# RE PORT

IN VITRO MICRONUCLEUS TEST IN CHINESE HAMSTER V79
CELLS 

# TEST GUIDELINE(S)

OECD 487 (201 0)
Commission Regulation (EC) No 640/2012 

# AUTHOR

STUDY COMPLETION DATE
May 17, 2013 

# TEST FACILITY

TEST FACILITY PROJECT IDENTIFICATION

SPONSOR

Page 1 of 39



### This document is directed to USEPA pursuant the Toxic Substances Control Act (TSCA). This document is the property of the sponsor and may be used only for that purpose intended by the sponsor and as authorized under applicable law. Every other or additional use, exploitation, reproduction, publication or submission to other parties requires the written permission of the sponsor, except as otherwise required by applicable law (e.g., TSCA). The use of these studies/documents for regulatory, registration or other commercial purposes [including but not limited to Registration, Evaluation, Authorization and Restriction of Chemical Substances (REACH)], without the express written permission of the sponsor is unauthorized and strictly prohibited.Rep ort - page 2 of 39 -



# 1 PAGE RESERVED FOR PROPRIETARY INFORMATION OR STATEMENT OF NO DATA CONFIDENTIALITY CLAIMSReport



# 2 STATEMENT OF COMPLIANCE - page 

### 3 of 

### 39

-



### This study performed in the test facility of was conducted in compliance with Good Labaratory Practice Regulations:
"Chemikaliengesetz" ( Chemieals Act) of the Federal Republic of Germany, "Anhang 1" (Annex 1 ), in its currently valid version.

These regulations are in accordance with GLP standards published as OECD Principles on Good Labaratory Practice (revised 1997, ENV/MC/CHEM(98)17), and are in accordance with, and implement, the requirements of Directives 2004/9/EC and 2004/1 0/EC.

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

Study Director

Date: May 17, 2013Report - page 4 of 39 -



# 3 PAGE RESERVED FOR FLAGGING STATEMENTSReport



# 4 S I GNATURE

Study Director

Date: May 17, 2013 - page 5 of 39

-Report



# 5 STATEMENT OF QUALITY ASSURANCE UNIT

Study: 1533001

Test ltem:
Study Director:

Title: ln vitro Micronucleus Test in Chinese Hamster 

### V79 Cells - page 6 o f 39

-



### The general facilities and activities of 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.

The General Study Plan 487

. MN79. was rev iewed for compliance on April18, 2012.

Phasesand Dates of QAU lnspections/ Audits Dates of Reports to the Study Director and to Management Study Specific Supplement: February 15, 2013 February 15, 2013

Process lns(2ection Test item preparation: March 13, 2013 March 13, 2013 Test system preparation and application: April 04
, 2013 April 04, 2013 Report: May 13, 2013 May 13, 2013

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

Head of Quality Assurance UnitRepor t - page 7 of 39 -



# 6 COPY OF THE GLP CERTIFICATE

Gute L aho r praxis/Good Laboratory Practice**GLP~Bescheinigung/Statcment** 

### of GLP Compliance (gemäß / accor di ng to § 19b Abs. I Chemikaliengesetz)

Ein e G L

P

- Inspektion zur Überwachung der Einhaltung der GLP

- Grundsätze gemäß Chemikaliengesetz bzw

. Richtli.iue 2004i9/EG w u rde durchgefuhr t in Assessment of conformity wi th GLP according to Chemikaliengesetz and Directive 200 4/ 9/EEC at:

(8:1 Prüfeinrich t ung

- Test facility 

### D Prüf s tandott 'T est site

(Unverwechselbare Be z eichnung und Adresse/Unequivocal narne and adre ss)
l'rüfuogeo nach Kategorie n /Areas ofEx p ert i se (gernäß /a ccording chemVwV

- GLP Nr. 5.3/0ECD guidance)

2 Prüfungen zur Be stimmung der toxiko l ogischen Eigenschaften 3 Prüfungen zur Be stimmung cler erbgutverän

d ernden Eigenschaften (in vit.ro u nd in vivo) 8 Ana ly tis c h e Prüfungen an bio l ogischen Materialien 2 Tax icity studies

3 Mutagenicity stuclies

8 Analytical studies on biological materials

25. April, 23./25

. un d 2 6 . .J uli 20 1 2 Datum de r Inspektion / Date of lnspection (Tag Monat Jalu· /d ay mouth year)

Die genannte Prüfeinrichtung befutdet sich im nationa l

en GLP

- Überwachungsv erf ahren und wird regelmäßig auf Einhalt u ng d er GLP

- Gmndsätze überwacht.
J\uf d e r Gnmdlagc des Inspektionsberichtes wird hiem1it bes t ätigt, dass in diese r Prüfeinrichtung die oben ge

nannten Priifimgcn unte r Ein h altung der GLP

- GrundsMtze dur.chgcflllut werden körnten. The above mentioned t es t facility is included in the national GLP Compliance Prograll1llle a nd is inspcctcd on a reg t dar basis.

Based on the in s pection report it can be c on llrroed, that this test. facility is able to conduct the aforementioned studies in compli ance with the Principles ofGLP.

(Name u nd J\dresse der GLP-Überwac h ungsbehörde/Na me and add r ess of the GLP Monitaring Autb.ori t yReport - page 8 of 39 -



# 7 CONTENTS

1 PAGE RESERVED FOR PROPRIETARY INFORMATION OR STATEMENT OF NO DATA CONFIDENTIALITY CLAIMS 2
2 STATEMENT OF COMPLIANCE 3
3 PAGE RESERVED FOR FLAGGING STATEMENTS 4
4 SIGNATURE 5
5 STATEMENT OF QUALITY ASSURANCE UNIT 6
6 COPY OF THE GLP CERTIFICATE 7
7 CONTENTS 8
8 PREFACE 10

8.1 General 10
8.2 Responsibilities 10
8.3 Schedule 10
8.4 Good Laboratory Practice 11
8.5 Guidelines 11
8.6 Archiving 11
8.7 Deviations from the Study Plan 12
9 SUMMARY OF RESULTS 13

9.1 Conclusion 15
10 INTRODUCTION 16

10.1 Aims of the Study 16
11 MATERIALS AND METHODS 17

11.1 Test Item 17
11.2 Controls 18
11.2.1 Solvent Controls 18
11.2.2 Positive Control Substances 18
11.3 Test System 19
11.3.1 Reasons for the Choice of the Cell Line V79 19
11.3.2 Cell Cultures 19
11.4 Mammalian Microsomal Fraction S9 Mix 19
11.4.1 S9 (Preparation by CCR) 19
11.4.2 S9 Mix 20
11.5 Dose Selection 20
11.6 Experimental Performance 21
11.6.1 Culture Medium and Conditions 21
11.6.2 Seeding of the Cultures 21Report - page 9 of 39 -



### 11.7 Treatment 21
11.7.1 Preparation of the Cultures 22
11.7.2 Analysis of Micronuclei and Cytotoxicity 22
11.8 Data Recording 22
11.9 Acceptability of the Assay 23
11.10 Evaluation of Results 23
12 RESULTS AND DISCUSSION 24
13 DISTRIBUTION OF THE REPORT 25
14 REFERENCES 26
15 ANNEX I: TABLES OF RESULTS 28

15.1 Experiment I 28
15.2 Experiment II 32
15.3 Biometry 36
16 ANNEX II 38

16.1 Historical laboratory control data 38
16.1.1 Percentage of micronucleated cells in Chinese hamster V79 cell cultures (October 2006 to August 2011) 38Report - page 10 of 39 -



# 8 PREFACE

8.1 General

Title: In vitro Micronucleus Test in Chinese Hamster V79 Cells
Sponsor:

Study Monitor:
Test Facility:



## 8.2 Responsibilities

Study Director: Deputy Study Director: Management:
Head of Quality Assurance Unit:



## 8.3 Schedule

Study Initiation Date: February 15, 2013 Experimental Starting Date: February 27, 2013 Experimental Completion Date: April 11, 2013Report - page 11 of 39 -



## 8.4 Good Laboratory Practice

The work described was performed in compliance with the Principles of GLP according to the “Chemikaliengesetz” (Chemicals Act) of the Federal Republic of Germany, “Anhang 1”, (Annex 1), in its currently valid version. These regulations are in accordance with GLP standards published as OECD Principles on Good Laboratory Practice (revised 1997, ENV/MC/CHEM(98)17), and are in accordance with, and implement, the requirements of Directives 2004/9/EC and 2004/10/EC.



## 8.5 Guidelines

This study followed the procedures indicated by the following internationally accepted guidelines and recommendations:
OECD Guideline for Testing of Chemicals, July 22, 2010, Guideline No. 487 “ In vitro Mammalian Cell Micronucleus Test.
Commission Regulation (EU) No 640/2012 of 6 July 2012, B49 “ In vitro Mammalian Cell Micronucleus Test”.



## 8.6 Archiving

The final report will be issued and sent to the Sponsor. The final report (or true copies) with original signatures will be archived at The raw data, specimens (as long as the quality permits evaluation), the study plan, and the final report (or true copies) will be sent by to the Sponsor. The Sponsor will archive the raw data, specimens (as long as the quality permits evaluation), the study plan and the final report for at least the period of time specified in the GLP principles at

will archive: Copies of raw data, study plan and report for at least 3 years at the test facility’s archive. Thereafter, the material will be transferred to the GLP archive of . in for archiving the remaining time up to a total archiving period of 15 years. No data will be discarded without the sponsor’s written consent.
A sample of the test item will be archived two years after the expiration date provided by the sponsor. If no expiration date is given, the archiving period will be the required 15 years. Thereafter the samples will be discarded without further notice.Report - page 12 of 39 -



## 8.7 Deviations from the Study Plan

Guidelines
This study followed the procedures indicated by the following internationally accepted guidelines and recommendations:
OECD Guideline for Testing of Chemicals, July 22, 2010, Guideline No. 487 “ In vitro Mammalian Cell Micronucleus Test.
Commission Regulation (EU) No 640/2012 of 6 July 2012, B49 “ In vitro Mammalian Cell Micronucleus Test”.
Reason for the deviation (printed in bold letters): Updating.
This deviation has no detrimental impact on the outcome of the study.Report - page 13 of 39 -



# 9 SUMMARY OF RESULTS

The test item , dissolved in THF, was assessed for its potential to induce micronuclei 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. I Exp. II Exp. I & II Exposure period 4 hrs 24 hrs 4 hrs Recovery 20 hrs

 20 hrs Preparation interval 24 hrs 24 hrs 24hrs



### In each experimental group two parallel cultures were analyzed and 1000 cells per culture were scored for micronuclei.
The highest applied concentration (2200.0 L g/mL; approx. 4.9 mM) was chosen with regard to the solubility properties of the test item and with respect to the OECD Guideline No. 487.
In the absence and presence of S9 mix no cytotoxic effects were observed up to the highest applied concentration.
No mutagenicity was observed either with or without S9 mix. However, in Experiment I in the presence of S9 mix one single increase (1.90 % micronucleated cells) slightly above the range of the laboratory historical control data (0.05 – 1.70 % micronucleated cells) was observed after treatment 9.0 L g/mL. Since the value was not statistically significant and no dose-dependency was observed, the finding has to be regarded as biologically irrelevant.
Appropriate mutagens were used as positive controls. They induced statistically significant increases in the percentage of micronucleated cells.Report - page 14 of 39 -



### Table 1: Summary of results of the micronucleus test with

Exp. Preparation Test item Proliferation Micronucleated interval concentration Index Cells* in L g/mL in % Exposure period 4 hrs without S9 mix

I 24 hrs Solvent control 1 2.94 0.75
Positive control 2 2.18 8.10 S

1.4 2.85 0.80
9.0 PS 2.84 0.90
22.5 PS 2.92 0.95
56.3 P/PS 2.85 0.50
2200.0 P/PS 2.83 n.e.
Exposure period 24 hrs without S9 mix

II 24 hrs Solvent control 1 2.75 0.50
Positive control 3 2.63 7.65 S

1.4 2.76 0.45
9.0 PS 2.78 0.35
22.5 PS 2.81 0.55
56.3 P/PS 2.72 0.35
2200.0 P/PS 2.78 n.e.

* The number of micronucleated cells was determined in a sample of 2000 cells
S Number of micronucleated cells statistically significantly higher than corresponding control values
P Precipitation occurred microscopically at the end of treatment
PS Phase separation occurred at the end of treatment n.e. Not evaluated since no cytotoxicity was observed up to the highest applied concentration
1 THF 0.5 % (v/v)
2 Mitomycin C 0.3 L g/mL
3 Griseofulvin 8.0 L g/mLReport - page 15 of 39 -



### Table 1, cont.: Summary of results of the micronucleus test with

Exp. Preparation Test item Proliferation Micronucleated interval concentration Index Cells* in L g/mL in % Exposure period 4 hrs with S9 mix

I 24 hrs Solvent control 1 1.98 1.20
Positive control 2 1.60 11.80 S

3.6 1.96 1.25
9.0 PS 1.96 1.90
22.5 PS 1.94 1.05
56.3 P/PS 2.00 1.20
2200.0 P/PS 2.03 n.e.
II 24 hrs Solvent control 1 2.01 0.75
Positive control 2 1.42 2.45 S

3.6 1.94 0.75
9.0 PS 2.06 1.15
22.5 P/PS 2.27 0.70
2200.0 P/PS 2.31 n.e.

* The number of micronucleated cells was determined in a sample of 2000 cells
S Number of micronucleated cells statistically significantly higher than corresponding control values
P Precipitation occurred microscopically at the end of treatment
PS Phase separation occurred at the end of treatment n.e. Not evaluated since no cytotoxicity was observed up to the highest applied concentration
1 THF 0.5 % (v/v)
2 CPA 15.0 L g/mL



## 9.1 Conclusion

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei in V79 cells (Chinese hamster cell line) in vitro in the absence and presence of metabolic activation.

Therefore, is considered to be non-mutagenic in this in vitro test system, when tested up to the highest applied concentration.Report - page 16 of 39 -



# 10 INTRODUCTION

According to national and international acts chemicals have to be tested before introduction to the market for a possible hazard to humans and environment. Genotoxicity studies provide important information for the assessment of the mutagenic potential of these substances (1, 4).

This in vitro test is an assay for the detection of micronuclei. The occurrence of micronuclei in interphase cells provides an indirect but easy and rapid measure of chromosomal damage and aneugenicity. Micronuclei arise from chromosomal fragments or whole chromosomes. Micronuclei rarely occur spontaneously but are inducible by clastogens or agents affecting the spindle apparatus (7, 8).
Micronuclei are generally evaluated in initial post treatment mitoses. Treatment of cell populations should be performed during 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 of V79 cells is 12 - 14 hours, a preparation time of approximately 24 hours is appropriate. Due to the limited capacity for metabolic activation of potential mutagens an exogenous metabolic activation system is necessary (2).
For validation of the test, reference mutagens were tested in parallel to the test item.



## 10.1 Aims of the Study

This in vitro assay was performed to assess the potential of to induce micronuclei in the absence and presence of metabolic activation (liver S9 mix from phenobarbital/ -naphthoflavone treated rats). Evaluation of micronucleated V79 cells (Chinese hamster cell line) was performed in two independent experiments at one preparation interval (24 hours).Report - page 17 of 39 -



# 11 MATERIALS AND METHODS

11.1 Test Item

Internal Test Item Number: S 1439411
The test item and the information concerning the test item were provided by the sponsor.

Test Item:
Test Item No.: 12/0275-1
Batch Number: 0008043725 Purity: 93.8 area-% (further details see analytical report No. AU124464-1, provided by the sponsor) Expiration Date: December 2013 Physical state, appearance: Solid, melt, white Molecular weight: 452.72 g/mol Storage conditions: At room temperature Stability in Solvent: Not indicated by the Sponsor
On the day of the experiment (immediately before treatment), the test item was dissolved in THF. The final concentration of THF in the culture medium was 0.5 % (v/v). The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
The osmolarity and pH-value were determined in the solvent control and the maximum concentration without metabolic activation:

Concentration [ L g/mL] Solvent control

Exp. I Osmolarity [mOsm] 2200.0 369 350
pH-value 7.5 7.5

Exp. II Osmolarity [mOsm] 2200.0 370 352
pH-value 7.5 7.4Report - page 18 of 39 -



## 11.2 Controls

11.2.1 Solvent Controls
Concurrent solvent controls (THF) were performed.
Name: THF Supplier: E. MERCK, 64293 Darmstadt, Germany Purity: 99.9 % Lot no.: 1648307240



### 11.2.2 Positive Control Substances

Without metabolic activation
Name: MMC; Mitomycin C Supplier: E. MERCK, 64293 Darmstadt Lot no.: 098 K 1681 Dissolved in: Deionised water Final Concentration: 0.3 L g/mL

Name: Griseofulvin Supplier: Sigma-Aldrich Chemie GmbH 82024 Taufkirchen, Germany Lot no.: 031 M 1289 V Dissolved in: DMSO Final Concentration: 8.0 L g/mL

With metabolic activation

Name: CPA; cyclophosphamide Supplier: Sigma-Aldrich Chemie GmbH 82024 Taufkirchen, Germany Lot no.: 120 M 1253 V Dissolved in: Saline Final Concentration: 15.0 L g/mL
The dilutions of the stock solutions were prepared on the day of the experiment. The stability of the positive control substances (Mitomycin C, Griseofulvin and CPA, respectively) in solution was proven by the mutagenic response in the expected range.Report - page 19 of 39 -



## 11.3 Test System

11.3.1 Reasons for the Choice of the Cell Line V79

The V79 cell line has been used successfully for many years in in vitro experiments. Especially the high proliferation rate (doubling time of V79 cells in stock cultures: 13 hours, determined on December 17, 2010) and a reasonable plating efficiency of untreated cells (in general  70 %) both necessary for the appropriate performance of the study, recommend the use of this cell line. 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 if they are metabolised by the mammalian organism. Metabolic activation of chemicals can be achieved at least partially by supplementing the cell cultures with liver microsome preparations (S9 mix).



### 11.3.2 Cell Cultures

Stocks of the V79 cells (obtained from ) are stored in liquid nitrogen in the cell bank of This allows the repeated use of the same cell culture batch in experiments. Before freezing each batch is screened for mycoplasm contamination and checked for karyotype stability. Consequently, the parameters of the experiments remain similar on time because of the reproducible characteristics of the cells. Thawed stock cultures were propagated at 37 °C in 80 cm 2 

### plastic flasks. About 5 x 10 5 

### cells per flask ere seeded in 15 mL of MEM (minimal essential medium) containing Hank’s salts, glutamine, Hepes (25 mM) and 10 % (v/v) fetal bovine serum (FBS). Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 L g/mL). The cells were sub-cultured twice a week. The cell cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % carbon dioxide (98.5 % air).



## 11.4 Mammalian Microsomal Fraction S9 Mix

11.4.1 S9 (Preparation by CCR)

Phenobarbital/ -naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from 8 - 12 weeks old male Wistar rats ( ) weight approx. 220 - 320 g induced by intraperitoneal applications of 80 mg/kg b.w. phenobarbital (Desitin, 22335 Hamburg, Germany) and by oral 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 KCl 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 °C for up to one week. Each batch of S9 is routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.Report - page 20 of 39 -



### The protein concentration was 25.3 mg/mL (Lot no. 141212) for Experiment I and 21.3 mg/mL (Lot no. 310113) for Experiment II.



### 11.4.2 S9 Mix
The S9 mix preparation was performed according to Ames et al. (1). Briefly, 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. S9 mix contained MgCl 2 

### (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4). During the experiment the S9 mix was stored on ice.



## 11.5 Dose Selection

Dose selection was performed following the current OECD Guideline No. 487. In general, the test item should be tested up to a maximum concentration of 5 mg/mL, 5 L L/mL or 10 mM if the test item is not toxic. The highest treatment concentration chosen for the evaluation of genotoxicity should reduce the proliferation index to 55 ± 5 % of the solvent control. The solubility of the test item and changes in the pH value and osmolarity influence the dose selection. With respect to the solubility of the test item, 2200.0 L g/mL of was applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations between 0.6 and 2200.0 L g/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. Precipitation of the test item was observed at 56.3 L g/mL and above. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I. Since no cytotoxicity was observed up to the highest applied concentration, 2200.0 L g/mL was chosen as top treatment concentration for Experiment II.
Table 2: Doses applied in the Micronucleus Test in Chinese Hamster V79 Cells with

Preparation

interval E x posure period Exp

. Concentration in L g/ mL

Without S9 mix

24 hrs 4 hrs I 0.6 1.4 3.6 PS 9.0 PS 22.5 PS 56.3 P/PS 140.8 P/PS 352.0 P/PS 880.0 P/PS 2200.0 P/PS

24 hrs 24 hrs II 0.6 1.4 3.6 PS 9.0 PS 22.5 PS 56.3 P/PS 140.8 P/PS 352.0 P/PS 880.0 P/PS 2200.0 P/PS

With S9 mix

24 hrs 4 hrs I 0.6 1.4 3.6 9.0 PS 22.5 PS 56.3 P/PS 140.8 P/PS 352.0 P/PS 880.0 P/PS 2200.0 P/PS

24 hrs 4 hrs II 0.6 1.4 3.6 9.0 PS 22.5 P/PS 56.3 P/PS 140.8 P/PS 352.0 P/PS 880.0 P/PS 2200.0 P/PS

Evaluated experimental points are shown in bold characters

P Precipitation occurred microscopically at the end of treatment

PS Phase separation occurred at the end of treatmentReport - page 21 of 39 -



## 11.6 Experimental Performance

11.6.1 Culture Medium and Conditions
For seeding and treatment of the cell cultures the culture medium was MEM (minimal essential medium) containing Hank’s salts, glutamine, Hepes (25 mM) and 10 % (v/v) fetal bovine serum (FBS). Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 L g/mL). All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO 2 

### (98.5 % air).



### 11.6.2 Seeding of the Cultures
Exponentially growing stock cultures more than 50 % confluent were rinsed with Ca-Mg-free salt solution containing 8000 mg/L NaCl, 200 mg/L KCl, 200 mg/L KH 2 

### PO 4 

### and 150 mg/L Na 2 

### HPO 4 

### . Afterwards the cells were treated with trypsin-EDTA-solution at 37 °C for approx. 5 minutes. Then, by adding complete culture medium including 10 % (v/v) FBS the enzymatic treatment was stopped and a single cell suspension was prepared. The trypsin concentration for all subculturing steps was 0.25 % (w/v) in Ca-Mg-free salt solution. The cells were seeded into Quadriperm dishes, which contained microscopic slides. The cells were seeded into Quadriperm dishes, which contained microscopic slides. Into each chamber 1.0 x 10 5



### – 1.5 x 10 5 

### cells were seeded with regard to the preparation time.



## 11.7 Treatment

Exposure period 4 hours The culture medium of exponentially growing cell cultures was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 L L S9 mix per mL medium was added. Concurrent solvent and positive controls were performed. After 4 hours the cultures were washed twice with "Saline G" (pH 7.2) containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose • 

### H 2 

### O, 192 mg/L Na 2 

### HPO 4 • 

### 2 H 2 

### O and 150 mg/L KH 2 

### PO 4 

### . Then the cells were cultured in complete medium containing 10 % (v/v) FBS for the remaining culture time of 20 hours.
Exposure period 24 hours The culture medium of exponentially growing cell cultures was replaced with complete medium containing 10 % (v/v) FBS including the test item without S9 mix. The medium was not changed until preparation of the cells. Concurrent solvent and positive controls were performed.Report - page 22 of 39 -



### 11.7.1 Preparation of the Cultures
For the micronucleus analysis, 24 hours after the start of the exposure, the cells were treated on the slides in the chambers of the quadriperm dishes with deionised water for 1 to 1.5 min at 37 °C. Afterwards the cells were fixed twice with a solution containing 3 parts ethanol, 1 part acetic acid and 1.25 % (v/v) formaldehyde. After preparation the cells were stained with Giemsa and labelled with a computer-generated random code to prevent scorer bias.



### 11.7.2 Analysis of Micronuclei and Cytotoxicity
Evaluation of the cultures was performed manually using NIKON microscopes with 40x objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (4). Briefly the micronuclei were stained in the same way as the main nucleus. The area of the micronucleus did not extend the third part of the area of the main nucleus. 1000 cells in two parallel cultures were scored for micronuclei, so that at least 2000 cells from clones with 2 - 8 cells were analysed per test group. The frequency of micronucleated cells was reported as % micronucleated cells. Cytotoxicity was assessed via counting the number of clones consisting of 1 cell (c1), 2 cells (c2), 3 - 4 cells (c4), and 5 - 8 cells (c8) among the cells that were scored for the presence of micronuclei. These clusters represented the cells that have divided 1, 2, or 3 times within the experiment. From these data, a proliferation index (PI) was calculated (see formula below [6,7]). Only those cultures were evaluated which showed a PI > 1.3, in order to guarantee for

a sufficient cell proliferation during treatment and recovery.



### c8)c4c2(c1 4) x(c83)x(c42)x(c21)x(c1 

# PI

+++ + + +

=

PI Proliferation index cx Number of clones with x cells (with x: 1, 2, 4, or 8)



## 11.8 Data Recording

The data generated was recorded in the raw data file. The results were presented in tabulated form, including experimental groups with the test item and the controls (positive and solvent controls).Report - page 23 of 39 -



## 11.9 Acceptability of the Assay

The micronucleus assay is considered acceptable if it meets the following criteria:
a) The number of micronucleated cells carrying one or more micronuclei found in the solvent controls falls within the range of the historical laboratory control data range (see ANNEX II).
b) The micronucleus frequency in the positive controls is statistically significantly increased.
c) The quality of the slides must allow the evaluation of a sufficient number of analyzable cells.



## 11.10 Evaluation of Results

A test item can be classified as mutagenic if:

 the number of micronucleated cells is not in the range of the historical control data (see ANNEX II) and

 either a concentration-related increase in three test groups or a statistically significant increase in the number of micronucleated cells is observed.

A test item can be classified as non-mutagenic if:

 the number of micronucleated cells in all evaluated test groups is in the range of the historical control data (see ANNEX II) and

 no statistically significant or concentration-related increase in the number of micronucleated cells is observed in comparison to the respective solvent control.
Statistical significance was confirmed by means of the Chi square test.Report - page 24 of 39 -



# 12 RESULTS AND DISCUSSION

The test item , dissolved in THF, was assessed for its potential to induce micronuclei 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 24 hours without S9 mix and 4 hours with metabolic activation. The cells were prepared 24 hours after start of treatment with the test item. In each experimental group two parallel cultures were set up and 1000 cells per culture were scored for micronuclei. The highest treatment concentration in this study, 2200.0 L g/mL (approx. 4.9 mM) was chosen with regard to the solubility properties of the test item and with respect to the OECD Guideline for in vitro mammalian cytogenetic tests. Visible precipitation of the test item in the culture medium was observed microscopically at the end of treatment at 56.3 L g/mL and above in Experiment I in the absence and presence of S9 mix and in Experiment II in the absence of S9 mix. In Experiment II in the presence of S9 mix precipitation was observed microscopically at the end of treatment at 22.5 L g/mL and above. Phase separation was observed in the absence of S9 mix at 3.6 L g/mL and above and in the presence of S9 mix at 9.0 L g/mL and above. No relevant influence on osmolarity or pH value was observed (see chapter 11.1). In the absence and presence of S9 mix, no cytotoxic effects were observed up to the highest applied concentration (Table 3, 4, 7 and 8, page 28, 29, 32 and 33). No mutagenicity was observed either with or without S9 mix. The rates of micronucleated cells after treatment with the test item in Experiment I without S9 mix (Table 5, page 30) and in Experiment II with and without S9 mix (Table 9 and 10, page 34 and 35) (0.35 - 1.15 %) were close to the rates of the solvent control values (0.50 - 0.75 %) and within the range of the laboratory historical solvent control data (4 hours treatment without S9 mix: 0.15 – 1.50 % micronucleated cells; 24 hours treatment without S9 mix: 0.05 – 1.50 % micronucleated cells; 4 hours treatment with S9 mix: 0.05 – 1.70 % micronucleated cells). In Experiment I in the presence of S9 mix one single increase (1.90 % micronucleated cells) slightly above the range of the laboratory historical control data (0.05 - 1.70 % micronucleated cells) was observed after treatment 9.0 L g/mL (Table 6, page 31). Since the value was not statistically significant and no dose-dependency was observed, the finding has to be regarded as biologically irrelevant. Mitomycin C (0.3 L g/mL), Griseofulvin (8.0 L g/mL) or CPA (15.0 L g/mL) were used as positive controls and showed a distinct increase in the percentage of micronucleated cells.Report - page 25 of 39 -



### In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei in V79 cells (Chinese hamster cell line) in vitro in the absence and presence of metabolic activation.

Therefore, has to be considered as non-mutagenic in this in vitro test system, when tested up to the highest applied concentration.



# 13 DISTRIBUTION OF THE REPORT

Sponsor 2x (1x original, 1x electronic copy as PDF-file) Study Director 1x (copy)Report - page 26 of 39 -



# 14 REFERENCES

1. Ames, B.N., McCann, J. und Yamasaki, E. (1975) 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. 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-tox program. Mutation Res., 87, 81-142 4. Countryman, P.I. and Heddle, J.A. (1976) The production of micronuclei from chromosome aberrations in irradiated cultures of human lymphocytes. Mutation Res., 41, 321-332 5. Glatt, H.R. (1993) Fremdstoffmetabolismus und Mutagenitätsprüfung. in: Fahrig, R. (ed.): Mutationsforschung und genetische Toxikologie, Wiss. Buchgesellschaft Darmstadt, 169-185 6. Kirsch-Volders, M., Sofuni, T., Aardema, M., Albertini, S., Eastmond, D., Fenech, M., Ishidate, M. Jr., Kirchner, S., Lorge, E., Morita, T., Norppa, H., Surallés, J., Vanhauwaert, A., Wakata, A. (2003) Report from the in vitro micronucleus assay working group. Mutation Res., 540, 153-163 7. Kirsch-Volders, Sofuni, T., Aardema, M., Albertini, S., Eastmond, D., Fenech, M., Ishidate, M. Jr., Kirchner, S., Lorge, E., Norppa, H., Surallés, J., Vanhauwaert, A., Wakata, A. (2004) Corrigendum to “Report from the in vitro micronucleus assay working group”. Mutation Res., 564, 97-100 8. Matsuoka, A., Yamazaki, N., Suzuki, T., Hayashi, M., Soffuni, T. (1993) Evaluation of the micronucleus test using a Chinese hamster cell line as an alternative to the conventional in vitro chromosomal aberration test. Mutation Res., 272, 223-236 9. Seelbach, A., Fissler, B., Strohbusch, A., Madle, S. (1993) Development of a modified micronucleus assay in vitro for the detection of aneugenic effects. Toxicol. In Vitro, 7, 185-193 10. Easterbrook, J., Lu, C., Sakai, Y. and Li, A.P. (2001)Effects of organic solvents on the activities of cytochrome P450 isoforms, UDPdependent glucuronyl transferase, and phenol sulfotransferase in human hepatocytes Drug Metabolism and Disposition, 29, 141-144Report - page 27 of 39 -

11. Mitchell, A.D., Auletta, A.E., Clive, D., Kirby, P.E., Moore, M.M. and Myhr, B.C. (1997) The L5178Y/ tk +/- mouse lymphoma specific gene and chromosomal mutation assay - A phase III report of the U.S. Environmental Protection Agency Gene-Tox Program Mutation Research, 394, 177-303Report- page 28 of 39 -15 ANNEX I: TABLES OF RESULTS15.1 Experiment ITable 3: Cytotoxicity indicated as proliferation index; exposure period 4 hrs without S9 mixTreatment Conc. S9 Exposure/Proliferationgroup per mL mix preparationIndexmeanSolv. control#0.5 % - 4 / 24 hrs2.94Pos. control##0.3Lg - 4 / 24 hrs2.18Test item1.4Lg - 4 / 24 hrs2.859.0Lg - 4 / 24 hrs2.8422.5Lg - 4 / 24 hrs2.9256.3Lg - 4 / 24 hrs2.852200.0Lg - 4 / 24 hrs2.83* Number of clones with 1 cell (c1), 2 cells (c2), 3 - 4 cells (c4), or 5 - 8 cells (c8)#THF##MMCReport- page 29 of 39 -Table 4: Cytotoxicity indicated as proliferation index; exposure period 4 hrs with S9 mixTreatment Conc. S9 Exposure/Proliferationgroup per mL mix preparationIndexmeanSolv. control#0.5 % + 4 / 24 hrs1.98Pos. control##15.0Lg + 4 / 24 hrs1.60Test item3.6Lg + 4 / 24 hrs1.969.0Lg + 4 / 24 hrs1.9622.5Lg + 4 / 24 hrs1.9456.3Lg + 4 / 24 hrs2.002200.0Lg + 4 / 24 hrs2.03* Number of clones with 1 cell (c1), 2 cells (c2), 3 - 4 cells (c4), or 5 - 8 cells (c8)#THF##CPAReport- page 30 of 39 -Table 5: Number of micronucleated cells; exposure period 4 hrs without S9 mixTreatment Conc. S9 Exposure/Micronucleated cellsgroup per mL mix preparationsum in 2000cells [%]Solv. control#0.5 % - 4 / 24 hrs15 0.75Pos. control##0.3Lg - 4 / 24 hrs162 8.10Test item1.4Lg - 4 / 24 hrs16 0.809.0Lg - 4 / 24 hrs18 0.9022.5Lg - 4 / 24 hrs19 0.9556.3Lg - 4 / 24 hrs10 0.50#THF##MMCReport- page 31 of 39 -Table 6: Number of micronucleated cells; exposure period 4 hrs with S9 mixTreatment Conc. S9 Exposure/Micronucleated cellsgroup per mL mix preparationsum in 2000cells [%]Solv. control#0.5 % + 4 / 24 hrs24 1.20Pos. control##15.0Lg + 4 / 24 hrs236 11.80Test item3.6Lg + 4 / 24 hrs25 1.259.0Lg + 4 / 24 hrs38 1.9022.5Lg + 4 / 24 hrs21 1.0556.3Lg + 4 / 24 hrs24 1.20#THF##CPAReport- page 32 of 39 -15.2 Experiment IITable 7: Cytotoxicity indicated as proliferation index; exposure period 24 hrs without S9 mixTreatment Conc. S9 Exposure/Proliferationgroup per mL mix preparationIndexmeanSolv. control#0.5 % - 24 / 24 hrs2.75Pos. control##8.0Lg - 24 / 24 hrs2.63Test item1.4Lg - 24 / 24 hrs2.769.0Lg - 24 / 24 hrs2.7822.5Lg - 24 / 24 hrs2.8156.3Lg - 24 / 24 hrs2.722200.0Lg - 24 / 24 hrs2.78* Number of clones with 1 cell (c1), 2 cells (c2), 3 - 4 cells (c4), or 5 - 8 cells (c8)#THF##GriseofulvinReport- page 33 of 39 -Table 8: Cytotoxicity indicated as proliferation index; exposure period 4 hrs with S9 mixTreatment Conc. S9 Exposure/Proliferationgroup per mL mix preparationIndexmeanSolv. control#0.5 % + 4 / 24 hrs2.01Pos. control##15.0Lg + 4 / 24 hrs1.42Test item3.6Lg + 4 / 24 hrs1.949.0Lg + 4 / 24 hrs2.0622.5Lg + 4 / 24 hrs2.272200.0Lg + 4 / 24 hrs2.31* Number of clones with 1 cell (c1), 2 cells (c2), 3 - 4 cells (c4), or 5 - 8 cells (c8)#THF##CPAReport- page 34 of 39 -Table 9: Number of micronucleated cells; exposure period 24 hrs without S9 mixTreatment Conc. S9 Exposure/Micronucleated cellsgroup per mL mix preparationsum in 2000cells [%]Solv. control#0.5 % - 24 / 24 hrs10 0.50Pos. control##8.0Lg - 24 / 24 hrs153 7.65Test item1.4Lg - 24 / 24 hrs9 0.459.0Lg - 24 / 24 hrs7 0.3522.5Lg - 24 / 24 hrs11 0.5556.3Lg - 24 / 24 hrs7 0.35#THF##GriseofulvinReport- page 35 of 39 -Table 10: Number of micronucleated cells; exposure period 4 hrs with S9 mixTreatment Conc. S9 Exposure/Micronucleated cellsgroup per mL mix preparationsum in 2000cells [%]Solv. control#0.5 % + 4 / 24 hrs15 0.75Pos. control##15.0Lg + 4 / 24 hrs49 2.45Test item3.6Lg + 4 / 24 hrs15 0.759.0Lg + 4 / 24 hrs23 1.1522.5Lg + 4 / 24 hrs14 0.70#THF##CPAReport - page 36 of 39 -



## 15.3 Biometry

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the Chi square test. Evaluation was performed only for test groups showing a higher number of micronucleated cells than the respective solvent control group.
Table 11: Biometry of Experiment I

Test group versus

solvent control Preparation interval E x posure period S9 mix Chi2

Test group 1.4 L g/mL 24 hours 4 hours - 0.033

" 9.0 L g/mL 24 hours 4 hours - 0.275

" 22.5 L g/mL 24 hours 4 hours - 0.475

" 56.3 L g/mL 24 hours 4 hours - n.c.

" 2200.0 L g/mL 24 hours 4 hours - n.c.

" 3.6 L g/mL 24 hours 4 hours + 0.021

" 9.0 L g/mL 24 hours 4 hours + 3.211

" 22.5 L g/mL 24 hours 4 hours + n.c.

" 56.3 L g/mL 24 hours 4 hours + n.c.

" 2200.0 L g/mL 24 hours 4 hours + n.c.

Positive control versus solvent control

MMC 0.3 L g/mL 24 hours 4 hours - 127.737 S

CPA 15.0 L g/mL 24 hours 4 hours + 184.879 S

n.c. Not calculated as the rate of micronucleated cells is equal or lower than the control rate
S Number of micronucleated cells is statistically significant higher than the control rateReport - page 37 of 39 -



### Table 12: Biometry of Experiment II

Test group versus

solvent control Preparation interval E x posure period S9 mix Chi2

Test group 1.4 L g/mL 24 hours 24 hours - n.c.

" 9.0 L g/mL 24 hours 24 hours - n.c.

" 22.5 L g/mL 24 hours 24 hours - 0.048

" 56.3 L g/mL 24 hours 24 hours - n.c.

" 2200.0 L g/mL 24 hours 24 hours - n.c.

" 3.6 L g/mL 24 hours 4 hours + n.c.

" 9.0 L g/mL 24 hours 4 hours + 1.700

" 22.5 L g/mL 24 hours 4 hours + n.c.

" 2200.0 L g/mL 24 hours 4 hours + n.c.

Positive control versus solvent control

Griseofulvin 8.0 L g/mL 24 hours 24 hours - 130.783 S

CPA 15.0 L g/mL 24 hours 4 hours + 18.356 S

n.c. Not calculated as the rate of micronucleated cells is equal or lower than the control rate
S Number of micronucleated cells is statistically significant higher than the control rateReport - page 38 of 39 -



# 16 ANNEX II
16.1 Historical laboratory control data
16.1.1 Percentage of micronucleated cells in Chinese hamster V79 cell cultures (October 2006 to August 2011)

1 Mean ± standard deviation 2 Aqueous solvents: deionised water (10 % v/v), 0.9 % (w/v) saline and culture medium MEM
3 Organic solvents: dimethyl sulfoxide, acetone, ethanol and tetrahydrofurane (0.5 % v/v)

1 Mean ± standard deviation 2 Aqueous solvents: deionised water (10 % v/v), 0.9 % (w/v) saline and culture medium MEM
3 Organic solvents: dimethyl sulfoxide, acetone, ethanol and tetrahydrofurane (0.5 % v/v)Report - page 39 of 39 -

1 Mean ± standard deviation 2 Aqueous solvents: deionised water (10 % v/v), 0.9 % (w/v) saline and culture medium MEM
3 Organic solvents: dimethyl sulfoxide, acetone, ethanol and tetrahydrofurane (0.5 % v/v)