Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
May 30, 2008
Deviations:
no
GLP compliance:
yes
Remarks:
Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
4-(4-methoxyphenyl)butan-2-one
EC Number:
203-184-2
EC Name:
4-(4-methoxyphenyl)butan-2-one
Cas Number:
104-20-1
Molecular formula:
C11H14O2
IUPAC Name:
4-(4-methoxyphenyl)butan-2-one

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Experiment II - strains TA100 and WP2 uvr A: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Experiment II - other strains: 33, 100, 333, 1000, 2500 and 5000 µg/plate
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylenediamine (4-NOPD) (without metabolic activation, TA1537 and TA98); 2-aminoanthracene (2-AA) (with metabolic activation, all strains)
Details on test system and experimental conditions:
CHARACTERISATION OF THE STRAINS:
- TA1537: his C 3076, rfa-, uvrB-; frame shift mutations
- TA98: his D 3052, rfa-, uvrB-, R-factor; frame shift mutations
- TA1535: his G 46, rfa-, uvrB-; base-pair substitutions
- TA100: his G 46, rfa-, uvrB-, R-factor; base-pair substitutions
- WP2 uvrA: trp-; uvrA-; base-pair substitutions and others

Strains were obtained from Trinova Biochem GmbH, 35394 Giessen, Germany.
Strains were regularly checked regarding membrane permeability, ampicillin resistance, UV sensitivity, amino acid requirements, and spontaneous mutation rates.

METHOD OF APPLICATION:
- Experiment I: plate incorporation
- Experiment II: pre-incubation

PRECULTURES:
Thawed bacterial suspensions were added to 50 mL nutrient medium (8 g nutrient broth and 5 g NaCl per litre). A solution of 50 µL of ampicillin (25 µg/mL) was added to strains TA98 and TA100. Incubation at 37°C for 4 hours. The optical density of the bacteria was determined by absorption measurement and the obtained values indicated that the bacteria were harvested at the late exponential or early stationary phase (10E8-10E9 cells/mL).

METABOLIC ACTIVATION SYSTEM:
Phenobarbital/beta-naphthoflavone induced rat liver S9 were used as the metabolic activation system. Each S9 batch was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test. The protein concentration of the S9 preparation was 35.0 mg/mL in experiment I and 30.7 mg/mL in experiment II.

S9 supernatant is thawed and mixed with S9 cofactor solution to result in a final concentration of approx. 10% v/v in the S9 mix. Co-factors are added to the S9 mix to reach the following concentrations: 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP in 100 mM sodium orthophosphate buffer, pH 7.4.

During the experiment, the S9 mix is stored in an ice bath.

The S9 mix substitution buffer contains 700 mL 100 mM sodium orthophosphate buffer, pH 7.4 and 300 mL 0.15 M KCl solution.
During the experiment the S9 mix substitution buffer is stored in an ice bath.

PRE-EXPERIMENT FOR TOXICITY:
- pre-experiment is reported as main experiment I

DOSE SELECTION
- Doses used in experiment I were in the range of 3-5000 µg/plate.
- Since toxic effects were observed in experiment I, at least 6 concentrations were tested in experiment II, with a maximum concentration of 5000 µg/plate.

EXPERIMENTAL PERFORMANCE

Experiment I (plate incorporation):
- 100 µL test solution at each dose level
- 500 µL S9 mix or S9 mix substitution buffer
- 100 µL bacteria suspension
- 2000 µL overlay agar
- Incubated for at least 48 hours at 37°C in the dark.

Experiment II (pre-incubation):
- 100 µL test solution at each dose level
- 500 µL S9 mix or S9 mix substitution buffer
- 100 µL bacteria suspension
- Mixed in a test tube and incubated at 37°C for 60 minutes
- 2000 µL overlay agar (45°C) added to each tube, and poured on minimal agar plates.
- Incubated for at least 48 hours at 37°C in the dark.

Sterile control:
- 100 µL test solution at each dose level
- 500 µL S9 mix or S9 mix substitution buffer
- 2000 µL overlay agar
- Incubated for at least 48 hours at 37°C in the dark.

Evaluation criteria:
ACCEPTABILITY OF THE ASSAY:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce an increase above the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control
- a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.

EVALUATION OF THE RESULTS:
- A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
- A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
- An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
- A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
Not mandatory according to OECD 471.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2500 µg/plate and above
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2500 µg/plate and above
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Summary of Experiment I:

Metabolic
Activation
Test
Group
Dose Level
(per plate)
Revertant Colony Counts (Mean ±SD)
      TA 1535 TA 1537 TA 98 TA 100 WP2 uvrA
               
Without DMSO   15 ± 2 13 ± 1 28 ± 7 180 ± 20 40 ± 9
  Untreated   14 ± 6 18 ± 5 26 ± 8 190 ± 11 41 ± 7
  Test item 3 μg 15 ± 6 12 ± 4 28 ± 5 173 ± 29 48 ± 2
    10 μg 15 ± 3 13 ± 2 24 ± 2 162 ± 10 40 ± 11
    33 μg 15 ± 2 13 ± 3 30 ± 4 172 ± 13 46 ± 2
    100 μg 17 ± 3 15 ± 2 27 ± 8 165 ± 13 43 ± 3
    333 μg 13 ± 5 12 ± 3 32 ± 4 195 ± 16 40 ± 1
    1000 μg 16 ± 4 9 ± 3 21 ± 6 182 ± 14 36 ± 8
    2500 μg 14 ± 1 7 ± 2 24 ± 2 150 ± 7 22 ± 6
    5000 μg 18 ± 3 8 ± 1 24 ± 4 75 ± 42R 12 ± 1
  NaN3 10 μg 1319 ± 68     2292 ± 81  
  4-NOPD 10 μg     384 ± 26    
  4-NOPD 50 μg   103 ± 13      
  MMS 2.0 μL         603 ± 51
With DMSO   13 ± 2 14 ± 1 29 ± 10 163 ± 4 53 ± 5
  Untreated   12 ± 4 21 ± 2 37 ± 10 179 ± 16 52 ± 8
  Test item 3 μg 15 ± 2 15 ± 1 33 ± 10 167 ± 89 53 ± 7
    10 μg 17 ± 4 14 ± 1 33 ± 9 163 ± 3 60 ± 8
    33 μg 12 ± 3 13 ± 3 33 ± 7 151 ± 15 56 ± 7
    100 μg 13 ± 2 15 ± 5 33 ± 8 154 ± 5 44 ± 4
    333 μg 11 ± 2 11 ± 4 37 ± 11 183 ± 15 53 ± 6
    1000 μg 12 ± 2 10 ± 3 36 ± 1 174 ± 4 41 ± 8
    2500 μg 12 ± 4 9 ± 1 32 ± 2 169 ± 14 25 ± 4
    5000 μg 9 ± 4 8 ± 1 28 ± 4 81 ± 9R 17 ± 1
  2-AA 2.5 μg 462 ± 22 149 ± 6 5767 ± 351 5421 ± 242  
  2-AA 10.0 μg         940 ± 39

Summary of Experiment II:

Metabolic
Activation
Test
Group
Dose Level
(per plate)
Revertant Colony Counts (Mean ±SD)
      TA 1535 TA 1537 TA 98 TA 100 WP2 uvrA
               
Without DMSO   10 ± 0 9 ± 3 28 ± 3 188 ± 29 39 ± 3
  Untreated   8 ± 4 9 ± 3 27 ± 1 206 ± 4 45 ± 16
  Test item 3 μg       186 ± 15 45 ± 12
    10 μg       204 ± 6 41 ± 8
    33 μg 12 ± 5 11 ± 0 24 ± 2 221 ± 18 51 ± 11
    100 μg 10 ± 3 10 ± 3 21 ± 7 213 ± 2 51 ± 4
    333 μg 12 ± 2 11 ± 2 30 ± 3 182 ± 7 39 ± 6
    1000 μg 16 ± 3 11 ± 5 23 ± 7 179 ± 21 26 ± 4
    2500 μg 17 ± 3 8 ± 3 26 ± 6 90 ± 17 18 ± 3
    5000 μg 15 ± 1 10 ± 4R 12 ± 3 95 ± 15 11 ± 1R
  NaN3 10 μg 1160 ± 53     1720 ± 142  
  4-NOPD 10 μg     424 ± 58    
  4-NOPD 50 μg   110 ± 25      
  MMS 2.0 μL         722 ± 57
With DMSO   12 ± 3 13 ± 2 44 ± 7 201 ± 19 58 ± 11
  Untreated   13 ± 7 15 ± 3 44 ± 13 212 ± 11 53 ± 6
  Test item 3 μg       175 ± 35 49 ± 2
    10 μg       175 ± 7 56 ± 9
    33 μg 12 ± 3 16 ± 4 41 ± 6 196 ± 14 61 ± 6
    100 μg 16 ± 1 13 ± 6 44 ± 10 178 ± 22 54 ± 5
    333 μg 13 ± 1 20 ± 1 42 ± 5 184 ± 14 47 ± 2
    1000 μg 11 ± 1 18 ± 6 36 ± 8 148 ± 3 44 ± 5
    2500 μg 9 ± 2 13 ± 3R 11 ± 3R 97 ± 33 15 ± 5R
    5000 μg 6 ± 3 5 ± 1R 2 ± 2MR 6 ± 2 6 ± 1MR
  2-AA 2.5 μg 407 ± 46 147 ± 21 4381 ± 574 4773 ± 268  
  2-AA 10.0 μg         338 ± 27

With:

- NaN3: sodium azide

- 2-AA: 2-aminoanthracene

- 4-NOPD: 4-nitro-o-phenylenediamine

- MMS: methyl methane sulfonate

- R: reduced background growth

- M: manual count

Applicant's summary and conclusion

Conclusions:
The test item was found to be not mutagenic under the conditions of the Ames test.
Executive summary:

The potential of the test item to induce gene mutation was assessed according to an Ames test in accordance with OECD 471 and GLP, using Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments, both with and without metabolic activation.

The following concentrations were tested, each in triplicate:

- Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate

- Experiment II - strains TA100 and WP2 uvr A: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate

- Experiment II - other strains: 33, 100, 333, 1000, 2500 and 5000 µg/plate

No precipitation of the test item occurred up to the highest investigated dose.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were observed in all strains, except of strain TA 1535.

The negative and positive controls were valid.

Under the experimental conditions reported, the test idem did not induce an increase in revertant colony numbers in any of the strains tested at any dose level, with or without metabolic activation. Hence, it can be stated that the substance is not mutagenic under the conditions of the test.