[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames OECD 471, Andres (2018)

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.

In Vitro Mammalian Cell Micronucleus Test OECD 487, Frühmesser (2018)

Under the conditions of this study, the test material did not show biologically relevant genotoxic activity for the induction of micronuclei.

Mammalian Gene Cell Mutation Test, OECD 476, Frühmesser (2018)

Under the conditions of this study, the test material was non mutagenic.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

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

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.

Target gene:

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

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.

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.

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

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

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

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

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.

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.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 October 2017 to 20 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EU) No. L 112/109, EU Method B.49: “In Vitro Mammalian Cell Micronucleus Test”

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human

Remarks:

Human whole blood treated with anti-coagulant (heparin).

Details on mammalian cell type (if applicable):

CELLS USED
- Blood Collection: Blood samples were obtained from healthy donors who neither smoke nor receive medication. The following 3 donors were chosen for the experimental part:
Pre-experiment with S9 mix: No.1 female, 23 years old, not examined for micronuclei ratio.
Pre-experiment without S9 mix: No. 1 female, 23 years old, not examined for micronuclei ratio.
Experiment I with S9 mix: No. 2 female, 23 years old, examined for micronuclei ratio.
Experiment I without S9 mix: No. 2 female, 23 years old, examined for micronuclei ratio.
Experiment II without S9: No. 3 female, 27 years old, examined for micronuclei ratio.
- Suitability of cells: Primary cultures of human peripheral lymphocytes are preferred for this type of study because of their low and stable background rate of micronuclei. In addition, human cells are generally the most relevant ones for risk assessment.
- Cell cultivation: The blood cultures were set up in defined time intervals within 24 h after collection in sterile culture vessels, each containing 1 part of heparinised blood and 9 parts of complete culture medium RPMI 1640 for cell proliferation. The cultures were incubated for 72 h (preexp.), 73 h (exp. II solvent and positive controls), 74 h (exp. II test material concentrations) or 48 h (exp. I) at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.

MEDIA USED
RPMI 1640, Supplier Biochrom AG, 12247 Berlin, Germany, served as base for:
- Complete Culture Medium: Foetal calf serum (FCS) 15 %, Penicillin/Streptomycin (per mL: 10 000 Units Pen/ 10 mg Strep) 1 % and Phytohaemagglutinin solution 2 %.
- Minimal Culture Medium (MCM): Penicillin/Streptomycin (per mL: 10 000 Units Pen/ 10 mg Strep) 1 % and Phytohaemagglutinin solution 2 %.
- All solutions and media were sterilized, either by autoclaving (121 °C, 20 minutes) or by membrane filtration.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

Pre-experiment nominal concentration of test material solution: 50, 25, 12.5, 6.25 and 3.13 mg/mL, resulting in nominal concentrations in the experiment of 250, 125, 62.5, 31.3 and 15.6 μg/mL

Due to cytotoxicity (3 highest test material concentrations) and precipitates (all test material concentrations) on the slides, an evaluation according to the demands of OECD 487 (≥ 3 evaluable test material concentrations) was not possible. Therefore, Experiment I was performed with a different range of concentrations.

Experiment I nominal concentration of test material solution: 3.13, 1.56, 0.78, 0.39 and 0.20 mg/mL resulting in nominal concentrations in the experiment of 15.6, 7.81, 3.91, 1.95 and 0.98 μg/mL.

Experiment II nominal concentration of test material solution: 3.13, 1.56, 0.78, 0.39 and 0.20 mg/mL, resulting in nominal concentrations in the experiment of 15.6, 7.81, 3.91, 1.95 and 0.98 μg/mL.
Since no cytotoxicity was observed and only the highest test material concentration showed slight precipitate on the slides, the 3 highest concentrations were selected for micronuclei scoring.


DOSE SELECTION
According to OECD 487, the maximum concentration of the test material should be 2 μL/mL, 2 mg/mL or 10 mM, whichever is the lowest. When cytotoxicity occurs, the highest concentration should aim to produce 55 ± 5 % cytotoxicity. For the test material the maximum concentration to be evaluated was 3.91 μg/mL (due to precipitate at the 2 higher test material concentrations). The test material concentrations 15.6 μg/mL and 7.81 μg/mL were evaluated additionally as the precipitate did not interfere with scoring and information from higher concentrations was obtained in this way.

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 determined in demineralised water, ethanol and dimethyl sulfoxide (DMSO). The test material was insoluble in demineralised water and DMSO. The best result was obtained in ethanol absolute. Nevertheless, the test material was also insoluble in this solvent at the required concentration of 1 g/mL. Therefore, a dilution series in ethanol absolute was performed. At a concentration of 50 mg/mL and 50 min at 37 °C, the test material was fully dissolved.
Based on these results, ethanol was used as solvent in the experiments. For the pre-experiment, a stock solution containing 50 mg/mL (± 10 %) of test material in ethanol absolute was prepared, corresponding to a maximum concentration of 250 μg/mL in the test. This stock solution was heated up to 37 °C for 50 min before it was used to prepare a geometric series of solutions in pre-warmed (37 °C) ethanol absolute. Due to the cytotoxic effects in the pre-experiment, the stock solutions (3.13 mg/mL) and the concentrations to be tested (highest one: 15.6 μg/mL) were adapted in experiment I and II. Five concentrations were used in each experimental part.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol for the test material, 0.9 % NaCl for the positive controls

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Colchicine: Without metabolic activation (Extended Exposure, Exp. II), at 0.035 μg/mL in 0.9 % NaCl

Details on test system and experimental conditions:

PRE-EXPERIMENT
Cytotoxicity is characterised by the cytokinesis-block proliferation index in comparison with the controls by counting 500 cells per culture in duplicate.
The experimental conditions in the pre-experiment were identical to those required and described for the genotoxicity assay; the cultures were set up in duplicates.
In the pre-experiment, 5 concentrations of the test item were used and tested with and without metabolic activation.
Exposure time was 4 h and culture harvest time was 23 h after start of exposure. Due to cytotoxicity (3 highest test item concentrations) and precipitates (all test item concentrations) on the slides, an evaluation according to the demands of OECD 487 (≥ 3 evaluable test item concentrations) was not possible. Therefore, Experiment I was performed with a different range of concentrations.

EXPERIMENT I
On the basis of the observations in the pre-experiment, 5 test material concentrations were used and tested in Experiment I without and with metabolic activation.
Exposure time was 4 h and the culture harvest time was 23 h after start of exposure. Since no cytotoxicity but precipitation on the slides of the 2 highest test item concentrations were observed, all concentrations used in the test were scored for the presence of micronuclei.
- Determination of Micronuclei
The following concentrations were selected for scoring of micronuclei:
With S9 mix: 15.6, 7.81, 3.91, 1.95 and 0.98 μg/mL.
Without S9 mix: 15.6, 7.81, 3.91, 1.95 and 0.98 μg/mL.
For each treatment, two cultures were evaluated, analysing at least 1 000 binucleated cells per culture.

EXPERIMENT II
In Experiment II, 5 concentrations of the test item were used and tested without metabolic activation.
Exposure time was 23 h for the test item concentrations and 23.5 h for the solvent and positive controls. Directly afterwards, preparation started. Since no cytotoxicity was observed and only the highest test item concentration showed slight precipitate on the slides, the 3 highest concentrations were selected for micronuclei scoring.
- Determination of Micronuclei
The following concentrations were selected for scoring of micronuclei: Without S9 mix 15.6, 7.81 and 3.91 μg/mL.
For each treatment, two cultures were evaluated, analysing at least 1 000 binucleated cells per culture.

CELL TREATMENT
- After the initial cell cultivation, duplicate cultures were prepared for each test group. After centrifugation (10 min, 500*g), the cells were resuspended in minimal culture medium and solvent control, positive control or the single test material concentrations were added. In the case of metabolic activation, 50 μL S9 mix per mL medium were used. The cell cultures were incubated at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2 for 4 h (exposure period).
- Contents of the cell cultures:
4 hour exposure with S9 mix: 8.5 mL minimal culture medium, 0.5 mL S9-Mix, 1 mL cultivated lymphocytes and 50 µL test material.
4 hour exposure without S9 mix: 9 mL minimal culture medium, 1 mL cultivated lymphocytes and 50 µL test material.
23.5/23 hour exposure without S9 mix: 9 mL complete culture medium, 1 mL cultivated lymphocytes, 50 µL test material and 50 µL cytochalasin B.
- After the exposure time of 4 h, the cells were spun down by gentle centrifugation for 5 min (500*g). The supernatant was discarded, the cells were re-suspended in 5 mL Saline G and centrifuged again. The washing procedure was repeated once as described.
- After washing, the cells were re-suspended in complete culture medium, cytochalasin B (final concentration 5 μg/mL) was added and the cells were incubated at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2 for 19 h until preparation.
- In Experiment II without metabolic activation, 73 h (controls) or 74 h (test material) after seeding, the blood cultures were centrifuged (10 min, 500*g). The cell pellet was resuspended in complete culture medium, cytochalasin B (final concentration 5 μg/mL) and solvent control, positive control and the single test material concentrations were added. The exposure duration was 23.5 h (controls) or 23 h (test material).

HARVESTING
- Each cell culture was harvested and processed separately. The cells were spun down by gentle centrifugation (10 min, 500*g). The supernatant was discarded and the cells were re-suspended in 12 mL hypotonic KCl solution. The cell suspension was allowed to stand for 10 min at room temperature (20 ± 5 °C). After removal of the hypotonic solution by centrifugation (10 min, 500*g), the cell pellet was fixed with a mixture of methanol and glacial acetic acid (3:1). After fixation at 2 – 8 °C for minimum 30 min, the cell suspension was spun down by gentle centrifugation (10 min, 500*g), the supernatant was discarded and the cell pellet was re-suspended in fixative again. The washing procedures were repeated until the cell pellet was white.
SLIDE PREPARATION
- The slides were prepared by dropping the cell suspension onto a clean microscope slide. The cells were then stained with a 10 % solution of Giemsa. All slides were independently coded before microscopic analysis.

DETERMINATION OF THE CYTOKINESIS-BLOCK PROLIFERATION INDEX
- In all replicates, the cytokinesis-block proliferation index (using at least 500 cells per culture) was determined in order to assess the cytotoxicity of the test material. From these determinations, the test material concentrations which were evaluated for scoring of micronuclei were defined.

DETERMINATION OF BINUCLEATED CELLS WITH MICRONUCLEI
- At least 1 000 binucleated cells per culture were scored for micronuclei. Only cells with sufficiently distinguishable cytoplasmic boundaries and clearly visible cytoplasm were included in the analysis.
- In Experiment II, the number of evaluated cells was doubled for the solvent control ethanol and for the test material concentration 15.6 μg/mL.

EVALUATION PROCEDURE
- Evaluation of the slides was performed using Zeiss microscopes with 40 x- and 100 x – oil immersion objectives. The generated data were recorded on raw data sheets. All calculations are performed with unrounded values. Therefore, re-calculation with rounded values may lead to slightly different results.
- Cytotoxicity Determination
The CBPI was calculated using the following equation:

CBPI = (MONC * 1 + BINC * 2 + MUNC * 3) / n

Where:
CBPI = Cytokinesis-block proliferation index
n = Total number of cells
MONC = Mononucleate cells
BINC = Binucleated cells
MUNC = Multinucleate cells

- Cytotoxicity was calculated as reduction in CBPI compared to the CBPI of the concurrent solvent control.

Cytotoxicity [%] = 100 – 100 * (CBPI test material / CBPI solvent control)

- The number of binucleated cells with and without micronuclei in each treatment group was compared with the solvent control value.

Evaluation criteria:

ACCEPTABILITY
The genotoxicity assay is considered acceptable if it meets the following criteria:
- All experimental conditions are tested (short exposure with and without metabolic activation, extended exposure without metabolic activation) unless a positive result is achieved in any experiment.
- In each experiment, an adequate number of cells is analysable both in the controls and in at least 3 test material concentrations.
- The micronucleus induction of the solvent and positive controls is compatible with the historical laboratory control data or the literature data.
- The positive control shows a statistically significant increase of binucleated cells with micronuclei compared with the concurrent solvent control.
- The criteria for cell proliferation and for the selection of concentrations are fulfilled.

CLASSIFICATION
The test material is considered to have no genotoxic effects if:
- Neither a statistically significant nor a concentration-related increase of the number of micronucleate cells in the evaluated test concentrations is observed.
- The obtained results lie within the range of the historical laboratory control data for solvent controls, considering also e.g. 95.5 % control limits where appropriate.

The test material is considered to have genotoxic effects if all of the following conditions are met:
- At least one test concentration shows a statistically significant increase of micronucleate cells compared to the concurrent solvent control.
- In at least one experimental condition a dose-related increase of micronucleate cells can be observed using trend analysis.
- Any of the results lies outside the range of the historical laboratory control data for solvent controls, considering also e.g. 95.5 % control limits where appropriate.

Statistics:

The number of binucleated cells with micronuclei in each treatment group was compared with the solvent control. Statistical significance was tested using Fisher’s exact test at the five per cent level (p <0.05) with the following equation:

φ(α) = [(a+b)!(c+d)!(a+c)!(b+d)!] / (n!a!b!c!d!)

Where:
a = number of binucleated cells with micronuclei of the solvent control
b = number of binucleated cells with micronuclei of the test material of the respective concentration
c = number of binucleated cells without micronuclei of the solvent control
d = number of binucleated cells without micronuclei of the test material of the respective concentration
n = total number of cells

For higher values of binucleated cells or binucleated cells with micronuclei, the chi-square test with the following equation was used.

χ^2 = [n · (a · d – c · b)^2] / (a + c) · (b + d) · (a + b) · (c + d)] a~ χ^2i

a = number of binucleated cells with micronuclei of the solvent control
b = number of binucleated cells with micronuclei of the test item of the respective concentration
c = number of binucleated cells without micronuclei of the solvent control
d = number of binucleated cells without micronuclei of the test item of the respective concentration
n = total number of cells

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRE-EXPERIMENT
- Since an evaluation according to the demands of OECD 487 was not possible, no genotoxicity test was performed. The concentrations used and tested in Experiment I were adjusted accordingly.

EXPERIMENT I
- In Experiment I without and with metabolic activation, no cytotoxic effect was observed at the tested concentrations.
- To avoid any influence of the precipitate on the outcome of the study, all 5 test material concentrations were scored for the presence of micronuclei.
- In the approach without metabolic activation, the test material concentration 15.6 μg/mL showed a statistically significant increased value of micronuclei (p = 0.032). In the experimental part with metabolic activation, the test material concentration 3.91 μg/mL also showed a statistically significant increased value of micronuclei (p = 0.024). However, both values lie inside the range of the historical laboratory control data for the solvent control ethanol, so these findings are considered as biologically irrelevant.

EXPERIMENT II
- In Experiment II, no cytotoxic effect was observed.
- The test material concentration 15.6 μg/mL showed a value of micronuclei lying above the historical control data for the solvent control ethanol but still inside the range of e.g. NaCl 0.9 %. Furthermore, this value was not statistically significantly increased compared with the concurrent solvent control ethanol. The results were confirmed by doubling the number of evaluated cells from ~ 2 000 to ~ 4 000 for the solvent control ethanol and the highest test material concentration. Overall, this finding is considered as biologically irrelevant.

Table 1: Experiment 1 Genotoxicity Results

Treatment	Mean CBPI	Cytotoxicity (%)	Total No. of BINC examined	Total No. of MBNC	% MBNC
Without metabolic activation
Solvent control Ethanol 0.5 % v/v	1.532	-	2006	5	0.25
Solvent control 0.9 % NaCl 0.5 % v/v	1.524	-	2006	6	0.30
Positive control MMC 0.3 μg/mL	1.321	13.3	2004	110	5.49**
Test material 15.6 μg/mL	1.511	1.4	2008	13	0.65*
Test material 7.81 μg/mL	1.496	2.4	2011	10	0.50
Test material 3.91 μg/mL	1.540	-0.5	2010	10	0.50
Test material 1.95 μg/mL	1.549	-1.1	2010	11	0.55
Test material 0.98 μg/mL	1.511	1.4	2006	6	0.30
With metabolic activation
Solvent control Ethanol 0.5 % v/v	1.589	-	2008	4	0.20
Solvent control 0.9 % NaCl 0.5 % v/v	1.503	-	2015	7	0.35
Positive control CPA 30 μg/mL	1.221	18.8	2018	42	2.08**
Test material 15.6 μg/mL	1.555	2.2	2013	7	0.35
Test material 7.81 μg/mL	1.557	2.0	2009	9	0.45
Test material 3.91 μg/mL	1.548	2.6	2000	12	0.60*
Test material 1.95 μg/mL	1.615	-1.6	2019	9	0.45
Test material 0.98 μg/mL	1.629	-2.5	2014	10	0.50

Asterisks indicate statistically significant differences to solvent control, with * p < 0.05, ** p < 0.01

 

Table 2: Experiment 2 Genotoxicity Results

Treatment	Mean CBPI	Cytotoxicity (%)	Total No. of BINC examined	Total No. of MBNC	% MBNC
Solvent control Ethanol 0.5 % v/v	1.710	-	4028	39	0.97
Solvent control 0.9 % NaCl 0.5 % v/v	1.702	-	2014	10	0.50
Positive control MMC 0.3 μg/mL	1.440	15.4	2042	151	7.39**
Positive control Colchicine 0.035 μg/mL	1.248	26.7	2016	106	5.26**
Test material 15.6 μg/mL	1.803	-5.4	4017	47	1.17
Test material 7.81 μg/mL	1.840	-7.6	2015	9	0.45
Test material 3.91 μg/mL	1.909	-11.6	2020	12	0.59

Asterisks indicate statistically significant differences to solvent control, with * p < 0.05, ** p < 0.01

 

Conclusions:

Under the conditions of this study, the test material did not show biologically relevant genotoxic activity for the induction of micronuclei.

Executive summary:

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 487 and EU Method B.49, under GLP conditions.

The study was performed to assess the genotoxic potential of the test material to induce micronuclei in human lymphocytes cultured in vitro in the absence and presence of an exogenous metabolic activation system (liver S9 mix from male rats, treated with Aroclor 1254).

The test material was dissolved in ethanol absolute and for the pre-experiment, a stock solution with a concentration of 50 mg/mL was prepared, corresponding to the highest concentration (250 μg/mL) in the pre-test. In addition, a geometric series of dilutions was prepared from the stock solution. Due to the cytotoxic effects in the pre-experiment, the stock solutions (3.13 mg/mL) and the concentrations to be tested (highest one: 15.6 μg/mL) were adapted in Experiment I and II.

Human peripheral blood lymphocytes, from a whole blood culture, were stimulated to divide by addition of phytohaemagglutinin and exposed to solvent control, test material and positive control.

After the culture harvest time, the cells were harvested and slides were prepared. Then, the proportion of cells containing micronuclei was determined.

Three independent and valid experiments were performed. In each experiment, all cell cultures were set up in duplicates. In order to assess the toxicity of the test material to cultivated human lymphocytes, the cytokinesis-block proliferation index (CBPI) was calculated for all cultures treated with solvent control, positive control and test material (pre-experiment: only solvent and positive controls). On the basis of the data of the cytokinesis-block proliferation index, the appropriate concentrations were selected for micronuclei scoring.

In the pre-experiment without and with metabolic activation, severe precipitation was observed on the slides at the 4 highest concentrations. Additionally, the 3 highest test material concentrations showed complete cytotoxicity, as far as could be determined due to the precipitate. Therefore, no genotoxicity test was performed, as an evaluation in accordance with OECD 487 was not possible (≥ 3 evaluable test material concentrations).

Due to these findings, Experiment I without and with metabolic activation and Experiment II (only without metabolic activation) were performed with lower test material concentrations.

The test material did not cause cytotoxicity at any of the evaluated test material concentrations.

In Experiment I without metabolic activation, the test material concentration 15.6 μg/mL showed a statistically significant increased value of micronuclei (p = 0.032). In Experiment I with metabolic activation, the test material concentration 3.91 μg/mL also showed a statistically significant increased value of micronuclei (p = 0.024). However, both values lie clearly within the range of the historical laboratory control data for the solvent control ethanol, so these findings are considered as biologically irrelevant.

In Experiment II, the test material concentration 15.6 μg/mL showed a value of micronuclei lying above the historical control data for the solvent control ethanol but still inside the range of e.g. the solvent control NaCl 0.9 %. Furthermore, this value was not statistically significantly increased compared with the concurrent solvent control ethanol. Therefore, this is also considered as biologically irrelevant.

All positive control compounds caused large, statistically significant increases in the proportion of binucleate cells with micronuclei, demonstrating the sensitivity of the test system.

Under the conditions of this study, the test material did not show biologically relevant genotoxic activity for the induction of micronuclei.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 November 2017 to 15 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test material was stored in the test facility at room temperature (20 ± 5 °C) protected from light.

Target gene:

The endogenous hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on chromosome X in Chinese Hamster cells (V79).

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: The cells were purchased from CLS (Eppelheim, Germany) and were sold under the name V79-4.
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years because of its sensitivity to chemical mutagens. Especially the high proliferation rate and a high cloning efficiency of untreated cells both necessary for the appropriate performance of the study, recommend the use of this cell line.
- Cell cycle length, doubling time or proliferation index: Doubling time 12 – 16 h in stock cultures.
- Methods for maintenance in cell culture if applicable: The cells were thawed 6 - 8 d prior treatment and cultivated in DMEM complete culture medium with 5 % HS in cell culture flasks at 37.0 ± 1.5 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.
- Modal number of chromosomes: The modal chromosome number was analysed and confirmed by the supplier of the cells.
- Normal (negative control) cell cycle time: The normal cell cycle time as well as the detection of the spontaneous mutant frequency of the used master cell stock is checked in each experiment.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Cultivated in DMEM complete culture medium with 5 % HS in cell culture flasks at 37.0 ± 1.5 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes. Cleansed and mycoplasma contamination screened stocks of cells were stored in liquid nitrogen in the cell bank of LAUS GmbH to allow a continuous working stock of cells, which guarantees similar parameters of the experiment and reproducible characteristics of the cells.
- Periodically checked for karyotype stability: The cells have a stable karyotype with a modal chromosome number of 22 (Bradley et al., 1981).
- Periodically 'cleansed' against high spontaneous background: Yes, and stored as described above.

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

Pre-Test I for Detection of Cytotoxicity (+S9 and -S9): 3.9, 7.8, 15.6, 31.3, 62.5, 125 and 250 µg/mL
Pre-Test II for Detection of Cytotoxicity (+S9): 0.24, 0.49, 0.98, 1.95, 3.91, 7.82 and 15.63 µg/mL
Pre-Test II for Detection of Cytotoxicity (-S9): 0.06, 0.12, 0.24, 0.49, 0.98, 1.95 and 3.91 µg/mL
Pre-Test III for Detection of Cytotoxicity (+S9 and -S9): 1.0, 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125 and 250 µg/mL

According to OECD 476, the highest concentration should be 0.01 M or 2 mg/mL or 2 μL/mL (whichever is the lowest), unless limited by the solubility or toxicity of the test material. Relative survival values below 20 % are considered toxic. For cytotoxic test materials the maximum concentrations should result in 10 to 20 % relative suspension growth (RSG) or relative total growth (RTG) and the analysed concentrations should cover a range from maximum to little or no cytotoxicity. According to the results of the pre-tests, 6 concentrations were chosen for the main experiment and tested with and without metabolic activation

Experiment I (+S9): 0.03, 0.06, 0.12, 0.24, 0.49 and 0.98 µg/mL
Experiment I (-S9): 0.98, 1.95, 3.91, 7.81, 15.63 and 31.25 µg/mL

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 determined in demineralised water, ethanol and dimethyl sulfoxide (DMSO). The test material is insoluble in demineralised water and DMSO. The best result was obtained in ethanol absolute. Nevertheless, the test material was also insoluble in this solvent at the required concentration of 1 g/mL. Therefore, a dilution series in ethanol absolute was performed. At a concentration of 50 mg/mL and 50 min at 37 °C, the test material was fully soluble. Based on these results, ethanol was used as the solvent in the experiments.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMEM without supplements for the positive control Ethylmethane sulfonate (EMS), DMSO for the positive control 7,12-dimethylbenz(a)anthracene (DMBA) and Ethanol absolute for the test material.

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

PREPARATION OF THE TEST SOLUTIONS
- Preparation was the same for pre-tests I and II. On the first day of the pre-tests, a stock solution containing of test material in ethanol absolute (pre-test I: 50 mg/mL, pre-test II: 3.13 mg/mL) was prepared. This stock solution was heated up to 37 °C for 50 min before it was used to prepare a geometric series of solutions in pre-warmed (37 °C) ethanol absolute. In pre-test III 9 stock solutions containing the test material in ethanol absolute were prepared and heated up to 37 °C for 50 min before they were used. The concentrations of the stock solutions were: 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39 and 0.20 mg/mL.
- Because of the low solubility of the test material for experiment I two stock solutions containing the test material in ethanol absolute were prepared (6.25 mg/mL and 3.125 mg/mL) and heated up to 37 °C for 50 min. The stock solution with the concentration 6.25 mg/mL was used directly in the experiment. The second stock solution (3.125 mg/mL) was used to prepare a geometric series of solutions in pre-warmed (37 °C) ethanol absolute.
- Osmolality and pH Values: The osmolality and the pH values of the solvent controls, the positive controls as well as the test material concentrations (in DMEM medium with 5 % horse serum (HS)) that were used in the pre-test were determined to exclude a negative influence on the assay by those parameters. The osmolality was determined using an osmometer and the pH value was determined using a pH meter. None of the tested positive controls or test material concentrations provoked a critical change of the osmolality and the pH value in comparison to the solvent controls. Therefore, a negative influence of these parameters on the assay can be excluded.


PERFORMANCE OF THE STUDY

Pre-Test for Detection of Cytotoxicity
- Because of the solubility of the test material, in total three pre-tests had to be performed in order to determine the concentration range applicable for mutagenicity experiments. As no CE II value was determined, the evaluation was done on CE I values. In respect of the pre-test the cytotoxicity was determined by measuring the survival of the cells after exposure to the test material (CE I). The survival is given as the cloning efficiency (CE) of treated cells in comparison to the controls. In context of the pre-test a clear reduction of the CE values below 20 % is regarded as cytotoxic.
- In the pre-tests, 7 concentrations of the test material were used and tested with and without metabolic activation, the exposure time was 4 hours.

Determination of Survival by Cloning Efficiency
- 500 cells were exposed to each concentration of the test material for 4 hours with and without metabolic activation (duplicate cultures per concentration and approach). Following treatment, the cells were washed twice with PBS Dulbecco (2.5 % HS). After an expression period of 7 d the cells were stained with methylene blue. Afterwards the colonies were counted and the absolute and the relative cloning efficiency values were calculated.

Experimental Performance
- The first experiment I was invalid and is not reported. All documentation is kept with the raw data.
- Experiment I was conducted with and without metabolic activation. The test material was incubated for 4 hours (with and without S9) and the cells were afterwards washed twice with PBS Dulbecco (2.5 % HS).
- First 1 * 10^6 cells per 10 cm culture dish and 500 cells (for the determination of the cytotoxicity) per 6 cm culture dish were seeded per tested concentration as well as for the solvent and positive controls and incubated for 23 hours and 45 min. The incubation conditions during the whole assay were 37.0 ± 1.5 °C in 5.0 ± 0.5 % CO2.
- The cells were treated with the test material after incubation. Each concentration was prepared in duplicate. Directly following to the treatment period the cells were washed with PBS Dulbecco (2.5 % HS) twice. Fresh complete culture medium (5 % HS) was added to the cells before the following incubation. The further implementation of the experiment was divided into the determination of the survival as well as the viability and the mutagenicity.
- For the determination of a cytotoxic effect of the test material, the survival of the cells was measured. For this purpose, the cells in the 6 cm dishes were stained with 0.1 % Löffler’s methylene blue solution in 0.01 % KOH solution after a 7 day-incubation time. The colonies were counted and the cloning efficiency (absolute and relative) was calculated.
- For the determination of the second part of the experiment (viability and mutagenicity), the cells in the 10 cm culture dishes were counted and adjusted to 1 * 10^6 cells per 10 cm culture dish after an incubation time of 68 hours + 30 min and afterwards further incubated. After a total expression time of at least 168 h, the cells were counted again and seeded into 10 cm culture dishes (5 * 10^5 ± 10^4 cells) for the evaluation of the mutagenicity in selection medium containing 6-TG (final concentration: 2 μg/mL) and into 6 cm culture dishes (500 ± 10 cells) for the evaluation of the viability in complete culture medium. Both plates were incubated for a further 7 days. After this incubation time the cell colonies were stained with 0.1 % Löffler’s methylene blue solution in 0.01 % KOH solution and counted for the calculation of cloning efficiency II and mutant frequency.

CALCULATIONS
Pre-Test (Cytotoxicity) directly after treatment:

Cloning efficiency absolute (CEabs), at the end of treatment = total number of colonies / total number of cells seeded

Cloning efficiency relative (CErel) = CEabs / CEabs of the corresponding solvent control * 100 %


Experiment I and II:

Cloning efficiency I absolute (CE Iabs), directly after treatment = number of cells at the end of treatment† / total number of cells at the beginning of treatment ††

Cloning efficiency I relative (CE Irel), at the end of treatment = CE Iabs / CE Iabs of the corresponding solvent control * 100 %

Cloning efficiency II absolute (CE IIabs), after expression time = total number of colonies / total number of seeded cells

Cloning efficiency II relative (CE IIrel), at the end of treatment = CE IIabs / CE IIabs of the corresponding solvent control * 100 %

Adjusted CE = CE IIabs * CE Iabs

Relative survival (RS) = Adjusted CE in treated culture / Adjusted CE in corresponding solvent control * 100 %

Cloning efficiency mutants (CEMut) = total number of mutant colonies found after plating in 6-TG medium / total number of plated cells in 6-TG medium

Mutant frequency (MF) = CEMut / CE in non-selective medium (=^CE IIabs)

† corresponds to the number of colonies after 7 days.
†† corresponds to the number of cells plated for treatment.

Note 1: The calculation of the relative cloning efficiency values (CE Irel and CE IIrel) is not required by the OECD 476 but is determined for better traceability of the results.
Note 2: Since there is no designation for the calculation “number of cells at the end of treatment+ / total number of cells at the beginning of treatment” in the OECD 476, this is defined as CE I in this study. The CE from the OECD 476 corresponds to the CE II in this study. This renaming has no influence on the calculation and is just for better traceability
Note 3: No calculation of the relative values (CE Irel, CE IIrel, and RS) was performed of the solvent controls. Since all relative values always refer to the respective solvent control, these values are always displayed as 100 %.

Evaluation criteria:

Providing that the study is acceptable:
A test material is considered to be clearly positive if in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
- the increase is concentration-related when evaluated with an appropriate trend test
- any of the results are outside the range of the historical laboratory control data for solvent controls, considering also e.g. 95.5 % control limits where appropriate.
When all of these criteria are met, the test material is then considered able to induce gene mutations in cultured mammalian cells in this test system.

A test material is considered clearly negative if in all experimental conditions examined:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
- there is no concentration-related increase when evaluated with an appropriate trend test
- the obtained results lie within the range of the historical laboratory control data for solvent controls, considering also e.g. 95.5 % control limits where the test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory’s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study is also taken into consideration. In cases when the response is neither clearly negative nor clearly positive as described above, or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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:

PRE-TESTS FOR DETECTION OF CYTOTOXICITY
- Pre-Test I: In the approach with metabolic activation, cytotoxic effects were determined at the concentrations 250 μg/mL to 15.63 μg/mL. At the two lowest test material concentrations the viability values were only slightly above the threshold of 20 % (23.8 % and 25.8 %). Precipitations were observed at the concentrations 250 μg/mL and 125 μg/mL. Turbidity was observed at the concentrations 250 μg/mL to 15.63 μg/mL. In the approach without metabolic activation cytotoxicity was detected in all tested concentrations. Precipitation and turbidity was observed at the concentrations 250 μg/mL and 125 μg/mL. Because of the strong cytotoxic effect in pre-test I, a second pre-test with lower test material concentrations was performed.
- Pre-Test II: In both approaches, no cytotoxic effects or precipitation/turbidity were observed in any of the tested concentrations. Since the results of pre-test I and pre-test II are contradictory, a third pre-test was performed. The different results between pre-test I and II are most probably due to the low solubility of the test material. Therefore in pre-test III no dilution series was prepared. For each concentration a single stock solution was prepared.
- Pre-test III: In the approach with metabolic activation, cytotoxic effects were determined at the concentrations 250 μg/mL to 31.3 μg/mL. In all lower test material concentrations the viability was not reduced below 69 %. Precipitations were observed at the concentrations 250 μg/mL and 125 μg/mL. Turbidity was observed in all tested concentrations. In the approach without metabolic activation, cytotoxic effects were determined at the concentrations 250 μg/mL to 31.3 μg/mL. At the test material concentration 15.6 μg/mL the viability was reduced to 20.3 % and therefore only very slightly above the threshold of 20 %. Even at the next lower test material concentration the viability was reduced to 33.2 %. Only in the three lowest test material concentrations the viability was above 51 %. Precipitations were observed at the concentrations 250 μg/mL and 125 μg/mL. Turbidity was observed at the concentrations 250 μg/mL to 62.5 μg/mL.
- Conclusion: In conclusion it can be stated that the test material induced a strong cytotoxic effect in both approaches and the solubility was also a limiting factor. Since in pre-test III turbidity was detected in all tested concentrations in the approach with metabolic activation, 0.98 μg/mL (lowest test material concentration in this approach) was chosen as highest test material concentration for experiment I +S9. Since a cytotoxic effect (without precipitation and/or turbidity) was detected in pre-test III in the four highest test material concentrations, 31.25 μg/mL (lowest test material concentration with a cytotoxic effect) was chosen as highest test material concentration for experiment I -S9.

EXPERIMENT I RESULTS AND DISCUSSION
- Since the result of experiment I was clearly negative, no second experiment (experiment II) was performed.
- In the approach with metabolic activation turbidity of the test material was observed at the highest test material concentration. In the approach without metabolic activation no precipitation/ turbidity was observed up to the highest tested concentration.
- EMS (experiment I: 300 μg/mL) and DMBA (1.5 μg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies but without reduction of the viability.
- In experiment I no cytotoxic effect was observed in any of the tested concentrations in the approach with metabolic activation. In the approach without metabolic activation cytotoxicity was only observed at the highest test material concentrations (relative survival: 0 %). Since the OECD guideline 476 indicates that concentrations that showed a cytotoxic effect or a strong reduction in viability cannot be analysed for mutagenicity, an evaluation of the mutagenicity was not possible in this concentration. However, since only 4 analysable concentrations a required by the OECD guideline 476, the study is still valid.
- No relevant increase in mutant colony numbers/10^6 cells was observed in the main experiment up to the maximum concentration. All mutant frequencies remained well within the historical range of solvent controls. None of the values was statistically significantly increased in comparison to the solvent control. In addition, no dose dependent effect was observed. Therefore the result of experiment I is clearly negative.
- In conclusion, it can be stated that under the experimental conditions reported, the test material did not induce mutations in the HPRT locus using the V79 cell line in the absence and the presence of metabolic activation.

ACCEPTABILITY OF THE TEST
- All acceptability criteria except the criteria for the selection of top concentration were met. Since the deviation of the acceptance criteria was declared as uncritical, the study is considered as acceptable.

Table 1: Summary of Results of Experiment I

Treatment (concentration in µg/mL)	S9 Mix	Culture	Relative survival (%)	Mutant frequency per 10^6 cells
					Mean
Test material solvent control	+	A	-	14	17
		B	-	20
DMBA solvent control	+	A	-	19	19
		B	-	20
DMBA (1.5)	+	A	70.7	192	191**
		B	99.0	189
Test material (0.98† )	+	A	78.8	12	10
		B	127.6	7
Test material (0.49)	+	A	84.7	15	17
		B	155.1	20
Test material (0.24)	+	A	100.8	13	15
		B	172.9	17
Test material (0.12)	+	A	102.9	14	10
		B	145.3	6
Test material (0.06)	+	A	110.2	20	14
		B	143.4	7
Test material (0.03)	+	A	123.1	10	12
		B	146.2	14
Test material solvent control	-	A	-	14	15
		B	-	16
EMS solvent control	-	A	-	20	14
		B	-	8
EMS (300)	-	A	95.6	184	147**
		B	77.5	111
Test material (31.25)	-	A	0.0	N/A	N/A
		B	0.0	N/A
Test material (15.63)	-	A	44.7	19	17
		B	30.1	15
Test material (7.81)	-	A	110.6	9	8
		B	65.1	7
Test material (3.91)	-	A	130.9	8	6
		B	72.3	4
Test material (1.95)	-	A	100.0	16	11
		B	64.0	6
Test material (0.98)	-	A	127.1	12	13
		B	101.3	14

n/a = not analysed for mutagenicity because of cytotoxicity

† = turbidity was observed at the end of treatment

Asterisks indicate statistically significant differences to solvent control, with ** p < 0.01

Conclusions:

Under the conditions of this study, the test material was non mutagenic.

Executive summary:

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 476 and EU Method B.17, under GLP conditions.

This study was performed to investigate the potential of the test material to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese Hamster cells (V79). The assay comprised 4 pre-tests (pre-test no. 2 was invalid) and two independent experiments I (first experiment I was invalid). Based on the results of the valid pre-tests the concentrations for the main experiment were determined. The invalid pre-test and first experiment I are not reported, all documentation is kept with the raw data and will be archived at the test facility.

Experiment I was performed with and without metabolic activation (liver S9 mix from male rats, treated with Aroclor 1254) and a treatment period of 4 h. Due to the clearly negative result in Experiment I, no second experiment (Experiment II) was performed.

The highest nominal concentration (approach with metabolic activation: 0.98 μg/mL, approach without metabolic activation: 31.25 μg/mL) applied was chosen with regard to the solubility of the test material in organic solvents and aqueous media, turbidity in the approaches as well as the results of the pre-tests.

Ethylmethane sulfonate (EMS) and 7,12-Dimethylbenzanthracene (DMBA) as appropriate reference mutagens were used as positive controls. Both induced a distinct increase in mutant colonies and thus, showed enough sensitivity of the testing procedure and the activity of the metabolic activation system.

No substantial and reproducible dose dependent increase in mutant colony numbers was observed up to the maximal concentration of the test material. The result of experiment I was clearly negative according to OECD 476. The test material did not induce gene mutations at the HPRT locus in V79 cells in the absence and presence of metabolic activation.

Under the conditions of this study, the test material was non mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames, Andres (2018)

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. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

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.

In Vitro Mammalian Cell Micronucleus Test, Frühmesser (2018)

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 487 and EU Method B.49, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The study was performed to assess the genotoxic potential of the test material to induce micronuclei in human lymphocytes cultured in vitro in the absence and presence of an exogenous metabolic activation system (liver S9 mix from male rats, treated with Aroclor 1254).

The test material was dissolved in ethanol absolute and for the pre-experiment, a stock solution with a concentration of 50 mg/mL was prepared, corresponding to the highest concentration (250 μg/mL) in the pre-test. In addition, a geometric series of dilutions was prepared from the stock solution. Due to the cytotoxic effects in the pre-experiment, the stock solutions (3.13 mg/mL) and the concentrations to be tested (highest one: 15.6 μg/mL) were adapted in Experiment I and II.

Human peripheral blood lymphocytes, from a whole blood culture, were stimulated to divide by addition of phytohaemagglutinin and exposed to solvent control, test material and positive control.

After the culture harvest time, the cells were harvested and slides were prepared. Then, the proportion of cells containing micronuclei was determined.

Three independent and valid experiments were performed. In each experiment, all cell cultures were set up in duplicates. In order to assess the toxicity of the test material to cultivated human lymphocytes, the cytokinesis-block proliferation index (CBPI) was calculated for all cultures treated with solvent control, positive control and test material (pre-experiment: only solvent and positive controls). On the basis of the data of the cytokinesis-block proliferation index, the appropriate concentrations were selected for micronuclei scoring.

In the pre-experiment without and with metabolic activation, severe precipitation was observed on the slides at the 4 highest concentrations. Additionally, the 3 highest test material concentrations showed complete cytotoxicity, as far as could be determined due to the precipitate. Therefore, no genotoxicity test was performed, as an evaluation in accordance with OECD 487 was not possible (≥ 3 evaluable test material concentrations).

Due to these findings, Experiment I without and with metabolic activation and Experiment II (only without metabolic activation) were performed with lower test material concentrations.

The test material did not cause cytotoxicity at any of the evaluated test material concentrations.

In Experiment I without metabolic activation, the test material concentration 15.6 μg/mL showed a statistically significant increased value of micronuclei (p = 0.032). In Experiment I with metabolic activation, the test material concentration 3.91 μg/mL also showed a statistically significant increased value of micronuclei (p = 0.024). However, both values lie clearly within the range of the historical laboratory control data for the solvent control ethanol, so these findings are considered as biologically irrelevant.

In Experiment II, the test material concentration 15.6 μg/mL showed a value of micronuclei lying above the historical control data for the solvent control ethanol but still inside the range of e.g. the solvent control NaCl 0.9 %. Furthermore, this value was not statistically significantly increased compared with the concurrent solvent control ethanol. Therefore, this is also considered as biologically irrelevant.

All positive control compounds caused large, statistically significant increases in the proportion of binucleate cells with micronuclei, demonstrating the sensitivity of the test system.

Under the conditions of this study, the test material did not show biologically relevant genotoxic activity for the induction of micronuclei.

Mammalian Cell Gene Mutation Test, Frühmesser (2018)

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 476 and EU Method B.17, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

This study was performed to investigate the potential of the test material to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese Hamster cells (V79). The assay comprised 4 pre-tests (pre-test no. 2 was invalid) and two independent experiments I (first experiment I was invalid). Based on the results of the valid pre-tests the concentrations for the main experiment were determined. The invalid pre-test and first experiment I are not reported, all documentation is kept with the raw data and will be archived at the test facility.

Experiment I was performed with and without metabolic activation (liver S9 mix from male rats, treated with Aroclor 1254) and a treatment period of 4 h. Due to the clearly negative result in Experiment I, no second experiment (Experiment II) was performed.

The highest nominal concentration (approach with metabolic activation: 0.98 μg/mL, approach without metabolic activation: 31.25 μg/mL) applied was chosen with regard to the solubility of the test material in organic solvents and aqueous media, turbidity in the approaches as well as the results of the pre-tests.

Ethylmethane sulfonate (EMS) and 7,12-Dimethylbenzanthracene (DMBA) as appropriate reference mutagens were used as positive controls. Both induced a distinct increase in mutant colonies and thus, showed enough sensitivity of the testing procedure and the activity of the metabolic activation system.

No substantial and reproducible dose dependent increase in mutant colony numbers was observed up to the maximal concentration of the test material. The result of experiment I was clearly negative according to OECD 476. The test material did not induce gene mutations at the HPRT locus in V79 cells in the absence and presence of metabolic activation.

Under the conditions of this study, the test material was non mutagenic.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.