[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:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers
of phenobarbital/β-naphthoflavone-induced rats.

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 had no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.

 

In vitro Micronucleus test, OECD 487:

The test item was tested in an in vitro micronucleus test using mouse lymphoma L5178Y TK+/-
3.7.2 C cells. The test item was formulated in dimethyl sulfoxide (DMSO) and it was examined
up to the cytotoxicity limit according to the OECD No. 487 guideline recommendations. In the
main tests using duplicate cultures, 2000 cells were scored for each evaluated test item treated,
negative (vehicle) and positive control samples.
The main tests included a 3-hour treatment with metabolic activation (in the presence of S9-
mix) and a 3-hour and 24-hour treatment without metabolic activation (in the absence of S9-
mix) were performed. Sampling was performed 24 hours after the beginning of the treatment.
The examined concentrations of the test item were 90, 30, 10, 3.33, 1.11, 0.37, 0.12, 0.041,
0.014 and 0.005 μg/mL (3-hour treatment with metabolic activation), 120, 60, 55, 50, 45, 40,
35, 30, 10 and 3.33 μg/mL (3-hour treatment without metabolic activation) and 60, 30, 25, 20,
15, 10, 3.33 and 1.11 μg/mL (24-hour treatment without metabolic activation).
In the main tests, there were no large changes in the pH and osmolality. No insolubility was
observed in the final treatment medium at the end of the treatment in all experiments.
Marked cytotoxicity was observed in the 3-hour treatment with metabolic activation at 90, 30,
10 and 3.33 μg/mL concentrations (survival rate values were ~0%, 18%, 48% and 65%,
respectively).
Marked cytotoxicity was observed in the 3-hour treatment without metabolic activation at 120,
60, 55, 50, 45 and 40 μg/mL concentrations (survival rate values were ~0%, ~0%, ~0%, 8%,
35% and 60%, respectively).
The same effect was observed in the 24-hour treatment without metabolic activation at 60, 30,
25, 20 and 15 μg/mL concentrations (survival rate values were ~0%, 21%, 41%, 46% and 57%,
respectively).
Therefore, concentrations of 10, 3.33 and 1.11 μg/mL (a total of three) were chosen for
evaluation in case of the short treatment with metabolic activation, concentrations of 45, 40,
30, 10 and 3.33 μg/mL (a total of five) were chosen for evaluation in case of the short treatment
without metabolic activation and concentrations of 25, 10 and 3.33 μg/mL (a total of three)
were chosen for evaluation in case of the long treatment without metabolic activation.
None of the treatment concentrations caused a biologically or statistically significant increase
in the number of micronucleated cells when compared to the appropriate negative (vehicle)
control value in the experiments with and without metabolic activation.
The negative (vehicle) control data were within the acceptable range for the spontaneous
frequency of micronucleated cells, the positive control substances caused a statistically
significant increase in the number of micronucleated cells in the experiments with or without
metabolic activation demonstrating the sensitivity of the test system. The evaluated
concentration range was considered to be adequate; three test item treated concentrations were
evaluated in the study.
In conclusion, the test item did not cause statistically or biologically significant
reproducible increases in the frequency of micronucleated mouse lymphoma L5178Y
TK+/- 3.7.2 C cells in the performed experiments with and without metabolic activation.
Therefore, the test item was considered as not being genotoxic in this test system under
the conditions of the study.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 February to 08 August 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

conducted under GLP conditions

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016

Deviations:

yes

Remarks:

Due to technical reasons, incorrect date of the main experiment was indicated in the Study Plan (23 April – 24 March 2022 instead of 23 – 24 March 2022). No adverse effect on the study.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Cell line: L5178Y TK+/- 3.7.2 C mouse lymphoma
Product No.: CRL-9518
Lot No.: 60797977
Supplier: American Type Culture Collection (Manassas, Virginia, USA)
Date of receipt: 27 February 2014
Date of working lot: 25 June 2021 (MP15)

The original L5178Y TK+/- 3.7.2 C mouse lymphoma cell line was obtained from the American
Type Culture Collection. Cells were stored as frozen stocks in liquid nitrogen. Each batch of
frozen cells was checked for the absence of mycoplasma. The modal chromosome number is
checked yearly. For each experiment, one or more vials was thawed rapidly, cells were diluted
in RPMI-10 medium and incubated at 37 ± 0.5 °C in a humidified atmosphere containing
approximately 5% CO2 in air. When cells were growing well, subcultures were established in
an appropriate number of flasks (after thawing, the cells were subcultured no more than five
times before used in the main assay).

Metabolic activation:

with and without

Metabolic activation system:

An advantage of using in vitro cell cultures is the accurate control of the concentration and
exposure time of cells to the test item under the study. However, due to the limited capacity of
cells growing in vitro for metabolic activation of potential genotoxic agents, an exogenous
metabolic activation system is necessary.
Many substances only develop genotoxic potential after they are metabolised. Metabolic
activation of substances can be achieved by supplementing the cell cultures with liver
microsome preparations (S9 mix). In the experiments with metabolic activation in this study,
a cofactor-supplemented post-mitochondrial S9 fraction prepared from activated rat liver was
used as an appropriate metabolic activation system.
The post-mitochondrial fraction (S9 fraction) was prepared by the Microbiological Laboratory
of the Test Facility according to Ames et al. and Maron and Ames. The documentation of the preparation of this post-mitochondrial fraction is stored in the reagent notebook in the Microbiological Laboratory which is archived yearly.
The supplier, batch number and expiry date of the used chemicals described in this section are summarized in Table 1 (Section 10. Other Chemicals of the final report).

Induction of Rat Liver Enzymes:
Male Wistar rats (423-747 g animals were 13-26 weeks old at the initiation) were treated with
Phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg bw/day by oral gavage for three
consecutive days. Rats were given drinking water and food ad libitum until 12 hours before
sacrifice when food was removed. Initiation date of the induction of liver enzymes used for
preparation S9 used in this study was 23 August 2021 (Charles River Laboratories Hungary Kft. code: E13644).

Preparation of Rat Liver Homogenate S9 Fraction:
On Day 4, the rats were euthanized (sacrifice was by ascending concentration of CO2,
confirmed by cutting through major thoracic blood vessels) and the livers were removed
aseptically using sterile surgical tools. After excision, livers were weighed and washed several
times in 0.15 M KCl. The washed livers were transferred to a beaker containing 3 mL of 0.15
M KCl per g of wet liver, and homogenized.
Homogenates were centrifuged for 10 minutes at 9000 g and the supernatant was decanted and
retained. The freshly prepared S9 fraction was aliquoted into 1-5 mL portions, frozen quickly
and stored at -80 ± 10ºC. The date of preparation of S9 fraction for this study was 26 August
2021 (Expiry date: 26 August 2023).
The protein concentration of the preparation was determined by a chemical analyser at 540 nm
in the Clinical Chemistry Laboratory of the test Facility. The protein concentration of the S9
fraction used in the study was determined to be 26.1 g/L. The sterility of the preparation was
confirmed.
The biological activity in the Salmonella assay of S9 was characterized using the two mutagens
(2-Aminoanthracene and Benzo(a)pyrene), that requires metabolic activation by microsomal
enzymes. The batch of S9 used in this study functioned appropriately.

Preparation of S9-mix:
The complete S9-mix was freshly prepared for the treatments on the day of use according to
the following ratio:
Concentration of the stock solution
Concentration in the mix
Potassium chloride (KCl) - Concentration of the stock solution 150 mM - Concentration in the mix - 0.2 mL/mL
NADP* (sodium salt) - Concentration of the stock solution 25 mg/mL - Concentration in the mix - 0.2 mL/mL
D-Glucose-6-phosphate Na (monosodium salt) - Concentration of the stock solution 180 mg/mL - Concentration in the mix - 0.2 mL/mL
S9 fraction - Concentration in the mix - 0.2 mL/mL
*NADP= β-Nicotinamide-dinucleotide-phosphate
Prior to addition to the culture medium the S9-mix was kept in an ice bath.

For all cultures treated in the presence of S9-mix, a 0.5 mL aliquot of the mix was added to
each cell culture (final volume was 10 mL). Thus, the final concentration of the liver
homogenate in the test system was 1.0%.

Test concentrations with justification for top dose:

Based on preliminary test, The highest examined concentration in the preliminary experiments was 2000 μg/mL (using a stock solution of 200 mg/mL and 0.1 mL treatment volume in the final volume of 10 mL).
In the preliminary test short treatment (3 hours) with metabolic activation, and short and long
treatment (3 and 24 hours, respectively) without metabolic activation were examined to
establish an appropriate concentration range for the main tests.
A total of ten test concentrations between 2000 and 3.906 μg/mL concentration were used to
evaluate toxicity in the presence and absence of metabolic activation in the preliminary test.
Detailed results of the cytotoxicity assays are presented in Table 2, Table 3 and Table 4 of
Appendix 3 in the study report.
Treatment concentrations for the main tests were selected on the basis of results of the
performed preliminary test and according to the OECD No. 487 guideline instructions (up to
the cytotoxicity limit). Higher concentrations than the selected high dose were expected to have
a survival rate lower than 40% (RICC).

The examined concentrations of the test item were 90, 30, 10, 3.33, 1.11, 0.37, 0.12,
0.041, 0.014 and 0.005 μg/mL (3-hour treatment with metabolic activation), 120, 60, 55, 50,
45, 40, 35, 30, 10 and 3.33 μg/mL (3-hour treatment without metabolic activation) and 60, 30,
25, 20, 15, 10, 3.33 and 1.11 μg/mL (24-hour treatment without metabolic activation).

Vehicle / solvent:

The vehicle for the test item was DMSO.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Main tests:
In Assay 1, in the presence of S9-mix (3-hour treatment), the selected concentration intervals
seemed to be not sufficiently refined to evaluate at least three test concentrations to meet the
acceptability criteria (appropriate cytotoxicity). Therefore, an additional experiment (Assay 2)
was performed to use different concentrations to give further information about the cytotoxic
effects and to meet the acceptability criteria.
The reported main tests (Assay 1 and Assay 2) were conducted as three independent
experiments. In Assay 1, 3-hour treatment was performed without metabolic activation (in the
absence of S9 mix) and a 24-hour treatment was performed without metabolic activation (in
the absence of S9 mix). In Assay 2, 3-hour treatment was performed with metabolic activation
(in the presence of S9 mix). Treatments were conducted in duplicate cultures; cells were
harvested 24-hour after the beginning of the treatment.

Evaluation of cytotoxicity was performed based on Relative Increase in Cell Counts (RICC);
survival rate values below 40% are considered to be too cytotoxic for evaluation as per the
OECD guidance. Selected concentration included the higher but acceptable levels of
cytotoxicity, plus non cytotoxic concentrations as appropriate.

Treatment of the Cells:
For the treatments, 1 x 10^6 cells were placed in each of a series of sterile flasks (culturing surface
25 cm2) (starting cell count = N0). Treatment medium contained a reduced serum level of 5%
(v/v) (RPMI-5). On the day of treatment, cells were exposed to the test item, the negative and
positive control items, with or without S9 mix at 37°C ± 1 °C in a humidified atmosphere
(approximately 5% CO2 in air). Untreated control was processed in a similar way.
A suitable volume of medium (0.1 mL), solvent (0.1 mL), test item (0.1 mL), positive control
solutions (0.1 mL) and 0.5 mL of S9-mix were added to a final volume of 10 mL per culture
per experiment (at least 1x106 cells). Duplicate cultures were used for each treatment.
Solubility of the test item, negative and positive control items in the final treatment medium was
visually examined at the beginning and end of the treatment in each case. Measurement of pH
and osmolality was also performed at the end of the treatment period in all experiments.
After incubation at 37 °C ± 1 °C (approximately 5% CO2 in air) with gentle shaking, the cell
cultures were centrifuged at 2000 rpm (approximately 836 g) for 5 minutes, washed with tissue
culture medium and suspended in 10 mL RPMI-10. Cells were transferred to flasks for growth
through the recovery period (21 hours) at 37°C ± 1 °C in a humidified atmosphere
(approximately 5% CO2 in air) in case of the three-hour treatments.
At the scheduled harvesting time (24-hour from the beginning of the treatment), the number of
surviving cells (N) was determined using a haemocytometer. In order to assess cytotoxicity,
results were expressed compared to the appropriate negative (vehicle) control as RICC (Relative
Increase in Cell Counts) and as RPD (Relative Population Doubling).

SLIDE PREPARATION:
After the final cell counting, the cells were treated with hypotonic KCl solution for about 3
minutes at room temperature, before being fixed (Methanol : Acetic-acid 3 : 1 (v : v) mixture
was used as fixative). Following the fixation, the cells were kept at 2-8°C for an overnight
period.
Then, after the cell count setting, a suspension of the fixed cells* was dropped onto clean
microscope slides and air-dried. The slides were stained with 5% (w/v) Giemsa solution for 5
minutes, then thoroughly rinsed with distilled water, air-dried at room temperature for at least
12 hours.

EXAMINATION OF SLIDES:
The stained slides were given random unique code numbers at the Test Facility by a person
who was not involved in the metaphase analysis. The code labels covered all unique
identification markings on the slides to ensure that they were scored without bias.
In the main tests, two coded slides were sent for evaluation to the attention of Principal
Investigator.
Natalie Danford, B.Sc., MPH, Ph.D.
Microptic Cytogenetics
2 Langland Close
Mumbles
Swansea, SA3 4LY, United Kingdom
Two thousand cells were scored per sample (1000 cells/replicate) to assess the ratio of
micronucleated cells. The frequency of micronucleated cells was expressed as percent of
micronucleated cells based on the first 2000 cells (1000 cells/replicate) observed in the optic
field. When the slide reading was completed for each test, the slide codes were broken and the
number of micronucleated cells were presented in tables. After the in life phase of the study had
been ended, the Principal Investigator issued a Work Phase Report.
The slide analysis was conducted under the control of the Principal Investigator in compliance
with Good Laboratory Practice as required by the United Kingdom GLP Compliance
Regulations 1999 (SI 1999 No. 3106, as amended 2004, SI No. 0994) and which are in
compliance with the OECD Principles of Good Laboratory Practice (as revised in 1997). These
principles are in conformity with other international GLP regulations. The results of the
evaluation in the form of a Phase Report (Work Phase Report) were included in the study report
as an appendix (Appendix 7 in the study report).
When slide reading had been performed, the original raw data (record sheets) for the analysis,
original Work Phase Report and the microscope slides were shipped back to the Test Facility.

Rationale for test conditions:

Test conditions were based on OECD guideline.

Evaluation criteria:

Interpretation of results:
The test item is considered to be clearly positive if all of the following criteria are met:
- Increases in the frequency of micronucleated cells are observed at one or more test
concentrations.
- The increases are reproducible between replicate cultures.
- The increases are statistically significant compared with the concurrent negative
control.
- The increases are not associated with large changes in pH or osmolality of the treated
cultures.
The test item is concluded to have given a negative response if no reproducible, statistically
significant increases in the number of micronucleated cells were observed when compared to
the negative (vehicle) control.
The historical control data for this laboratory is also considered in the evaluation. Evidence of
a dose-response relationship is considered to support the conclusion.

Statistics:

For each condition of each experiment, the number of micronucleated cells in treated cultures
was compared to that of the negative (vehicle) control cultures. Statistical analysis (Fisher’s
exact test) was performed by the Principal Investigator at the Test Site (as documented in the
raw data and reported). The frequency of micronucleated cells referring 1000 cells are reported
in the study report.

Key result

Species / strain:

mouse lymphoma L5178Y cells

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 valid

Positive controls validity:

valid

Additional information on results:

In Assay 1, a 3-hour and 24-hour treatment without metabolic activation (in the absence of S9-
mix) were performed. In Assay 2, a 3-hour treatment with metabolic activation (in the presence
of S9-mix) was performed. Sampling was performed 24 hours after the beginning of the
treatment. The examined concentrations of the test item were 90, 30, 10, 3.33, 1.11, 0.37, 0.12,
0.041, 0.014 and 0.005 μg/mL (3-hour treatment with metabolic activation), 120, 60, 55, 50,
45, 40, 35, 30, 10 and 3.33 μg/mL (3-hour treatment without metabolic activation) and 60, 30,
25, 20, 15, 10, 3.33 and 1.11 μg/mL (24-hour treatment without metabolic activation).
In Assays, there were no large changes in the pH and osmolality. No insolubility was observed
in the final treatment medium at the end of the treatment in all experiments.
Marked cytotoxicity was observed in the 3-hour treatment with metabolic activation at 90, 30,
10 and 3.33 μg/mL concentrations (survival rate values were ~0%, 18%, 48% and 65%,
respectively) (for more details see Table 5 of Appendix 4 in the study report).
Marked cytotoxicity was observed in the 3-hour treatment without metabolic activation at 120,
60, 55, 50, 45 and 40 μg/mL concentrations (survival rate values were ~0%, ~0%, ~0%, 8%,
35% and 60%, respectively) (for more details see Table 6 of Appendix 4 in the study report).
The same effect was observed in the 24-hour treatment without metabolic activation at 60, 30,
25, 20 and 15 μg/mL concentrations (survival rate values were ~0%, 21%, 41%, 46% and 57%,
respectively) (for more details see Table 7 of Appendix 4 in the study report).
Therefore, concentrations of 10, 3.33 and 1.11 μg/mL (a total of three) were chosen for
evaluation in case of the short treatment with metabolic activation, concentrations of 45, 40,
30, 10 and 3.33 μg/mL (a total of five) were chosen for evaluation in case of the short treatment
without metabolic activation and concentrations of 25, 10 and 3.33 μg/mL (a total of three)
were chosen for evaluation in case of the long treatment without metabolic activation.
None of the treatment concentrations caused a biologically or statistically significant increase
in the number of micronucleated cells when compared to the appropriate negative (vehicle)
control value in the experiments with and without metabolic activation. Summarized data are
shown in Table 8-Table 10 of Appendix 5 in the study report.
The work phase report of the slide reading (micronucleus analysis) is presented in Appendix 7 in the study report.

Validity of the Study
The tested concentrations in the in vitro micronucleus test were selected based on the results
of the preliminary test. Insolubility was not detected in Assay 1 and Assay 2; while marked
cytotoxicity were detected in Assay 1 and Assay 2 with and/or without metabolic activation.
The evaluated concentration ranges of the main tests were considered to be adequate, as they
covered the range from toxicity to no or little toxicity.
At least three test item concentrations were evaluated in all experiments, this meets the criteria
of the OECD No. 487 guideline.
The spontaneous frequency of micronucleated cells of the negative (vehicle) controls in the
performed experiments were within the acceptable range.
In the performed experiments, the positive control substances (Cyclophosphamide in the
experiment with metabolic activation; Mitomycin C and Colchicine in the experiments without
metabolic activation) caused the expected statistically significant increase in the number of
micronucleated cells (Table 8-Table 10 of Appendix 5 in the study report) demonstrating the sensitivity of the test
system in each assay.

Conclusions:

The test item was tested for potential genotoxic activity using the in vitro micronucleus test in
mammalian cells. The study included a preliminary test for dose selection and two main tests.
The performed experiments were considered to be valid and to reflect the real potential of the
test item to cause cytogenetic damage in the cultured mouse lymphoma L5178Y TK+/- 3.7.2 C
cells used in this study.
Treatment with the test item did not result in a statistically or biologically significant,
reproducible increase in the frequency of the micronucleated cells in the presence or absence of a metabolic activation system which was a cofactor-supplemented post-mitochondrial S9
fraction prepared from the livers of phenobarbital/β-naphthoflavone induced rats.
In conclusion, the test item did not cause statistically or biologically significant
reproducible increases in the frequency of micronucleated mouse lymphoma L5178Y
TK+/- 3.7.2 C cells in the performed experiments with and without metabolic activation.
Therefore, the test item was considered as not being genotoxic in this test system under
the conditions of the study.

Executive summary:

The test item was tested in an in vitro micronucleus test using mouse lymphoma L5178Y TK+/-
3.7.2 C cells. The test item was formulated in dimethyl sulfoxide (DMSO) and it was examined
up to the cytotoxicity limit according to the OECD No. 487 guideline recommendations. In the
main tests using duplicate cultures, 2000 cells were scored for each evaluated test item treated,
negative (vehicle) and positive control samples.
The main tests included a 3-hour treatment with metabolic activation (in the presence of S9-
mix) and a 3-hour and 24-hour treatment without metabolic activation (in the absence of S9-
mix) were performed. Sampling was performed 24 hours after the beginning of the treatment.
The examined concentrations of the test item were 90, 30, 10, 3.33, 1.11, 0.37, 0.12, 0.041,
0.014 and 0.005 μg/mL (3-hour treatment with metabolic activation), 120, 60, 55, 50, 45, 40,
35, 30, 10 and 3.33 μg/mL (3-hour treatment without metabolic activation) and 60, 30, 25, 20,
15, 10, 3.33 and 1.11 μg/mL (24-hour treatment without metabolic activation).
In the main tests, there were no large changes in the pH and osmolality. No insolubility was
observed in the final treatment medium at the end of the treatment in all experiments.
Marked cytotoxicity was observed in the 3-hour treatment with metabolic activation at 90, 30,
10 and 3.33 μg/mL concentrations (survival rate values were ~0%, 18%, 48% and 65%,
respectively).
Marked cytotoxicity was observed in the 3-hour treatment without metabolic activation at 120,
60, 55, 50, 45 and 40 μg/mL concentrations (survival rate values were ~0%, ~0%, ~0%, 8%,
35% and 60%, respectively).
The same effect was observed in the 24-hour treatment without metabolic activation at 60, 30,
25, 20 and 15 μg/mL concentrations (survival rate values were ~0%, 21%, 41%, 46% and 57%,
respectively).
Therefore, concentrations of 10, 3.33 and 1.11 μg/mL (a total of three) were chosen for
evaluation in case of the short treatment with metabolic activation, concentrations of 45, 40,
30, 10 and 3.33 μg/mL (a total of five) were chosen for evaluation in case of the short treatment
without metabolic activation and concentrations of 25, 10 and 3.33 μg/mL (a total of three)
were chosen for evaluation in case of the long treatment without metabolic activation.
None of the treatment concentrations caused a biologically or statistically significant increase
in the number of micronucleated cells when compared to the appropriate negative (vehicle)
control value in the experiments with and without metabolic activation.
The negative (vehicle) control data were within the acceptable range for the spontaneous
frequency of micronucleated cells, the positive control substances caused a statistically
significant increase in the number of micronucleated cells in the experiments with or without
metabolic activation demonstrating the sensitivity of the test system. The evaluated
concentration range was considered to be adequate; three test item treated concentrations were
evaluated in the study.
In conclusion, the test item did not cause statistically or biologically significant
reproducible increases in the frequency of micronucleated mouse lymphoma L5178Y
TK+/- 3.7.2 C cells in the performed experiments with and without metabolic activation.
Therefore, the test item was considered as not being genotoxic in this test system under
the conditions of the study.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 February to 01 July 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

conducted under GLP conditions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

2020

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

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

Target gene:

Histidine and Tryptophan

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Metabolic Activation System:
Test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system, which is a cofactor-supplemented post-mitochondrial S9 fraction.
The post-mitochondrial fraction (S9 fraction) was prepared by the Microbiological Laboratory of Charles River Laboratories Hungary Kft. according to Ames et al. and Maron and Ames. The documentation of the preparation of this post-mitochondrial fraction is stored in the reagent notebook in the Microbiological Laboratory which is archived yearly. The composition of solution refers to 1000 mL.

- Induction of Liver Enzymes
Male Wistar rats (502-719 g, animals were 26-29 weeks old and 410-708 g, animals were 9-25 weeks old) were treated with phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg/day by oral gavage for three consecutive days. Rats were given drinking water and food ad libitum until 12 h before sacrifice when food was removed. Sacrifice was by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels. Initiation of the induction of liver enzymes used for preparation S9 used in this study was 28 June 2021 (Charles River Laboratories Hungary code: E13612) and 11 January 2021 (Charles River Laboratories Hungary code: E13455).

- Preparation of Rat Liver Homogenate S9 Fraction
On Day 4, the rats were euthanized, and the livers were removed aseptically using sterile surgical tools. After excision, livers were weighed and washed several times in 0.15 M KCl. The washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenized. Homogenates were centrifuged for 10 min at 9000 g and the supernatant was decanted and retained. The freshly prepared S9 fraction was aliquoted into 1-5 mL portions, frozen quickly and stored at -80 ± 10ºC. The dates of preparation of S9 fractions for this study was 01 July 2021 (E13612, Expiry date: 01 July 2023).
The sterility of the preparation was confirmed in each case. The protein concentration of the preparation was determined by a chemical analyzer at 540 nm in the Clinical Chemistry Laboratory of Charles River Laboratories Hungary Kft. The mean protein concentration of the S9 fraction used was determined to be 25.1 g/L.
The biological activity in the Salmonella assay of S9 was characterized in each case using the two mutagens 2-Aminoanthracene and Benzo(a)pyrene, that requires metabolic activation by microsomal enzymes. The batches of S9 used in this study functioned appropriately.

- The S9 Mix (containing 10 % (v/v) of S9)
Salt solution for S9 Mix:
NADP Na 7.66 g
D-glucose-6 phosphate Na 3.53 g
MgCl2 x 6 H2O 4.07 g
KCl 6.15 g
Distilled water q.s. ad 1000 mL
Sterilization was performed 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 (see above): 400 mL
Prior to addition to the culture medium the S9 mix was kept in an ice bath.

- 0.2 M Sodium Phosphate Buffer, pH 7.4

Solution A:
Na2HPO4 x 12 H2O 71.63 g
Distilled water q.s. ad 1000 mL
Sterilization was performed at 121°C in an autoclave.

Solution B:
NaH2PO4 x H2O 27.6 g
Distilled water q.s. ad 1000 mL
Sterilization was performed at 121°C in an autoclave.

0.2M Sodium phosphate buffer pH 7.4:
Solution A 880 mL
Solution B 120 mL

Test concentrations with justification for top dose:

In the Preliminary Range Finding Test, the plate incorporation method was used. The
preliminary test was performed using Salmonella typhimurium TA98 and Salmonella
typhimurium TA100 tester strains in the presence and absence of metabolic activation system
(±S9 Mix) with appropriate untreated, negative (solvent) and positive controls. Each sample
(including the controls) was tested in triplicate.
Following concentrations were examined: 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate.
No precipitate was detected on the plates in the preliminary experiment in both examined
bacterial strains with and without metabolic activation
Inhibitory, cytotoxic effect of the test item (absent / reduced / slight reduced background lawn
development) was observed in the preliminary experiment in both examined bacterial strains
with and without metabolic activation at 5000, 2500, 1000 and 316 μg/plate concentrations.
Based on the results of the preliminary experiment, the examined test concentrations in Assay
1 were 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate and in Assay 2 were 1581, 500, 158.1,
50, 15.81, 5, 1.581 and 0.5 μg/plate.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO) was selected as vehicle for the study (based on the Sponsor's information). The obtained stock formulation (50 μL) with the solution of top agar (section 10.2.4. of the final report) and phosphate buffer (section 10.3.4. of the final report) was examined in a test tube without test bacterium suspension.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO and distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other:

Remarks:

with S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO and distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other:

Remarks:

without S9

Details on test system and experimental conditions:

TEST SYSTEM
BACTERIAL STRAINS
1) Origin
Date of arrival and origin:
Salmonella typhimurium TA98 17 February 2021, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA
Salmonella typhimurium TA100 17 February 2021, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA
Salmonella typhimurium TA1535 17 February 2021, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA
Salmonella typhimurium TA1537 17 February 2021, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA
Escherichia coli WP2 uvrA 17 February 2021, MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA
The true copies of original certificates and other documents of the strains are collected and stored in the Microbiological Laboratory of Charles River Laboratories Hungary Kft.

2) Genotypes
In addition to histidine or tryptophan mutation, each strain has additional mutations, which enhances its sensitivity to mutagens. The uvrB (uvrA) strains are defective in excision repair, making them more sensitive to the mutagenic and lethal effects of a wide variety of mutagens because they cannot repair DNA damages. The presence of rfa mutation increases the permeability of the bacterial lipopolysaccharide wall for larger molecules. The plasmid pKM101 (TA98, TA100) carries the muc+ gene which participates in the error-prone "SOS" DNA repair pathway induced by DNA damage. This plasmid also carries an ampicillin resistance transfer factor (R-factor) which is used to identify its presence in the cell. The Escherichia coli strain used in this test (WP2 uvrA) is also defective in DNA excision repair. The genotypes of the tester strains used for mutagenicity testing are summarized in Table 4 of the final report attached.

3) Storage
The strains are stored at -80 ± 10ºC in the Culture Collection of the Microbiological Laboratory of Charles River Laboratories Hungary Kft. Frozen permanent cultures of the tester strains were prepared from fresh, overnight cultures to which DMSO was added as a cryoprotective agent.

4) 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 are checked regularly according to Ames et al. and Maron and Ames.
Established procedures (Standard Operating Procedures) for the preparations of each batch of frozen stock culture, raw data and reports of phenotype confirmation are stored in the Microbiological Laboratory of Charles River Laboratories Hungary Kft.

5) Spontaneous Reversion of Tester Strains
Each test strain reverts spontaneously at a frequency that is characteristic of the strain. Spontaneous reversion of the test strains to histidine (Salmonella typhimurium strains) or tryptophan (Escherichia coli strain) independence is measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate.
Historical control values for spontaneous revertants (revertants/plate) for untreated control sample without and with metabolic activation were in the period of 2015-2020 were (as guide) as follows: Salmonella typhimurium TA98: 11-50, 13-54, TA100: 67-152, 67-152, TA1535: 1-33, 3-39, TA1537: 2-26, 1-29, Escherichia coli WP2 uvrA: 14-77, 16-89. More detailed historical control data are shown in Appendix 6 of the study report attached.

6) Procedure for Growing Cultures
The frozen bacterial cultures were thawed at room temperature and 200 μL inoculum were used to inoculate each 50 mL of Nutrient Broth No.2 (Section 10.2.2. of the final report) for the overnight cultures in the assay. The cultures were incubated for 10-14 hours at 37°C in a Gyrotory water bath shaker.

7) Viability of the Testing Cultures
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 prepared by sterile physiological saline on Nutrient Agar (Section 10.2.3. of the final report) plates. The viable cell number of the cultures was determined by manual counting after approximately 24-hour incubation at 37°C.

MEDIA
The supplier, batch number and expiry date of the used chemicals described in sections 10.2.2.-10.2.5. are summarized in Table 2 (Chemicals used in the experiments table in Section 8. of the final report). The composition of media refers to 1000 mL.

1) The Typical Composition (g/1000 mL) of Minimal Glucose Agar
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
Distilled water q.s. ad1 1000 mL
Minimal glucose agar plates (Batch number: 716351, Expiry date: 08 March 2022 was used in the Preliminary Range Finding Test and in Assay 1; Batch number: 766269, Expiry date: 31
May 2022 was used in the Assay 2) were provided by Merck. Certificate of Analysis were obtained from the Supplier.

2) Nutrient Broth No.2
Nutrient Broth No.2 25.0 g
Distilled water q.s. ad 1000 mL
Sterilization was performed at 121°C in an autoclave.

3) Nutrient Agar
Nutrient Agar 20.0 g
Distilled water q.s. ad 1000 mL
Sterilization was performed at 121°C in an autoclave.

4) Top Agar for Salmonella typhimurium Strains

Agar solution:
Agar Bacteriological 4.0 g
NaCl 5.0 g
Distilled water q.s. ad 1000 mL
Sterilization was performed at 121°C in an autoclave.

Histidine – Biotin solution (0.5 mM):
D-Biotin (F.W. 244.31) 122.2 mg
L-Histidine x·HCl x H2O (F.W. 209.63) 104.8 mg
Distilled water q.s. ad 1000 mL
Sterilization was performed by filtration using a 0.22 μm membrane filter.

Complete Top Agar for Salmonella typhimurium strains:
Histidine – Biotin solution (0.5 mM) 100 mL
Agar solution 900 mL

5) Top Agar for Escherichia coli Strain
Tryptophan solution (2 mg/mL):
L-Tryptophan (F.W. 204.23) 2000 mg
Distilled water q.s. ad 1000 mL
Sterilization was performed by filtration using a 0.22 μm membrane filter.
Complete Top Agar for Escherichia coli strain:
Nutrient Broth 2 (see section 10.2.2.) 50 mL
Tryptophan solution (2 mg/mL) 2.5 mL
Agar solution (see section 10.2.4.) 947.5 mL


TEST PROCEDURE
1) Concentrations
Concentrations for the main tests were selected on the basis of the available information. In the main tests different concentrations were used.

2) Preliminary Compatibility Test
Dimethyl sulfoxide (DMSO) was selected as vehicle for the study (based on the Sponsor's information). The obtained stock formulation (50 μL) with the solution of top agar (section 10.2.4. of the final report) and phosphate buffer (section 10.3.4. of the final report) was examined in a test tube without test bacterium suspension. The results of the Preliminary Compatibility Test are summarized in Table 5 of the final report.

3) Test Item Concentrations in the Mutagenicity Tests (Assay 1 and 2)
Based on the results of the preliminary test, a 100 mg/mL stock solution was prepared in DMSO, which was diluted by serial dilutions in several steps to obtain the dosing formulations
for lower doses. The maximum test concentration was 1581 μg test item/plate.
Examined concentrations in Assay 1 were 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate.
Examined concentrations in Assay 2 were 1581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate.

4) Control Groups Used in the Tests
Strain-specific positive and negative (solvent) controls, both with and/or without metabolic activation were included in Assay 1 and Assay 2. In addition, an untreated control was used demonstrating that the chosen vehicle induced no deleterious or mutagenic effects. The control groups used in the tests are listed in Table 6 of the final report.

5) Experimental Method
The experimental methods were conducted according to the methods described by Ames et al. and Maron and Ames, Kier et al. , Venitt and Parry , OECD Guideline No. 471, 2020 , Commission Regulation (EC) No. 440/2008, 2008, EPA Guidelines, OPPTS 870.5100, 1998, 1996 and according to the relevant SOPs of Charles River Laboratories Hungary Kft.

6) Procedure for Exposure in the Assay 1
Assay 1 followed the standard plate incorporation procedure. Bacteria (cultured in Nutrient Broth No.2 as described in Section 10.2.2.) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system.
Molten top agar was prepared and kept at 45°C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates was properly labelled. The test item and other components were prepared freshly and added to the overlay (45°C).
The content of the tubes:
top agar 2000 μL
vehicle or test item formulation (or reference controls) 50 μL
overnight culture of test strain 100 μL
phosphate buffer (pH 7.4) or S9 mix 500 μL

This solution was mixed and poured on the surface of minimal agar plates. For activation studies, 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, with the addition of untreated, negative (vehicle/solvent) and positive controls. After preparation, the plates were incubated at 37°C for 48(+/- 1) hours.

7) Procedure for Exposure in the Assay 2
Assay 2 followed the standard pre-incubation procedure since no biologically relevant increase in the number of revertant colonies was observed in the Assay 1 (see details in
Section 16. of the final report).
Bacteria (cultured in Nutrient Broth No.2. as described in section 10.1.6.) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45°C.
Before the overlaying, the test item formulation (or vehicle/solvent or reference control), the bacterial culture (Section 10.1.6.) and the S9 mix or phosphate buffer was added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle/solvent). The tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 minutes at 37ºC in a shaking incubator.
After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37°C for 48(±1) hours.

Rationale for test conditions:

Test conditions follow the OECD 471 guideline.

Evaluation criteria:

- Criteria for Validity
The study was considered valid if:
• the number of revertant colonies of the negative (vehicle/solvent) and positive controls are in the relevant historical control range, generated at the test facility, in all tester strains of the main tests (with or without S9-mix);
• at least five analysable concentrations are presented in all strains of the main tests.
• the highest tested concentration should be at the limit test concentration, or the highest
acceptable concentration based on cytotoxicity and/or precipitation.

- Criteria for a Positive Response
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:
• the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
• the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.
According to the guidelines, statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.

- Criteria for a Negative Response
A test article is considered non-mutagenic if it produces neither a concentration-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the concentration groups, with or without metabolic activation.

Statistics:

Not used.

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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

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

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True 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:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In Assay 1, the plate incorporation method was used. In Assay 2 the pre-incubation method was
used. The assays were carried out using four Salmonella typhimurium strains (TA98, TA100,
TA1535 and TA1537) and the Escherichia coli WP2 uvrA strain. The assays were performed
in the presence and absence of a metabolic activation system. Each test was performed with
appropriate untreated, negative (solvent) and positive controls. In the main tests each sample
(including the controls) was tested in triplicate.
Based on the results of the preliminary experiment, the examined test concentrations in Assay
1 were 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate and in Assay 2 were 1581, 500, 158.1,
50, 15.81, 5, 1.581 and 0.5 μg/plate.
No precipitate was detected on the plates in the main test in all examined bacterial strains with
and without metabolic activation.
Inhibitory, cytotoxic effect of the test item (absent / reduced / slight reduced background lawn
development) was observed in the Assay 1 in the examined bacterial strains with and/or without
metabolic activation at 1581 and 500 μg/plate concentrations and slight reduced background
lawn development was observed in Salmonella typhimurium TA100, TA1535 and TA1537
bacterial strains without metabolic activation at 158.1 μg/plate concentration. Same cytotoxic
effect of the test item (absent / reduced / slight reduced background lawn development) was
observed in the Assay 2 in the examined bacterial strains with and/or without metabolic
activation at 1581, 500 and 158.1 μg/plate concentrations and slight reduced background lawn
development was observed in the examined bacterial strains without metabolic activation at
50 μg/plate concentration.
In Assay 1 (plate incorporation method), the highest revertant rate was observed in Escherichia coli
WP2 uvrA bacterial strain at 158.1 μg/plate concentration without metabolic activation (the
observed mutation factor value was: MF: 1.38). However, there was no dose-response relationship,
the observed mutation factor values were below the biologically relevant threshold limit and the
number of revertant colonies was within the historical control range.
In Assay 2 (pre-incubation method), the highest revertant rate was observed in Salmonella
typhimurium TA1535 bacterial strain at 15.81 μg/plate concentration without metabolic activation
(the observed mutation factor value was: MF: 1.52). However, there was no dose-response
relationship, the observed mutation factor values were below the biologically relevant threshold
limit and the number of revertant colonies was within the historical control range.
In the main assays the number of revertant colonies did not show any biologically relevant
increase compared to the solvent controls. There were no reproducible dose-related trends and
there was no indication of any treatment-related effect.
Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected
in the main tests in some other sporadic cases. However, no dose-dependence was observed in
those cases, and they were below the biologically relevant threshold value. The numbers of
revertant colonies were within the historical control range in each case, so they were considered
to reflect the biological variability of the test.
Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed
in the main tests at some non-cytotoxic concentrations. However, no background inhibition was
recorded and the mean numbers of revertant colonies were in the historical control range in all
cases, thus they were considered as biological variability of the test system.

Validity of the tests:

Untreated, negative (vehicle/solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range in all strains. At least five analysable concentrations were presented in all strains with and without metabolic activation.

The reference mutagens showed a distinct increase of induced revertant colonies in each strain with and without metabolic activation. The viability of the bacterial cells was checked by a plating experiment in each test. The study was considered to be valid.

Conclusions:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation
Assay.
The experiments were carried out using histidine-requiring auxotroph strains of Salmonella
typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the
tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the
presence and absence of a metabolic activation system, which was a cofactor-supplemented
post-mitochondrial S9 fraction prepared from the livers of phenobarbital/β-naphthoflavoneinduced
rats.
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 had no mutagenic activity on the growth of the
bacterial strains under the test conditions used in this study.

Executive summary:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.
The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers
of phenobarbital/β-naphthoflavone-induced rats.
The study included a Preliminary Compatibility Test, a Preliminary Concentration Range Finding Test (Plate Incorporation Method), an Assay 1 (Plate Incorporation Method) and an Assay 2 (Pre-Incubation Method).
Based on the results of the Compatibility Test, the test item was dissolved in DMSO.
Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate were examined in the Range Finding Test in Salmonella typhimurium TA98 and TA100 tester strains in the absence and presence of metabolic activation. Based on the results of the preliminary experiment, the examined test concentrations in Assay 1 were 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate
and in Assay 2 were 1581, 500, 158.1, 50, 15.81, 5, 1.581 and 0.5 μg/plate.
No precipitate was detected on the plates in the main tests in all examined bacterial strains with and without metabolic activation.
Inhibitory, cytotoxic effect of the test item (absent / reduced / slight reduced background lawn development) was observed in the Assay 1 in the examined bacterial strains with and/or without metabolic activation at 1581 and 500 μg/plate concentrations and slight reduced background lawn development was observed in Salmonella typhimurium TA100, TA1535 and TA1537
bacterial strains without metabolic activation at 158.1 μg/plate concentration. Same cytotoxic effect of the test item (absent / reduced / slight reduced background lawn development) was observed in the Assay 2 in the examined bacterial strains with and/or without metabolic activation at 1581, 500 and 158.1 μg/plate concentrations and slight reduced background lawn
development was observed in the examined bacterial strains without metabolic activation at 50 μg/plate concentration.
In the assays the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no reproducible dose-related trends and there was no indication of any treatment-related effect.
The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of
the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.
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 had no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the study results which are negatives, the test substance should not be classified as mutagenic nor clastogenic according to the EU CLP regulation (No 1272/2008 and its adaption 286/2011) and GHS.