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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From November 12th to 21st, 2019
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:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of trimethyl-3-[(1-oxooctadecyl)amino]propylammonium methyl sulphate, Propane-1,2-diol and Trimethyl-3-[(1-oxohexadecyl)amino]propylammonium methyl sulphate
EC Number:
947-471-7
Molecular formula:
C22H47N2O.CH3O4S - C3H8O2 - C24H51N2O.CH3O4S
IUPAC Name:
Reaction mass of trimethyl-3-[(1-oxooctadecyl)amino]propylammonium methyl sulphate, Propane-1,2-diol and Trimethyl-3-[(1-oxohexadecyl)amino]propylammonium methyl sulphate
Test material form:
solid
Remarks:
yellowish-brown solid

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
histidine-requiring auxotroph
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
tryptophan-requiring auxotroph
Metabolic activation:
with and without
Metabolic activation system:
The S9 Mix (with Rat Liver S9)
Salt solution for S9 mix Final concentration in S9 mix
NADP Na 7.66 g 4 mM
D-glucose-6 phosphate Na 3.53 g 5 mM
MgCl2 1.90 g 8 mM
KCl 6.15 g 33 mM
Ultrapure water ad 1000 ml
Sterilized by filtration through a 0.22 µm membrane filter.

The complete S9 mix was freshly prepared containing components as follows:
Ice cold 0.2 M sodium phosphate-buffer, pH 7.4 500 ml
Rat liver homogenate (S9) 100 ml
Salt solution for S9 mix 400 ml
The S9 mix (containing 10 % S9) was kept in an ice bath before it was added to the culture medium.

Sodium Phosphate Buffer (0.2 M, pH 7.4)
Solution A:
Na2HPO4 × 12H2O 71.63 g
Ultrapure water ad 1000 ml
Solution B:
NaH2PO4 × H2O 27.6 g
Ultrapure water ad 1000 ml
Solution A 880 ml
Solution B 120 ml*
* The components were mixed in the above ratio; thereafter the pH was checked and corrected. The correction was performed with admixture of the solution A or B.
After the pH setting the sterilization was performed by filtration through a 0.22 µm membrane filter.


Test concentrations with justification for top dose:
Initial mutation test - concentrations based on solubility test and range finding test:
-S9: 120, 80, 50, 16, 5, 1.6 and 0.5 µg/plate;
+S9: 500, 160, 50, 16, 5, 1.6 and 0.5 µg/plate;
Based on the results of the initial mutation test (noticed strong inhibitory effect of the test item), revision of the examined concentration range was done. In the confirmatory mutation test the following concentration levels were examined:
- S9: 50, 16, 5, 1.6, 0.5, 0.16 and 0.05 µg/plate;
+S9: 320, 160, 50, 16, 5, 1.6 and 0.5 µg/plate.
Vehicle / solvent:
Ultrapure water (ASTM Type I)
This solvent is compatible with the survival of the bacteria and the S9 activity.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-nitro-1,2-phenylenediamine
Remarks:
without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9
Details on test system and experimental conditions:
Procedure for bacterial cultures
The frozen bacterial cultures were thawed at room temperature and 200 µl inoculum was used to inoculate each 50 ml of Nutrient Broth No. 2 for the overnight cultures in the assay. The cultures were incubated for approximately 10-12 hours in a 37 °C Benchtop Incubator Shaker.

Viability and the cell count of the testing bacterial cultures
Fresh cultures of bacteria were grown up to the late exponential or early stationary phase of growth (approximately 10^9 cells per ml). Cultures in late stationary phase were not used. The titer was demonstrated in each assay through the determination of viable cell numbers by a plating experiment.
The viability of each testing culture was determined by plating 0.1 ml of the 10^-5, 10^-6, 10^-7 and 10^-8 dilutions of cultures on nutrient agar plates. The viable cell number of the cultures was determined by manual colony counting.

Media
An appropriate minimal agar and an overlay agar containing histidine and biotin or tryptophan, to allow for a few cell divisions were used.
Media composition refers to 1000 ml.

The Minimal Glucose Agar (MGA) Plates
Ready-to-use minimal glucose agar (MGA) plates were used in the study. Typical composition (g/1000 ml) of MGA plates:
Glucose 20.0 g
Magnesium sulfate 0.2 g
Citric acid 2.0 g
di-Potassium hydrogenphosphate 10.0 g
Sodium ammonium hydrogenphosphate 3.5 g
Agar agar 13.0 g

Nutrient Broth No. 2
Nutrient broth No. 2 25.0 g
Ultrapure water ad 1000.0 ml
Sterilization for 20 minutes was performed at 121 ˚C in an autoclave.

Nutrient Agar
Nutrient Agar 20.0 g
Ultrapure water ad 1000.0 ml
Sterilization for 20 minutes was performed at 121 ˚C in an autoclave.

Top Agar for Salmonella typhimurium Strains
Agar solution:
Agar Bacteriological 4.0 g
NaCl 5.0 g
Ultrapure water ad 1000.0 ml
Sterilization for 20 minutes was performed at 121 ˚C in an autoclave.

Histidine – Biotin solution (0.5 mM):
D-Biotin 122.2 mg
L-Histidine•HCl H2O 104.8 mg
Ultrapure water ad 1000.0 ml
Sterilization was performed by filtration through a 0.22 µm membrane filter.

Complete Top Agar for Salmonella typhimurium strains:
Histidine – Biotin solution (0.5 mM) 100.0 ml
Agar solution 900.0 ml

Top Agar for Escherichia coli Strain
Tryptophan solution (2 mg/ml):
L-Tryptophan 2000.0 mg
Ultrapure water ad 1000.0 ml
Sterilization was performed by filtration through a 0.22 µm membrane filter.

Complete Top Agar for Escherichia coli strain:
Nutrient Broth 50.0 ml
Tryptophan solution (2 mg/mL) 2.5 ml
Agar solution 947.5 ml

Test procedure
The study included a preliminary solubility test, a preliminary concentration range finding test (informatory toxicity test) an initial mutation test (plate incorporation test), and a confirmatory mutation test (pre-incubation test). In the preliminary concentration range finding test as well as in the initial mutation test the plate incorporation method was used.
The applied method as well as the investigated concentrations for the confirmatory mutation test were decided after having evaluated the results of initial mutation test.

Solubility Test
In the solubility test the test item behavior was investigated in the applied test system when formulated in ultrapure water (ASTM Type I). The test item was dissolved in ultrapure water; and the obtained solution with the solution of top agar and phosphate buffer were examined in a test tube without test bacterium suspension.

Conc. of test item in the solvent (mg/ml): 50
Solubility, visible behavior in the solvent: clear solution
Solubility in the top solution (final treatment mixture) (test item solution 100 µl + phosphate buffer 500 µl + top agar 2 ml): clear solution
Test item concentration in the test tube µg/tube: 5000

Concentration Range Finding Test (Informatory Toxicity Test)
Based on the solubility test, the stock solution with a concentration of 50 mg/ml was prepared in ultrapure water and diluted in at least 6 steps by factor of approximately √10.
The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined at the concentrations of 5000, 1600, 500, 160, 50, 16 and 5 µg/plate of the test item.
Most of the revertant colony numbers of vehicle control plates with and without S9 were in line with the corresponding historical control data ranges. In the case of Salmonella typhimurium TA98, in the presence of exogenous metabolic activation (+S9) the revertant colony numbers of the water control were slightly above the corresponding historical control data range (actual mean value: 43; historical control data range: 13-41), without any effect on the results of the study. The positive control treatments showed the expected, biological relevant increases in induced revertant colonies in both tester strains.
In the concentration range finding test strong inhibitory, cytotoxic effect of the test item was observed in S. typhimurium TA98 down to and including the concentration level of 160 µg/plate, in the absence ( S9), in the range of 5000-500 µg/plate (+S9); in TA100 in the range of 5000-50 µg/plate ( S9) and in the range of 5000-160 µg/plate (+S9).
The inhibitory effect was indicated by affected background lawn development: absent or reduced background lawn and/or affected colony development: absent or decreased revertant colony counts (below the corresponding historical control data ranges).
No precipitation of the test item was observed on the plates after about 48 hours incubation in the above bacterial strains at any examined concentration level (±S9).

Controls
The tests were performed with parallel running controls: untreated, solvent and positive reference.
Summary of Controls
Type of control Activation with S9 mix Solvent Top agar Strain-specific positive chemical solutions Phosphate buffer
Untreated - - + - +
Untreated + - + - -
Solvent + + + - -
Positive control - - + + +
Positive control + - + + -

Procedure for the Initial Mutation Test
A standard plate incorporation procedure was performed as an initial mutation test. Bacteria (cultured in Nutrient Broth No.2) were exposed to the test item both in the presence and absence of rat liver S9 as metabolic activation system. Molten top agar was prepared and kept at 45 °C. Two ml of top agar was aliquoted into individual test tubes (3 tubes per both control and each concentration level). The equivalent number of minimal glucose agar plates was properly labelled. Conditions were investigated in triplicate. The test item and other components were prepared fresh and added to the overlay (45 °C).
The content of the tubes:
top agar 2000 µl
solvent or solution of test item or positive controls 100 µl
overnight culture of test strain* (containing approximately 10^8 viable cells) 100 µl
phosphate buffer (pH: 7.4) or S9 mix 500 µl
*: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA.
This solution was mixed and poured on the surface of the properly labeled minimal agar plates (3 plates per both control and each concentration level). For incubations with metabolic activation, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions and each with the addition of negative and positive controls. The plates were incubated at 37 °C for about 48 hours.

Procedure for the Confirmatory Mutation Test
A pre-incubation procedure was performed, as a confirmatory mutation test. Before the overlaying of the test item, the bacterial culture (0.1 ml, containing approximately 10^8 viable cells) and the S9 mix or phosphate buffer (0.5 ml) was added into appropriate tubes to allow direct contact between bacteria and the test item (in its solvent). These tubes were gently mixed and incubated for 20 min at 37 °C in a shaking incubator. After the incubation period, 2 ml of molten top agar was added to the tubes, and the content was mixed and poured onto minimal glucose agar plates as described for the standard plate incorporation method. Tubes were aerated during pre-incubation by using a shaker. The entire test consisted of non-activation and activation test conditions and each of them with the addition of negative and positive controls. For an adequate estimate of variation, triplicate plating was used at each dose level. After preparation the plates were incubated at 37 °C for about 48 hours.
Evaluation criteria:
Evaluation of Experimental Data
The evaluation of revertant colony count, background lawn development and test item precipitate were performed manually by unaided eye.
The colony numbers on the untreated, solvent control, positive control and the test item treated plates were determined, the mean values, standard deviations and the mutation rates were calculated.
Mutation Rate = Mean number of revertants at test item (or control*) treatments / Mean number of revertants of solvent control
* : untreated, solvent or positive control

In general, equivocal results are clarified by further testing preferably using a modification of experimental conditions.
Negative results need to confirmed on a case-by-case basis. In those cases where confirmation of negative results is not considered necessary, justification is provided.

Evaluation and Interpretation of Results
A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the solvent control,
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the solvent control.

According to the guidelines, the biological relevance of the results will be the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Criteria for a Negative Response: a test article is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Results and discussion

Test resultsopen allclose all
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:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
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:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
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:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Validity of the performed experiments
The tester strains used in this study demonstrated the specific phenotype characteristics, were in line with the corresponding historical control data ranges, and showed the adequate strain culture titer.
Each batch of the S9 fraction used in this test had the appropriate biological activity and was active in the applied system.
The numbers of revertant colonies in the solvent control (ultrapure water (ASTM Type I)) showed characteristic mean numbers agreed with the actual historical control data ranges in all strains in all experimental phases.
The positive control reference items (diagnostic mutagens) induced the expected, biological relevant increases (more than 3-fold increase) in revertant colonies and most of the number of revertant colonies was within the historical control data range per strain, thereby meeting the criteria for a valid positive control in all experimental phases and all tester strains.
In the initial mutation test, in the case of Salmonella typhimurium TA1537 at the 9-Aminoacridine (9AA) positive control the obtained revertant colony numbers were above the corresponding historical control data range (actual mean value: 3888, the historical control data range: 137-2265); however at these high revertant colony numbers it was considered as having no relevance and any effect on the validity of this experimental part of the study.
Seven concentration levels were investigated in the informatory toxicity test and in the main mutation experiments. In initial and confirmatory mutation tests the concentration levels were investigated in a shifted way. For these experiments in summary nine concentration levels were prepared, but seven investigated at each experimental part.
In the performed experimental phases there were at least five analyzable concentrations and a minimum of three non-toxic and non-precipitated dose levels at each tester strain.
All criteria for the validity of the performed experiments have therefore been met.

Controls
The spontaneous revertant colony numbers of the ultrapure water (ASTM Type I) solvent control plates showed characteristic mean numbers in line with the actual historical control data ranges in all strains in both main experimental phases of the study.
Each of the investigated reference mutagens (positive controls) showed the expected increase (at least a 3-fold increase) in induced revertant colonies over the mean value of the respective solvent control in main experimental phases and additionally most of the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in main experimental phases, in all tester strains.
The revertant colony numbers of the untreated and dimethyl sulfoxide (DMSO) control plates in the main experiments were slightly higher or lower than the ultrapure water control plates. The higher or lower revertant counts of these controls remained in the corresponding historical control data ranges.
In summary, the actual values of untreated, solvent and positive controls were in line with the criteria for validity of the assay.

Initial and Confirmatory Mutation Tests (Plate Incorporation and Pre-Incubation Tests)
No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with test item at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments.
In the main experiments, occasional increases in the revertant colony numbers were observed. These increases did not show a dose-response relationship, were of minor intensity, and all of the increases remained far below the biologically relevant thresholds for being positive. The obtained increases were therefore considered as biologically not relevant, being in the range of the biological variability of the applied test system.
The highest mean revertant colony number increase was observed in the initial mutation test (plate incorporation test) in Salmonella typhimurium TA1537 strain at 0.5 μg/plate, in the absence of metabolic activation (-S9). The revertant colony numbers at this concentration remained within the corresponding solvent historical control data range; the value was unique, was not accompanied with dose-relationship; therefore, this higher value was considered rather as being within the biological variability range of the applied system than a test item effect. The mutation rate was 1.50, far below the genotoxicological threshold for being positive.

In the initial and confirmatory mutation tests unequivocal inhibitory effect of the test item on bacterial growth was observed. The cytotoxicity was indicated by affected background lawn development (absent, reduced or slightly reduced background lawn) and/or affected colony development: absent revertants or decreased number of revertants, below the corresponding vehicle and/or historical control data ranges. In general, 5 µg/plate (noticed in absence of exogenous metabolic activation in S. typhimurium TA98 following the pre-incubation procedure) was considered as lowest concentration showing cytotoxicity.

In the performed experiments no precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9).

Any other information on results incl. tables

Signs of cytotoxicity in the plate incorporation and pre-incubation tests

Plate Incorporation Test 
Conc. Salmonella typhimurium E. coliWP2uvrA
(µg/plate) TA98 TA100 TA1535 TA1537
‑S9 0 ‑S9 0 ‑S9 0 ‑S9 0 ‑S9 0
500   B0   A   B0   B0   <<B
160     <<B   *    
120 <<B   A   B0   B0   <<B  
80 <<B   B0   <<B   B0   SB  
50 SB <<B <SB <SB *
16 * *
5 *
1.6 **
0.5 * **
Pre-Incubation Test
Conc. Salmonella typhimurium E. coliWP2uvrA
(µg/plate) TA98 TA100 TA1535 TA1537
‑S9 0 ‑S9 0 ‑S9 0 ‑S9 0 ‑S9 0
320   <<B   <<B   B   <B   <SB
160     <<B   <B   *  
50 <<B <<B <B <<B * <B *
16 <<SB <<SB ** <SB <SB *
5 <<SB ** *
1.6 * *
0.5
0.16          
0.05 *          

A:          Absent background lawn development;

B0:       No revertant growth and reduced background lawn development;

B:          Reduced background lawn development;

SB:        Slightly reduced background lawn development;

<<:        Revertant colony numbers below the vehicle and historical control data ranges;

<:           Revertant colony numbers below the vehicle control data range, evaluated as unequivocal sign of inhibitory effect of the test item;

*:           Revertant colony numbers below the corresponding vehicle control range; however, within the      biological variability range of the applied test system, evaluated as no inhibition;

**:         Revertant colony numbers below the corresponding historical control data range, within the           corresponding vehicle control range; within the biological variability range of the applied test system,        evaluated as no inhibition;

‑:            No inhibition

Cells with dark grey filling: The concentration was not tested for the corresponding strain.

Applicant's summary and conclusion

Conclusions:
No mutagenic activity.
Executive summary:

Mutagenic activity of test item was evaluated according to OECD guideline 471 in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and E. colu WP2 uvrA in presence and absence of metabolic activation by S9 -mix.

A preliminary solubility test and a preliminary range-finding test (using the plate incorporation method) were run.

In the initial mutation test (plate incorporation method), test concentrations were:

-S9: 120, 80, 50, 16, 5, 1.6, 0.5 µg/plate;

+S9: 500, 160, 50, 16, 5, 1.6, 0.5 µg/plate.

In the confirmatory mutation test (plate incubation method), test concentrations were:

-S9: 50, 16, 5, 1.6, 0.5, 0.16, 0.05 µg/plate;

+S9: 320, 160, 50, 16, 5, 1.6, 0.5 µg/plate.

No precipitation was seen in any concentration level.

Unequivocal inhibitory effect of test item on bacterial growth was observed. The cytotoxicity was indicated by affected background lawn development and/or affected colony development: absent revertants or decreased number of revertants below the corresponding vehicle and/or historical control data ranges. In general, 5 µg/plate (noticed in absence of exogenous metabolic activation in TA98 following the pre-incubation procedure) was considered as lowest concentration showing cytotoxicity.

Positive and negative controls were valid.

No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Therefore, the item is considered not mutagenic in this assay.