TITLE PAGE

REPORT

SALMONELLA TYPHIMURIUM / ESCHERICHIA COLI REVERSE MUTATION ASSAY

TEST GUIDELINES

OECD 471 Commission Regulation (EC) No 440/2008; B.13 / B.14 US EPA OPPTS 870.5100

This document contains manufac turing and trade secrets of the Sponsor(s). It is the property of the Sponsor(s) and may be used only for that purpose for which it was intended by the Sponsor(s ). Every other or additional use, exploitation, reproduction, publication or submission to other parties require the written permission of the Sponso r(s), with the exception of regulatory a gencies acting within the limits of their administrative authority.

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FLAGGING CRITERIA

The flagging criteria of 40 CFR 158.34/40 CFR161.34 are not applicable for this study type

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- CERTIFICATE (FROM THE COMPETENT AUTHORITY)

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TITLE PAGE BLANK PAGE GLP COMPLIANCE STATE ME NT FLAGGING CRITERIA SIGNATURE PAGE CONTRIBUTORS TO THE STUDY / SUPERVISORY LABORATORY PERSONNEL STATEMENT OF THE QUA LIT Y ASSURANCE UNIT GLP

- CERTIFICATE (FROM THE COMPETENT AUTHORIT Y) 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 typhimurium

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

3.7.3. Titer determination

3.7.4. Controls

Page 9 of 47 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors3.7.4.1. Negative controls / Vehicle controls

3.7.4.2. Positive controls

3.7.5. Scope of tests and test conditi ons
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

4.2. TOXICITY

4.3. SOLUBILITY

5. DISCUSSION

6. CONCLUSION

7. REFERENCES

APPENDIX 1 TABLES 1 - 7 (STANDARD PLATE T EST) AP PENDIX 2 STERILITY CONTROL APPENDIX 3 S9 FRACT ION CHARACTERIZATION APPENDIX 4 HISTORIC AL NEGATIVE CONTROL DATA APPENDIX 5 HISTORICAL POSITIVE CONTROL DATA

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### 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 rev erse mutation assay.

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

DOSE RANGE: 33 μg - 5 2 00 μg/plate (SPT)

TEST CONDITIONS: Standard plate test (SPT) with and without metabolic activation (liver S9 mix from induced rats ).

SOLUBILITY: Pr ecipitation of the test substance was found from 4 600 μg/plate and above w ith and without S9 mix.

TOXICITY: No bacteriotoxic effect was observed under all test conditions (for details see item 4.2 .).

MUTAGENICITY:

A biologically relevant increase in the number of his + or trp + revertants was not observed in the standard plate test test either without S9 mix or after the addition of a metabolizing system using the tester strains TA 1535, TA 100, TA 1537 and E. coli WP2uvrA

.

Co ntrary, using the tester strain TA 98 a reproducible and dose

- dependent increase in the numbers of his + revertants exceeding a revertant factor of 2 compared to the concurrent vehicle control was observed after the ad dition of a metabolizing system.

TA 98 without S9 mix:

with S9 mix: No increase in the number of his + or trp + revertants

Increase at 333

, 1 000, 2 600 and 5 200 μg/plate (Exp. 1 and Exp. 2).

CONCLUSION:

Thus, under the experimental conditions of this study, the test substance is mutagenic in the Salmonella typhimurium /Escherichia coli reverse mutation assay in the presence of metabolic activation

.

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### 2. INTRODUCTION

2.1. OBJECTIVES

Bacterial reverse mutation assays using amino

- acid requiring strains of Salmonella typhi murium 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 Mutation Test using Bact eria, No B. 13 / B. 14 ; No L 142

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

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Test dates: Study initiation date: 20 Feb 2012
Experimental starting date (1st administration of the test substance ): 21 Feb 2012

Experime ntal completion date (evaluation last experiment): 02 Mar 2012

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 MUAMES system, they are stored on electronic data media.

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### 3. MATERIAL AND METHODS

3.1. TEST ITEM

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

Name of test substance :

Test substance No.:

Batch identification:

CAS No.:

Purity/composition: 94.4 g/100 g

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 throughout the study period was guaranteed until 31 May 2014 as indicated by the manufacturer, and the manufacturer holds this responsibility.

ADDITIONAL TEST SUBSTANCE INFORMATION

Date of production : May 2011

Physical state, appearance: Solid, brown

Storage condi tions: Room temperature

More detailed information may be requested from the sponsor (

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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 nutrient 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 47 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors3.2.3. Checking the tester strains

The Salmonella strains are checked for the following characteristics at regular intervals: deep rough character (rfa); UV sensitivity ( Ä uvrB); ampicillin resistance (R factor plasmid).

E. coli WP2 uvrA is checked for UV sensi tivity. 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).

St andardized 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 minutes 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 3

.

Page 16 of 47 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors3.3.2. 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 concentratio ns 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 an Na 2 HPO 4 solution with an 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 3 ).

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 5.2 mg/plate was used as top dose in both experiments.

3.5. TEST SUBSTANCE PREPARATION

The test substanc e was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution s.
The test substance was dissolved in dimethylsulfoxide (DMSO).

To achieve a clear solution of the test substance in the vehicle, the test subst ance preparation was 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 tes t substance in the vehicle DMSO was not determined analytically.

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3.7.1. Choice of the vehicle

Due to the limited solubility of the test substance in ultrapure water

, DMSO 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 consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid soluti on 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 c ulture

0.5 mL S9 mix ( with metabolic activation )

or 0.5 mL phosphate buffer ( without metabolic activation ) After mixing, the samples are poured onto Vogel

- Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds. Composition of the minimal glucose agar:

980 mL ultrapure water

20 mL Vogel

- Bonner E medium 15 g Difco bacto agar 20 g D- glucose, monohydrate.

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

Page 18 of 47 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors• Escherich ia 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 buff er ( without metabolic activation ) After mixing, the samples are poured onto minimal agar plates within approx. 30 seconds. The composition of the minimal agar (SA1 selective agar) is based on the description of Green, M.H.L. and Muriel, W.J. ( 5 ), with the exception of the amino acid solution, which has previously been added to the soft agar:

300 mL solution B (agar) 100 mL solution A (saline solution) 8 mL solution C (glucose solution) 10 mL solution D (casein soluti on)

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

Page 19 of 47 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors3.7.3. Titer determination

The titer was 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. 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 solution (5 mM tryptophan or 5 mM histid ine + 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

After mixin g, 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.

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 2 ).

• 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 47 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors3.7.4.2. Positive c ontrols

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

Without 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 (AAC) (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

Th e 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.

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1st Experiment

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

Doses: 0; 33; 100; 333; 1 000; 2 6 00 and 5 2 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 1537 and TA 98

Doses: 0; 33; 100; 333; 1 000; 2 600 and 5 200 μg/plate (TA 98)
0; 2 600; 3 200; 3 600; 4 200; 4 600 and 5 200 μg/plate (TA 1537) Type of test: Standard plate test test with S9 mix Number of plates: 3 test plates per dose or per control

Reason: M utagenicity was observed in the standard plate test

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3.8.1. Mutagenicity

Individual plate counts, the mean number of revertant colonies per plate and 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 r epeat 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 expe riments.

3.8.3. Toxicity

Toxicity detected by a

• decrease in the number of revertants

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

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

3.8.4. Solubility

Precipitation of the test material is recorded and indicated in the tables. As long 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 detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate might also be tested in repeat experiments for further clarification/substantiation.

Page 23 of 47 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors3.8.5. 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 4 ).

• The sterility controls revealed no indication of bacterial contamination (see Appendix 2).

• 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 5 ).

• Fresh 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.

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### 4. RESULTS ( Appendix 1

, Tables 1 - 7)

The test substance was tested for mutagenicity in the Salmonella typhimurium / Escherichia coli reverse mutation assay both in the standard plate 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 - 7)

4.1.1.1. Tests without S9 mix

TA 1535:

TA 100:

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

TA 98:

E. coli WP2 uvrA:

4.1.1.2. Tests with S9 mix

TA 1535: No relevant increase in the number of his + revertants.

TA 100: No relevant increase in the number of his + revertants.

TA 1537: 1st Exp.: Slight increase in the number of his + revertants only in the first experiment at the top dose of 5 200 μg/plate (factor 1.5)

. 2nd Exp.: No relevant increase in the number of his + revertants.

TA 98: 1st Exp.: I ncrease in the number of his + revertants at 333, 1 000, 2 600 and 5 200 μg/plate (factors 2.2 up to 4. 9 , respectively ). 2nd Exp.: I ncrease in the number of his + revertants at 333, 1 000, 2 600 and 5 200 μg/plate ( factors 1.7 up to 4.7, respectively ).

E. coli WP2 uvrA: No relevant increase in the number of trp + revertants.

Page 25 of 47 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsors4.2. TOXICITY

No bacteriotoxic effect (reduced his - or trp - background growth, decreas e in the number of his + or trp + revertants, reduction in the titer) was observed in the standard plate test up to the highest required concentration.

4.3. SOLUBILITY

Test substance precipitati on was found from 4 600 μg/plate onward with and without S9 mix.



### 5. DISCUSSION

According to the results of the present study, t he test substance led to a distinct and dosedependent increase in the number of his + revertants with the strain TA 98 with S9 mix only. The increase of his + revertants was reproducible in two experiments carried out independently of each other. Bas ed on the recent assessment crit eria the test substance has to be considered positive. The slight increase of revertants in the first standard plate test using the strain TA 1537 with metabolic activation at the top dose of 5 200 μg/plate was not reproducible in a second experiment, so it has to be regar ded as biologically not relevant. 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 the acceptance criteria of this study. In this study with and without S9 mix, the num ber of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain (see Appendix 4). 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 5).



### 6. CONCLUSION

Thus, under the experimental condition s chosen here, it is concluded that is a mutagenic test substance in the bacterial reverse mutation test in the the presence of metabolic activation.

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### 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.; Am es, B.N.: Revised method for the Salmonella mutagenicity 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 testin g using trp+ reversions in Escherichia coli. Mut. Res., 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.; S eino, 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: Nor poth, 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 27 of 47 This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsA PPENDIX 1

TABLES 1 - 7

(STANDARD PLATE TEST )

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This document is the property of the sponsors (specified on page 1), use by other parties requires written permission by the sponsorsA PPENDIX 2

STERILITY CONTROL

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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
5 200 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
5 200 μg test substance No growth

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S9 FRACTION CHARACTE RIZATION

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Preparation of S9 fraction: 02 Dec 2011

Content of protein: 2 6.4 g/L

Characterization of the S9 mix with benzo[a]pyrene: 23 Dec 2011

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 89 11 1.0 DMSO 19 2 1.0

0.625 μg 191 32 2.1 0.625 μg 82 5 4.4

1.25 μg 248 24 2.8 1.25 μg 143 8 7.6

2.5 μg 318 36 3.6 2.5 μg 205 75 11.0

5.0 μg 564 43 6.3 5.0 μg 560 40 30.0

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

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HISTORICAL NEGATIVE CONTROL DATA

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### Historical Negative Control Data

Strain: TA 1535

Method S9 mix Negative control Period No. of plates Min* Max* Mean* SD
SPT 

### - Water 10.04-11.11 400 8 48 17 10
SPT 

### - DMSO 07.08-11.11 400 8 51 18 6
SPT 

### - Acetone 01.96-12.11 330 9 31 18 3
SPT 1 : 9 Water 08.04-11.11 400 10 42 18 5
SPT 1 : 9 DMSO 07.08-12.11 400 10 58 18 6
SPT 1 : 9 Acetone 01.96-12.11 327 10 35 18 3



### Strain: TA 100

Method S9 mix Negative control Period No. of plates Min* Max* Mean* SD
SPT 

### - Water 07.04-11.11 400 57 138 103 14
SPT 

### - DMSO 06.08-12.11 400 50 160 101 18
SPT 

### - Acetone 01.96-12.11 371 70 160 115 15
SPT 1 : 9 Water 06.04-11.11 400 76 156 112 15
SPT 1 : 9 DMSO 06.08-12.11 400 63 158 109 19
SPT 1 : 9 Acetone 01.96-12.11 372 66 172 118 17

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

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### Historical Negative Control Data

Strain: TA 1537

Method S9 mix Negative control Period No. of plates Min* Max* Mean* SD
SPT 

### - Water 07.04-11.11 400 4 16 9 2
SPT 

### - DMSO 04.08-12.11 400 5 16 8 2
SPT 

### - Acetone 01.96-12.11 327 4 18 10 2
SPT 1 : 9 Water 07.04-11.11 400 4 16 9 2
SPT 1 : 9 DMSO 04.08-12.11 400 4 18 9 2
SPT 1 : 9 Acetone 01.96-12.11 327 5 20 11 3



### Strain: TA 98

Method S9 mix Negative control Period No. of plates Min* Max* Mean* SD
SPT 

### - Water 07.04-11.11 400 14 53 28 5
SPT 

### - DMSO 05.08-12.11 400 12 45 26 6
SPT 

### - Acetone 01.96-12.11 333 15 52 29 5
SPT 1 : 9 Water 10.04-11.11 400 16 58 35 7
SPT 1 : 9 DMSO 06.08-12.11 400 16 53 33 7 SPT 1 : 9 Acetone 01.96-12.11 333 20 54 38 6

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

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### Historical Negative Control Data

Strain: WP2 uvrA

Method S9 mix Negative control Period No. of plates Min* Max* Mean* SD
SPT 

### - Water 12.03-11.11 400 21 60 37 7
SPT 

### - DMSO 03.08-12.11 400 24 61 39 7
SPT 

### - Acetone 01.96-12.11 312 18 56 43 6
SPT 1 : 9 Water 12.03-11.11 400 26 60 42 8
SPT 1 : 9 DMSO 02.08-12.11 400 25 64 44 7 SPT 1 : 9 Acetone 01.96-12.11 312 15 63 38 8

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

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HISTORICAL POSITIVE CONTROL D ATA

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### Historical Positive Control Data

Strain: TA 1535

Method S9 mix Positive control μg/plate Period No. of plates Min* Max* Mean* SD
SPT 

### - MNNG 5.0 08.09-12.11 400 507 1164 863 190
SPT 1 : 9 2-AA 2.5 08.09-12.11 400 74 1170 189 132



### Strain: TA 100

Method S9 mix Positive control μg/plate Period No. of plates Min* Max* Mean* SD
SPT 

### - MNNG 5.0 07.09-12.11 400 512 1429 835 166
SPT 1 : 9 2-AA 2.5 07.09-12.11 400 468 2117 858 199

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

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

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### Strain: TA 1537

Method S9 mix Positive control μg/plate Period No. of plates Min* Max* Mean* SD
SPT 

### - AAC 100 07.09-12.11 400 208 981 389 78
SPT 1 : 9 2-AA 2.5 07.09-12.11 400 80 590 158 48



### Strain: TA 98

Method S9 mix Positive control μg/plate Period No. of plates Min* Max* Mean* SD
SPT 

### - NOPD 10 07.09-12.11 400 307 1185 569 153
SPT 1 : 9 2-AA 2.5 08.07-12.11 400 438 1412 725 152

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

2 - AA = 2-aminoanthracene
AAC = 9-aminoacridine NOPD = 4-nitro-o-phenylendiamine 

### Historical Positive Control Data

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### Historical Positive Control Data

Strain: WP2 uvrA

Method S9 mix Positive control μg/plate Period No. of plates Min* Max* Mean* SD
SPT 

### - 4-NQO 5.0 06.09-12.11 400 428 1508 843 203
SPT 1 : 9 2-AA 60.0 06.09-12.11 400 152 301 237 31

* = revertants/plate SPT = standard plate test

SD = standard deviation

2 - AA = 2-aminoanthracene

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