DR.U.NOACK-LABORATORIEN Report Page 4 of 33 DOBA Bacterial Reverse Mutation Test (Amestest) Project-No. 060601CL acc. OECD 471 / Directive 2000/32/EC Method B. 13/14 Study-No. USO108573



# Personnel Involved

Study Director: Silke Fiebig (Engineer, Biotechnologist)

Deputy: Gunda Winkelmann (Engineer of Horticulture)

Technical Staff: Karin Ruthenberg Doris Streichhardt Barbara Wienker Henriette Winkler

Quality Assurance Unit: Gudrun Möhrmann-Kalabokidis (Biologist)

Deputy: Ute Kutzner

Head of Testing Facility: Dr. Udo Noack (Biologist)

Sponsor representative Dr. Dieter LämmermannDR.U.NOACK-LABORATORIEN Report Page 5 of 33 DOBA Bacterial Reverse Mutation Test (Amestest) Project-No. 060601CL acc. OECD 471 / Directive 2000/32/EC Method B. 13/14 Study-No. USO108573



### Table of Content

Page

Title Page 1 Statement of GLP Compliance 2 Statement of the Quality Assurance Unit 3 Personnel Involved 4 Table of Content 5 List of Tables 6

1 Summary 7 2 Characterisation Data of the Test Item 8 3 Method 9 4 Results 16 4.1 Test of the Reference Items 16 4.2 Results of the Test Item 17 4.2.1 Definitive Tests 17 5 Validity Criteria 32 6 Conclusions 32 7 Literature / References 32 8 GLP-Certificate of D R .U.N OACK -L ABORATORIEN 33DR.U.NOACK-LABORATORIEN Report Page 6 of 33 DOBA Bacterial Reverse Mutation Test (Amestest) Project-No. 060601CL acc. OECD 471 / Directive 2000/32/EC Method B. 13/14 Study-No. USO108573



### List of Tables

Page

Table 1: Mutagenic and Cytotoxic Effects of the Test Item 7 Table 2: Induction Rates of Referenc e Items (Positive Controls) 16 Table 3: Spontaneous Revertants with DMSO (Plate Incorporation Method) 18 Table 4: Spontaneous Revertants with Double Distilled Water (Plate Incorporation Method) 19 Table 5: Spontaneous Revertants with DMSO (Preincubation Method) 20 Table 6: Spontaneous Revertants with D ouble Distilled Water (Preincubation Method) 21 Table 7: Induction Rates TA 1537 - S9 (Plate Incorporation Method) 22 Table 8: Induction Rates TA 1537 - S9 (Preincubation Method) 22 Table 9: Induction Rates TA 1537 + S9 (Plate Incorporation Method) 23 Table 10: Induction Rates TA 1537 + S9 (Preincubation Method) 23 Table 11: Induction Rates TA 98 - S9 (Plate Incorporation Method) 24 Table 12: Induction Rates TA 98 - S9 (Preinc ubation Method) 24 Table 13: Induction Rates TA 98 + S9 (Plate Incorporation Method) 25 Table 14: Induction Rates TA 98 + S9 (Preinc ubation Method) 25 Table 15: Induction Rates TA 100 - S9 (Plate Incorporation Method) 26 Table 16: Induction Rates TA 100 - S9 (Preinc ubation Method) 26 Table 17: Induction Rates TA 100 + S9 (Plate Incorporation Met hod) 27 Table 18: Induction Rates TA 100 + S9 (Preinc ubation Method) 27 Table 19: Induction Rates TA 1535 - S9 (Plate Incorporation Met hod) 28 Table 20: Induction Rates TA 1535 - S9 (Preinc ubation Method) 28 Table 21: Induction Rates TA 1535 + S9 (Plate Incorporation Met hod) 29 Table 22: Induction Rates TA 1535 + S9 (Preinc ubation Method) 29 Table 23: Induction Rates E. coli WP2 (uvrA) - S9 (Plate Incorporation Method) 30 Table 24: Induction Rates E. coli WP2 (uvrA) - S9 (Preincubation Method) 30 Table 25: Induction Rates E. coli WP2 (uvrA) + S9 (Plate Incorporation Method) 31 Table 26: Induction Rates E. coli WP2 (uvrA) + S9 (Preincubation Method) 31DR.U.NOACK-LABORATORIEN Report Page 7 of 33 DOBA Bacterial Reverse Mutation Test (Amestest) Project-No. 060601CL acc. OECD 471 / Directive 2000/32/EC Method B. 13/14 Study-No. USO108573



## 1 Summary

The mutagenic effects of the test item DOBA we re determined in a bacterial reverse mutation test according to OECD Guideline No. 471 fo r Testing of Chemicals (1997) / Directive 2000/32/EC Method B. 13/14 (2000) in two independent studies (plate incorporation method and preincubation method) from June 21, 2006 to July 06, 2006 at D R .U.N OACK -L ABORATORIEN in 31157 Sarstedt, Germany. Test systems were the Salmonella typhimurium strains TA 1537, TA 98, TA 100 and TA 1535 and Escherichia coli WP2 (uvrA) with (+) and without (-) the metabolic activation system S9 (from male Wi star rats), respectively. Positive and negative controls were included in each study. The test item was dissolved in DMSO and applied once at test initiation with the concentration ranges as given in Table 1. Duration of each study was 48 - 72 h. Mutagenic and cytotoxic effects are summarized in Table 1.

Table 1: Mutagenic and Cytotoxic Effects of the Test Item

Tested Concentration Range (factor √ 10)

[mg/plate] Lowest Cytotoxic Concentration [mg/plate] Strain S9 Plate Incorporation Method Preincubation Method Lowest Mutagenic Concentration

[mg/plate] Plate Incorporation Method Preincubation Method

- 1.6 0.5 TA 1537 + 1.6 – 0.016 1.6 1.6 - > 5.0 0.5 TA 98 + > 5.0 > 5.0 - 5.0 0.5 TA 100 + 5.0 5.0 - 1.6 0.5 TA 1535 + > 5.0 > 5.0 - > 5.0 > 5.0 E. coli WP2 (uvrA) + 5.0 - 0.05 5.0 - 0.05 none

> 5.0 > 5.0



### The test item is regarded to be not mutagenic under test conditions.DR.U.NOACK-LABORATORIEN Report Page 8 of 33 DOBA Bacterial Reverse Mutation Test (Amestest) Project-No. 060601CL acc. OECD 471 / Directive 2000/32/EC Method B. 13/14 Study-No. USO108573



## 2 Characterisation Data of the Test Item

TEST ITEM DOBA

Batch Number BES06024

Purity 98.1 %

Appearance Colourless solid

Water solubility < 1 g/L

Stability Not specified

Density 0.55 g/L (20 °C)

Melting point 143 – 145 °C

Expiry date 2007-03-06

Recommended storage Room temperature (20 °C)

Storage at testing facility Room temperat ure, protected from moisture and light

Retention At least 1 g has been retained.

Identification parameter Name, batch number, st ate, consistency, colour and melting point at testing facility

The test item and the information concerning t he test item were provided by the sponsor.DR.U.NOACK-LABORATORIEN Report Page 9 of 33 DOBA Bacterial Reverse Mutation Test (Amestest) Project-No. 060601CL acc. OECD 471 / Directive 2000/32/EC Method B. 13/14 Study-No. USO108573



## 3 Method

TEST GUIDELINES OECD Guideline No. 471 for Testing of Chemicals (1997) / Directive 2000/32/EC Method B. 13/14 (2000)

TYPE AND PURPOSE Determination of the mutagenic effe cts of the test item to four OF THE STUDY different Salmonella typhimurium strains and one Escherichia coli strain over 48 / 72 h.

TEST SYSTEM Salmonella typhimurium strains TA 1537, TA 98, TA 100 and TA 1535 and Escherichia coli WP2 (uvrA) with (+) and without (-) metabolic activation system (S9).

Reason for the selection Salmonella typhimurium strains and Escherichia coli WP2 of the test system were sele cted according to the guidelines.

Origin University of California, Berkeley Divison of Biochemistry & Molecular Biology, CA 94720 USA (TA 98, TA 100, TA 1535)

T RINOVA B IOCHEM G MB H, Kerkrader Straße 10, 35394 Gießen ( E. coli WP2 (uvrA), TA 1537 and TA 100 derived from master culture obtained directly from University of California)

Storage at test facility Under liquid nitrogen

Inoculum An overnight culture wa s prepared. Incubation was done for 18 h at 37 °C. Titer of the overnight culture was ≥ 1 × 10 8 cells/mL.

Metabolic activation As met abolic activation system a certified post-mitochondrial (S9) system (S9) fraction from livers of male Wistar rats, which were induced with Phenobarbital intraperitoneally and β -Naphtoflavone orally was used. The S9 fraction was received from CCR (In den Leppsteinwiesen 19, D-64380 Roßdorf). At test start an aliquot of the S9-fraction was thawed and enriched with cofactors according to DIN Guideline 38415 part 4 (S9-Mix). Batch number of S9- fraction was 230905 and 020606 (31.2 mg protein/mL S9), respectively.DR.U.NOACK-LABORATORIEN Report Page 10 of 33 DOBA Bacterial Reverse Mutation Test (Amestest) Project-No. 060601CL acc. OECD 471 / Directive 2000/32/EC Method B. 13/14 Study-No. USO108573

TEST ITEM DOBA

Stock solution 50 g/L, prepared with DMSO

Dispersion treatment Agitation

Test concentrations Test strain [mg/plate] TA 98, TA 100, TA 1535, E. coli WP2 (uvrA) 5.0 – 1.6 – 0.5 – 0.16 – 0.05

TA 1537 Plate Incorporation Method: 5.0 – 1.6 – 0. 5 – 0.16 – 0.05 Preincubation Method: 1.6 – 0.5 – 0. 16 – 0.05 – 0.016

CONTROLS Untreated controls with double distilled water were tested.

SOLVENT CONTROLS Controls with DMSO were tested.

REFERENCE ITEMS

Reference item Batch No. Purity [%] Salmonella / E. coli strain Test conc. [μg/plate]

ICR 191 026K1228 99.6 TA 1537 - S9 2.0

4-Nitro-ophenylendiamine 10625LB355 98 TA 98 - S9 0.5

Benzo(a)pyrene 1224946 97.2 (rel.) TA 98 + S9 10

Nitrofurantoine 014K1284 100 TA 100 - S9 0.2

Sodium azide 448503/1 10405368 99.9 TA 1535 - S9 0.25

4-Nitroquinolin 1- oxide 084K0988 100 E. coli WP2 (uvrA) -S9 1

TA 1537, TA 100, TA 1535 + S9 22-Aminoanthracen S29093- 215 99.8

E. coli WP2 (uvrA) + S9 25DR.U.NOACK-LABORATORIEN Report Page 11 of 33 DOBA Bacterial Reverse Mutation Test (Amestest) Project-No. 060601CL acc. OECD 471 / Directive 2000/32/EC Method B. 13/14 Study-No. USO108573

TEST METHOD

Media According to DIN Guideline 38415 Part 4 (1999)

Test container Petri dishes with cams (ø 94 mm, 16 mm high) with about 25 mL V OGEL -B ONNER minimal medium

Application Application was ca rried out once at test start.

Composition of the test Plate Incorporation Method media 2.0 mL top agar with 0.5 mM Histidin-/Biotin solution and L-Tryptophan solution (for E. coli WP (uvrA)), respectively, for plates without metabolic activation system 1.5 mL top agar with 0.5 mM Histidin-/Biotin solution and L-Tryptophan solution (for E. coli WP (uvrA)), respectively, for plates with metabolic activation system 0.5 mL S9-Mix for plates with metabolic activation system 0.1 mL test / reference item solution, double distilled water and DMSO, respectively 0.1 mL bacteria (overnight culture) were mixed and poured over the surface of a Vogel-Bonner minimal agar plate

Preincubation Method 0.35 mL test / reference item solution, double distilled water and DMSO, respectively 1.75 mL buffer solution or S9-Mix 0.35 mL bacteria (overnight culture) were preincubated in a microtiter plate for at least 20 min at 35 ± 2 °C on a shaker.

0.7 mL of the preincubated solution1.5 mL top agar with 0.5 mM Histidin-/Biotin- and L-Tryptophansolution (for E. coli WP (uvrA)), respectively, were mixed and poured over the surface of a Vogel-Bonner minimal agar plate

Titer control Plate Incorporation Method 2.0 mL top agar with 5 mM Histidin- / 0.5 mM Biotin-solution and 5 mM L-Tryptophan-solution (for E. coli WP (uvrA)), respectively 0.5 mL S9-Mix 0.1 mL bacteria (10 -5 dilution from overnight culture)DR.U.NOACK-LABORATORIEN Report Page 12 of 33 DOBA Bacterial Reverse Mutation Test (Amestest) Project-No. 060601CL acc. OECD 471 / Directive 2000/32/EC Method B. 13/14 Study-No. USO108573

were mixed and poured over the surface of a Vogel-Bonner minimal agar plate

Preincubation Method 0.35 mL double distilled water 1.75 mL S9-Mix 0.35 mL bacteria (overnight culture) were preincubated in a microtiter plate for at least 20 min at 35 ± 2 °C on a shaker.

0.7 mL of the preincubated solution was mixed with1.5 mL top agar with 0.5 mM Histidin-/Biotin- and L-Tryptophansolution (for E. coli WP (uvrA)), respectively, were mixed and poured over the surface of a Vogel-Bonner minimal agar plate

Replicates Three replicates per concentration level and control. Titer control replicates were prepared 2-fold.

Confirmation of genotypes The genotypes of the tested strains were checked within each study: histidine and tryptophan auxotrophy, respectively, ampicillin resistance, UV-sensitiv ity and growth inhibition with crystal violet.

Temperature (Min-Max) 36.0 - 36.9 °C Preincubation temperature (Min-Max): 34.0 – 36.9 °C

Test duration 48 - 72 h

KIND AND FREQUENCY Genotypes were evaluated for each study. Plates of the OF MEASUREMENT AND mutagenicity test were inspected for present and reduced OBSERVATION background lawn after an incubation time of 48 h. Plates with colonies which correspond not with the typical shape and colour of Salmonella typhimurium and Escherichia coli

, respectively, were regarded as contaminated and were not included in calculations. Colonies per plate (revertants) we re counted. Plates with reduced background were considered carefully for evaluation as appropriate.

Equipment Microflow biological safety cabinet (MDH) Colony counter (manual counting), BZG 30 (WTW) Thermometer, Oregon Scientific Analysis balance, LA230-S, S ARTORIUSDR.U.NOACK-LABORATORIEN Report Page 13 of 33 DOBA Bacterial Reverse Mutation Test (Amestest) Project-No. 060601CL acc. OECD 471 / Directive 2000/32/EC Method B. 13/14 Study-No. USO108573

Balance, PJ Precisa junior 2000 C, D IGITANA Laboratory incubators, B 5042 E and B 6060 (H ERAEUS ) Water bath, WB 22 (M EMMERT )

COURSE OF THE STUDY Each study was conducted with 5 different concentration levels of the test item in a geometrical series of factor √ 10. Application was carried out as specified above. Pl ates for confirmation of the genotypes were incubated for 24 h and plates of the mutagenicity test for 48 – 72 h, respectively, at 37 ± 1 °C. The results were confirmed in a second independent study (preincubation method). Evaluations were performed as described below.

EVALUATIONS AND Since a reduced background lawn is regarded to be a cytotoxic STATISTICS effect, plates with reduced background lawn normally were not included into evaluation procedures. Arithmetic mean values and standard deviations were calculated out of colonies per plate of three replicates.

For evaluation of the results the induction rate of the mean values was calculated (1):

revertant colonies of test item Induction rate =

 (1) revertant colonies of the corresponding control

Corresponding control for the reference item was double distilled water and DMSO for the test item.

The test item is to be classified mutagenic if there is a concentration effect relationship over at least three concentrations and / or the induction rate is ≥ 2.

Software The data presented in the tables are computer generated and rounded for presentation. Thus m anual calculation of results based on the data in this report may yield in minor deviations from these figures.

Calculations were carried out using software

- Excel 2000 (2000), M ICROSOFT C ORPORATIONDR.U.NOACK-LABORATORIEN Report Page 14 of 33 DOBA Bacterial Reverse Mutation Test (Amestest) Project-No. 060601CL acc. OECD 471 / Directive 2000/32/EC Method B. 13/14 Study-No. USO108573

VALIDITY CRITERIA Following genotypes of the test ed strains have to be confirmed: - Histidine- and tryptophan auxotrophy, respectively - Ampicillin resistance - UV-sensitivity - growth inhibition with crystal violet (rfa-mutation)

Titer of the overnight culture has to be ≥ 1

⋅ 10 8 cells/mL

Spontaneous revertants (negative c ontrols) should be within the following ranges:

Test strain Metabolic system MV ± SD Valid range ( ± S9)

- S9 8 ± 4 TA 1537 + S9 10 ± 7 2 - 40

- S9 27 ± 8 TA 98 + S9 32 ± 14 15 - 50

- S9 118 ± 34 TA 100 + S9 101 ± 33 60 - 200

- S9 25 ± 15 TA 1535 + S9 16 ± 14 5 - 40

- S9 45 ± 12 E. coli WP2 (uvrA) + S9 57 ± 17 30 - 100

MV = Mean of all reference tests until January 2006 SD = standard deviation

The induction rates of the positve controls had to be ≥ 2.

DATES Study initiation 2006-06-20 Experimental starting 2006-06-21 Experimental completion 2006-07-06 Study completion Please refer to page 1

DEVIATIONS FROM Validity criteria were taken from the literature and DIN guideline, THE GUIDELINE since in OECD and EC guideline no validity criteria are described.DR.U.NOACK-LABORATORIEN Report Page 15 of 33 DOBA Bacterial Reverse Mutation Test (Amestest) Project-No. 060601CL acc. OECD 471 / Directive 2000/32/EC Method B. 13/14 Study-No. USO108573

DEVIATIONS FROM None THE STUDY PLAN

ARCHIVING The following will be retained in the archive of the testing facility for the period as specified in the operative national GLP regulations:

• all raw data

• study plan

• final report

• all records performed by the quality assurance programme including master schedules

• sample of test and reference items

Additionally microfilms will be retained in a safe-deposit by Volksbank Sarstedt, D-31157 Sarstedt.DR.U.NOACK-LABORATORIEN Report Page 16 of 33 DOBA Bacterial Reverse Mutation Test (Amestest) Project-No. 060601CL acc. OECD 471 / Directive 2000/32/EC Method B. 13/14 Study-No. USO108573



## 4 Results

4.1 Test of the Reference Items

Positive controls were tested parallel to the test item.

Table 2: Induction Rates of Reference Items (Positive Controls)

Strain Reference item [μg/plate] S9 Induction Rate*

Plate Incorporation Method Preincubation Method

ICR 191 2.0 - 112.1 > 240.0 TA 1537

2-Aminoanthracene 2.0 + 55.4 26.9 4-Nitro-o-phenylenediamine 0.5 - 3.9 4.0 TA 98 Benzo(a)pyrene 10 + 22.4 10.9

Nitrofurantoine 0.2 - 2.7 4.6 TA 100 2-Aminoanthracene 2.0 + > 6.9 > 6.5

Sodium azide 0.25 - 5.7 6.8 TA 1535 2-Aminoanthracene 2.0 + 10.6 13.0 4-Nitroquinolin-1-oxid 1.0 - 8.3 > 22.2 E. coli WP2 (uvrA) 2-Aminoanthracene 25.0 + 9.3 13.3

Plates with number of colonies higher than 1000/ 1200 colonies were not counted, induction rates were calculated with this number and given as > values. *) Induction Rates were calculated wi th the double distilled water controlDR.U.NOACK-LABORATORIEN Report Page 17 of 33 DOBA Bacterial Reverse Mutation Test (Amestest) Project-No. 060601CL acc. OECD 471 / Directive 2000/32/EC Method B. 13/14 Study-No. USO108573

4.2 Results of the Test Item

4.2.1 Definitive Tests

The test item was clearly dissolved in DMSO. Du ring the application the te st item precipitated, fine white particles occured in the petridish. To di stinguish the particles from revertant colonies, the incubation was extended to 72 h. Neverthel ess cytotoxic effects (reduced background lawn) were not unequivocal determinable for all strains.

Revertant colonies of the test item plates are given in Table 7 to Table 26. Spontaneous revertants are listed in Table 3 to Table 6. The arithmetic mean value and the standard dev iation were calculated out of the three replicates of the revertant colony plates. For evaluation of the results t he induction rates of the mean values were calculated with the DMSO control (Table 7 to Table 26). Mutagenic and cytotoxic effects are summarized in Table 1.DR.U.NOACK-LABORATORIEN Report Page 18 of 33 DOBA Bacterial Reverse Mutation Test (Amestest) Project-No. 060601CL acc. OECD 471 / Directive 2000/32/EC Method B. 13/14 Study-No. USO108573

Table 3: Spontaneous Revertants with DMSO (Plate Incorporation Method)

colonies / plate standard strain S9 valid range counted mean value deviation

7 - 4 7 2.5 9 TA 1537 2 - 40 4 + 6 7 3.1 10 17 - 15 17 2.5 20 TA 98 15 - 50 24 + 18 25 7.0 32 103 - 121 103 18.0 85 TA 100 60 - 200 157 + 172 165 10.6

contaminated 31 - 39 32 6.1 27 TA 1535 5 - 40 14 + 10 14 3.5 17 49 - 52 45 10.2 33 EC WP2 30 - 100

(uvrA) 36 + 43 46 12.3 60DR.U.NOACK-LABORATORIEN Report Page 19 of 33 DOBA Bacterial Reverse Mutation Test (Amestest) Project-No. 060601CL acc. OECD 471 / Directive 2000/32/EC Method B. 13/14 Study-No. USO108573

Table 4: Spontaneous Revertants with Double Distilled Water (Plate Incorporation Method)

colonies / plate standard strain S9 valid range counted mean value deviation

3 - 4 5 2.6 8 TA 1537 2 - 40 8 + 7 8 0.6 8 23 - 20 22 1.5 22 TA 98 15 - 50 23 + 17 18 4.2 15 91 - 89 99 15.6 117 TA 100 60 - 200 156 + 139 145 9.3 141 25 - 36 32 5.9 34 TA 1535 5 - 40 18 + 10 17 7.0 24 47 - 52 46 6.6 39 EC WP2 30 - 100

(uvrA) 48 + 61 53 7.2 49DR.U.NOACK-LABORATORIEN Report Page 20 of 33 DOBA Bacterial Reverse Mutation Test (Amestest) Project-No. 060601CL acc. OECD 471 / Directive 2000/32/EC Method B. 13/14 Study-No. USO108573

Table 5: Spontaneous Revertants with DMSO (Preincubation Method)

colonies / plate standard strain S9 valid range counted mean value deviation 8 - 3 6 2.6 7 TA 1537 2 - 40 2 + 5 5 3.0 8 31 - 31 30 2.3 27 TA 98 15 - 50 34 + 21 25 7.5 21 122 - 128 124 3.8 121 TA 100 60 - 200 188 + 118 142 39.6 121 31 - 33 35 5.9 42 TA 1535 5 - 40 14 + 21 16 4.4 13 37 - 31 34 3.1 33 EC WP2 30 - 100

(uvrA) 41 + 58 51 9.1 55DR.U.NOACK-LABORATORIEN Report Page 21 of 33 DOBA Bacterial Reverse Mutation Test (Amestest) Project-No. 060601CL acc. OECD 471 / Directive 2000/32/EC Method B. 13/14 Study-No. USO108573

Table 6: Spontaneous Revertants with Double Distilled Water (Preincubation Method)

colonies / plate standard strain S9 valid range counted mean value deviation 7 - 4 5 1.7 4 TA 1537 2 - 40 11 + 11 10 1.7 8 38 - 43 41 2.9 43 TA 98 15 - 50 42 + 42 42 0.6 41 151 - 161 191 61.4 262 TA 100 60 - 200 130 + 145 184 81.5 278 22 - 42 33 10.1 34 TA 1535 5 - 40 19 + 17 15 5.3 9 52 - 35 45 8.9 48 EC WP2 30 - 100

(uvrA) 51 + 52 52 1.5 54DR.U.NOACK-LABORATORIEN Report Page 22 of 33 DOBA Bacterial Reverse Mutation Test (Amestest) Project-No. 060601CL acc. OECD 471 / Directive 2000/32/EC Method B. 13/14 Study-No. USO108573

Table 7: Induction Rates TA 1537 - S9 (P late Incorporation Method)

test item background colonies / mean value standard induction rate concentration lawn plate deviation

[mg/plate]

+ 7 control + 4 7 2.5 - + 9 - 0 5 - 0 1 1.2 0.1 - 2 - 2 1.6 - 0 1 1.2 0.1 - 0 + 4 0.5 + 8 8 3.5 1.2

+ 11 + 5 0.16 + 6 6 0.6 0.9

+ 6 + 3 0.05 + 4 4 1.5 0.7

+ 6

Table 8: Induction Rates TA 1537 - S9 (Preincubation Method) test item background colonies / mean value standard induction rate concentration lawn plate deviation

[mg/plate] + 8 control + 3 6 2.6 - + 7 - 0 1.6 - 0 0 0.0 0.0

- 0 - 0 0.5 - 4 2 2.0 0.3

- 2 + 3 0.16 + 4 3 1.0 0.5

+ 2 + 6 0.05 + 3 4 1.7 0.7

+ 3 + 9 0.016 + 6 8 1.5 1.3 + 8 + = present - = reducedDR.U.NOACK-LABORATORIEN Report Page 23 of 33 DOBA Bacterial Reverse Mutation Test (Amestest) Project-No. 060601CL acc. OECD 471 / Directive 2000/32/EC Method B. 13/14 Study-No. USO108573

Table 9: Induction Rates TA 1537 + S9 (Plate Incorporation Method) test item background colonies / mean value standard induction rate concentration lawn plate deviation

[mg/plate] + 4 control + 6 7 3.1 - + 10 - 6 5 - 4 6 2.0 0.9 - 8 - 1 1.6 - n.d. 1 n.d. 0.2 - n.d. + 5 0.5 + 5 5 0.6 0.7

+ 4 + 1 0.16 + 2 3 2.6 0.5

+ 6 + 5 0.05 + 5 5 0.0 0.8

+ 5

Table 10: Induction Rates TA 1537 + S9 (Preincubation Method)

test item background colonies / mean value standard induction rate concentration lawn plate deviation

[mg/plate] + 2 control + 5 5 3.0 - + 8 - n.d. 1.6 - n.d. n.d. n.d. n.d. - n.d. + 9 0.5 + 4 8 3.6 1.6

+ 11 + 8 0.16 + 8 7 2.3 1.3

+ 4 + 8 0.05 + 7 8 0.6 1.5

+ 8 + 10 0.016 + 7 8 1.7 1.6

+ 7 + = present - = reduced n.d. = not determinableDR.U.NOACK-LABORATORIEN Report Page 24 of 33 DOBA Bacterial Reverse Mutation Test (Amestest) Project-No. 060601CL acc. OECD 471 / Directive 2000/32/EC Method B. 13/14 Study-No. USO108573

Table 11: Induction Rates TA 98 - S9 (Plate Incorporation Method)

test item background colonies / mean value standard induction rate concentration lawn plate deviation

[mg/plate] + 17 control + 15 17 2.5 - + 20 + 3 5 + 6 7 4.0 0.4 + 11 + 3 1.6 + 6 3 2.5 0.2 + 1 + 11 0.5 + 19 18 7.0 1.1

+ 25 + 10 0.16 + 12 13 3.6 0.8

+ 17 + 23 0.05 + 26 23 3.0 1.3

+ 20

Table 12: Induction Rates TA 98 - S9 (Preincubation Met hod)

test item background colonies / mean value standard induction rate concentration lawn plate deviation

[mg/plate] + 31 control + 31 30 2.3 - + 27 - n.d. 5 - n.d. n.d. n.d. n.d. - n.d. - 3 1.6 - 4 4 1.5 0.1 - 6 - 11 0.5 - 6 7 3.2 0.2

- 5 + 24 0.16 + 19 22 2.6 0.7

+ 23 + 23 0.05 + 24 21 3.8 0.7

+ 17 + = present - = reduced n.d. = not determinableDR.U.NOACK-LABORATORIEN Report Page 25 of 33 DOBA Bacterial Reverse Mutation Test (Amestest) Project-No. 060601CL acc. OECD 471 / Directive 2000/32/EC Method B. 13/14 Study-No. USO108573

Table 13: Induction Rates TA 98 + S9 (Plate Incorporation Method)

test item background colonies / mean value standard induction rate concentration lawn plate deviation

[mg/plate] + 24 control + 18 25 7.0 - + 32 + 7 5 + 8 6 2.6 0.2 + 3 + 18 1.6 + 15 17 1.7 0.7 + 18 + 14 0.5 + 20 18 3.2 0.7

+ 19 + 17 0.16 + 19 20 3.6 0.8

+ 24 + 19 0.05 + 18 20 2.6 0.8

+ 23

Table 14: Induction Rates TA 98 + S9 (Preincubation Met hod)

test item background colonies / mean value standard induction rate concentration lawn plate deviation

[mg/plate] + 34 control + 21 25 7.5 - + 21 + 3 5 + 6 6 3.0 0.2 + 9 + 27 1.6 + 17 21 5.1 0.8 + 20 + 36 0.5 + 25 28 7.4 1.1

+ 22 + 33 0.16 + 33 31 3.5 1.2

+ 27 + 20 0.05 + 34 27 7.0 1.1

+ 27 + = presentDR.U.NOACK-LABORATORIEN Report Page 26 of 33 DOBA Bacterial Reverse Mutation Test (Amestest) Project-No. 060601CL acc. OECD 471 / Directive 2000/32/EC Method B. 13/14 Study-No. USO108573

Table 15: Induction Rates TA 100 - S9 (P late Incorporation Method)

test item background colonies / mean value standard induction rate concentration lawn plate deviation

[mg/plate] + 103 control + 121 103 18.0 - + 85 - n.d. 5 - n.d. n.d. n.d. n.d. - n.d. + 23 1.6 + 28 25 2.9 0.2 + 23 + 80 0.5 + 84 85 5.6 0.8

+ 91 + 49 0.16 + 31 38 9.9 0.4

+ 33 + 132 0.05 + 91 101 27.4 1.0

+ 80

Table 16: Induction Rates TA 100 - S9 (Preincubation Met hod)

test item background colonies / mean value standard induction rate concentration lawn plate deviation

[mg/plate] + 122 control + 128 124 3.8 - + 121 - 54 5 - 44 52 6.8 0.4 - 57 -/+ 72 1.6 -/+ 61 69 7.0 0.6 -/+ 74 - n.d. 0.5 - n.d. n.d. n.d. n.d.

- n.d. + 99 0.16 + 108 95 14.8 0.8

+ 79 + 136 0.05 + 111 118 15.4 1.0

+ 108 + = present - = reduced n.d. = not det erminable + / - = slight cytotoxicityDR.U.NOACK-LABORATORIEN Report Page 27 of 33 DOBA Bacterial Reverse Mutation Test (Amestest) Project-No. 060601CL acc. OECD 471 / Directive 2000/32/EC Method B. 13/14 Study-No. USO108573

Table 17: Induction Rates TA 100 + S9 (Plate Incorporation Met hod)

test item background colonies / mean value standard induction rate concentration lawn plate deviation

[mg/plate] + 157 control + 172 165 10.6 -

+ contaminated - 18 5 - n.d. n.d. n.d. n.d. - n.d. + 81 1.6 + 76 77 4.0 0.5 + 73 + 106 0.5 + 86 97 10.1 0.6

+ 98 + 107 0.16 + 115 109 5.3 0.7

+ 105 + 166 0.05 + 137 138 27.0 0.8

+ 112

Table 18: Induction Rates TA 100 + S9 (Preincubation Met hod)

test item background colonies / mean value standard induction rate concentration lawn plate deviation

[mg/plate] + 188 control + 118 142 39.6 - + 121 - n.d. 5 - n.d. n.d. n.d. n.d. - n.d. + 36 1.6 + 42 43 8.1 0.3 + 52 + 97 0.5 + 167 121 39.6 0.9

+ 100 + 121 0.16 + 100 109 10.8 0.8

+ 106 + 116 0.05 + 110 110 5.5 0.8

+ 105 + = present - = reduced n.d. = not determinableDR.U.NOACK-LABORATORIEN Report Page 28 of 33 DOBA Bacterial Reverse Mutation Test (Amestest) Project-No. 060601CL acc. OECD 471 / Directive 2000/32/EC Method B. 13/14 Study-No. USO108573

Table 19: Induction Rates TA 1535 - S9 (Plate Incorporation Met hod)

test item background colonies / mean value standard induction rate concentration lawn plate deviation

[mg/plate] + 31 control + 39 32 6.1 - + 27 - 3 5 - 6 4 1.5 0.1 - 4 +/- 13 1.6 +/- 13 12 2.3 0.4 +/- 9 + 12 0.5 + 21 18 5.2 0.6

+ 21 + 30 0.16 + 59 40 16.7 1.2

+ 30 + 26 0.05 + 30 29 3.1 0.9

+ 32

Table 20: Induction Rates TA 1535 - S9 (Preincubation Met hod)

test item background colonies / mean value standard induction rate concentration lawn plate deviation

[mg/plate] + 31 control + 33 35 5.9 - + 42 - n.d. 5 - n.d. n.d. n.d. n.d. - n.d. - n.d. 1.6 - n.d. n.d. n.d. n.d. - n.d. - 1 0.5 - 2 2 0.7 0.0

- n.d. + 19 0.16 + 21 19 2.5 0.5

+ 16 + 30 0.05 + 42 37 6.4 1.1

+ 40 + = present - = reduced n.d. = not deter minable + / - = slight cytotoxicityDR.U.NOACK-LABORATORIEN Report Page 29 of 33 DOBA Bacterial Reverse Mutation Test (Amestest) Project-No. 060601CL acc. OECD 471 / Directive 2000/32/EC Method B. 13/14 Study-No. USO108573

Table 21: Induction Rates TA 1535 + S9 (Plate Incorporation Met hod)

test item background colonies / mean value standard induction rate concentration lawn plate deviation

[mg/plate] + 14 control + 10 14 3.5 - + 17 + 2 5 + 4 5 3.1 0.3 + 8 + 11 1.6 + 3 7 4.0 0.5 + 6 + 16 0.5 + 15 16 1.5 1.2

+ 18 + 10 0.16 + 8 10 1.5 0.7

+ 11 + 13 0.05 + 20 13 6.5 1.0

+ 7

Table 22: Induction Rates TA 1535 + S9 (Preincubation Met hod)

test item background colonies / mean value standard induction rate concentration lawn plate deviation

[mg/plate] + 14 control + 21 16 4.4 - + 13 + 1 5 + 2 1 0.6 0.1 + 1 + 7 1.6 + 9 8 1.2 0.5 + 9 + 9 0.5 + 8 10 2.1 0.6

+ 12 + 7 0.16 + 17 14 5.8 0.9

+ 17 + 20 0.05 + 12 15 4.4 0.9

+ 13 + = presentDR.U.NOACK-LABORATORIEN Report Page 30 of 33 DOBA Bacterial Reverse Mutation Test (Amestest) Project-No. 060601CL acc. OECD 471 / Directive 2000/32/EC Method B. 13/14 Study-No. USO108573

Table 23: Induction Rates E. coli WP2 (uvrA) - S9 (Plate Incorporation Method)

test item background colonies / mean value standard induction rate concentration lawn plate deviation

[mg/plate] + 49 control + 52 45 10.2 - + 33 + 51 5 + 42 45 4.9 1.0 + 43 + 44 1.6 + 48 44 4.0 1.0 + 40 + 44 0.5 + 42 45 3.6 1.0

+ 49 + 44 0.16 + 54 48 5.1 1.1

+ 47 + 38 0.05 + 51 41 8.5 0.9

+ 35

Table 24: Induction Rates E. coli WP2 (uvrA) - S9 (Preincubation Method)

test item background colonies / mean value standard induction rate concentration lawn plate deviation

[mg/plate] + 37 control + 31 34 3.1 - + 33 + 33 5 + 20 30 9.3 0.9 + 38 + 38 1.6 + 37 36 3.2 1.1 + 32 + 43 0.5 + 49 43 6.5 1.3

+ 36 + 36 0.16 + 24 29 6.1 0.9

+ 28 + 41 0.05 + 44 41 2.5 1.2

+ 39 + = presentDR.U.NOACK-LABORATORIEN Report Page 31 of 33 DOBA Bacterial Reverse Mutation Test (Amestest) Project-No. 060601CL acc. OECD 471 / Directive 2000/32/EC Method B. 13/14 Study-No. USO108573

Table 25: Induction Rates E. coli WP2 (uvrA) + S9 (Plate Incorporation Method)

test item background colonies / mean value standard induction rate concentration lawn plate deviation

[mg/plate] + 36 control + 43 46 12.3 - + 60 + 49 5 + 50 52 3.8 1.1 + 56 + 58 1.6 + 63 53 12.7 1.2 + 39 + 46 0.5 + 46 50 7.5 1.1

+ 59 + 63 0.16 + 55 59 4.0 1.3

+ 58 + 62 0.05 + 61 61 1.5 1.3

+ 59

Table 26: Induction Rates E. coli WP2 (uvrA) + S9 (Preincubation Method)

test item background colonies / mean value standard induction rate concentration lawn plate deviation

[mg/plate] + 41 control + 58 51 9.1 - + 55 + 43 5 + 33 39 5.5 0.8 + 42 + 47 1.6 + 51 47 3.5 0.9 + 44 + 42 0.5 + 67 50 15.0 1.0

+ 40 + 48 0.16 + 42 41 8.1 0.8

+ 32 + 44 0.05 + 35 39 4.7 0.8

+ 37 + = presentDR.U.NOACK-LABORATORIEN Report Page 32 of 33 DOBA Bacterial Reverse Mutation Test (Amestest) Project-No. 060601CL acc. OECD 471 / Directive 2000/32/EC Method B. 13/14 Study-No. USO108573



## 5 Validity Criteria

• The genotypes of the tested strains have been confirmed: - Histidine- and tryptophan auxotrophy, respectively - Ampicillin resistance - UV-sensitivity - growth inhibition with crystal violet (rfa-mutation)

• Titer of the overnight cultures was > 1

⋅ 10 8 cells/mL.

• Spontaneous mutation rates (negative c ontrols) met the requirements.

• The induction rates of the positive controls was ≥ 2.



## 6 Conclusions

In this study DOBA was found to have no mutagenic effects on Salmonella typhimurium strains TA 1537, TA 98, TA 100, TA 1535 and Escherichia coli WP2 (uvrA) with (+) and without (-) the metabolic activation system S9 from male Wistar rats.



## 7 Literature / References

OECD Guideline 471 for testing of chemicals: Salmonella typhimurium . Reverse Mutation Assay (adopted July 21. 1997)

Richtlinie 2000/32/EC Methode B. 13/14.: Rü ckmutationsversuch unter Verwendung von Bakterien (Juni 2000)

DIN-Richtlinie 38415 Teil 4 (Dezember 1999)

L EVIN et al. (1982): A new Salmonella te ster strain (TA 102) with A

⋅ T base pairs at the site of mutation detects oxidative mutagens. Proc. Natl. Acad. Sci.. USA. Vol. 79. pp 7445-7449

M ARON . D.M. and A MES . B.N. (1983): Revised methods for the Salmonella mutagenicity test. Mutation Research 113. 173-215DR.U.NOACK-LABORATORIEN Report Page 33 of 33 DOBA Bacterial Reverse Mutation Test (Amestest) Project-No. 060601CL acc. OECD 471 / Directive 2000/32/EC Method B. 13/14 Study-No. USO108573



## 8 GLP-Certificate of D R 

## .U.N OACK 

## -L ABORATORIENDR.U.NOACK-LABORATORIEN Robust Summary Page 1 of 3 DOBA Bacterial Reverse Mutation Test (Amestest) Project-No. 060601CL acc. OECD 471 / EC Directive 2000/32/EC Method B. 13/14 Study-No. USO108573



## Robust Summary

Test Item DOBA (batch number: BES06024) Remarks none

Testing Facility D R .U.N OACK -L ABORATORIEN Käthe-Paulus-Str. 1, D-31157 Sarstedt Tel. +49(0)5066 70670, email: info@noack-lab.de Author Silke Fiebig Report issued 2006 - 08 - 11

Method/Guideline followed OECD Guideline No. 471 for Testing of Chemicals (1997) / EC Directive 2000/32/EC Method B. 13/14 (2000) Type Bacterial reverse mutation test GLP Yes [X] No [ ] Year 2006 Test system Salmonella typhimurium strains TA 1537, TA 98, TA 100, TA 1535, and Escherichia coli WP2 (uvrA) with (+) and without (-) metabolic activation system (S9) Exposure period 48 - 72 h Metabolic activation As metabolic acti vation system a post-mitochondrial (S9) system (S9) fraction from livers of male Wistar rats, which were induced with Phenobarbital intraperitoneally and β -Naphtoflavone orally was used. The S9 fraction was received from CCR (In den Leppsteinwiesen 19, D-64380 Roßdorf). At test start an aliquot of the S9-fraction was thawed and enriched with cofactors according to DIN Guideline 38415 part 4 (S9-Mix). Batch number of S9- fraction was 230905 and 020606 (31.2 mg protein/mL S9), respectively. Concentrations tested Test strain [mg/plate] TA 98, TA 100, TA 1535, E. coli WP2 (uvrA) 5.0 – 1.6 – 0.5 – 0.16 – 0.05

TA 1537 Plate Incorporation Method: 5.0 – 1.6 – 0. 5 – 0.16 – 0.05 Preincubation Method: 1.6 – 0.5 – 0. 16 – 0.05 – 0.016

Statistical Methods Arithmetic mean val ues and standard deviations were calculated out of colonies per plate of thr ee replicates. Induction rates of the mean values were calculated out of the relation of revertant colonies of control and test item plates.DR.U.NOACK-LABORATORIEN Robust Summary Page 2 of 3 DOBA Bacterial Reverse Mutation Test (Amestest) Project-No. 060601CL acc. OECD 471 / EC Directive 2000/32/EC Method B. 13/14 Study-No. USO108573

Test Conditions / Test Design Three replicates per concent ration level and control. Titer control replicates were prepared 2-fold. Frequency of dosing: Once at test start. Composition of test media: Plate Incorporation Method 2.0 mL top agar with 0.5 mM Histidin-/Biotin solution and L-Tryptophan solution (for E. coli WP (uvrA)), respectively, for plates without metabolic activation system 1.5 mL top agar with 0.5 mM Histidin-/Biotin solution and L-Tryptophan solution (for E. coli WP (uvrA)), respectively, for plates with metabolic activation system 0.5 mL S9-Mix for plates with metabolic activation system 0.1 mL test / reference item solution, double distilled water and DMSO, respectively 0.1 mL bacteria (overnight culture) were mixed and poured over the surface of a Vogel-Bonner minimal agar plate

Preincubation Method 0.35 mL test / reference item solution, double distilled water and DMSO, respectively 1.75 mL buffer solution or S9-Mix 0.35 mL bacteria (overnight culture) were preincubated in a microtiter plate for at least 20 min at 35 ± 2 °C on a shaker.

0.7 mL of the preincubated solution1.5 mL top agar with 0.5 mM Histidin-/Biotin- and L-Tryptophansolution (for E. coli WP (uvrA)), respectively, were mixed and poured over the surface of a Vogel-Bonner minimal agar plate

Description of follow up The results were confirmed in two independent studies. repeat study Plate incorporation method was carried out in the first study and preincubation method in the second study.DR.U.NOACK-LABORATORIEN Robust Summary Page 3 of 3 DOBA Bacterial Reverse Mutation Test (Amestest) Project-No. 060601CL acc. OECD 471 / EC Directive 2000/32/EC Method B. 13/14 Study-No. USO108573

Criteria for evaluating Genotypes were evaluated for each study. Plates of the mutagenicity test re sults were inspected for present and reduced background lawn after incubation. Plates with colonies which correspond not with the typical shape and colour of Salmonella typhimurium or Escherichia coli , respectively, were regarded as contaminated and were not included in calculations. Colonies per plate (revertants) we re counted. Plates with reduced background were considered carefully for evaluation as appropriate.

Results

Table 1: Mutagenic and Cytotoxic Effects of the Test Item

Tested Concentration Range (factor √ 10)

[mg/plate] Lowest Cytotoxic Concentration [mg/plate] Strain S9 Plate Incorporation Method Preincubation Method Lowest Mutagenic Concentration

[mg/plate] Plate Incorporation Method Preincubation Method

- 1.6 0.5 TA 1537 + 1.6 – 0.016 1.6 1.6 - > 5.0 0.5 TA 98 + > 5.0 > 5.0 - 5.0 0.5 TA 100 + 5.0 5.0 - 1.6 0.5 TA 1535 + > 5.0 > 5.0 - > 5.0 > 5.0 E. coli WP2 (uvrA) + 5.0 - 0.05 5.0 - 0.05 none

> 5.0 > 5.0



## Conclusions

The test item is regarded to be not mutagenic under test conditions.