[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 November 2017 to 12 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Octadecanoic acid, reaction products with tetraethylenepentamine

Cas Number:

71799-54-7

Molecular formula:

Not applicable - UVCB substance

IUPAC Name:

Octadecanoic acid, reaction products with tetraethylenepentamine

Test material form:

solid

Details on test material:

- Appearance: Amber solid
- Storage: Room temperature (20 ± 5 °C); protected from light.

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- On the day of the start of the experiment 1a and 1b, a stock solution containing 50 g/L (nominal) of the test material in ethanol was prepared (50 minutes in the water bath at 37 °C). The test material solution was not sterile filtrated before use. The stock solution was used to prepare the geometric series of the concentrations to be tested. The following nominal concentrations were prepared for the experiment 1a: 5000, 1500, 500, 150 and 50 µg/plate. The following nominal concentrations were prepared for the experiment 1b: 1500, 500, 150, 50, 15, 5 and 1.5 µg/plate.
- On the day of the start of the experiments 1c, 2a and 2b, a stock solution containing 15 g/L (nominal), additionally for experiment 2b a stock solution containing 5 g/L of the test material in ethanol were prepared.
- The following nominal concentrations were prepared for the experiment 1c for the bacteria strains TA97a with and without metabolic activation, TA98 with and without metabolic activation and TA100 with and without metabolic activation: 1500, 500, 150, 50, 15, 5 and 1.5 µg/plate.
- The following nominal concentrations were prepared for the experiment 2a for the bacteria strains TA102 and TA1535 with and without metabolic activation: 1500, 750, 375, 188, 94, 47 and 23 µg/plate. The following nominal concentrations were prepared for the experiment 2b for the bacteria strains TA97a with metabolic activation, TA98 with and without metabolic activation and TA100 with metabolic activation: 1500, 750, 375, 188, 94, 47, 23 and 12 µg/plate. The following nominal concentrations were prepared for the experiment 2b for the bacteria strains TA97a and TA100 without metabolic activation: 500, 250, 125, 62.5, 31.3, 15.6, 7.8 and 3.9 µg/plate.

Method

Target gene:

- Histidine requirement in Salmonella typhimurium strains (Histidine operon).

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: TRINOVA BioChem GmbH (batch: TA97a: 4997D, TA98: 5011D, TA100: 4996D, TA102: 4982D, TA1535: 5012D)
- Cells were stored as lyophilizates in the refrigerator at 2-8 °C. The lyophilizates were used to prepare permanent cultures which were filled into vials and stored at < - 75 °C.
- Eight hours before the start of each experiment, an aliquot of a permanent culture per strain to be used was taken from the deep freezer to inoculate a culture vessel containing nutrient broth. After incubation overnight for eight hours at 37 ± 1 °C, the cultures were used in the experiment. During the test, the cultures were stored at room temperature to prevent changes in the titre.
- On the day of the test, the bacteria cultures were checked for growth visually.

MEDIA USED
The purity of the chemicals which were used were either “analytical grade“ or “for microbiological purposes“. All solutions and media were sterilized, either by autoclaving (121 °C, 20 minutes) or by membrane filtration.
Composition is stated as nominal composition, exact weights differed by max. ± 10 %.
The given volumes are exemplary for the composition of the media/solutions. The real volumes/weights are stated in the raw data.

Nutrient Broth for Overnight Culture
2.8 g Nutrient broth Merck 5443
H2O demineralized ad 350 Ml

Isotonic Sodium Chloride Solution for Dilution Purposes
0.9 g Sodium chloride
H2O demineralized ad 100 mL

Vogel-Bonner-Medium 20fold
4.0 g Magnesium sulphate (MgSO4·7H2O)
40.0 g Citric acid mono hydrate (MR 210.14 g/mol)
200.0 g Potassium phosphate, dibasic (anhydrous) (K2HPO4)
70.0 g Sodium ammonium phosphate, monobasic, tetra hydrate (Na(NH4)HPO4·4H2O)
H2O demineralized ad 1000.0 mL

Glucose Solution 40 %
440.0 g Glucose monohydrate (MR 198.17g/mol)
H2O demineralized ad 1000.0 mL

Minimal Glucose Agar
500.0 mL Vogel-Bonner-Solution 20-fold
500.0 mL Glucose solution 40 %
H2O demineralized 9000.0 mL
Agar 150.0 g

Biotin Agar
500.0 mL Minimal-Glucose-Agar. 80 °C
3.0 mL Biotin solution 0.5 mM

Histidine-Biotin-Agar
350.0 mL Biotin-Agar, 80 °C
3.5 mL Histidine solution 0.5 %

Ampicillin-Agar
200.0 mL Histidine-biotin agar, 80 °C
0.6 mL Ampicillin solution 0.8 %

Ampicillin-Tetracycline Plates
50.0 mL Ampicillin agar, 80 °C
0.01 mL Tetracycline solution 0.8 %

Nutrient Agar Plates
0.8 g Nutrient broth Merck 5443
0.5 g Sodium chloride (NaCl)
Agar 1.52 g
H2O demineralized 100 mL

Basis for Top-Agar and Maximal-Soft-Agar
6 g Agar
5 g Sodium chloride (NaCl)
H2O demineralized ad 1000 mL

Histidine-Biotin-Solution 0.5/0.5 mM (Use: Top Agar)
12.2 mg D-Biotin (MR 244.3 g/mol)
10.5 mg L-Histidine· HCl· H2O (MR 209.7 g/mol)
H2O demineralized 90 °C ad 100.0 mL

To 100 mL basis, 10 mL histidine-biotin-solution 0.5/0.5 mM were added.

Histidine-Biotin-Solution 5 mM/ 0.5 mM (Use: Maximal-Soft-Agar)
12.2 mg D-Biotin (MR 244.3 g/mol)
105 mg L-Histidine·HCl·1H2O (MR 209.7 g/mol)
H2O demineralized 90 °C ad 100.0 mL
To 100 mL basis,10 mL histidine-biotin-solution 5/0.5 mM were added.

Phosphate Buffer
0.184 g Sodium di-hydrogen phosphate monohydrate NaH2PO4· H2O
1.722 g Di-sodium hydrogen phosphate dihydrate Na2HPO4 · 2H2O
H2O demineralized ad 100.0 mL
The pH of the solution was adjusted to 7.4 with HCl 1M.

Salt Solution for S9-Mix
1.23 g Potassium chloride (KCl)
0.814 g Magnesium chloride hexahydrate MgCl2·6H2O
H2O demineralized ad 10.0 mL

NADP-Solution for S9-Mix, 0.1 M
787.4 mg NADP disodium salt (MR = 787.4 g/mol)
10 mL H2O demineralized


Glucose-6-Phosphate (G6P) Solution for S9-Mix, 1 M
680.3 mg Glucose-6-phosphate disodiumsalt dihydrat (MR = 340.13 g/mol)
2 mL H2O demineralized ad

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

Experiment 1a: 5000, 1500, 500, 150 and 50 µg/plate
Experiment 1b: 1500, 500, 150, 50, 15, 5 and 1.5 µg/plate
Experiment 1c: 1500, 500, 150, 50, 15, 5 and 1.5 µg/plate
Experiment 2a: 1500, 750, 375, 188, 94, 47 and 23 µg/plate
Experiment 2b: 1500, 750, 375, 188, 94, 47, 23 and 12 µg/plate (+ and - S9); 500, 250, 125, 62.5, 31.3, 15.6, 7.8 and 3.9 µg/plate (- S9)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: In a non-GLP pre-test, the solubility of the test material was tested at a concentration of 50 g/L in demineralised water, dimethyl sulfoxide (DMSO) and ethanol. Ethanol was chosen as vehicle, because the test material was sufficiently soluble in it after 50 minutes in the water bath at 37 °C, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethylsulfoxide (DMSO), Demineralised water and Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-Nitro-1,2-phenylene Diamine (20 µg/plate in DMSO for TA97a, TA98 and TA102 -S9) and 2-Amino-anthracene (1 µg/plate in DMSO for TA97a, TA100, TA102 and TA1535 +S9)

Details on test system and experimental conditions:

EXPERIMENTAL PREPARATIONS
- On the day of the test, the bacteria cultures were checked for growth visually. The incubation chambers were heated to 37 ± 1 °C. The water bath was turned to 43 ± 1 °C. The table surface was disinfected. The S9 mix was freshly prepared and stored at 0 °C.
- General preparation: Per bacterial strain and concentration, three plates with and three plates without metabolic activation (-S9) were used. For the top agar 100 mL agar basis was melted in a microwave oven, 10 mL of the histidine-biotin-solution 0.5 mM was added, then the mixture was placed in the water bath at 43 ± 1 °C.

PLATE INCORPORATION METHOD
The following materials were gently vortexed in a test tube and poured onto the selective agar plates:
- 100 µL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
- 500 µL S9 mix (for test with metabolic activation) or phosphate buffer (for test without metabolic activation).
- 100 µL bacteria suspension
- 2000 µL overlay agar (top agar)
The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incubator at 37 ± 1 °C.

PRE-INCUBATION METHOD
The following materials were gently vortexed in a test tube and incubated at 37 ± 1 °C for 20 minutes:
- 100 µL test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control)
- 500 µL S9 mix (for test with metabolic activation) or phosphate buffer (for test without metabolic activation).
- 100 µL bacteria suspension
After the pre-incubation for 20 minutes, 2000 µL top agar was added and the tube was gently vortexed. The mixture was poured onto the selective agar plate.
The plates were closed and left to solidify for a few minutes, then inverted and placed in the incubator at 37 ± 1 °C.

EXPERIMENT 1A
- Concentrations: 5000, 1500, 500, 150 and 50 µg/plate
- Incubation: 48 h at 37 ± 1°C
- Strains: TA97a, TA98, TA100, TA102, TA1535
- Method: Plate incorporation

EXPERIMENT 1B
- Concentrations: 1500, 500, 150, 15, 5 and 1.5 µg/plate
- Incubation: 48 h at 37 ± 1°C
- Strains: TA97a, TA98, TA100, TA102, TA1535
- Method: Plate incorporation

EXPERIMENT 1C
- Concentrations: 1500, 500, 150, 15, 5 and 1.5 µg/plate
- Incubation: 48 h at 37 ± 1°C
- Strains: TA97a, TA98, TA100
- Method: Plate incorporation

EXPERIMENT 2A
- Concentrations: 1500, 750, 375, 188, 94, 47 and 23 µg/plate
- Incubation: 48 h at 37 ± 1°C
- Strains: TA102, TA1535
- Method: Pre-incubation

EXPERIMENT 2B
- Concentrations: 1500, 750, 375, 188, 94, 47 23 and 12 µg/plate
- Incubation: 48 h at 37 ± 1°C
- Strains: TA97a (+S9), TA98(+S9, -S9), TA100 (+S9)
- Method: pre-incubation
- Concentrations: 500, 250, 125, 62.5, 31.3, 15.6, 7.8 and 3.9 µg/plate
- Incubation: 48 h at 37 ± 1 °C
- Strains: TA97a (-S9), TA100 (-S9)
- Method: Pre-incubation

REFERENCES AND VIABILITY
- Genotype Confirmation: Genotype confirmation is performed for each batch of lyophilised bacteria before stock culture preparation.
- Histidine requirement: Each strain was streaked on a biotin and a histidine-biotin-plate, using a sterilised wire loop. The plates were incubated for 24 hours at 37 ± 1 °C.
- Ampicillin/Tetracycline-Resistance (pKM 101, pAQ1): Each strain was streaked on an ampicillin agar plate and on an ampicillin-tetracycline agar plate. TA1535 was used as control strain, since it is not ampicillin resistant. The plates were incubated for 24 hours at 37 ± 1 °C.
- UV-sensitivity (uvrB): Each strain was streaked on a plate, and one half of the plate covered with aluminium foil so that one half of each streak was protected against light. The plates for the strain TA97a, TA100 and TA102 were irradiated for 8 seconds, the plates for the strain TA 98 were irradiated for 10 seconds and the plates for the strain TA1535 were irradiated for 6 seconds with a germicidal lamp (254 nm, 30W). Keeping a distance of 33 cm for the strains TA97a, TA102 and TA1535. Keeping a distance of 66 cm for the following strains: TA98, TA100. Incubation for 24 hours at 37 ± 1 °C followed.
- Crystal violet sensitivity (deep rough/rfa): For each strain, two plates were used. 0.1 mL of bacteria suspension were mixed with 2 mL Top-Agar and poured on nutrient agar. Sterile paper discs (Ø 9 mm), each soaked with 10 µL of crystal violet solution (0.1 %) were placed into the middle of each plate, followed by incubation for 24 hours at 37 ± 1 °C.
- Spontaneous Revertants: Three replicates, with/without S9, for each solvent which was used in the test, incubation for 48 hours at 37 ± 1 °C.
- Determination of Titre : The titre was determined by dilution of the overnight culture using sodium chloride solution and placing 0.1 mL on maximal-soft agar. Incubation for 48 hours at 37 ± 1 °C followed. It should give a density of 109 cells/mL (at the least), two replicates with and without metabolic activation.
- Toxicity Control: Performed in experiment 1a only analogously to the titre control with the maximum dose of test material on maximal-soft agar, two replicates with and without metabolic activation, incubation for 48 hours at 37 ± 1 °C.
- Sterility Control : Performed analogously to the test with solvent only and S9 (without adding bacteria) on top agar, incubation for 48 hours at 37 ± 1 °C, four replicates. - Solubility: Plates were checked for precipitation of test material at the end of the incubation by visual inspection.

Evaluation criteria:

- The colonies were counted visually and the numbers were recorded. A validated spread-sheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
- A substance is considered to have mutagenic potential, if a reproducible increase of re-vertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Statistics:

- The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test material solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous re-vertants) was given.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

EXPERIMENT 1A
- Confirmation of the Criteria and Validity
All strains met the criterion of at least 10^9 bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and nearly all (one exception) were within the historical control data ranges.

- Solubility and Toxicity
In this experiment, the test material showed no precipitates on the plates in all tested concentrations.
No bacterial background lawn and no bacteria growth were observed at the highest concentration (5000 µg/plate).
In the second concentration (1500 µg/plate), the bacterial background lawn was not reduced, but a decrease in the number of revertants was observed in all bacteria strains.
In the next lower concentrations, the bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains.
The test material showed signs of toxicity towards all bacteria strains in both the absence and presence of metabolic activation in the two highest concentrations (5000 and 1500 µg/plate).
In the third concentration (500 µg/plate) signs of toxicity were observed towards the bacteria strains TA97a and TA100 in the treatment without metabolic activation.
In the lower concentrations, no toxicity was observed.

- Mutagenicity
No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found. Therefore, the test material is stated as not mutagenic under the test conditions.
Based on the toxicity results, a repetition of the experiment was performed with lower concentrations.


EXPERIMENT 1B
- Confirmation of the Criteria and Validity
All strains met the criterion of at least 10^9 bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.

- Solubility and Toxicity
In the experiment, the test material showed no precipitates on the plates in all tested concentrations. The bacterial background was observed at all evaluated concentrations.
In the highest concentration (1500 µg/plate), a decrease in the number of revertants (TA102) resp. no bacteria growth (TA1535) was observed.
Due to a contamination on the agar plates, no evaluation of the bacteria strains TA97a, TA98 and TA100 was possible.

- Mutagenicity
No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found. Therefore, the test material is stated as not mutagenic under the test conditions of this experiment.

EXPERIMENT 1C
- Confirmation of the Criteria and Validity
All strains met the criterion of at least 10^9 bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.

- Solubility and Toxicity
In this experiment, the test material showed no precipitates on the plates in all tested concentrations.
The bacterial background was observed at all tested concentrations.
In the highest concentration (1500 µg/plate), a decrease in the number of revertants was observed in the bacteria strains TA97a with metabolic activation and TA98 with and without metabolic activation. Additionally, signs of toxicity were observed towards the bacteria strain TA97a and TA100 without metabolic activation in the next lower concentration (500 µg/plate).

- Mutagenicity
No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found. Therefore, the test material is stated as not mutagenic under the test conditions.

EXPERIMENT 2A
- Confirmation of the Criteria and Validity
The tested bacteria strains (TA102 und TA1535) met the criterion of at least 10^9 bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and nearly all were within the historical control data ranges.

- Solubility and Toxicity
In this experiment, the test material showed no precipitates on the plates in all tested concentrations.
The bacterial background lawn was not reduced at all tested concentrations.
Signs of toxicity were observed in the two highest concentrations; at 1500 µg/plate no bacteria growth was observed and in the next lower concentration (750 µg/plate) a decrease in the number of spontaneous revertants was observed.
In the lower concentrations, no toxicity was observed.

- Mutagenicity
No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.

EXPERIMENT 2B
- Confirmation of the Criteria and Validity
All strains met the criterion of at least 10^9 bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.

- Solubility and Toxicity
In this experiment, the test material showed no precipitates on the plates in all tested concentrations.
Based on the results of the experiments with the plate incorporation method, the test material was tested up to concentrations of 1500 µg/plate in the absence and the presence of S9- mix in the strain TA98 and in the presence of metabolic activation in the strains TA97a and TA100 using the pre-incubation method. However, the strains TA97a and TA100 were tested up to 500 µg/plate in the treatment without metabolic activation.
The test material showed no precipitates on the plates at any of the concentrations.
The bacterial background lawn was not reduced at all tested concentrations, excepted in the highest concentration towards the bacteria strains TA97a and TA100 without metabolic activation.
Signs of toxicity were observed in the highest concentration (1500 or 500 µg/plate respectively). In the next lower concentration, a decrease in the number of spontaneous revertants was observed for the bacteria strain TA100 with and without metabolic activation.
For the bacteria strain TA97a without metabolic activation, the three highest concentrations showed cytotoxicity (500, 250 and 125 µg/plate).

- Mutagenicity
No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found. Therefore, the test material is stated as not mutagenic under the test conditions.

MUTAGENICITY OF THE TEST MATERIAL
The test material showed no increase in the number of revertants in all bacteria strains in all five experiments.
All negative and nearly all strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Based on the results of this study it is concluded that the test material is not mutagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the present study.

ACCEPTABILITY OF THE STUDY
In all experiments, no precipitation of the material was observed at any of the tested concentrations up to 5000 µg/plate.
In all experiments, the test material caused cytotoxicity towards the bacteria strains in different concentrations.
The confirmation tests of the genotype did not show any irregularities. The control of the titre was above the demanded value of 10^9 bacteria/mL.
All of the means of all replicates of the spontaneous revertants (in negative and solvent controls) were within the range of the historical data of the test facility. Nearly all numbers of revertant colonies of the positive controls were within the range of the historical data of the laboratory, but all were in-creased in comparison with the negative controls, which demonstrated the mutagenic potential of the diagnostic mutagens.
Since all criteria for acceptability have been met, the study is considered valid.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the test material is not mutagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation.

Executive summary:

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 471 and EU Method B.13/14, under GLP conditions using the Bacterial Reverse Mutation Assay. Five valid experiments were performed.

The test material was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535). The test was performed in five experiments in the presence and absence of metabolic activation, with +S9 standing for presence of metabolic activation, and –S9 standing for absence of metabolic activation.

Experiment 1a:

In this experiment, the test material (dissolved in ethanol) was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method.

The test material showed no precipitates on the plates at any of the concentrations.

No bacterial background lawn and no bacteria growth were observed at the highest concentration (5000 µg/plate).

In the second concentration (1500 µg/plate), the bacterial background lawn was not reduced, but a decrease in the number of revertants was observed in all bacteria strains.

In the next lower concentrations, the bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains.

The test material showed signs of toxicity towards all bacteria strains in both the absence and presence of metabolic activation in the two highest concentrations (5000 and 1500 µg/plate).

In the third concentration (500 µg/plate) signs of toxicity towards the bacteria strains TA97a and TA100 in the treatment without metabolic activation were observed.

The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

Experiment 1b:

In this experiment, the test material was tested up to concentrations of 1500 µg/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method, except for the bacteria strains TA97a and TA100 in the treatment without metabolic activation where the highest tested concentration was 500 µg/plate.

Due to a contamination on the agar plates, no evaluation of the bacteria strains TA97a, TA98 and TA100 was possible.

The test material showed no precipitates on the plates at any of the concentrations.

The bacterial background was observed at all tested concentrations.

In the highest concentration (1500 µg/plate), a decrease (TA102) resp. no bacteria growth (TA1535) in the number of revertants was observed.

The results of this experiment showed that none of the tested and evaluable concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

This experiment was repeated on the strains TA97a, TA98 and TA100 (Exp. 1c).

Experiment 1c:

In this experiment, the test material was tested up to concentrations of 1500 µg/plate in the absence and presence of S9-mix in the strains TA97a, TA98 and TA100 using the plate incorporation method, except for the bacteria strains TA97a and TA100 in the treatment without metabolic activation where the highest tested concentration was 500 µg/plate.

The test material showed no precipitates on the plates at any of the concentrations.

The bacterial background was observed at all tested concentrations.

In the highest concentration (1500 µg/plate), a decrease in the number of revertants was observed in the bacteria strains TA97a and TA100 with metabolic activation and TA98 with and without metabolic activation. Additionally, signs of toxicity were observed towards the bacteria strain TA97a and TA100 without metabolic activation in the next lower concentration (500 µg/plate).

In the lower concentrations, no signs of toxicity were observed.

The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

Experiment 2a:

Based on the results of the experiments with the plate incorporation method, the test material was tested up to concentrations of 1500 µg/plate in the absence and presence of S9-mix in the bacteria strains TA102 and TA1535 using the pre-incubation method.

The test material showed no precipitates on the plates at any of the concentrations.

The bacterial background lawn was not reduced at all tested concentrations.

Signs of toxicity were observed in the two highest concentrations: at 1500 µg/plate no bacteria growth was observed and in the next lower concentration (750 µg/plate) a decrease in the number of spontaneous revertants was observed.

The results of this experiments showed that the test material caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation. The test material did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation.

Experiment 2b:

Based on the results of the experiments with the plate incorporation method, the test material was tested up to concentrations of 1500 µg/plate in the absence and presence of S9-mix in the strains TA97a, TA98 and TA100 using the pre-incubation method, excepted for the bacteria strains TA97a and TA100 in the treatment without metabolic activation where the highest tested concentration was 500 µg/plate.

The test material showed no precipitates on the plates at any of the concentrations.

The bacterial background lawn was not reduced at all tested concentrations, excepted in the two highest concentration towards the bacteria strains TA97a and TA100 without metabolic activation.

Signs of toxicity were observed in the highest concentrations for all strains (1500 or 500 µg/plate respectively). In the next lower concentration a decrease in the number of spontaneous revertants was also observed for the bacteria strain TA100 with and without metabolic activation. 

For the bacteria strain TA97a without metabolic activation the three highest concentrations showed cytotoxicity (500, 250 and 125 µg/plate).

The results of this experiments showed that the test material caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation. The test material did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation.

Under the conditions of this study, the test material is not mutagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation.