TITLE PAGE

REPORT

SALMONELLA TYPHIMURIUM / ESCHERICHIA COLI REVERSE MUTATION ASSAY

TEST GUIDELINE(S)

OECD 471 Co mmission Regulation (EC) No 4 40/2008; B.13 / B.14 US EPA OPPTS 870.5100

AUTHOR (S tudy Director)

STUDY COMPLETION DATE 23 Sep 2013

TEST FACILITY

TEST FACILITY PROJECT IDENTIFICATION
Project No. :

SPONSOR

Page 1 of 52 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.THIS PAGE IS INTENTIONALLY LEFT BLANK

BLANK PAGE

Page 2 of 52GLP

- CERTIFICATE (FROM THE COMPETENT AUTHORITY)

Page 8 of 52CONTENTS

TITLE PAGE BLANK PAGE GLP COMPLIANCE STATE MENT FLAGGING CRITERIA SIGNATURE PAGE CONTRIBUTORS TO THE STUDY / SUPERVISORY LABORATORY PERSONNEL STATEMENT OF THE QUA LITY ASSURANCE UNIT GLP

- CERTIFICATE (FROM THE COMPETENT AUTHORITY) CONTENTS
1. SUMMARY

2. INTRODUCTION

2.1. OBJECTIVES

2.2. SELECTION OF DOSES

2.3. TEST GUIDELINES

2.4. STUDY DATES

2.5. RETENTION OF RECORDS

3. MATERIAL AND METHODS

3.1. TEST ITEM

3.2. TEST SYSTEM

3.2.1. Salmonella typhimur ium

3.2.2. Escherichia coli

3.2.3. Checking the tester strains

3.3. EXOGENOUS METABOLIC ACTIVATION

3.3.1. S9 fraction

3.3.2. S9 mix

3.4. DOSES

3.5. TEST SUBSTANCE PREPA RATION

3.6. ANALYSIS OF TEST SUB STANCE PREPARATION

3.7. EXPERIMENTAL PROCEDU RE

3.7.1. Choice of the vehicle

3.7.2. Mutagenicity tests

3.7.2.1. Standard plate test

3.7.2.2. Preincubation Test

3.7.3. Titer determination

3.7.4. Controls

Page 9 of 523.7.4.1. Negative controls / Vehicle controls

3.7.4.2. Positive controls

3.7.5. Scope of tests and test conditions

3.8. EVALUATION

3.8.1. Mutagenicity

3.8.2. Titer

3.8.3. Toxicity

3.8.4. Solubility

3.8.5. Acceptance criteria

3.8.6. Assessment criteria

4. RESULTS

4.1. MUTAGENICITY

4.1.1. Standard plate test

4.1.1.1. Tests without S9 mix

4.1.1.2. Tests with S9 mix

4.1.2. Preincubation test

4.1.2.1. Tests without S9 mix

4.1.2.2. Tests with S9 mix

4.2. TOXICITY

4.3. SOLUBILITY

5. DISCUSSION

6. CONCLUSION

7. REFERENCES
APPENDIX 1 TABLES 1 - 5 (STANDARD PLATE T EST) APPENDIX 2 TABLES 6 - 10 (PREINCUBATION T EST) APPENDIX 3 STERILIT Y CONTROL APPENDIX 4 S9 FRACT ION CHARACTERIZATION APPENDIX 5 HISTORICAL NEGATI VE CONTROL DATA APPENDIX 6 HISTORICAL POSITIVE CONTROL DATA

Page 10 of 52

### 1. SUMMARY

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay.

STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA

DOSE RANGE: 33 μg - 5 400 μg/plate (SPT) 33 μg - 5 400 μg/plate (PIT)

T EST CONDITIONS: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats ).

SOLUBILITY: Precipitation of the test substance was found from about 333 μg/plate onward with and without S 9 mix.

TOXICITY: A weak bacteriotoxic effect was occasionally observed depending on the strain and t est conditions from about 2 700 μg/plate onw ard (for details see item 4.2 .).

MUTAGENICITY:

A relevant increase in the number of his + or trp + revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.

CONCLUSION:

Thus, under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation
assay in the absence and the presence of metabolic activation

.

Page 11 of 52

### 2. INTRODUCTION

2.1. OBJECTIVES

Bacterial reverse mutation assays using amino

- acid requiring strain s of Salmonella typhimurium and Escherichia coli are commonly employed as initial screening methods for detecting a point mutagenic activity of chemical substance s ( 1

, 2

, 5 ) and are used to screen for possible mammalian mutagens and carcinogens. The principle of these assays is that mutations are detected by the reversion of mutations present in the bacterial strains. This leads to a restoration of the functional capability of the bacteria to synthesize the essential amino acid and thus to the ability to grow in the absence of the amino acid required by the parent strains.

Most of the substance s are not mutagenic or carcinogenic themselves, but only after metabolic transformation, which in vivo is catalyzed mainly by the enzyme systems of the liver. Therefore, the tests are carried out not only directly, but also in the presence of an exogenous metabolic activation system. The most commonly used system is a cofactor

supplemented postmitochondrial fraction (S9) obtained from livers of rats treated with an
enzyme

- inducing agent.

2.2. SELECTION OF DOSES

See section 3.7.5

.

2.3. TEST GUIDELINES

The tests were carried out in accordance with the following test guidelines:

- OECD Guideline for the Testing of Chemicals No. 471, 21 Jul 1997, “Bacterial Reverse Mutation Test”

- Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), Part B: Methods for the determination of toxicity and other health effects: Mutagenicity – Reverse Mut ation Test using Bacteria, No B. 13 / B. 14 ; No L 142

- US EPA OPPTS 870.5100, Aug 1998, “Bacterial Reverse Mutation Test”

Page 12 of 522.4. STUDY DATES

Test dates: Study initiation date: 17 May 2013

Experimental starting date (1st administration of the test substance ): 21 May 2013

Experimental completion date (evaluation last experiment): 17 Jun 2013

2.5. RETENTION OF RECORDS

GLP

- relevant records and materials are stored at for at least the period of time specified in the GLP principles. Details concerning responsibilities or locations of archiving can be seen from the respective SOPs and from the raw data. In so far as raw data were recorded in the system, they are stored on electronic data media.

Page 13 of 52

### 3. MATERIAL AND METHODS

3.1. TEST ITEM

The analyses of the test item (= test substance) were carried out at

Name of test substance :

Test substance No.: 12/0275 -1

Batch identification: 0008043725

CAS No.: Not indicated by the sponsor

Purity/composition: : 93.8 area -% (see analytical report; project no.: AU124464 -1)

Homogeneity: The homogeneity of the test substance was ensured by mixing before preparation of the test substance solutions

. Storage stability: The stability of the test substance under storage conditions was guaranteed until Dec 2013 as indicated by the
sponsor , and the sponsor holds this responsibility.

ADDITIONAL TEST SUBSTANCE INFORMATION

Date of production/supply: Jan 2012

Physical state, appearance: Solid , melt, white

Storage conditions: Room temperature

The sponsor is responsible for compliance for all test substance information and their storage, except for those test substance investigations commissioned by the test facility.

Page 14 of 523.2. TEST SYSTEM

For testing, deep

- frozen (

- 70°C to

- 80°C) bacterial cultures (Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA) are thawed at room temperature, and 0.1 mL of this bacterial suspension is inoculated in nutrient broth solution (8 g/L Difco

nu trient broth + 5 g/L NaCl) and incubated in the shaking water bath at 37°C for about 12 - 16 hours. As a rule, a germ density of ≥ 10 8 bacteria/mL is reached. These cultures grown overnight are kept in iced water from the beginning of the experiment until the end in order to prevent further growth.

The use of the strains mentioned is in accordance with the current scientific

recomme ndations for the conduct of this assay.

The Salmonella strains TA 98, TA 100 and TA 1537 were obtained from (former) KNOLL Aktien gesell schaft, Ludwigshafen, Germany, on 30 Oct 1989. The Salmonella strain TA 1535 and the Escherichia coli strain were obtained from Merck KGaA, Darmstadt, Germany (22 Apr 2010 and 09 Sep 1991, respectively).

3.2.1. Salmonella typhimurium

The rate of induced back mutations of several bacteria mutants from histidine auxotrophy (his - ) to histidine prototrophy (his + ) is determined ( 2

, 3

, 4 ). The tester strains TA 1535, TA 1537, TA 98 and TA 100 selected by Ames and coworkers are derivatives of Salmonella typhimurium LT2 and have GC base pairs at the primary reversion site. All strains have a defective excision repair system (uvrB), which prevents the repair of lesions which are induced in the DNA, and this deficiency results in greatly enhanced sensitivity of some mutagens. Furthermore, all strains show a considerably reduced hydrophilic polysaccharide layer (rfa), which leads to an increase in permeability to lipophilic substance s.
The strains TA 1535 and TA 100 are derived from histidine

- prototrophic Salmonella strains by the substitution mutation his G 46 and are used to detect base pair substitutions. TA 1537 and TA 98 are strains for the detection of frameshift mutagens. These strains carry different frameshift markers, i.e. the +1 mutant his C 3076 in the case of TA 1537 and the +2 type his D 3052 in the case of TA 98.

The strains TA 98 and TA 100 carry an R factor plasmid pKM 101 ( 4 ) and, in addition to having genes resistant to antibiotics, they have a modified postreplication DNA repair system, which increases the mutation rate by inducing a defective repair in the DNA; this again leads to a considerable increase in sensitivity.

3.2.2. Escherichia coli

Escherichia coli WP2 uvrA which has an AT base pair at the primary reversion site is a derivative of E. coli WP2 with a deficient excision repair and is used to detect substance s which induce base pair substitutions ( 5 ). The rate of induced back mutations from tryptophan auxotrophy (trp - ) to tryptophan independence (trp + ) is determined.

Page 15 of 523.2.3. Checking the tester strains

The Salmonella strains are checked for the following characteristics at regular intervals: deep rough character (rfa); UV sensitivity ( ∆ uvrB); ampicillin resistance (R factor plasmid). E. coli WP2 uvrA is checked for UV sensitivity.
Histidine and tryptophan auxotrophy is checked in each experiment via the spontaneous rate.

3.3. EXOGENOUS METABOLIC ACTIVATION

3.3.1. S9 fraction

The S9 fraction is prepared according to Ames et al. ( 1

, 2 ) at in an AAALAC

approved laboratory in accordance with the German Animal Welfare Act and the effective European Council Directive

. At least 5 male Wistar rats [Crl:WI(Han)] (200 - 300 g; Charles River Laboratories Germany GmbH) received 80 mg/kg b.w. phenobarbital i.p. and β

- naphtho flavone orally (both supplied by Sigma

- Aldrich, 82024 Taufkirchen, Germany) each on three consecutive days.
During this time, the animals are housed in Makrolon cages: central air conditioning with a fixed range of temperature of 20 - 24°C and a relative humidity of 30 - 70%. The day/night rhythm is 12 hours (light period from 6.00 - 18.00 hours and dark period from 18.00 - 6.00 hours). Standardized pelleted feed and drinking water from bottles were available ad libitum.

24 hours after the last administration, the rats are sacrificed, and the livers are prepared using sterile solvents and glassware at a temperature of +4°C. The livers are weighed and washed in a weight

- equivalent volume of a 150 mM KCl solution, then cut into small pieces and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9 000 x g for 10 mi nutes at +4°C, 5 mL portions of the supernatant (so

- called S9 fraction) are stored at

- 70°C to

- 80°C.
The preparation date and protein content of the S9 fraction are given in Appendix 4

.

Page 16 of 523.3.2. S9 mix

The S9 mix is prepared freshly prior to each experiment ( 1

, 2 ). F or this purpose, a sufficient amount of S9 fraction is thawed at room temperature and 1 volume of S9 fraction is mixed with 9 volumes of S9 supplement (cofactors). This preparation, the so

- called S9 mix, is kept on ice until used. The concentrations of the cofactors in the S9 mix are:

MgCl 2 8 mM KCl 33 mM glucose

- 6

- phosphate 5 mM NADP 4 mM phosphate buffer (pH 7.4) 15 mM

The phosphate buffer ( 6 ) is prepared by mixing a Na 2 HPO 4 solution with a NaH 2 PO 4 solution in a ratio of about 4:1. To demonstrate the efficacy of the S9 mix in this assay, the S9 batch was characterized with benzo(a)pyrene (see Appendix 4 ).

3.4. DOSES

In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate are generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose will be tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 μL/plate might also be tested in repeat experiments for further clarification/substantiation. In this study, due to the purity of the test substance 5.4 mg/plate was used as top dose in both experiments.

3.5. TEST SUBSTANCE PREPARATION

The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution s.
The test substance was dissolved acetone.

To achieve c lear solution s of the test substance in the vehicle, the test substance preparation s w ere treated with ultrasonic waves and w ere shaken thoroughly.

The further concentrations were diluted according to the planned doses.

All test substance formulations were prepared immediately before administration.

3.6. ANALYSIS OF TEST SUBSTANCE PREPARATION

The stability of the test substance in the vehicle acetone was not determined analytically.

Page 17 of 523.7. EXPERIMENTAL PROCEDU RE

3.7.1. Choice of the vehicle

Due to the insolubility of the test substance in ultrapure water

, acetone was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available (9)

.

3.7.2. Mutagenicity tests

3.7.2.1. Standard plate test

The experimental procedure of the standard plate test (plate incorporation method) is based on the method of Ames et al. ( 1

, 2 ).

• Salmonella typhimurium
Test tubes containing 2

- mL portions of soft agar (overlay agar), which consist s of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at about 42 - 45°C, and the remaining components are added in the following order:

0.1 mL test solution or vehicle (negative control) 0.1 mL fresh bacterial culture

0.5 mL S9 mix ( with metabolic activation )

or 0.5 mL phosphate buffer ( without metabolic activation ) After mixing, the samples are poured onto Merckoplate® plates (minimal glucose agar plates from Merck KGaA, Darmstadt.Germany) within approx. 30 seconds.

After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his + revertants) are counted.

Page 18 of 52• Escherichia coli

Test tubes containing 2

- mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) are kept in a water bath at about 42 - 45°C, and the remaining components are added in the following order:

0.1 mL test solution or vehicle (negative control) 0.1 mL fresh bacterial culture

0.5 mL S9 mix ( with metabolic activation )

or 0.5 mL phosphate buffer ( without metabol ic activation ) After mixing, the samples are poured onto Merckoplate® plates (minimal glucose agar plates from Merck KGaA, Darmstadt.Germany) within approx. 30 seconds.

After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies (trp + re vertants) are counted.

3.7.2.2. Preincubation Test

The experimental procedure is based on the method described by Yahagi et al. ( 7 ) and Matsushima et al. ( 8 ).

0.1 mL test solution or vehicle, 0.1 mL bacteria l suspension and 0.5 mL S9 mix ( with metabolic activation ) or phosphate buffer ( without metabolic activation ) are incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar is added and, after mixing, the samples are poured onto the agar plates within approx. 30 seconds.

After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies are counted.

Page 19 of 523.7.3. Titer determination

The titer was determined only in the experimental parts with S9 mix both for the ne gative controls (vehicle only) and for the two highest doses in all experiments.
In the standard plate test, 0.1 mL of the overnight cultures is diluted to 10 -6 in each case. Test tubes containing 2

- mL portions of soft agar containing maximal amino acid so lution (5 mM tryptophan or 5 mM histidine + 0.5 mM biotin) are kept in a water bath at about 42 - 45°C, and the remaining components are added in the following order:

0.1 mL vehicle (without and with test substance) 0.1 mL fresh bacterial culture (dilution: 10 -6 ) 0.5 mL S9 mix
In the preincubation test, 0.1 mL of the overnight cultures is diluted to 10 - 6 in each case. 0.1 mL vehicle (with and without test substance ), 0.1 mL bacterial suspension and 0.5 mL S9 mix are incubated at 37°C for about 20 minutes using a shaker. Subsequently, 2 mL of soft agar containing maximal amino acid solution for titer determination ( 5 mM tryptophan or 5 mM histidine + 0.5 mM biotin) is added. After mixing, the samples are poured onto the agar plates within approx. 30 seco nds. After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies are counted.

3.7.4. Controls

3.7.4.1. Negative controls / Vehicle controls

Each experiment includes negative controls in order to check for possible contaminants (sterility control) and to determine the spontaneous mutation rate (vehicle control).

• Sterility control Additional plates are treated with soft agar, S9 mix, buffer, vehicle or the test substance but without the addition of tester strains (see Appendix 3 ).

• Vehicle control

The vehicle control with and without S9 mix only contains the vehicle used for the test substance at the same concentration and volume for all tester strains.

Page 20 of 523.7.4.2. Positive controls

The following positive controls are used to check the mutability of the bacteria and the activity of the S9 mix:

With S9 mix

• 2- aminoanthracene (2

- AA) (SIGMA, A

- 1381) 1)

- 2.5 μg/plate, dissolved in DMSO

- strains: TA 1535, TA 100, TA 1537, TA 98

- 60 μg/plate, dissolved in DMSO

- strain: Escherichia coli WP2 uvrA

W ithout S9 mix

• N- methyl

- N'

- nitro -N- nitrosoguanidine (MNNG) (FLUKA, 68051) 1)

- 5 μg/plate, dissolved in DMSO

- strains: TA 1535, TA 100

• 4- nitro -o- phenylen e diamine (NOPD) (SIGMA, N

- 9504) 1)

- 10 μg/plate, dissolved in DMSO

- strain: TA 98

• 9- aminoacridine (AA C) (SIGMA, A

- 1135) 1)

- 100 μg/plate, dissolved in DMSO

- strain: TA 1537

• 4- nitroquinoline -N- oxide (4

- NQO) (SIGMA, N

- 8141) 1)

- 5 μg/plate, dissolved in DMSO

- strain: E. coli WP2 uvrA The stability of the selected positive controls is well

- defined under the selected culture conditions, since they are well

- established reference mutagens.

1) Supplier, catalogue No.

Page 21 of 523.7.5. Scope of tests and test conditions

1st Experiment

Strains: TA 1535, TA 100, TA 1537, TA 98, E. coli WP2 uvrA

Doses: 0; 33; 100; 333; 1 000; 2 70 0 and 5 4 00 μg/plate Type of test: Standard plate test with and without S9 mix
Number of plates: 3 test plates per dose or per control

2nd Experiment

Strains: TA 1535, TA 100, TA 1537, TA 98, E. coli WP2 uvrA

Doses: 0; 33; 100; 333; 1 000; 2 700 and 5 400 μg/plate Type of test: Preincubation test with and without S9 mix
Number of plates: 3 test plates per dose or per control

Reason: No mutagenicity was observed in the standard plate test.

3rd Experiment

St rains: TA 1535, TA 100, TA 1537 and TA 98

Doses: 0; 33; 100; 333; 1 000; 2 700 and 5 4 00 μg/plate Type of test: Preincubation test with and without S9 mix
Number of plates: 3 test plates per dose or per control

Reason: Due to technical reason, an evaluation of the TA strain in the 2n d experiment was not po ssible. The 2n d experiment with the TA strain was not reported. The raw data were recorded in the system and are stored on electronic data media.

Page 22 of 523.8. EVALUATION

3.8.1. Mutagenicity

Individual plate counts, the mean number of revertant colonies per plate an d the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments. In general, six doses of the test substance are tested with a maximum of 5 mg/plate, and triplicate plating is used for all test groups at least in the 1st Experiment. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments are based on the findings of the 1st Experiment.

3.8.2. Titer

The titer is generally determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.

3.8.3. Toxicity

Toxicity detected by a

• decrease in the number of revertants

• clearing or diminution of the background lawn (= reduced h is - or trp - background growth)

• reduction in the titer is recorded for all test groups both with and without S9 mix in all experiments and indicated in the tables.

3.8.4. Solubility

Precipitation of the test material is recorded and indicated in the tables. As lon g as precipitation does not interfere with the colony scoring, 5 mg/plate is generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose at least in the 1st Experiment even in the case of relatively insoluble test compounds to dete ct possible mutagenic impurities. Furthermore, doses > 5 mg/plate might also be tested in repeat experiments for further clarification/substantiation.

Page 23 of 523.8.5. Acceptance criteria

Generally, the experiment is considered valid if the following criteria are met:

• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain (see A ppendix 5 ).

• The sterility controls revealed no indication of bacterial contamination (see Appendix 3)

.

• The positive control substance s both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above (see Appendix 6 ).

• Fre sh bacterial culture containing approximately 10 9 cells per mL were used. For approval the titer of viable bacteria was ≥ 10 8 colonies per mL.

3.8.6. Assessment criteria

The test substance is considered positive in this assay if the following criteria are met:

• A dose

- related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

A test substance is generally considered non

- mutagenic in this test if:

• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Page 24 of 52

### 4. RESULTS ( Appendix 1 / 2, Tables 1 - 10 )

The test substance was tested for mutagenicity in the Salmonella typhimurium / Escherichia coli reverse mutation assay both in the standard plate test and in the preincubatio n test with and without the addition of a metabolizing system (S9 mix) obtained from rat liver using the Salmonella strains TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA.

4.1. MUTAGENICITY

4.1.1. Standard plate test ( Appendix 1 , Tables 1 - 5)

4.1.1.1. Tests without S9 mix

TA 1535:

TA 100:

TA 1537: No relevant increase in the number of his + or trp + revertants.

TA 98:

E. coli WP2 uvrA:

4.1.1.2. Tests with S9 mix

TA 1535:

TA 100:

TA 1537: No relevant increase in the nu mber of his + or trp + revertants.

TA 98:

E. coli WP2 uvrA:

Page 25 of 524.1.2. Preincubation test ( Appendix 2 , Tables 6 - 10 )

4.1.2.1. Tests without S9 mix

TA 1535:

TA 100:

TA 1537: No relevant increase in the number of his + or trp + revertants.

TA 98:

E. coli WP2 uvrA:

4.1.2.2. Tests with S9 mix

TA 1535:

TA 100:

TA 1537: No relevant increase in the number of his + or trp + revertants.

TA 98:

E. coli WP2 uvrA:

Page 26 of 524.2. TOXICITY

A weak bacteriotoxic effect (slight decrease in the number of his + or trp + revertants, slight reduction in the titer) was observ ed in the standard plat e test and in the preincubation test depending on the strain and test conditi ons from about 2 700 μg/plate onward.

4.3. SOLUBILITY

Test substance precipitati on was found from about 333 μg/plate onward with and without S9 mix.



### 5. DISCUSSION

According to the results of the present study, the test substanc e did not lead to a relevant increase in the number of revertant coloni es either without S9 mix or after adding a metabolizing system in three experiments carri ed out independently of each other (standard plate test and prei ncubation assay). Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this st udy, since the values fulfilled th e acceptance criteria of this study.
In this study with and without S9 mix, the num ber of revertant colonies in the negative controls was nearby the range of the historical negative contro l data for each tester strain (see Appendix 5).
In addition, the positive control substances both with a nd without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above (see Appendix 6).



### 6. CONCLUSION

Thus, under the experimental conditions chosen here, it is concluded that is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Page 27 of 52

### 7. REFERENCES

1. Ames, B.N.; McCann, J.; Yamasaki, E.: Methods for detecting carcinogens and mutagens with the Salmonella/mammalian

- microsome mutagenicity test. Mut. Res., 31 , 347 - 364 (1975)

2. Maron, D.M.; Ames, B.N.: Revised method for the Salmonella mutagen icity test. Mut. Res., 113 , 173 - 215 (1983)

3. Ames, B.N.; Lee, F.D.; Durston, W.E.: An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proc. Nat. Acad. Sci. USA, 70 , 782 - 786 (1973)

4. McCann, J.; Spingarn, N.E .; Kobori, J.; Ames, B.N.: Detection of carcinogens as mutagens: Bacterial tester strains with R factor plasmids. Proc. Nat. Acad. Sci. USA, 72 , 979 - 983 (1975)

5. Green, M.H.L.; Muriel, W.J.: Mutagen testing using trp+ reversions in Escherichia coli. Mut. R es., 38 , 3 - 32 (1976)

6. DeMarini, D.M.; Dallas, M.M.; Lewtas, J.: Cytotoxicity and effect on mutagenicity of buffers in a microsuspension assay. Ter. Carc. Mut., 9 , 287 - 295 (1989)

7. Yahagi, T.; Nagao, M.; Seino, Y.; Matsushima, T.; Sugimura, T.; Okada, M.: Mutagenicities of N

- nitrosoamines in Salmonella. Mut. Res., 48 , 121 - 129 (1977)

8. Matsushima, T.; Sugimura, T.; Nagao, M.; Yahagi, T.; Shirai, A.: Factors modulating mutagenicity in microbial tests. In: Norpoth, K.H. and R.C. Garner, Short

- Term Test Systems for Detecting Carcinogens. Springer Verlag Berlin, Heidelberg, New York (1980)

9. Maron, D.; Katzenellenbogen, J.; Ames, B.N.: Compatibility of organic solvents with the Salmonella/Microsome Test. Mut. Res., 88 , 343 – 350 (1981).

Page 28 of 52A PPENDIX 1

TABLES 1 - 5

(STANDARD PLATE TEST )

Page 29 of 52Page 30 of 52Page 31 of 52Page 32 of 52Page 33 of 52Page 34 of 52A PPENDIX 2

TABLES 6 - 10

(PREINCUBATION TEST)

Page 35 of 52Page 36 of 52Page 37 of 52Page 38 of 52Page 39 of 52Page 40 of 52A PPENDIX 3

STERILITY CONTROL

Page 41 of 52STERILITY CONTROL

1st Experiment

Components Results

2.0 mL soft agar No growth

0.5 mL S9 mix No growth

0.5 mL buffer No growth

0.5 mL vehicle No growth

5 400 μg test substance No growth

2nd Experiment

Components Results

2.0 mL soft agar No growth

0.5 mL S9 mix No growth

0.5 mL buffer No growth

0.5 mL vehicle No growth

5 400 μg test substance No growth

3rd Experiment

Components Results

2.0 mL soft agar No growth

0.5 mL S9 mix No growth

0.5 mL buffer No growth

0.5 mL vehicle No growth

5 400 μg test substance No growth

Page 42 of 52A PPENDIX 4

S9 FRACTION CHARACTE RIZATION

Page 43 of 52S9 fraction

Preparation of S9 fraction: 19 O ct 2012

Content of protein: 27 .01 g/L

Characterization of the S9 mix with benzo[a]pyrene: 08 N ov 2012

METHOD: Standard Plate Test

S-9 FRACTION/COFACTORS: 1:9

STRAIN: TA 100 STRAIN: TA 98

DOSE/PLATE M REV SD FAC DOSE/PLATE M REV SD FAC

DMSO 53 6 1.0 DMSO 22 5 1.0

0.625 μg 112 7 2.1 0.625 μg 52 9 2.3

1.25 μg 351 191 6.6 1.25 μg 157 23 7.0

2.5 μg 374 38 7.0 2.5 μg 221 16 9.9

5.0 μg 578 51 10.8 5.0 μg 440 43 19.7

(REV = revertants/plate, M = mean,SD = standard deviation, FAC = factor)

Page 44 of 52A PPENDIX 5

HISTORICAL NEGATIVE CONTROL DATA

Page 45 of 52

### Historical Negative Control Data

Strain: TA 1535

Strain: TA 100

* = revertants/plate PIT = preincubation test SPT = standard plate test SD = standard deviation

Page 46 of 52

### Historical Negative Control Data

Strain: TA 1537

Strain: TA 98

* = revertants/plate PIT = preincubation test SPT = standard plate test SD = standard deviation

Page 47 of 52

### Historical Negative Control Data

Strain: WP2 uvrA

* = revertants/plate PIT = preincubation test SPT = standard plate test SD = standard deviation

Page 48 of 52A PPENDIX 6

HISTORICAL POSITIVE CONTROL DATA

Page 49 of 52

### Historical Positive Control Data

Strain: TA 1535

Strain: TA 100

* = revertants/plate SPT = standard plate test PIT = preincubation test

SD = standard deviation

2 - AA = 2-aminoanthracene MNNG = N-methyl-N'-nitro-N-nitrosoguanidine

Page 50 of 52

### Strain: TA 1537

Strain: TA 98

* = revertants/plate SPT = standard plate test PIT = preincubation test

SD = standard deviation

2 - AA = 2-aminoanthracene

AAC = 9-aminoacridine NOPD = 4-nitro-o-phenylendiamine 

### Historical Positive Control Data

Page 51 of 52

### Historical Positive Control Data

Strain: WP2 uvrA

* = revertants/plate SPT = standard plate test

PIT = preincubation test
SD = standard deviation

2 - AA = 2-aminoanthracene

4-NQO = 4-Nitroquinoline-N-oxide

Page 52 of 52