Aim, material and method – Mutagenic potential – STABILGREEN® K
Aim
Cited from OECD guideline 471:
“The bacterial reverse mutation test uses amino-acid requiring strains of Salmonella typhimurium to detect point mutations, which involve substitution, addition or deletion of one or a few DNA base pairs. The principle of this bacterial reverse mutation test is that it detects mutations which revert mutations present in the test strains and restore the functional capability of the bacteria to synthesize an essential amino acid.”
The bacterial reverse mutation test is commonly employed as an initial screen for genotoxic activity and, in particular, for point mutation-inducing activity.
Principle of the test method: Suspensions of bacterial cells are exposed to the test substance in the presence and in the absence of an exogenous metabolic activation system. In the plate incorporation method, these suspensions are mixed with an overlay agar and plated immediately onto minimal medium. In the pre-incubation method, the treatment mixture is incubated and then mixed with an overlay agar before plating onto minimal medium. For both techniques, after 2 or 3 days of incubation, revertant colonies are counted and compared to the number of spontaneous revertant colonies on solvent control plates.
This study was performed in order to evaluate the mutagenic potential of STABILGREEN®K (Polyglyceryl-4 Oleyl Ether, Dioleyl Phosphate) in the Bacterial Reverse Mutation Test using five strains of Salmonella typhimurium.
MATERIALS AND METHODS
Test Item
|
Designation in Test Facility: |
15102801G |
|
Date of Receipt: |
28 January 2020 |
|
Condition at Receipt |
Room temperature, in proper conditions |
Specification
The following information concerning identity and composition of the test item was provided by the sponsor.
|
Name |
STABILGREENK (Polyglyceryl-4 Oleyl Ether, Dioleyl Phosphate) |
|
Batch no.: |
0136CL |
|
Appearance: |
AMBER liquid |
|
Composition: |
Liquid ester of Polyglyceryl-3 and olive fatty acids which has undergone a partial phosphatization |
|
Purity: |
Not applicable, mixture of olive oil fatty acids esters |
|
Homogeneity: |
HOMOGENEOUS |
|
Expiry date: |
31. Jul. 2019 |
|
Storage: |
Room Temperature (20 ± 5°C) |
The following additional information is relevant to the conduct of the study, according to OECD 471:
Stability: H2O: 96h; EtOH: 96h; acetone: 96h; CH3CN: 96h; DMSO: 96h
Solubility: H2O: <0.1g/L; EtOH: >1 g/L; acetone: >1 g/L; CH3CN: >1g/L; DMSO: >1g/L
It is provided by the sponsor as well.
Storage
The test item was stored in the test facility in a closed vessel at room temperature (20±5°C).
Preparation
In a non-GLP pre-test, the solubility of the test item was tested in a concentration of 50 g/L in demineralized H2O, dimethyl sulfoxide (DMSO) and ethanol.
On the day of the start of the first experiment, a stock solution containing 50 g/L of the test item in DMSO was prepared. The test item solution was not sterile filtrated before use.
DMSO was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertant in the tested concentrations.
The stock solution was used to prepare the geometric series of the concentrations to be tested. The following nominal concentrations were prepared for the first experiment:
5000 μg/plate, 1500 μg/plate, 500 μg/plate, 150 μg/plate and 50 μg/plate.
On the day of the start of the second experiment, a stock solution containing 50 g/L of the test item in DMSO was prepared.
The following nominal concentrations were prepared for the second experiment:
5000 μg/plate, 2500 μg/plate, 1250 μg/plate, 625 μg/plate, 313 μg/plate and 156 μg/plate.
Each solution was used for the experiments within 2 h.
Positive Controls
The following mutagenic substances were used as positive controls in both experiments:
| 4-Nitro-1,2-phenylene diamine, C6H7N3O2; CAS-No.: 99-56-9 | |
| Concentration per plate: | 20 μg |
| Solvent: | DMSO |
| Strains: | TA97a, TA98 and TA102 |
| Metabolic activation: | none |
| Sodium azide, NaN3; CAS-No.: 26628-22-8 | |
| Concentration per plate: | 1 μg |
| Solvent: | H2O |
| Strains: | TA100 and TA1535 |
| Metabolic activation: | none |
| 2-Amino-anthracene, C14H11N; CAS-No.: 613-13-8 | |
| Concentration per plate: | 1 μg |
| Solvent: | DMSO |
| Strain: | TA97a, TA100, TA102 and TA1535. |
| Metabolic activation: | S9 |
| Benzo-a-pyrene, C20H12; CAS-No.: 50-32-8 | |
| Concentration per plate: | 20 μg |
| Solvent: | DMSO |
| Strain: | TA98 |
| Metabolic activation: | S9 |
Test System
Specification
Species: Salmonella typhimurium LT2
Strains: TA97a, TA98, TA100, TA102 and TA1535
Mutations of the strains are listed in the following table:
Table Mutation Details
|
Mutation |
present in strain |
||||||
|
Name |
Category |
Effect |
TA97a |
TA98 |
TA100 |
TAI 02 |
TAI 535 |
|
hisD6610 |
frame shift |
histidine deficiency |
x |
|
|
|
|
|
hisD3052 |
frame shift |
histidine deficiency |
x |
|
|
|
|
|
hisG46 |
base pair substitution |
histidine deficiency |
|
x |
|
x |
|
|
hisG428 |
base pair substitution |
histidine deficiency |
|
|
x |
|
|
|
uvrB |
deletion |
UV sensitivity, biotine deficiency |
x |
x |
x |
|
x |
|
rfa |
deletion |
lipopolysaccharide side chain deficiency |
x |
x |
x |
x |
x |
|
pKM101 |
plasmide |
ampicillin resistance |
x |
x |
x |
x |
|
|
pAQ1 |
plasmide |
tetracyclin resistance |
|
|
x |
|
|
Origin and Culture
Salmonella typhimurium (all strains used) were obtained from TRINOVA BioChem (batch of the bacteria strains: TA97a: 4987D, TA98: 5011D, TA100: 4996D, TA102: 4982D, TA1535: 5012D) and were stored as lyophilizates in the fridge at 2-8 °C.
8 h 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 8 h at 37 ± 1 °C, the cultures were used in the experiment. During the test, the cultures were stored at room temperature as to prevent changes in the titre.
Chemicals
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. The weights may differ from the theoretical value (difference <10 %).
Nutrient Broth for Overnight Culture
|
Nutrient broth Merck 5443 |
2.8 g ad |
|
H2O demineralised |
350 mL |
Isotonic Sodium Chloride Solution for Dilution Purposes
|
Sodium chloride |
0.9 g |
|
H2O demineralised |
ad 100 mL |
Vogel-Bonner-Medium 20fold
|
Magnesium sulphate (MgSO4*7H2O) |
4.0 g |
|
Citric acid mono hydrate (MR210.14 g/mol) |
40.0 g |
|
Potassium phosphate, dibasic (anhydrous) (K2HPO4) |
200.0 g |
|
Sodium ammonium phosphate, monobasic, tetra hydrate (Na(NH4)HPO4*4H2O) |
70.0 g |
|
H2O demineralised |
ad 1000.0 mL |
Glucose Solution 40%
|
Glucose monohydrate (MR 198.17g/mol) |
440.0 g |
|
H2O demineralised |
ad 1000.0 mL |
Minimal Glucose Agar
|
Vogel-Bonner-Solution 20fold |
500.0 mL |
|
Glucose solution 40% |
500.0 mL |
|
H2O demineralised |
9000.0 mL |
|
Agar |
150.0 g |
Biotin Agar
|
Minimal-Glucose-Agar. 80 °C Biotin solution 0.5 mM |
500.0 mL |
|
Biotin solution 0.5 mM |
3.0 mL |
Histidine-Biotin-Agar
|
Biotin-Agar, 80 °C |
350.0 mL |
|
Histidine solution 0.5% |
3.5 mL |
Ampicillin-Agar
|
Histidine-biotin agar, 80 °C |
200.0 mL |
|
Ampicillin solution 0.8% |
0.6 mL |
Ampicillin-Tetracycline Plates
|
Ampicillin agar, 80 °C |
50.0 mL |
|
Tetracycline solution 0.8% |
0.01 mL |
Nutrient Agar Plates
|
Nutrient broth Merck 5443 Sodium chloride (NaCI) Agar |
0.8 g |
|
Sodium chloride (NaCI) |
0.5 g |
|
Agar |
1.52 g |
|
H2O demineralised |
100 mL |
Basis for Top-Agar and Maximal-Soft-Agar
|
Agar |
6g |
|
Sodium chloride (NaCI) |
5g |
|
H2O demineralised |
ad 1000 mL |
Histidine-Biotin-Solution 0.5 mM/0.5 mM (Use: Top Agar)
|
D-Biotin (MR 244.3 g/mol) |
12.2 mg |
|
L-Histidine* HCI*1H2O (MR 209.7 g/mol) |
10.5 mg |
|
H2O demineralised 90°C |
100.0 mL |
To 100 mL basis (see 6.4.11), 10 mL histidine-biotin-solution 0.5 mM/0.5 mM were added.
Histidine-Biotin-Solution 5 mM/ 0.5 mM (Use: Maximal-Soft-Agar)
|
D-Biotin (MR 244.3 g/mol) |
12.2 mg |
|
L-Histidine* HCI*1H2O (MR 209.7 g/mol) |
105 mg |
|
H2O demineralised 90°C |
ad 100.0 mL |
To 100 mL basis (see 6.4.11), 10 mL histidine-biotin-solution 5 mM/0.5 mM were added.
Phosphate Buffer
|
Sodium di-hydrogen phosphate monohydrate NaH2PO4*H2O |
0.184 g |
|
Di-sodium hydrogen phosphate dihydrate Na2HPO4*2H2O H2O demineralised |
1.722 g |
|
H2O demineralised |
ad 100.0 mL |
The pH of the solution was adjusted to 7.4.
Salt Solution for S9-Mix
|
Potassium chloride (KCI) |
1.23 g |
|
Magnesium chloride hexahydrate MgCI2*6H2O |
0.814 g |
|
H2O demineralised |
ad 10.0 mL |
NADP-Solution for S9-Mix, 0.1 M
|
NADP disodium salt (MR = 787.4 g/mol) |
787.4 MG |
|
H2O demineralised |
10 mL |
Glucose-6-Phosphate (G6P) Solution for S9-Mix, 1 M
|
Glucose-6-phosphate (MR = 340.13 g/mol) |
680.3 mg |
|
H2O demineralised |
ad 3 mL |
S9-Mix
|
Phosphate buffer |
22.5 mL |
|
0.1 M NADP-solution |
1.0 mL |
|
1 M G6P-solution |
0.125 mL |
|
Salt solution |
0.5 mL |
|
Rat liver S9 |
1.0 mL |
S9: was obtained by Trinova Biochem. Gießen.
Batch nos.: 3509, 3425, 3449
Specification: produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intraperitoneally.
Test Vessels
All vessels used are made of glass or sterilizable plastic. They were sterilized before use by autoclaving.
The following vessels were used:
- Schott-bottles, glass vials, and culture flasks for solutions and media
- Plastic petri plates
- test tubes for top-agar-bacteria-substance mix
Instruments and Devices
The following instruments and devices were used in the performance of the study.
- Autoclave 3870 ELV-B
- Precision scales Mettler Toledo PB 5001-SO2
- Precision scales Mettler Toledo XS 6001S
- Analytical scales Mettler Toledo XS 205 DU
- Analytical scales Mettler Toledo AB 184 SA
- Incubation chambers Memmert, adjustable to 37 °C
- Table water bath neoLAB, adjustable 43°C
- deep freezer
- Orbital shakers GFL 3005
- Piston-driven pipettes with sterile tips
- Pipetting device Accu Jet
- Repeater pipette Auto Rep S
- pH-meter 340i wtw
- Glass thermometer
- T ally counters
- Microwave
- fridge and freezer
- stop watch
- tweezer
Standard laboratory material (glassware) and equipment was also used.
Usage and, if applicable, calibration of all instruments following the corresponding SOP in the current edition.
PERFORMANCE OF THE STUDY
Culture of Bacteria
8 h before the start of each experiment, one vial permanent culture of each strain was taken from the deep freezer and an aliquot was put into a culture flask containing nutrient broth. After incubation for 8 h at 37 ±1 °C, the cultures were used in the experiment. During the test, the cultures were stored at room temperature as to prevent changes in the titre.
Conduct of Experiment
Preparations
In the days before each test (exact production dates are documented in the raw data), the media and solutions were prepared.
On the day of the test, the bacteria cultures were checked for growth. 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.
Experimental Parameters
First Experiment
|
Date of treatment |
02 February 2020 |
|
Concentrations tested |
5000 /1500 / 500 /150/50 μg/plate |
|
Incubation time |
48 h |
|
Incubation temperature |
37 ±1 °C |
|
Tested strains |
TA97a, TA98, TA100, TA102, TA1535 |
|
Method |
plate incorporation method |
Second Experiment
|
Date of treatment |
05 February 2020 |
|
Concentrations tested |
5000 / 2500 /1250 / 625 / 313 μg/plate |
|
Incubation time |
48 h |
|
Incubation temperature |
37 ±1 °C |
|
Tested strains |
TA97a, TA98, TA100, TA102, TA1535 |
|
Method |
pre-incubation method |
Description of the Method
General preparation
Per strain and dose, 3 plates with and 3 plates without S9 mix were used.
The test item solutions were prepared according to chapter 6.1.3.
Top agar basis was melted in a microwave oven, after melting, 10 mL of histidine-biotin- solution 0.5 mM per 100 mL basis was added and the bottle 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 mu tagen solution (positive control)
- 500 μL S9 mix (see chapter 6.4.18, for test with metabolic activation) or phosphate buffer (for test without metabolic activation).
- 100 μL bacteria suspension (see chapter 6.3.2, test system, culture of the strains)
- 2000 μL overlay agar (top agar)
The plates were closed and left to harden 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 min:
- 100 μL test suspension at each dose level, solvent (negative control) or reference mutagen solution (positive control)
- 500 μL S9 mix (see chapter 6.4.18, for test with metabolic activation) or phosphate buffer (for test without metabolic activation).
- 100 μL bacteria suspension (see chapter 6.3.2, test system, culture of the strains) After pre-incubation, 2000 μL overlay agar (top agar) was added, the tube was gently vortexed and the mixture was poured onto the selective agar plate.
The plates were closed and left to harden for a few minutes, then inverted and placed in the dark incubator at 37 ±1 °C.
Evaluation
The colonies were counted visually and the numbers were recorded. A validated spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertant divided by mean spontaneous revertant) of the test item solutions and the positive controls. Additionally, the absolute number of revertant (Rev. Abs.) (mean revertant minus mean spontaneous revertant) was given.
A substance is considered to have mutagenic potential, if a reproducible increase of revertant 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.