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.
Report - page 2 of 39 - 

1 PAGE RESERVED FOR PROPRIETARY INFORMATION 

OR STATEMENT OF NO DATA CONFIDENTIALITY 

CLAIMS 
Report 

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, 2013 
Report - page 4 of 39 - 

3 PAGE RESERVED FOR FLAGGING STATEMENTS 
Report 

4 SIGNATURE 

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 of 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 reviewed 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 Unit 
Report - page 7 of 39 - 

6 COPY OF THE GLP CERTIFICATE 

Gute Lahorpraxis/Good Laboratory Practice 

GLP~Bescheinigung/Statcment of GLP Compliance 

(gemäß/according to § 19b Abs. I Chemikaliengesetz) 

Eine GLP-Inspektion zur Überwachung der Einhaltung 

der GLP-Grundsätze gemäß Chemikaliengesetz bzw. 

Richtli.iue 2004i9/EG wurde durchgefuhrt in 

Assessment of conformity with GLP according to 

Chemikaliengesetz and Directive 2004/9/EEC at: 

(8:1 Prüfeinrichtung-Test facility D Prüfstandott'Test site 

(Unverwechselbare Bezeichnung und Adresse/Unequivocal narne and adress) 

l'rüfuogeo nach Kategorien/Areas ofExpertise 

(gernäß/according chemVwV-GLP Nr. 5.3/0ECD guidance) 

2 Prüfungen zur Bestimmung der toxikologischen 

Eigenschaften 

3 Prüfungen zur Bestimmung cler erbgutverändernden Eigenschaften (in vit.ro u nd in vivo) 

8 Analytische Prüfungen an biologischen Materialien 

2 Taxicity studies 

3 Mutagenicity stuclies 

8 Analytical studies on biological materials 

25. April, 23./25. und 26 . .Juli 2012 

Datum der Inspektion/Date of lnspection 

(Tag Monat Jalu·/day mouth year) 

Die genannte Prüfeinrichtung befutdet sich im nationalen GLP-Überwachungsverfahren und wird regelmäßig auf Einhaltung der GLP-Gmndsätze überwacht. 

J\uf der Gnmdlagc des Inspektionsberichtes wird hiem1it 

bestätigt, dass in dieser Prüfeinrichtung die oben genannten Priifimgcn unter Einhaltung der GLP- GrundsMtze dur.chgcflllut werden körnten. 

The above mentioned test facility is included 

in the national GLP Compliance Prograll1llle and is 

inspcctcd on a regtdar basis. 

Based on the inspection report it can be conllrroed, 

that this test. facility is able to conduct the 

aforementioned studies in compliance with the 

Principles ofGLP. 

(Name und J\dresse der GLP-Überwachungsbehörde/Name and address of the GLP Monitaring Autb.ority 
Report - 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 21
Report - 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) 38
Report - 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, 2013 
Report - 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 Lg/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 Lg/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 Lg/mL in % 

Exposure period 4 hrs without S9 mix 

I 24 hrs Solvent control1 2.94 0.75 

Positive control2 2.18 8.10S

1.4 2.85 0.80 

9.0PS 2.84 0.90 

22.5PS 2.92 0.95 

56.3P/PS 2.85 0.50 

2200.0P/PS 2.83 n.e. 

Exposure period 24 hrs without S9 mix 

II 24 hrs Solvent control1 2.75 0.50 

Positive control3 2.63 7.65S

1.4 2.76 0.45 

9.0PS 2.78 0.35 

22.5PS 2.81 0.55 

56.3P/PS 2.72 0.35 

2200.0P/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 Lg/mL 

3 Griseofulvin 8.0 Lg/mL 
Report - 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 Lg/mL in % 

Exposure period 4 hrs with S9 mix 

I 24 hrs Solvent control1 1.98 1.20 

Positive control2 1.60 11.80S

3.6 1.96 1.25 

9.0PS 1.96 1.90 

22.5PS 1.94 1.05 

56.3P/PS 2.00 1.20 

2200.0P/PS 2.03 n.e. 

II 24 hrs Solvent control1 2.01 0.75 

Positive control2 1.42 2.45S

3.6 1.94 0.75 

9.0PS 2.06 1.15 

22.5P/PS 2.27 0.70 

2200.0P/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 Lg/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 

[Lg/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 7.4 
Report - 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 Lg/mL 

Name: Griseofulvin 

Supplier: Sigma-Aldrich Chemie GmbH 

82024 Taufkirchen, Germany 

Lot no.: 031 M 1289 V 

Dissolved in: DMSO 

Final Concentration: 8.0 Lg/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 Lg/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 cm2 plastic flasks. About 5 x 105 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 Lg/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 MgCl2 (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 LL/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 Lg/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 Lg/mL (with and without S9 mix) were chosen for 

the evaluation of cytotoxicity. Precipitation of the test item was observed at 56.3 Lg/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 Lg/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 

Exposure

period 

Exp. Concentration 

in Lg/mL 

Without S9 mix

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

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

With S9 mix

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

24 hrs 4 hrs II 0.6 1.4 3.6 9.0PS 22.5P/PS 56.3P/PS 140.8P/PS 352.0P/PS 880.0P/PS 2200.0P/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 treatment 
Report - 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 Lg/mL). All cultures were incubated at 37 °C in a humidified atmosphere with 

1.5 % CO2 (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 KH2PO4 and 150 mg/L 

Na2HPO4. 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 105 – 1.5 x 105 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 LL 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 • H2O, 192 mg/L Na2HPO4 • 2 H2O and 150 mg/L KH2PO4. 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

x(c83)x(c42)x(c21)x(c1 4) 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 Lg/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 Lg/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 Lg/mL and 

above. 

Phase separation was observed in the absence of S9 mix at 3.6 Lg/mL and above and in the 

presence of S9 mix at 9.0 Lg/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 Lg/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 Lg/mL), Griseofulvin (8.0 Lg/mL) or CPA (15.0 Lg/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, 141-144 
Report - 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, 177-303 
Report 

15 ANNEX I: TABLES OF RESULTS15.1 Experiment ITable 3: Cytotoxicity indicated as proliferation index; exposure period 4 hrs without S9 mix

Treatment Conc. S9 Exposure/ 

group per mL mix preparation 

Solv. control# 0.5 % - 4 / 24 hrs Pos. control## 0.3 Lg - 4 / 24 hrs Test item 1.4 Lg - 4 / 24 hrs 

 9.0 Lg - 4 / 24 hrs  22.5 Lg - 4 / 24 hrs  56.3 Lg - 4 / 24 hrs  2200.0 Lg - 4 / 24 hrs 

* Number of clones with 1 cell (c1), 2 cells (c2), 3 - 4 cells (c4), or 5 - 8 cells (c8)# THF## MMC
Report 

Table 4: Cytotoxicity indicated as proliferation index; exposure period 4 hrs with S9 mix

Treatment Conc. S9 Exposure/ 

group per mL mix preparation 

Solv. control# 0.5 % + 4 / 24 hrs Pos. control## 15.0 Lg + 4 / 24 hrs Test item 3.6 Lg + 4 / 24 hrs 

 9.0 Lg + 4 / 24 hrs  22.5 Lg + 4 / 24 hrs  56.3 Lg + 4 / 24 hrs  2200.0 Lg + 4 / 24 hrs 

* Number of clones with 1 cell (c1), 2 cells (c2), 3 - 4 cells (c4), or 5 - 8 cells (c8)# THF## CPA
Report 

Table 5: Number of micronucleated cells; exposure period 4 hrs without S9 mix

Treatment Conc. S9 Exposure/ Micronucleated cells

group per mL mix preparation 

Solv. control# 0.5 % - 4 / 24 hrs Pos. control## 0.3 Lg - 4 / 24 hrs Test item 1.4 Lg - 4 / 24 hrs 

 9.0 Lg - 4 / 24 hrs  22.5 Lg - 4 / 24 hrs  56.3 Lg - 4 / 24 hrs 

# THF## MMC
Report 

Table 6: Number of micronucleated cells; exposure period 4 hrs with S9 mix

Treatment Conc. S9 Exposure/ Micronucleated cells

group per mL mix preparation 

Solv. control# 0.5 % + 4 / 24 hrs Pos. control## 15.0 Lg + 4 / 24 hrs Test item 3.6 Lg + 4 / 24 hrs 

 9.0 Lg + 4 / 24 hrs  22.5 Lg + 4 / 24 hrs  56.3 Lg + 4 / 24 hrs 

# THF## CPA
Report 

15.2 Experiment IITable 7: Cytotoxicity indicated as proliferation index; exposure period 24 hrs without S9 mix

Treatment Conc. S9 Exposure/ 

group per mL mix preparation 

Solv. control# 0.5 % - 24 / 24 hrs Pos. control## 8.0 Lg - 24 / 24 hrs Test item 1.4 Lg - 24 / 24 hrs 

 9.0 Lg - 24 / 24 hrs  22.5 Lg - 24 / 24 hrs  56.3 Lg - 24 / 24 hrs  2200.0 Lg - 24 / 24 hrs 

* Number of clones with 1 cell (c1), 2 cells (c2), 3 - 4 cells (c4), or 5 - 8 cells (c8)# THF## Griseofulvin
Report 

Table 8: Cytotoxicity indicated as proliferation index; exposure period 4 hrs with S9 mix

Treatment Conc. S9 Exposure/ 

group per mL mix preparation 

Solv. control# 0.5 % + 4 / 24 hrs Pos. control## 15.0 Lg + 4 / 24 hrs Test item 3.6 Lg + 4 / 24 hrs 

 9.0 Lg + 4 / 24 hrs  22.5 Lg + 4 / 24 hrs  2200.0 Lg + 4 / 24 hrs 

* Number of clones with 1 cell (c1), 2 cells (c2), 3 - 4 cells (c4), or 5 - 8 cells (c8)# THF## CPA
Report 

Table 9: Number of micronucleated cells; exposure period 24 hrs without S9 mix

Treatment Conc. S9 Exposure/ Micronucleated cells

group per mL mix preparation 

Solv. control# 0.5 % - 24 / 24 hrs Pos. control## 8.0 Lg - 24 / 24 hrs Test item 1.4 Lg - 24 / 24 hrs 

 9.0 Lg - 24 / 24 hrs  22.5 Lg - 24 / 24 hrs  56.3 Lg - 24 / 24 hrs 

# THF## Griseofulvin
Report 

Table 10: Number of micronucleated cells; exposure period 4 hrs with S9 mix

Treatment Conc. S9 Exposure/ Micronucleated cells

group per mL mix preparation 

Solv. control# 0.5 % + 4 / 24 hrs Pos. control## 15.0 Lg + 4 / 24 hrs Test item 3.6 Lg + 4 / 24 hrs 

 9.0 Lg + 4 / 24 hrs  22.5 Lg + 4 / 24 hrs 

# THF## CPA
Report - 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 

Exposure 

period 

S9 mix Chi2 

Test group 1.4 Lg/mL 24 hours 4 hours - 0.033 

" 9.0 Lg/mL 24 hours 4 hours - 0.275 

" 22.5 Lg/mL 24 hours 4 hours - 0.475 

" 56.3 Lg/mL 24 hours 4 hours - n.c. 

" 2200.0 Lg/mL 24 hours 4 hours - n.c. 

" 3.6 Lg/mL 24 hours 4 hours + 0.021 

" 9.0 Lg/mL 24 hours 4 hours + 3.211 

" 22.5 Lg/mL 24 hours 4 hours + n.c. 

" 56.3 Lg/mL 24 hours 4 hours + n.c. 

" 2200.0 Lg/mL 24 hours 4 hours + n.c. 

Positive control versus 

solvent control 

MMC 0.3 Lg/mL 24 hours 4 hours - 127.737S

CPA 15.0 Lg/mL 24 hours 4 hours + 184.879S

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 rate 
Report - page 37 of 39 - 

Table 12: Biometry of Experiment II 

Test group versus 

solvent control 

Preparation

interval 

Exposure 

period 

S9 mix Chi2 

Test group 1.4 Lg/mL 24 hours 24 hours - n.c. 

" 9.0 Lg/mL 24 hours 24 hours - n.c. 

" 22.5 Lg/mL 24 hours 24 hours - 0.048 

" 56.3 Lg/mL 24 hours 24 hours - n.c. 

" 2200.0 Lg/mL 24 hours 24 hours - n.c. 

" 3.6 Lg/mL 24 hours 4 hours + n.c. 

" 9.0 Lg/mL 24 hours 4 hours + 1.700 

" 22.5 Lg/mL 24 hours 4 hours + n.c. 

" 2200.0 Lg/mL 24 hours 4 hours + n.c. 

Positive control versus 

solvent control 

Griseofulvin 8.0 Lg/mL 24 hours 24 hours - 130.783S

CPA 15.0 Lg/mL 24 hours 4 hours + 18.356S

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 rate 
Report - 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)