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Toxicological information

Genetic toxicity: in vitro

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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:
November from 15 to 24, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21st July, 1997
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Dilasoft TF
IUPAC Name:
Dilasoft TF
Test material form:
liquid

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Trinova Biochem GmbH
- Storage: the strains were stored at -80 ± 10 ºC in the testing facility in the form of lyophilized discs and in frozen permanent copies. Frozen permanent cultures of the tester strains were prepared from fresh, overnight cultures to which DMSO (8 % (v/v)) was added as a cryoprotective agent.

BACTERIA 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 10-13 hours in a 37 °C Benchtop Incubator Shaker

CONFIRMATION OF PHENOTYPES OF TESTER STRAINS
The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies were checked regularly according to Ames et al.

SPONTANEOUS REVERSION OF TESTER STRAINS
Each tester strain reverts spontaneously at a frequency that is characteristic for the strain. Spontaneous reversions of the test strains to histidine or tryptophan prototrophs were measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate.
Metabolic activation:
with and without
Metabolic activation system:
Rat Liver S9 Fraction
Test concentrations with justification for top dose:
MAIN EXPERIMENTS: 5000, 1600, 500, 160, 50 and 16 µg/plate.
CONCENTRATION RANGE FINDING: 5000, 1600, 500, 160, 50, 16 and 5 µg/plate.
Vehicle / solvent:
- Vehicle used: ultrapure water (ASTM Type I)
- Justification for choice of solvent/vehicle: the vehicle was chosen due to its non-toxicity to the bacteria and the compatibility to the S9 activity based on the available laboratory’s historical control database.
- Stock suspension: based on the solubility test, a stock suspension with a concentration of 50 mg/ml was prepared in ultrapure water (ASTM Type I) and diluted accordingly.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-Nitro-1,2-phenylenediamine // 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION
EXPERIMENT 1 - in agar (plate incorporation)
- Culturing: bacteria were cultured in Nutrient Broth No.2
- Medium: molten top agar was prepared and kept at 45 °C. 2 ml of top agar was aliquoted into individual test tubes.
- 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.
- Incubation: the plates were incubated at 37 °C for about 48 hours.
- Replicates: 3 tubes per both control and each concentration level.

EXPERIMENT 2 - pre-incubation test
- Medium: 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).
- Preincubation: the tubes were gently mixed and incubated for 20 min at 37 ºC in a shaking incubator.
- Content of the tubes: 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.
- Aeration: tubes were aerated during pre-incubation by using a shaker.
- Incubation: after preparation the plates were incubated at 37 °C for about 48 hours.
- Replicates: triplicate plating was used at each dose level.

PRELIMINARY CONCENTRATION RANGE FINDING
- Strains: TA 98 and TA 100
- Concentrations: 5000, 1600, 500, 160, 50, 16 and 5 µg/plate.

VIABILITY AND CELL COUNT
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 titre 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.

METABOLIC ACTIVATION
Rat Liver S9 Fraction
The S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF)-induced rat liver was provided by Trinova Biochem GmbH.

VALIDITY OF THE TEST
The tests (initial and confirmatory mutation tests) are considered valid if:
- all of the Salmonella tester strains demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene
- the Salmonella typhimurium TA 98 and TA 100 tester strains demonstrate the presence of the pKM101 plasmid R-factor
- the Escherichia coli WP2 uvrA culture demonstrate the deletion in the uvrA gene
- the bacterial cultures demonstrate the characteristic mean number of spontaneous revertants in the solvent controls
- the tester strain culture titers are in the 10^9 cells/ml order
- the batch of S9 used in this study shows the appropriate biological activity
- the positive control reference items (known mutagens) show the expected increase (at least a 3.0-fold increase) in induced revertant colonies over the mean value of the respective solvent control, and the number of induced revertant colonies are in accordance with the corresponding historical control ranges
- there are at least five analyzable concentrations (at each tester strain) (a minimum of three non-toxic dose levels is required to evaluate assay data)

A dose level is considered toxic if
- reduced revertant colony numbers are observed as compared to the mean solvent control value and the reduction shows a dose-dependent relationship, and / or
- the reduced revertant colony numbers are below the historical control data range and / or
- pinpoint colonies appear and / or
- reduced background lawn development occurs.
Evaluation criteria:
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 TA 100 the number of reversions is at least twice as high as the reversion rate of the solvent control,
- in strain Salmonella typhimurium TA 98, TA 1535, TA 1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the solvent control.

Criteria for a negative response: a test item 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 results
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
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 performed experiments, sporadically increased revertant colony numbers were observed. These increases did not show a clear dose-response relationship, were mostly 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 revertant colony number increase was observed in the initial mutation test (plate incorporation test) in S. typhimurium TA1535 at the concentration of 50 µg/plate, in the presence of an exogenous metabolic activation (+S9 mix). The increased revertant colony numbers at this treatment remained within the corresponding solvent historical control data range and the appearance of this value was considered to be unique without concentration related increases. The mutation rate was 1.66, which was 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 not observed. All of the noticed lower revertant colony numbers (when compared to the revertant colony numbers of the corresponding solvent control) remained in the range of the biological variability of the applied test system and the background lawn development was not affected in any case.
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 mix).

PRELIMINARY CONCENTRATION RANGE FINDING
In the informatory toxicity test the revertant colony numbers of solvent control plates with and without S9 mix were in line with the corresponding historical control data ranges. The positive control treatments showed the expected biological relevant increases in induced revertant colonies in both tester strains.
In the informatory toxicity test a cytotoxic effect of the test item on the bacterial strains was not observed. The colony and background lawn development was not affected in any case. All of the observed slight revertant colony number decreases (compared to the revertant colony numbers of the solvent control) remained within the biological variability range of the applied test system.
The obtained revertant colony numbers were sporadically higher than the revertant colony numbers of the solvent control; however all of the observed higher colony numbers remained within the biological variability range of the applied test system and far below the biologically relevant threshold for being positive.
No precipitation of the test item was observed on the plates in the above bacterial strains at any examined concentration level (±S9 mix).

VALIDITY OF THE PERFROMED 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.
Each of the investigated reference mutagens showed the expected increase (at least a 3-fold increase) in induced revertant colonies over the mean value of the respective solvent control in all main experimental phases and the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in the main experimental phases, in the tester strains.
The spontaneous revertant colony numbers of the ultrapure water (ASTM Type I) solvent control plates showed characteristic mean numbers agreed with the actual historical control data ranges in all strains in both main experimental phases.
Seven concentration levels were investigated in the informatory toxicity test and six concentration levels were investigated in the main mutation experiments (initial and confirmatory mutation tests).
In the performed main 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
In the initial and confirmatory mutation tests the revertant colony numbers of the ultrapure water (ASTM Type I) solvent control plates with and without S9 mix were in line with the corresponding historical control data ranges.
The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains.
The revertant colony numbers of the untreated and dimethyl sulfoxide (DMSO) control plates in different experimental phases 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.

Applicant's summary and conclusion

Conclusions:
The test item has no mutagenic activity on the applied bacterium tester strains under the tested conditions.
Executive summary:

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay, in accordance with the testing method and procedures outlined into the OECD guideline 471.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (strains TA 98, TA 100, TA 1535 and TA 1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (strain WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/β-naphthoflavone-induced rats.

The study included a preliminary solubility tests, a preliminary concentration range finding tests (informatory toxicity tests), an initial mutation test (plate incorporation test), and a confirmatory mutation test (pre-incubation test).

Based on the results of the solubility test and the concentration range finding test the test item was dissolved in ultrapure water (ASTM Type I). At the formulation of test item solutions a correction of the concentrations for the organic content of test item was made in the experiments.

Based on the cytotoxicity and solubility results of the preliminary concentration range finding test (informatory toxicity test) and based on the recommendations in OECD guideline 471, the following concentrations of the test item were prepared and investigated in the initial and confirmatory mutation tests: 5000, 1600, 500, 160, 50 and 16 µg/plate.

The results of the preliminary concentration range finding test allowed the applying of the recommended maximum test concentration of 5000 µg/plate.

No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 mix) throughout the study.

The test item did not show inhibitory, cytotoxic effects in the performed experiments. The colony and background lawn development was not affected in any case; the obtained revertant colony number decreases (compared to the revertant colony numbers of the solvent control) remained within the biological variability range of the applied test system.

The revertant colony numbers of solvent control ultrapure water (ASTM Type I) plates with and without S9 mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges.

The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant colonies and the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains.

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.

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

Conclusion

In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the tested conditions.