TITLE PAGE

REPORT

SALMONELLA TYPHIMURIUM / ESCHERICHIA COLI

REVERSE MUTATION ASSAY

TEST GUIDELINE(S)

OECD 471

Commission Regulation (EC) No 440/2008; B.13 / B.14

US EPA OPPTS 870.5100

AUTHOR

(Study 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 52





GLP-CERTIFICATE (FROM THE COMPETENT AUTHORITY)

Page 8 of 52
CONTENTS

TITLE PAGE

BLANK PAGE

GLP COMPLIANCE STATEMENT

FLAGGING CRITERIA

SIGNATURE PAGE

CONTRIBUTORS TO THE STUDY / SUPERVISORY LABORATORY PERSONNEL

STATEMENT OF THE QUALITY 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 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 PREPARATION

3.6. ANALYSIS OF TEST SUBSTANCE PREPARATION

3.7. EXPERIMENTAL PROCEDURE

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 52
3.7.4.1. 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 TEST)

APPENDIX 2 TABLES 6 - 10 (PREINCUBATION TEST)

APPENDIX 3 STERILITY CONTROL

APPENDIX 4 S9 FRACTION CHARACTERIZATION

APPENDIX 5 HISTORICAL NEGATIVE 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)

TEST 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 S9 mix.

TOXICITY: A weak bacteriotoxic effect was occasionally observed 

depending on the strain and test conditions from about 

2 700 μg/plate onward (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 strains of Salmonella 

typhimurium and Escherichia coli are commonly employed as initial screening methods for 

detecting a point mutagenic activity of chemical substances (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 substances 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 cofactorsupplemented 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 Bacteria, No B. 13 / B. 14; No L 142

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

Page 12 of 52
2.4. 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 52
3.2. 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 

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 ≥ 108 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 

recommendations for the conduct of this assay.

The Salmonella strains TA 98, TA 100 and TA 1537 were obtained from (former) KNOLL 

Aktiengesellschaft, 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 substances.

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 substances 

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 52
3.2.3. 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 AAALACapproved 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-naphthoflavone 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 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 4. 

Page 16 of 52
3.3.2. S9 mix

The S9 mix is prepared freshly prior to each experiment (1, 2). For 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:

MgCl2 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 Na2HPO4 solution with a NaH2PO4 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 solutions. 

The test substance was dissolved acetone.

To achieve clear solutions of the test substance in the vehicle, the test substance

preparations were treated with ultrasonic waves and were 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 52
3.7. EXPERIMENTAL PROCEDURE

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 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 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 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 

(trp+ revertants) 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 bacterial 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 52
3.7.3. 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 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 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 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 52
3.7.4.2. 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

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-phenylenediamine (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

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 52
3.7.5. 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; 2 700 and 5 400 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

Strains: TA 1535, TA 100, TA 1537 and TA 98

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: Due to technical reason, an evaluation of the TA strain in the 

2nd experiment was not possible. The 2nd 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 52
3.8. EVALUATION

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 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 his- 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 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 52
3.8.5. 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 Appendix 5).

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

• The positive control substances 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).

• Fresh bacterial culture containing approximately 109 cells per mL were used. For approval 

the titer of viable bacteria was ≥ 108 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 preincubation 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 number of his+ or trp+ revertants.

TA 98:

E. coli WP2 uvrA:

Page 25 of 52
4.1.2. 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 52
4.2. TOXICITY 

A weak bacteriotoxic effect (slight decrease in the number of his+

 or trp+ revertants, slight 

reduction in the titer) was observed in the standard plate test and in the preincubation test 

depending on the strain and test conditions from about 2 700 μg/plate onward. 

4.3. SOLUBILITY 

Test substance precipitation 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 substance did not lead to a relevant 

increase in the number of revertant colonies either without S9 mix or after adding a 

metabolizing system in three experiments carried out independently of each other (standard 

plate test and preincubation assay). 

Besides, the results of the negative as well as the positive controls performed in parallel 

corroborated the validity of this study, since the values fulfilled the acceptance criteria of this 

study. 

In this study with and without S9 mix, the number of revertant colonies in the negative 

controls was nearby the range of the historical negative control data for each tester strain 

(see Appendix 5). 

In addition, the positive control substances both with and 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 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 testing 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.; 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 52
APPENDIX 1 

TABLES 1 - 5 

(STANDARD PLATE TEST) 

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APPENDIX 2 

TABLES 6 - 10

(PREINCUBATION TEST)

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APPENDIX 3 

STERILITY CONTROL

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STERILITY 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

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APPENDIX 4 

S9 FRACTION CHARACTERIZATION

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S9 fraction

Preparation of S9 fraction: 19 Oct 2012

Content of protein: 27.01 g/L

Characterization of the S9 mix with benzo[a]pyrene: 08 Nov 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)

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APPENDIX 5 

HISTORICAL NEGATIVE CONTROL DATA

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Historical Negative Control Data 

Strain: TA 1535

Strain: TA 100

 * = revertants/plate PIT = preincubation test

 SPT = standard plate test SD = standard deviation

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Historical Negative Control Data 

Strain: TA 1537

Strain: TA 98

 * = revertants/plate PIT = preincubation test

 SPT = standard plate test SD = standard deviation

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Historical Negative Control Data

Strain: WP2 uvrA

 * = revertants/plate PIT = preincubation test

 SPT = standard plate test SD = standard deviation

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APPENDIX 6 

HISTORICAL POSITIVE CONTROL DATA

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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

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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

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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

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