 

Slide Cells % Aberrant cells Aberrations 

no. scored incl. excl. with ex-

- page 28 of 36 -

gaps* gaps* changes 

Gaps I 

g ig 

Chromatid type I Chromosome type I Other 

b f d ex ib if id ex ma cd 

Without 59 mix 

Solvent control: Deionised water 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 I mL 

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 I mL 

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 of chromatid or chromosome type where no or only 

a minimal misalignment of chromosomal 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= chromosome type exchange, cd =chromosomal 

disintegration (= pulverization) 
Study Number 1234902 

Leuco Sulfur Black 1 

Report 

Table 5: Structural chromosome aberrations Experiment I; 

preparation interval18 hrs with S9 mix: exposure period 4 hrs 

Slide Cells % Aberrant cells Aberrations 

no. scored incl. excl. with ex-

- page 29 of 36 -

gaps* gaps* changes 

Gaps I 

g ig 

Chromatid type I Chromosome type I Other 

b f d ex ib if id ex rna cd 

With 59 mix 

Solvent control: De ionised 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 of chromatid or chromosome type where no or only 

a minimal misalignment of chromosomal 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= chromosome type exchange, cd =chromosomal 

disintegration (= pulverization) 
Study Number 1234902 

Leuco Sulfur Black 1 

12.4 Experiment II 

Table 6: Mitotic index; 

Report 

preparation interval 18 hrs with 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 

- page 30 of 36 -

%** 

100.0 

70.5 

96.9 

90.3 

112.8 

100.0 

87.0 

104.0 

106.0 

113.0 

* 

** 

The mitotic index was determined in a sample of 1000 cells per culture of each test group in % 

For the positive control groups and the test item groups, the relative values of the mitotic index are 

related to the solvent controls 

# Deionised water 

" EMS 

### CPA 
Study Number 1234902 

Leuco Sulfur Black 1 

Report 

Table 7: Structural chromosome aberrations Experiment II; 

preparation interval18 hrs without S9 mix: exposure period 18 hrs 

Slide Cells % Aberrant cells Aberrations 

no. scored incl. excl. with ex-

- page 31 of 36 -

gaps* gaps* changes Gap~ l 

g tg 

Chromatid type I Chromosome type I Other 

b f d ex ib if id ex rna cd 

Without S9 mix 

Solvent control: 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: EMS 400.0 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 of chromatid or chromosome type where no or only 

a minimal misalignment of chromosomal 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 = chromosome type exchange, cd = chromosomal 

disintegration (= pulverization) 
Study Number 1234902 

Leuco Sulfur Black 1 

Report 

Table 8: Structural chromosome aberrations Experiment II; 

preparation interval18 hrs with S9 mix: exposure period 4 hrs 

Slide Cells % Aberrant cells Aberrations 

no. scored incl. excl. with ex-

- page 32 of 36 -

gaps* gaps* changes 

Gaps I Chromatid type I Chromosome type I Other 

g ig b f d ex ib if id ex ma cd 

With 59 mix 

Solvent control: 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 of chromatid or chromosome type where no or only 

a minimal misalignment of chromosomal 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= chromosome type exchange, cd =chromosomal 

disintegration (= pulverization) 
Study Number 1234902 

Leuco Sulfur Black 1 

12.5 Biometry 

Report - page 33 of 36 -

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the 

Fisher's exact test. Evaluation was performed only for cells carrying aberrations excluding 

gaps. 

Table 9: Biometry of Experiment I 

Test group versus Preparation Exposure S9 mix p-value 

solvent control interval period 

Test group 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 

solvent control 

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. Not calculated as the aberration rate is equal or lower than the corresponding control rate 

s Aberration rate is statistically significant higher than the control rate 

Table 10: Biometry of Experiment II 

Test group versus Preparation Exposure S9 mix p-value 

solvent control interval period 

Test group 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 

solvent control 

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. Not calculated as the aberration rate is equal or lower than the corresponding control rate 

5 Aberration rate is statistically significant higher than the control rate 
Study Number 1234902 

Leuco Sulfur Black 1 

13 ANNEX II 

Report 

13.1 Chromosome Aberrations: Classification and Criteria 

1. Gaps 

- page 34 of 36 -

Gaps are small areas of the chromosome, which are unstained. The chromatids remain 

aligned as normal and the gap does not extend along the chromatid for a distance greater than 

the width of a chromatid. If the gap occurs 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 if the chromosome of origin 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 of the chromosome. A fragment with 

two chromatids is formed and this may be displaced by varying degrees. Breaks are 

distinguished from gaps by the size of the unstained region. A chromosome break is evaluated 

if the fragment is associated with a chromosome from which it was probably derived. 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 the assessed 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 different forms 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 of which can be associated with a fragment, which if 

possible should be evaluated as part of the exchange. 

5. Multiple Aberrations 

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 greater than 4 then 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. If less than 22 ± 1 chromosomes 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. If the 

chromosomes are arranged in closely apposed pairs, i.e. 4 chromatids instead of 2, the cell is 

evaluated as endoreduplicated (e). 
Study Number 1234902 

Leuco Sulfur Black 1 

14 ANNEX Ill 

Report - page 35 of 36 -

14.1 Historical laboratory control data 

14.1.1 Percentage of aberrant cells in Chinese hamster V79 cell cultures 

(January 2006 to January 2008) 

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) 

Without 59 mix 

Aberrant cells excluding gaps (%) 

No. of performed 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. of performed 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 

6.2 
Study Number 1234902 

Leuco Sulfur Black 1 

Report - page 36 of 36 -

14.1.2 Percentage of polyploid cells in Chinese hamster V79 cell cultures 

(January 2006 to January 2008) 

Negative control 

Polyploid cells (%) 

TreatmenU preparation No. of performed Range Mean Standard 

interval (hours) studies (Min.- Max.) deviation 

Without S9 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 

/