GENE MUTATION ASSAY 

IN CHINESE HAMSTER V79 CELLS 

IN VITRO 

(V79/ HPRT) 

WITH 

Study Number: 1533002 

Test ltem: 

Project Number: 

RE PORT 

Author: 

Test Facility: 

Test Guidelines: OECD 476 

Commission Regulation (EC) No. 440/2008; 8. 17 

US EPA OPPTS 870.5300 

Final Report lssued: April 30, 2013 

Sponsor: 

Page 1 of39 

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" (Chemicals Act) of the Federal Republic of Germany, "Anhang 1" 

(Annex 1), in its currently valid version. 

"OECD Principles of Good Labaratory 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 

To be filled for USA EPA submission only: 

Sponsor 

Date 

Submitter 

Date 
Report Page 4 of 39 

3 PAGE RESERVED FOR FLAGGING STATEMENTS 
4 SIGNAlURE 

Report 

Study Director 

Page 5 of 39 

........ ............ . 

Date: April 30, 2013 
Report Page 6 of 39 

5 STATEMENT OF QUALITY ASSURANCE UNIT 

Study: 

Test ltem: 

Study Director: 

Title: 

1533002 

Gene Mutation Assay 

in Chinese Hamster V79 Cells 

in vitro (V79/HPRT) 

with 

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 476.HPRT was reviewed for compliance on 

December 27, 2011 . 

Phasesand Dates of QAU lnspections/ Audits Dates of Reports to the Study 

Director and to Management 

Study Specific Supplement: February 04, 2013 February 04, 2013 

Process I nsQection 

Preparation for Application: March 12, 2013 March 12, 2013 

Application: April 03, 2013 April 03, 2013 

Report: April 30, 2013 April 30, 2013 

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

Head of Quality Assurance Unit 

Date: April 30, 2013 
Report Page 7 of 39 

6 COPY OF GLP CERTIFICATE 

Gute Laborpraxis/Good Laboratory Practice 

GLP-Bescbeinigung!Statement of GLP Compliance 

(gemäß/according to § 19b Abs. 1 Chemikaliengesetz) 

Eine GLP-lnspektion zur Überwachung der Einhaltung 

der GLP-Grundsätze gemäß Chemikaliengesetz bzw. 

Richtlinie 2004/9/EG wurde durchgeflihrt in 

Assessment of confonnity with GLP according 10 

Chemikaliengesetz aod Directive 2004/9/EEC at: 

I8J Prüfeinrichtung/Test facility 0 PrUfstaodortrrest site 

(Unverwechselbare Bezeichnung und Adresse!Unequivocal narne and adress) 

Prüfungen nach Katcgoricn/Arcas of Expertise 

(gemäß/accordiog chemVwV-GLP Nr. 5.3/0ECD guidance) 

2 Prüfungen zur Bestimmung der toxikologischen 

Eigenschaften 

3 Prüfungen zur Bestimmung der erbgutverändernden Eigenschaften (in vitro und in vivo) 

6 Prüfungen zur Bestimmung von Rückständen 

8 Analytische Prüfungen an biologischen Materialien 

2 Toxicity studies 

3 Mutagenicity studjes 

6 Residues 

8 Analytical studies on biolog.ical mate rials 

15.08. und 27.-29. 10.2008 

Datum der Inspektion/Date of lnspection 

(Tag Monat Jahr/day month year) 

Die genannte PrOfeinrichtung befindet sich im nationalen GLP-Überwachungsverfahren und wird regelmäßig auf Einhaltung der GLP-Grundsätze Oberwacht. 

Auf der Grundtage des Inspektionsberichtes wird hiermit 

bestätigt, dass in dieser Prüfeinrichtung die oben genannten PrOfungen unter Einhaltung der GLP- Grundsätze durchgefilhrt werden können. 

The above mentioned test facility is included 

in Lhe national GLP Compliance Programme and is 

inspected on a regular basis. 

Basedon the inspection report it can be confinoed, 

tbat this test facility is able to conduct the 

aforementioned studies in compliance with the 

J>rinciples ofGLP. 

Jm Auftrag 

(Name und Adresse der GLP-Überwachungsbehörde!Name and address ofthe GLP Monitoring Authority 
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 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 11

9 SUMMARY 12

10 OBJECTIVE 14

10.1 Aims of the Study 14

10.2 Relevance of the Test System 14

11 MATERIALS AND METHODS 15

11.1 Test Item 15

11.2 Controls 16

11.2.1 Solvent Controls 16

11.2.2 Positive Control Substances 16

11.3 Test System 17

11.3.1 Reasons for the Choice of the Cell Line V79 17

11.3.2 Cell Cultures 17

11.4 Mammalian Microsomal Fraction S9 Mix 17

11.4.1 S9 (Preparation by 17

11.4.2 S9 Mix 18

11.5 Pre-Test on Toxicity 18

11.6 Dose Selection 18

11.7 Experimental Performance 19

11.7.1 Culture Medium 19

11.7.2 Seeding 19

11.7.3 Treatment 20

11.8 Data Recording 20

11.9 Acceptability of the Assay 21

11.10 Evaluation of Results 21

11.11 Statistical Analysis 22

12 CALCULATION AND PROCESSING OF THE DATA 23
Report Page 9 of 39 

CONTENTS CONTINUED 

13 RESULTS AND DISCUSSION 24

14 REFERENCES 25

15 DISTRIBUTION OF THE REPORT 25

16 ANNEX I: TABLES OF RESULTS 26

16.1 Pre-test 26

16.2 Main Experiment 27

16.2.1 Experiment I Culture I 27

16.2.2 Experiment I Culture II 30

16.2.3 Experiment II, Culture I 33

16.2.4 Experiment II, Culture II 36

17 ANNEX II: HISTORICAL DATA 39
Report Page 10 of 39 

8 PREFACE 

8.1 General 

Title: Gene Mutation Assay 

in Chinese Hamster V79 Cells 

in vitro (V79/HPRT) 

with 

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 04, 2013 

Experimental Starting Date: March 07, 2013 

Experimental Completion Date: April 29, 2013 
Report Page 11 of 39 

8.4 Good Laboratory Practice 

The study was performed in compliance with: 

”Chemikaliengesetz“ (Chemicals Act) of the Federal Republic of Germany, ”Anhang 1“ 

(Annex 1), in its currently valid version. 

”OECD Principles of Good Laboratory Practice“, as revised in 1997 [C(97)186/Final]. 

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 was conducted according to the procedures indicated by the following internationally accepted guidelines and recommendations: 

OECD Guidelines for Testing of Chemicals No. 476 “In vitro Mammalian Cell Gene 

Mutation Test” (adopted July 21, 1997) 

Commission Regulation (EC) 440/2008; B17 “Mutagenicity – In vitro Mammalian Cell 

Gene Mutation Test”, dated May 30, 2008 

US EPA (TSCA) OPPTS 870.5300 “In Vitro Mammalian Cell Gene MutationTest”, EPA 

712-C-98-221, August 1998 

8.6 Archiving 

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, report, and specimens (if any) for at least 3 years at the 

test facility’s archive. Thereafter, the material will be transferred to the GLP archive of 

 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. 

8.7 Deviations from the Study Plan 

There were no deviations to the study plan. 
Report Page 12 of 39 

9 SUMMARY 

The study was performed to investigate the potential of to induce 

gene mutations at the HPRT locus in V79 cells of the Chinese hamster. 

The assay was performed in two independent experiments, using two parallel cultures 

each. The first main experiment was performed with and without liver microsomal 

activation and a treatment period of 4 hours. The second experiment was performed with 

a treatment time of 4 hours with and 24 hours without metabolic activation. 

The highest concentration used in the range finding pre-experiment was 2200 μg/mL of 

the test item based on the solubility properties of the test item. The concentration range of 

the main experiments was limited by precipitation of the test item. The test item was 

dissolved in THF. 

The tested concentrations of the main experiments are described in chapter 11.6

(page 18). The evaluated experimental points and the results are summarised in Table 1 

(page 13). 

No substantial and reproducible dose dependent increase of the mutation frequency was 

observed up to the maximum concentration with and without metabolic activation. 

Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a 

distinct increase in mutant colonies and thus, showed the sensitivity of the test system 

and the activity of the metabolic activation system. 

Conclusion 

In conclusion it can be stated that under the experimental conditions reported the test item 

did not induce gene mutations at the HPRT locus in V79 cells. 

Therefore, is considered to be non-mutagenic in this HPRT assay. 
Report Page 13 of 39 

Table 1: Summary of results 

relative relative relative mutant relative relative relative mutant

conc. P S9 cloning cell cloning colonies/ induction cloning cell cloning colonies/ induction

μg/mL mix efficiency I density efficiency II 106

 cells factor efficiency I density efficiency II 106 cells factor

%% % %% %

Column 1 2 3 4 5 6 7 8 9 10 11 12 13

Experiment I / 4 h treatment

Solvent control with THF - 100.0 100.0 100.0 12.3 1.0 100.0 100.0 100.0 19.6 1.0

Positive control (EMS) 150.0 - 97.1 86.5 91.6 118.3 9.6 97.7 89.0 106.3 129.3 6.6

Test item 1.0 - 98.9 86.3 80.3 19.8 1.6 97.1 86.3 89.9 16.1 0.8

Test item 3.0 - 97.9 80.8 90.8 17.5 1.4 99.2 77.9 97.9 10.5 0.5

Test item 9.0 - 97.0 74.7 88.2 20.1 1.6 97.9 75.7 93.0 9.4 0.5

Test item 27.0 P - 97.9 74.5 88.0 23.3 1.9 97.2 76.5 89.4 15.3 0.8

Test item 81.0 P - 96.7 78.2 95.6 30.3 2.5 96.6 80.9 96.2 13.1 0.7

Test item 243.0 P - 97.3 96.6

Solvent control with THF + 100.0 100.0 100.0 4.1 1.0 100.0 100.0 100.0 4.9 1.0

Positive control (DMBA) 1.1 + 95.4 69.7 76.4 350.3 86.1 88.7 69.1 62.2 806.5 166.2

Test item 1.0 + 95.5 68.7 90.1 7.4 1.8 100.6 87.0 67.7 20.7 4.3

Test item 3.0 + 94.5 77.7 85.9 9.8 2.4 99.7 79.7 75.4 15.3 3.2

Test item 9.0 + 95.2 70.8 102.3 8.1 2.0 99.0 95.7 103.6 19.4 4.0

Test item 27.0 P + 93.5 97.6 91.2 11.8 2.9 101.0 63.8 86.2 13.1 2.7

Test item 81.0 P + 95.0 78.7 97.7 9.6 2.4 105.9 95.7 114.9 12.0 2.5

Test item 243.0 P + 93.0 102.7

Experiment II / 24 h treatment

Solvent control with THF - 100.0 100.0 100.0 12.1 1.0 100.0 100.0 100.0 6.4 1.0

Positive control (EMS) 150.0 - 87.2 99.7 98.0 136.5 11.3 99.0 108.8 86.7 187.4 29.2

Test item 1.0 - 77.7 90.8 93.3 14.3 1.2 96.5 83.0 74.9 14.2 2.2

Test item 3.0 - 84.8 114.3 89.9 6.2 0.5 91.2 120.4 80.1 9.0 1.4

Test item 9.0 - 86.1 101.9 110.4 7.9 0.7 95.5 89.1 91.5 12.4 1.9

Test item 27.0 P - 84.5 113.1 100.5 4.5 0.4 100.5 86.8 82.8 5.9 0.9

Test item 81.0 P - 80.9 96.0 86.1 10.2 0.8 96.4 93.5 91.1 7.5 1.2

Test item 243.0 P - 65.0 85.7

Experiment II / 4 h treatment

Solvent control with THF + 100.0 100.0 100.0 10.5 1.0 100.0 100.0 100.0 8.7 1.0

Positive control (DMBA) 1.1 + 79.9 82.3 93.4 107.2 10.2 116.0 101.3 99.4 82.1 9.4

Test item 1.0 + 98.8 103.7 103.7 5.2 0.5 104.0 96.0 96.2 6.9 0.8

Test item 3.0 + 97.3 94.7 97.0 12.5 1.2 116.0 121.6 105.2 6.9 0.8

Test item 9.0 + 94.3 97.2 98.2 8.4 0.8 104.9 145.6 107.3 6.7 0.8

Test item 27.0 P + 90.6 102.1 101.1 3.8 0.4 106.2 122.9 107.1 6.1 0.7

Test item 81.0 P + 96.1 103.1 102.9 9.7 0.9 101.3 141.9 93.5 13.2 1.5

Test item 243.0 P + 94.1 105.1

culture I culture II

culture I culture II

culture was not continued# culture was not continued#

culture was not continued# culture was not continued#

culture was not continued# culture was not continued#

culture was not continued# culture was not continued#

# culture was not continued to avoid analysis of too many concentrations showing precipitation 

P = Precipitation 
Report Page 14 of 39 

10 OBJECTIVE 

10.1 Aims of the Study 

This in vitro experiment was performed to assess the potential of the test item to induce 

gene mutations using the Chinese hamster cell line V79. Two parallel cultures were used 

throughout the assay. The first experiment was performed with a treatment time of 4 hours 

with and without metabolic activation. The second experiment was performed with a 

treatment time of 4 hours with and 24 hours without metabolic activation. 

10.2 Relevance of the Test System 

In vitro methods are valuable when it is desired to accurately control the concentration 

and exposure time of cells to the test item under study. However, the limited capacity of 

metabolic activation of potential mutagens requires an exogenous metabolic activation 

system. 

This in vitro test is an assay for the detection of forward gene mutations in mammalian 

cells. Gene mutations are discussed as an initial step in the carcinogenic process. 

The V79 cells are exposed to the test item both with and without exogenous metabolic 

activation. At a defined time interval after treatment the descendants of the treated original 

population are monitored for the loss of functional HPRT enzyme. 

HPRT (hypoxanthine-guanine phosphoribosyl transferase) catalyzes the conversion of the 

nontoxic 6-TG (6-thioguanine) to its toxic ribophosphorylated derivative. Therefore, cells 

deficient in HPRT due to a forward mutation are resistant to 6-TG. These cells are able to 

proliferate in the presence of 6-TG whereas the non-mutated cells die. However, the 

mutant phenotype requires a certain period of time before it is completely expressed. The 

phenotypic expression is achieved by allowing exponential growth of the cells for 7 - 9 

days. The expression period is terminated by adding 6-TG to the culture medium. 

Mutant frequency is determined by seeding known numbers of cells in medium containing 

the selective agent to detect mutant cells, and in medium without selective agent to determine the surviving cells. After a suitable period the colonies are counted. Mutant frequencies are calculated from the number of mutant colonies corrected for cell survival. 

In order to establish a concentration response effect of the test item at least four concentration levels are tested. These concentration levels should yield a concentration related 

toxic effect. The highest concentration level should induce a reduced level of survival. 

To demonstrate the sensitivity of the test system reference mutagens are tested in parallel 

to the test item. 
Report Page 15 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-% determined by 

non-GLP GC-FID (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 osmolarity and pH-value was determined in the solvent control and at the highest 

concentration of the pre-experiment without metabolic activation: 

solvent control 2200 μg/mL 

Osmolarity mOsm 370 351 

pH-value 7.51 7.55
Report Page 16 of 39 

11.2 Controls 

11.2.1 Solvent Controls 

Concurrent solvent controls (THF) were performed. 

Name: THF (tetrahydrofuran)

Supplier: Merck, 64293 Darmstadt, Germany 

Purity: 99.9%

Lot No.: I648307240 

Expiry Date: July 2015 

11.2.2 Positive Control Substances 

Without metabolic activation 

Name: EMS; ethylmethane sulfonate 

Supplier: Acros Organics, 2440 Geel, Belgium 

Lot No.: A 0315915 (experiment I) 

A 0319868 (experiment II) 

Expiry date: May 2013 (experiment I) 

February 2015 (experiment II) 

Dissolved in: Nutrient medium 

Final concentration: 0.15 mg/mL = 1.2 mM 

With metabolic activation 

Name: DMBA; 7,12-dimethylbenz(a)anthracene 

Supplier: Sigma Chemie GmbH, 82041 Deisenhofen, Germany 

Lot No.: 061M1197V 

Expiry date: May 2017 

Dissolved in: DMSO; dimethyl sulfoxide 

Supplier: Merck, 64293 Darmstadt, Germany, 

final concentration in nutrient medium 0.5 % 

Final concentration: 1.1 μg/mL = 4.3 μM 

The stability of EMS and DMBA is well-defined under the selected culture conditions, 

since both positive control substances are well-established reference mutagens and 

showed a mutagenic response with the expected mutation rate. 

The dilutions of the stock solutions were prepared on the day of the experiment and used 

immediately. 
Report Page 17 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 in in vitro experiments for many years. 

Especially the high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good 

cloning efficiency of untreated cells (as a rule more than 50%) both necessary for the 

appropriate performance of the study, recommend the use of this cell line. The cells have 

a stable karyotype with a modal chromosome number of 22 (3). 

11.3.2 Cell Cultures 

Large stocks of the V79 cell line (supplied by 

 are stored in liquid nitrogen in the cell bank of 

 allowing the repeated use of the same cell culture batch in experiments. 

Before freezing, the level of spontaneous mutants was depressed by treatment with HATmedium as described in (4). Each batch is screened for mycoplasm contamination and 

checked for karyotype stability and spontaneous mutant frequency. Consequently, the

parameters of the experiments remain similar because of the reproducible characteristics 

of the cells. 

Thawed stock cultures are propagated at 37 °C in 80 cm2

 plastic flasks. About 5105 cells 

were seeded into each flask with 15 mL of MEM (minimal essential medium) containing 

Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 μg/mL) and 

amphotericin B (1%). The cells were sub-cultured twice weekly. The cell cultures were 

incubated at 37 °C in a humidified atmosphere with 1.5% CO2. 

11.4 Mammalian Microsomal Fraction S9 Mix 

Lacking metabolic activities of cells under in vitro conditions are a disadvantage of assays 

with cell cultures as many chemicals only develop a mutagenic potential when they are 

metabolized by the mammalian organism. However, metabolic activation of chemicals can 

be achieved at least partially by supplementing the cell cultures with mammalian liver 

microsome preparations (S9 mix). 

11.4.1 S9 (Preparation by 

Phenobarbital/-naphthoflavone induced rat liver S9 was used as metabolic activation 

system. The S9 were 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 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 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 mix was routinely tested with 2-aminoanthracene as 

well as benzo(a)pyrene. 
Report Page 18 of 39 

The protein concentration of the S9 preparation was 25.6 mg/mL (Lot. No.: 141212) in the 

pre-experiment and 21.3 mg/mL (Lot. No.: 310113) in the main experiments. 

11.4.2 S9 Mix 

An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor 

solution to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors 

were added to the S9 supernatant to reach following concentrations in the S9 mix: 

8 mM MgCl2

33 mM KCl

5 mM glucose-6-phosphate

4 mM NADP

in 100 mM sodium-phosphate-buffer, pH 7.4. 

During the experiment, the S9 mix was stored in an ice bath. 

11.5 Pre-Test on Toxicity 

A pre-test was performed in order to determine the concentration range for the 

mutagenicity experiments. The general culture conditions and experimental conditions in 

this pre-test were the same as described for the mutagenicity experiment below. In this 

pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per 

concentration level) after treatment with the test item was observed and compared to the 

controls. Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE). 

11.6 Dose Selection 

According to the current OECD Guideline for Cell Gene Mutation Tests at least four 

analysable concentrations should be used in two parallel cultures. For freely-soluble and 

non-cytotoxic test items the maximum concentration should be 5 mg/mL, 5 μL/mL or 10 

mM, whichever is the lowest. For cytotoxic test items the maximum concentration should 

result in approximately 10 to 20% relative survival or cell density at sub-cultivation and the 

analysed concentrations should cover a range from the maximum to little or no 

cytotoxicity. Relatively insoluble test items should be tested up to the highest 

concentration that can be formulated in an appropriate solvent as solution or homogenous 

suspension. These test items should be tested up or beyond their limit of solubility. 

Precipitation should be evaluated at the end of treatment by the unaided eye. 

The highest concentration used in the pre-test was 2200 μg/mL limited by the solubility of 

the test item in THF and aqueous medium. Test item concentrations between 17.2 μg/mL 

and 2200 μg/mL were used to evaluate toxicity in the presence (4 hours treatment) and 

absence (4 hours and 24 hours treatment) of metabolic activation. 

No cytotoxic effects indicated by a cloning efficiency below 50% were noted up to the 

maximum concentration with and without metabolic activation following 4 and 24 hours 

treatment. 

The test medium was checked for precipitation or phase separation at the end of each 

treatment period (4 or 24 hours) prior to removal to the test item. Precipitation was 

observed at 34.4 μg/mL and above after 4 hours treatment with and without metabolic 
Report Page 19 of 39 

activation. Following 24 hours treatment phase separation was noted at 34.4 μg/mL. At 

68.8 μg/mL and above precipitation was observed. 

There was no relevant shift of the osmolarity and pH value even at the maximum 

concentration of the test item. 

Based on the results of the pre-experiment, the individual concentrations of the main 

experiments were selected. The individual concentrations were spaced by a factor of 3 to 

cover soluble and insoluble concentrations. 

Doses applied in the gene mutation assay with (concentrations given 

in bold letters were chosen for mutation rate analysis): 

exposure 

period 

S9 

mix 

concentrations 

in μg/mL 

Experiment I

 4 hours - 1.0 3.0 9.0 27.0 P 81.0 P 243.0 P

 4 hours + 1.0 3.0 9.0 27.0 P 81.0 P 243.0 P

Experiment II

24 hours - 1.0 3.0 9.0 27.0 P 81.0 P 243.0 P

 4 hours + 1.0 3.0 9.0 27.0 P 81.0 P 243.0 P

P = Precipitation 

In experiment I and II the cultures at the highest concentration with and without metabolic 

activation were not continued to avoid analysis of too many precipitating concentrations. 

11.7 Experimental Performance 

11.7.1 Culture Medium 

For seeding and treatment of the cell cultures the complete culture medium was MEM 

(minimal essential medium) containing Hank’s salts, neomycin (5 μg/mL) and 

amphotericin B (1%). For the selection of mutant cells the complete medium was 

supplemented with 11 μg/mL 6-thioguanine. All cultures were incubated at 37 °C in a 

humidified atmosphere with 1.5% CO2. 

11.7.2 Seeding 

Two to three days after sub-cultivation stock cultures were trypsinized at 37 °C for 5 

minutes. Then the enzymatic digestion was stopped by adding complete culture medium 

with 10% FBS and a single cell suspension was prepared. The trypsin concentration for all 

sub-culturing steps was 0.2% in PBS. 
Report Page 20 of 39 

The PBS is composed as follows (per litre): 

NaCl 8000 mg

KCl 200 mg 

KH2PO4 200 mg 

Na2HPO4 150 mg 

Prior to the trypsin treatment the cells were rinsed with Ca-Mg-free salt solution containing 

200 mg/L EDTA (ethylene diamine tetraacetic acid). Approximately 1.5106

 (single 

culture) and 5102

 cells (in duplicate) were seeded in plastic culture flasks. The cells were 

grown for 24 hours prior to treatment. 

11.7.3 Treatment 

After 24 hours the medium was replaced with serum-free medium containing the test item, 

either without S9 mix or with 50 μL/mL S9 mix. Concurrent solvent and positive controls 

were treated in parallel. After 4 hours this medium was replaced with complete medium 

following two washing steps with "saline G". In the second experiment the cells were 

exposed to the test item for 24 hours in complete medium, supplemented with 10% FBS, 

in the absence of metabolic activation. 

The "saline G" solution had the following constituents (per litre): 

NaCl 8000 mg

KCl 400 mg 

Glucose 1100 mg

Na2HPO42H2O 192 mg 

KH2PO4 150 mg 

The pH was adjusted to 7.2 

The colonies used to determine the cloning efficiency (survival) were fixed and stained 

approx. 7 days after treatment as described below. 

Three or four days after treatment 1.5106

 cells per experimental point were subcultivated in 175 cm2 flasks containing 30 mL medium. Following the expression time of 7 

days five 80 cm2 cell culture flasks were seeded with about 3 - 5105

 cells each in medium 

containing 6-TG. Two additional 25 cm2 flasks were seeded with approx. 500 cells each in 

non-selective medium to determine the viability. 

The cultures were incubated at 37 °C in a humidified atmosphere with 1.5% CO2 for about 

8 days. The colonies were stained with 10% methylene blue in 0.01% KOH solution. 

The stained colonies with more than 50 cells were counted. In doubt the colony size was 

checked with a preparation microscope. 

11.8 Data Recording 

The data generated were recorded in the raw data. The results are presented in tabular 

form, including experimental groups with the test item, solvent, and positive controls. 
Report Page 21 of 39 

11.9 Acceptability of the Assay 

The gene mutation assay is considered acceptable if it meets the following criteria: 

The numbers of mutant colonies per 106

 cells found in the solvent controls falls within the 

laboratory historical control data (see Annex II). 

The positive control substances should produce a significant increase in mutant colony 

frequencies (see Annex II). 

The cloning efficiency II (absolute value) of the solvent controls should exceed 0.5. 

The data of this study comply with the above mentioned criteria [see Annex I (tables of 

results, mutation rate and factor calculated referring to the cloning efficiency of the 

untreated cultures) and Annex II (Historical data)]. 

11.10 Evaluation of Results 

A test item is classified as positive if it induces either a concentration-related increase of 

the mutant frequency or a reproducible and positive response at one of the test points. 

A test item producing neither a concentration-related increase of the mutant frequency nor 

a reproducible positive response at any of the test points is considered non-mutagenic in 

this system. 

A positive response is described as follows: 

A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is 

three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment. 

The test item is classified as mutagenic if there is a reproducible concentration-related 

increase of the mutation frequency. Such evaluation may be considered also in the case 

that a threefold increase of the mutant frequency is not observed. 

However, in a case by case evaluation this decision depends on the level of the 

corresponding solvent control data. If there is by chance a low spontaneous mutation rate 

within the laboratory ́s historical control data range, a concentration-related increase of 

the mutations within this range has to be discussed. The variability of the mutation rates of 

solvent controls within all experiments of this study was also taken into consideration. 
Report Page 22 of 39 

11.11 Statistical Analysis 

A linear regression (least squares) was performed to assess a possible dose dependent 

increase of mutant frequencies. The number of mutant colonies obtained for the groups 

treated with the test item were compared to the solvent control groups. A trend is judged 

as significant whenever the p-value (probability value) is below 0.05. However, both, 

biological relevance and statistical significance was considered together. 

experimental group p-value 

experiment I, culture I without S9 mix 0.015S

experiment I, culture II without S9 mix 0.774

experiment I, culture I with S9 mix 0.377

experiment I, culture II with S9 mix 0.748

experiment II, culture I without S9 mix 0.841

experiment II, culture II without S9 mix 0.469

experiment II, culture I with S9 mix 0.997

experiment II, culture II with S9 mix 0.043S

S = Significant trend 
Report Page 23 of 39 

12 Calculation and Processing of the Data 

The data listed in the tables (Annex I) are calculated and processed as described below. 

Pre-test 

cloning efficiency, absolute mean number of colonies per flask divided by the 

number of cells seeded  100

cloning efficiency, relative (mean number of colonies per flask divided by the 

mean number of colonies per flask of the 

corresponding control)  100 

Main test 

cloning efficiency I (survival) cloning efficiency determined immediately after 

treatment to measure toxicity. 

cloning efficiency II (viability) cloning efficiency determined after the expression 

period to measure viability of the cells without selective 

agent. 

cloning efficiency I 

(survival, absolute)

mean number of colonies per flask divided by the 

number of cells seeded 

cloning efficiency I 

(survival, relative)

(mean number of colonies per flask divided by the 

mean number of colonies per flask of the 

corresponding control)  100 

cell density % of control (cell density at 1st subcultivation divided by the cell 

density at 1st subcultivation of the corresponding 

control)  100

cloning efficiency II 

(viability, absolute)

mean number of colonies per flask divided by the 

number of cells seeded 

cloning efficiency II 

(viability, relative)

cloning efficiency II absolute divided by the cloning 

efficiency II absolute of the corresponding control 

 100

cells survived (after plating in 

TG containing medium) number of cells seeded  cloning efficiency II absolute 

mutant colonies / 106

 cells mean number of mutant colonies per flask found 

after plating in TG medium  106 divided by the number 

of cells survived 

induction factor mutant colonies per 106

 cells / mutant colonies 

per 106

 cells of the corresponding solvent control 
Report Page 24 of 39 

13 RESULTS AND DISCUSSION

The test item was assessed for its potential to induce gene mutations 

at the HPRT locus using V79 cells of the Chinese hamster. 

The study was performed in two independent experiments, using identical experimental 

procedures. In the first experiment the treatment period was 4 hours with and without 

metabolic activation. The second experiment was performed with a treatment time of 

4 hours with and 24 hours without metabolic activation. 

In experiment I and II the cell cultures were assayed at the following concentrations: 

exposure 

period 

S9 

mix 

concentrations 

in μg/mL 

Experiment I

 4 hours - 1.0 3.0 9.0 27.0 P 81.0 P

 4 hours + 1.0 3.0 9.0 27.0 P 81.0 P

Experiment II

24 hours - 1.0 3.0 9.0 27.0 P 81.0 P

 4 hours + 1.0 3.0 9.0 27.0 P 81.0 P

P = Precipitation 

In the main experiments precipitation was observed at 27.0 μg/mL and above in all of the 

experimental parts. At almost all of the concentrations showing precipitation additional 

small droplets were noted as phase separation. 

No cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% 

was observed up to the maximum concentration with and without metabolic activation. 

No relevant and reproducible increase in mutant colony numbers/106

 cells occurred in the 

main experiments up to the maximum concentration. The induction factor exceeded the 

threshold of three times the corresponding solvent control in culture II of the first 

experiment at 1.0, 3.0, and 9.0 μg/mL with metabolic activation. These effects however, 

where judged as biologically irrelevant as they are based on the rather low solvent control 

of 4.9 mutant colonies/106

 cells. The actual values of the mutation frequency did not 

exceed the historical range of solvent controls. The increase was not dose dependent as 

indicated by a lacking statistical significance. 

A linear regression analysis (least squares) was performed to assess a possible dose 

dependent increase of mutant frequencies. A significant dose dependent trend of the 

mutation frequency indicated by a probability value of <0.05 was determined in the first 

culture of experiment I without metabolic activation and in the second culture of 

experiment II with metabolic activation. However, the trends were judged as biologically 

irrelevant fluctuations as the mutation frequency did not exceed the threshold described 

above. 

In both experiments of this study (with and without S9 mix) the range of the solvent 

controls was from 4.1 up to 19.6 mutants per 106

 cells; the range of the groups treated 

with the test item was from 3.8 up to 30.3 mutants per 106

 cells. 
Report Page 25 of 39 

EMS (150 μg/mL) and DMBA (1.1 μg/mL) were used as positive controls and showed a 

distinct increase in induced mutant colonies. 

Conclusion 

In conclusion it can be stated that under the experimental conditions reported the test item 

did not induce gene mutations at the HPRT locus in V79 cells. 

Therefore, is considered to be non-mutagenic in this HPRT assay. 

14 REFERENCES

1. 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

2. 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

3. P. Howard-Flanders

Mutagenesis in mammalian cells

Mutation Research 86, 307-327, 1981

4. M.O. Bradley, B. Bhuyan, M.C. Francis, R. Langenbach, A. Peterson,

and E. Huberman

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

of the literature. A report of the gene-tox program

15 DISTRIBUTION OF THE REPORT

Sponsor 2x (1x original, 1x electronic copy as PDF-file) 

Study Director 1x (copy) 
Report 

16 ANNEX I: TABLES OF RESULTS 16.1 Pre-test Table 2: Toxicity data 

conc. S9 Duration of cells

μg/mL mix treatment seeded

Test group (h) I/IIColumn 1 2 3 4

Solvent control with THF - 4 520

Test item 17.2 - 4 520

Test item 34.4 - 4 520Test item 68.8 - 4 520Test item 137.5 - 4 520Test item 175.0 - 4 520Test item 550.0 - 4 520Test item 1100.0 - 4 520Test item 2200.0 - 4 520Solvent control with THF + 4 520

Test item 17.2 + 4 520

Test item 34.4 + 4 520Test item 68.8 + 4 520Test item 137.5 + 4 520Test item 175.0 + 4 520Test item 550.0 + 4 520Test item 1100.0 + 4 520Test item 2200.0 + 4 520Solvent control with THF - 24 520

Test item 17.2 - 24 520

Test item 34.4 - 24 520

Test item 68.8 - 24 520

Test item 137.5 - 24 520

Test item 175.0 - 24 520

Test item 550.0 - 24 520Test item 1100.0 - 24 520

Test item 2200.0 - 24 520
Report 

16.2 Main Experiment 16.2.1 Experiment I Culture I Table 3: Cloning Efficiency I (Survival) 

conc. P S9 cells

Test group μg/mL mix seeded

I/II

Column 123Solvent control with THF - 514Positive control with EMS 150.0 - 514

Test item 1.0 - 514Test item 3.0 - 514Test item 9.0 - 514Test item 27.0 P - 514

Test item 81.0 P - 514

Test item 243.0 P - 514

Solvent control with THF + 514Positive control with DMBA 1.1 + 514Test item 1.0 + 514Test item 3.0 + 514Test item 9.0 + 514Test item 27.0 P + 514

Test item 81.0 P + 514

Test item 243.0 P + 514

# culture was not continued to avoid analysis of too many concentrations showing precipitation P Precipitation 
Report 

Table 4: Cloning Efficiency II (Viability), experiment I, culture I 

conc. P S9 cells

Test group μg/mL mix seeded

I/II

Column 1 2 3 4Solvent control with THF - 507Positive control with EMS 150.0 - 525Test item 1.0 - 555Test item 3.0 - 512Test item 9.0 - 513Test item 27.0 P - 511Test item 81.0 P - 510Test item 243.0 P -Solvent control with THF + 567Positive control with DMBA 1.1 + 535Test item 1.0 + 569Test item 3.0 + 562Test item 9.0 + 515Test item 27.0 P + 519Test item 81.0 P + 570Test item 243.0 P +

culture wculture w

# culture was not continued to avoid analysis of too many concentrations showing precipitation P Precipitation 
Report 

Table 5: Mutagenicity data (Mutation rates), experiment I, culture I 

conc. P S9

Test group μg/mL mixColumn 1 2

Solvent control with THFPositive control with EMS 150.0 -Test item 1.0Test item 3.0Test item 9.0Test item 27.0 P -Test item 81.0 P -Test item 243.0 P -Solvent control with THFPositive control with DMBA 1.1 +Test item 1.0Test item 3.0Test item 9.0Test item 27.0 PTest item 81.0 PTest item 243.0 P +

# culture was not continued to avoid analysis of too many concentrations showing precipitation P Precipitation 
Report 

16.2.2 Experiment I Culture II Table 6: Cloning Efficiency I (Survival), culture II 

conc. P S9 cells

Test group μg/mL mix seeded

I/II

Column 123 4Solvent control with THF - 514Positive control with EMS 150.0 - 514

Test item 1.0 - 514

Test item 3.0 - 514

Test item 9.0 - 514

Test item 27.0 P - 514

Test item 81.0 P - 514

Test item 243.0 P - 514Solvent control with THF + 514Positive control with DMBA 1.1 + 514Test item 1.0 + 514Test item 3.0 + 514

Test item 9.0 + 514

Test item 27.0 P + 514Test item 81.0 P + 514Test item 243.0 P + 514

# culture was not continued to avoid analysis of too many concentrations showing precipitation P Precipitation 
Report 

Table 7: Cloning Efficiency II (Viability), experiment I, culture II 

conc. P S9 cells

Test group μg/mL mix seeded

I/II

Column 1 2 3Solvent control with THF - 569Positive control with EMS 150.0 - 507Test item 1.0 - 534Test item 3.0 - 522Test item 9.0 - 540Test item 27.0 P - 560Test item 81.0 P - 551Test item 243.0 P -Solvent control with THF + 501Positive control with DMBA 1.1 + 552Test item 1.0 + 532Test item 3.0 + 583Test item 9.0 + 590Test item 27.0 P + 520Test item 81.0 P + 553Test item 243.0 P +

culture w

culture w

# culture was not continued to avoid analysis of too many concentrations showing precipitation P Precipitation 
Report 

Table 8: Mutagenicity data (Mutation rates), exper

conc. P S9

Test group μg/mL mix

Column 1 2

Solvent control with THF -Positive control with EMS 150.0 -Test item 1.0 -

Test item 3.0 -

Test item 9.0 -

Test item 27.0 P -

Test item 81.0 P -

Test item 243.0 P -Solvent control with THF +Positive control with DMBA 1.1 +Test item 1.0 +

Test item 3.0 +

Test item 9.0 +

Test item 27.0 P +

Test item 81.0 P +

Test item 243.0 P +

# culture was not continued to avoid analysis of too many concentrations showing precipitation P Precipitation 
Report 

16.2.3 Experiment II, Culture I Table 9: Cloning Efficiency I (Survival) 

conc. P S9 cells

Test group μg/mL mix seeded

I/II

Column 123Solvent control with THF - 509Positive control with EMS 150.0 - 509Test item 1.0 - 509Test item 3.0 - 509Test item 9.0 - 509Test item 27.0 P - 509Test item 81.0 P - 509Test item 243.0 P - 509Solvent control with THF + 509Positive control with DMBA 1.1 + 509Test item 1.0 + 509Test item 3.0 + 509

Test item 9.0 + 509

Test item 27.0 P + 509Test item 81.0 P + 509Test item 243.0 P + 509

# culture was not continued to avoid analysis of too many concentrations showing precipitation P Precipitation 
Report 

Table 10: Cloning Efficiency II (Viability), experiment II, culture I 

conc. P S9 cells

Test group μg/mL mix seeded

I/II

Column 123 4Solvent control with THF - 516Positive control with EMS 150.0 - 541Test item 1.0 - 517Test item 3.0 - 549Test item 9.0 - 504Test item 27.0 P - 507Test item 81.0 P - 544Test item 243.0 P -Solvent control with THF + 522Positive control with DMBA 1.1 + 505Test item 1.0 + 531Test item 3.0 + 545Test item 9.0 + 510Test item 27.0 P + 538Test item 81.0 P + 533Test item 243.0 P +

culture wculture w

# culture was not continued to avoid analysis of too many concentrations showing precipitation P Precipitation 
Report 

Table 11: Mutagenicity data (Mutation rates), expe

conc. P S9

Test group μg/mL mix

Column 1 2

Solvent control with THFPositive control with EMS 150.0 -Test item 1.0Test item 3.0Test item 9.0Test item 27.0 PTest item 81.0 PTest item 243.0 P -Solvent control with THFPositive control with DMBA 1.1 +Test item 1.0Test item 3.0Test item 9.0Test item 27.0 PTest item 81.0 PTest item 243.0 P +

# culture was not continued to avoid analysis of too many concentrations showing precipitation P Precipitation 
Report 

16.2.4 Experiment II, Culture II Table 12: Cloning Efficiency I (Survival) 

conc. P S9 cells

Test group μg/mL mix seeded

I/II

Column 123Solvent control with THF - 509Positive control with EMS 150.0 - 509Test item 1.0 - 509Test item 3.0 - 509Test item 9.0 - 509Test item 27.0 P - 509Test item 81.0 P - 509Test item 243.0 P - 509Solvent control with THF + 509

Positive control with DMBA 1.1 + 509Test item 1.0 + 509Test item 3.0 + 509Test item 9.0 + 509Test item 27.0 P + 509Test item 81.0 P + 509Test item 243.0 P + 509

# culture was not continued to avoid analysis of too many concentrations showing precipitation P Precipitation 
Report 

Table 13: Cloning Efficiency II (Viability), experiment II, culture II 

conc. P S9 cells

Test group μg/mL mix seeded

I/II

Column 123Solvent control with THF - 513Positive control with EMS 150.0 - 507Test item 1.0 - 553Test item 3.0 - 521Test item 9.0 - 517Test item 27.0 P - 504Test item 81.0 P - 518Test item 243.0 P -Solvent control with THF + 546Positive control with DMBA 1.1 + 525Test item 1.0 + 511Test item 3.0 + 512Test item 9.0 + 520Test item 27.0 P + 504Test item 81.0 P + 548Test item 243.0 P +

culture wculture w

# culture was not continued to avoid analysis of too many concentrations showing precipitation P Precipitation 
Report 

conc. P S9

Test group μg/mL mix

Column 1 2

Solvent control with THFPositive control with EMS 150.0 -Test item 1.0Test item 3.0Test item 9.0Test item 27.0 PTest item 81.0 PTest item 243.0 P -Solvent control with THFPositive control with DMBA 1.1 +Test item 1.0Test item 3.0Test item 9.0Test item 27.0 PTest item 81.0 PTest item 243.0 P +

# culture was not continued to avoid analysis of too many concentrations showing precipitation P Precipitation Table 14: Mutagenicity data (Mutation rates), experiment II, culture II 
Report Page 39 of 39 

17 ANNEX II: Historical Data 

These values represent the historical control data from 2010 – 2011