## 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.Repo rt Page 2 of 39



# 1 PAGE RESERVED FOR PROPRIETARY INFORMATION OR STATEMENT OF NO DAT A 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:

"Chemika l iengesetz" (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

DateRepo rt Page 4 of 39



# 3 PAGE RESERVED FOR FLAGGING STATEMENTS

## 4 SIGNAlURE Report

Study Director Page 5 of 39



# ...... .. ............ .

Date: April 30, 2013Report 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, 2013Repo rt Page 7 of 39



# 6 COPY OF GLP CERTIFICATE

G u te Laborpraxis/Good Laboratory Practice

GLP-Bescbei ni gung!Statement of GLP Complia n ce (gemäß/acco rdin g to § 19b Abs. 1 Chemikaliengesetz)

Eine GLP-lnspektion zur Überwachung der Einhaltung der GLP-Grundsätze gemäß Chemi kalien gesetz bzw. Richtlinie 2004 / 9/EG wurde durchgeflihrt in Assessment of confonnity with GLP according 10 Chemi kalien gesetz aod Di r ective 2004 / 9 /EEC at:



### I8J Prüfeinrichtung / Test facility 

### 0 PrUfstaodortrrest site

(Unve r wec h se lb are Bezeichnung und Adre ss e!Unequivocal narn e and adress)

Prüfun ge n na c h Katc goric n / Arca s o f Ex perti se (gemäß / accordiog c h emVwV-GLP Nr. 5.3 / 0ECD guida n ce)

2 Prüfungen zur B e stimmung der toxiko l ogischen Eigenschaften 3 Prüfungen zur Bestimmung der erbgutverän

dernden Eigenschaften (in vitro und in vivo) 6 Prüfungen zur Bestimmung von Rückständen 8 Analytische Prüfungen an biologischen Materialien 2 Toxicity studies

3 Mutagenicit y studjes

6 Residues 8 Analytical studies on biolog

. ical ma te rials

1 5 .08

. und 27.-29. 10 .2008 Datum der Ins pek tio n/Date of lnspection (Tag Monat Jahr / day month year)

Die genann te PrOfeinrichtung befindet sich im natio

nalen GLP-Überwachungsverfahren und wird regelmäßig auf Einhalt un g der GLP-Grundsätze Oberwacht.

Auf der Grundtage des In spektionsberichtes wird hiermit bestä ti gt, dass in dieser Prüfeinrichtung die oben genannten PrOfungen unter Ei nhaltun g der GLP

- Grundsätze durchgefilhrt we rden können. The above mentioned test facility i s incl ud ed in Lhe national GLP Compliance Programme and is inspected on a regular basis.

Basedon the inspection report it can be confinoed

, tbat this test facili t y is able to conduct the aforementioned studies in compliance with the J>rincip l es ofGLP.

Jm Auftrag

(Name und Adresse der GLP

- Überwac hun gsbehö r de!Name and address ofthe GLP M onitoring AuthorityRepo rt 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 23Repo rt Page 9 of 39



### CONTENTS CONTI NUED

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 39Repo rt 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, 2013Repo rt Page 11 of 39



## 8.4 Good Laboratory Practice
The study was performed in compliance with: ”Chemikaliengesetz“ (Chemicals Act) of t he 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.Repo rt 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 mutati on 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.Repo rt 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 10 6 cells factor efficiency I density efficiency II 10 6 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 II culture I 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 n ot continued to avoid analysis of too many concentrations showing precipitation P = PrecipitationRepo rt 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 containingthe 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 bef ore 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 7.55Repo rt 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 be cause of the reproducible characteristics of the cells. Thawed stock cultures are propagated at 37 °C in 80 cm 2 

### plastic flasks. About 5  10 5 

### 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% CO 2 

### .



## 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.Repo rt 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 MgCl 2 

### 33 mM KCl

5 mM glucose-6-p hosphate 4 mM NADP

in 100 mM sodium-phosphate-buffer, pH 7.4. During the experiment, t he 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 metabolicRepo rt Page 19 of 39



### activation. Following 24 hours treat ment 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

Experim ent 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 = Precip itation 

### 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% CO 2 

### .



### 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 KH 2 

### PO 4 

### 200 mg Na 2 

### HPO 4 

### 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  10 6 

### (single culture) and 5  10 2 

### 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 Na 2 

### HPO 4 

###  2H 2 

### O 192 mg KH 2 

### PO 4 

### 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  10 6 

### 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  10 5 

### 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% CO 2 

### 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.Repo rt 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 10 6 

### 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 isthree 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.Repo rt 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 0.015 S



### 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 0.043 S

S = Significant trendRepo rt 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 / 10 6 

### cells mean number of mutant colonies per flask found after plating in TG medium  10 6 

### divided by the number of cells survived

induction factor mutant colonies per 10 6 

### cells / mutant colonies per 10 6 

### cells of the corresponding solvent controlRepo rt 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

Experim ent 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/10 6 

### 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/10 6 

### 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 10 6 

### cells; the range of the groups treated with the test item was from 3.8 up to 30.3 mutants per 10 6 

### cells.Repo rt 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)ReportPage 26 of 3916ANNEX I: TABLES OF RESULTS16.1Pre-testTable2: Toxicity dataconc. S9Duration of cellsμg/mL mix treatment seededTest group(h)I/IIColumn1 2 34Solvent control with THF-4 520Test item17.2-4 520Test item34.4-4 520Test item68.8-4 520Test item137.5-4 520Test item175.0-4 520Test item550.0-4 520Test item1100.0-4 520Test item2200.0-4 520Solvent control with THF+4 520Test item17.2 +4 520Test item34.4 +4 520Test item68.8 +4 520Test item137.5 +4 520Test item175.0 +4 520Test item550.0 +4 520Test item1100.0 +4 520Test item2200.0 +4 520Solvent control with THF-24 520Test item17.2 -24 520Test item34.4 -24 520Test item68.8 -24 520Test item137.5 -24520Test item175.0 -24 520Test item550.0 -24 520Test item1100.0 -24 520Test item2200.0 -24 520ReportPage 27 of 3916.2Main Experiment16.2.1Experiment I Culture ITable 3: Cloning Efficiency I (Survival)conc. P S9 cellsTest groupμg/mL mix seededI/IIColumn123Solvent control with THF- 514Positive control with EMS 150.0 - 514Test item1.0 - 514Test item3.0 - 514Test item9.0 - 514Test item27.0 P - 514Test item81.0 P - 514Test item243.0 P - 514Solvent control with THF+ 514Positive control with DMBA 1.1 + 514Test item1.0 + 514Test item3.0 + 514Test item9.0 + 514Test item27.0 P + 514Test item81.0 P + 514Test item243.0 P + 514# culture was not continued to avoid analysis oftoo many concentrations showing precipitationP PrecipitationReportPage 28 of 39Table 4: Cloning Efficiency II (Viability), experiment I, culture Iconc. P S9 cellsTest group μg/mL mix seededI/IIColumn 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 oftoo many concentrations showing precipitationP PrecipitationReportPage 29 of 39Table 5: Mutagenicity data (Mutation rates), experiment I, culture Iconc. P S9Test groupμg/mL mixColumn1 2Solvent control with THFPositive control with EMS 150.0 -Test item1.0Test item3.0Test item9.0Test item27.0 P -Test item81.0 P -Test item243.0 P -Solvent control with THFPositive control with DMBA 1.1 +Test item1.0Test item3.0Test item9.0Test item27.0 PTest item81.0 PTest item243.0 P +# culture was not continued to avoid analysis oftoo many concentrations showing precipitationP PrecipitationReportPage 30 of 3916.2.2Experiment I Culture IITable 6: Cloning Efficiency I (Survival), culture IIconc. P S9 cellsTest groupμg/mL mix seededI/IIColumn123 4Solvent control with THF- 514Positive control with EMS150.0- 514Test item1.0- 514Test item3.0- 514Test item9.0- 514Test item27.0 P - 514Test item81.0 P - 514Test item243.0 P - 514Solvent control with THF+ 514Positive control with DMBA 1.1 + 514Test item1.0 + 514Test item3.0 + 514Test item9.0 + 514Test item27.0 P + 514Test item81.0 P + 514Test item243.0 P + 514# culture was not continued to avoid analysis oftoo many concentrations showing precipitationP PrecipitationReportPage 31 of 39Table 7: Cloning Efficiency II (Viability), experiment I, culture IIconc. P S9 cellsTest group μg/mL mix seededI/IIColumn 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 wculture w# culture was not continued to avoid analysis oftoo many concentrations showing precipitationP PrecipitationReportPage 32 of 39Table 8: Mutagenicity data (Mutation rates), experconc. P S9Test groupμg/mL mixColumn1 2Solvent control with THF-Positive control with EMS 150.0 -Test item1.0 -Test item3.0 -Test item9.0 -Test item27.0 P -Test item81.0 P -Test item243.0 P -Solvent control with THF+Positive control with DMBA 1.1 +Test item1.0 +Test item3.0 +Test item9.0 +Test item27.0 P +Test item81.0 P +Test item243.0 P +# culture was not continued to avoid analysis oftoo many concentrations showing precipitationP PrecipitationReportPage 33 of 3916.2.3Experiment II, Culture ITable 9: Cloning Efficiency I (Survival)conc. P S9 cellsTest groupμg/mL mix seededI/IIColumn123Solvent control with THF- 509Positive control with EMS150.0 - 509Test item1.0 - 509Test item3.0 - 509Test item9.0 - 509Test item27.0 P - 509Test item81.0 P - 509Test item243.0 P - 509Solvent control with THF+ 509Positive control with DMBA 1.1 + 509Test item1.0 + 509Test item3.0 + 509Test item9.0 + 509Test item27.0 P + 509Test item81.0 P + 509Test item243.0 P + 509# culture was not continued to avoid analysis oftoo many concentrations showing precipitationP PrecipitationReportPage 34 of 39Table 10: Cloning Efficiency II (Viability), experiment II, culture Iconc. P S9 cellsTest groupμg/mL mix seededI/IIColumn123 4Solvent control with THF- 516Positive control with EMS 150.0 - 541Test item1.0 - 517Test item3.0 - 549Test item9.0 - 504Test item27.0 P - 507Test item81.0 P - 544Test item243.0 P -Solvent control with THF+ 522Positive control with DMBA 1.1 + 505Test item1.0 + 531Test item3.0 + 545Test item9.0 + 510Test item27.0 P + 538Test item81.0 P + 533Test item243.0 P +culture wculture w# culture was not continued to avoid analysis oftoo many concentrations showing precipitationP PrecipitationReportPage 35 of 39Table 11: Mutagenicity data (Mutation rates), expeconc. P S9Test groupμg/mL mixColumn1 2Solvent control with THFPositive control with EMS 150.0 -Test item1.0Test item3.0Test item9.0Test item27.0 PTest item81.0 PTest item243.0 P -Solvent control with THFPositive control with DMBA 1.1 +Test item1.0Test item3.0Test item9.0Test item27.0 PTest item81.0 PTest item243.0 P +# culture was not continued to avoid analysis oftoo many concentrations showing precipitationP PrecipitationReportPage 36 of 3916.2.4Experiment II, Culture IITable 12: Cloning Efficiency I (Survival)conc. P S9 cellsTest groupμg/mL mix seededI/IIColumn123Solvent control with THF- 509Positive control with EMS150.0 - 509Test item1.0 - 509Test item3.0 - 509Test item9.0 - 509Test item27.0 P - 509Test item81.0 P - 509Test item243.0 P - 509Solvent control with THF+ 509Positive control with DMBA 1.1 + 509Test item1.0 + 509Test item3.0 + 509Test item9.0 + 509Test item27.0 P + 509Test item81.0 P + 509Test item243.0 P + 509# culture was not continued to avoid analysis oftoo many concentrations showing precipitationP PrecipitationReportPage 37 of 39Table 13: Cloning Efficiency II (Viability), experiment II, culture IIconc. P S9 cellsTest groupμg/mL mix seededI/IIColumn123Solvent control with THF- 513Positive control with EMS 150.0 - 507Test item1.0 - 553Test item3.0 - 521Test item9.0 - 517Test item27.0 P - 504Test item81.0 P - 518Test item243.0 P -Solvent control with THF+ 546Positive control with DMBA 1.1 + 525Test item1.0 + 511Test item3.0 + 512Test item9.0 + 520Test item27.0 P + 504Test item81.0 P + 548Test item243.0 P +culture wculture w# culture was not continued to avoid analysis oftoo many concentrations showing precipitationP PrecipitationReportPage 38 of 39conc. P S9Test groupμg/mL mixColumn1 2Solvent control with THFPositive control with EMS 150.0 -Test item1.0Test item3.0Test item9.0Test item27.0 PTest item81.0 PTest item243.0 P -Solvent control with THFPositive control with DMBA 1.1 +Test item1.0Test item3.0Test item9.0Test item27.0 PTest item81.0 PTest item243.0 P +# culture was not continued to avoid analysis oftoo many concentrations showing precipitationP PrecipitationTable 14: Mutagenicity data (Mutation rates), experiment II, culture IIRepo rt Page 39 of 39



# 17 ANNEX II: Historical Data

These values represent the historical control data from 2010 – 2011