Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid: viscous

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 97
Remarks:
a
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 102
Species / strain / cell type:
S. typhimurium TA 1535
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 mix:
22.5 mL phosphate buffer, 1.0 mL 0.1M NADP-solution, 0.125 mL 1M G6P-solution, 0.5 mL salt solution, 1.0 mL Rat liver S9.
S9 batch numbers 4008 and 4052 from Trinova Biochem GmbH, Giessen; produced from livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg bw intraperitoneally.
Test concentrations with justification for top dose:
Experiment I: 0.05, 0.15, 0.5, 1.5 and 5 µL/plate.
Experimetn II: 0.16, 0.31, 0.63, 1.25, 2.5 and 5 µL/plate.
Testing up to a maximum concentration of 5µL/plate as described in the OECD guideline.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: a pre-test showed that the test item is sufficiently soluble in DMSO.
Controls
Untreated negative controls:
yes
Remarks:
demineralised water
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 4-nitro-1,2-phenylene diamine (TA 97a, TA98 and TA 102); 2-amino-anthracene (TA97a, TA100, TA102 and TA 1535)
Details on test system and experimental conditions:
All Salmonella typhimurium strains were obtained from TRINOVA BioChem GmbH (batch: TA97a: 5033D, TA98: 5136D, TA100: 5141D, TA102: 5145D, TA1535: 5138D) and 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 as to prevent changes in the titre.

Experiment I:
- Method: plate incorporation method
- Concentrations tested: 5 / 1.5 / 0.5 / 0.15 / 0.05 µL/plate
- Incubation time: 48 h
- Incubation temperature: 37 ±1 °C
- Tested strains: TA97a, TA98, TA100, TA102, TA1535

Experiment II:
- Method: pre-incubation method
- Concentrations tested: 5 / 2.5 / 1.25 / 0.63 / 0.31 / 0.16 µL/plate
- Incubation time: 48 h
- Incubation temperature: 37 ±1 °C
- Tested strains: TA97a, TA98, TA100, TA102, TA1535

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate

METHOD OF TREATMENT/ EXPOSURE:

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 (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 mu-tagen 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.

REFERENCES AND VALIDITY
- Genotype Confirmation: Genotype confirmation is performed for each batch of lyophilized bacteria before stock culture preparation.
- Histidine requirement: Each strain was streaked on a biotin and a histidine-biotin-plate, using a sterilized 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 (diameter 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 solu-tion 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 1 only analogously to the titre control with the maximum dose of test item 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 item at the end of the incubation by visual inspection.
- Positive Controls: Using diagnostic mutagens, three replicates were prepared. The stock solutions of the substances were diluted to achieve an application volume of 0.1 mL/plate, incubation for 48 hours at 37 ±1°C.
Evaluation criteria:
Five different analysable and non-toxic concentrations should be used for the evaluation of the mutagenic potential of the test item.
The colonies were counted visually and the numbers were recorded. The mean values and standard deviations of each threefold determination were calculated as well as the increase factor of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (mean revertants less mean spontaneous revertants) was given.

A substance is considered to be mutagenic, if a reproducible increase with or without metabolic activation of revertant colonies per plate exceeding an increase factor of 2 for the bacteria strains TA97a, TA98, TA100, TA102 and TA1535 compared to vehicle controls in at least one strain can be observed and there is a concentration-related increase.

A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

A substance is not mutagenic if it does not meet these criteria.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 97
Remarks:
a
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
The test item is not mutagenic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and in the presence of metabolic activation under the experimental conditions in the present study.
Executive summary:

The genetic toxicity of the "Oligomerisation product obtained by plasma treatment of vegetable oil mono- and polyunsaturated C16-C22 triglycerides" was assessed by means of an Ames test. The study was performed in accordance with OECD Guideline 471 and EU method B.13/14 and in accordance with the GLP criteria.

The test was performed in 2 experiments with 5 strains of Salmonella typhimurion (TA97a, TA98, TA100, TA102 and TA1535) in the presence and the absence of metabolic activation with S9 mix.

Experiment 1:

In the first experiment,the test item (dissolved in DMSO) was tested up to concentrations of 5 µL/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 item showed no precipitates on the plates at any of the 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 itemshowed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation.

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 2:

Based on the results of the first experiment,the test item was tested up to concentrations of 5 µL/plate in the absence and presence of S9 mix in all bacteria strains using the pre-incubation method.

The test item showed no precipitates on the plates at any of the 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 itemshowed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation.

The results of this experiments showed that the test item 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 item did not induce a dose-related increase in the number of revertant colonies in all strains, in the presence and absence of metabolic activation.

 

Based on the results of this study it is concluded that Oligomerisation product obtained by plasma treatment of vegetable oil mono- and polyunsaturated C16-C22 triglycerides.is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.