[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECDTG471): not mutagenic
Chromosome aberration test(OECDTG473): not clastogenic
Mouse lymphoma test (OECDTG490): not mutagenic

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 May 2021 - 05 July 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

Thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: L5178Y/TK+/--3.7.2C mouse lymphoma cells. American Type Culture Collection, (ATCC, Manassas, USA) (2001).
- Suitability of cells: recommended test system in international guidelines

For cell lines:
- Absence of Mycoplasma contamination: Yes
- Methods for maintenance in cell culture: stock cultures of the cells were stored in liquid nitrogen (- 196°C).
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: Basic medium
RPMI 1640 Hepes buffered medium (Dutch modification) (Life Technologies) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively) (Life Technologies), 1 mM sodium pyruvate (Sigma, Zwijndrecht, The Netherlands) and 2 mM L-glutamin (Life Technologies).
Growth medium
Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum.
Exposure medium
Cells will be exposed to the test item in basic medium supplemented with 5% to 10% (v/v)
heat-inactivated horse serum.
Selective medium
Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum and 5 µg/mL trifluorothymidine (TFT) (Sigma).
Non-selective medium
Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum.
Environmental conditions
All incubations were carried out in a humid atmosphere (80 - 100%, actual range 50 - 98%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.1 - 39.9 °C).

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and was prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight).
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom); 4 µmol HEPES (Life technologies). The above solution was filter (0.22 µm)-sterilized. To 0.5 mL S9-mix components 0.5 mL.
- concentration or volume of S9 mix and S9 in the final culture medium: The concentration of the S9-fraction in the exposure medium was 4% (v/v).

Test concentrations with justification for top dose:

- Dose-range finding test, with S9 (3h) and without S9 (3h): 1, 5, 10, 25, 75 and 125 μg/mL
- Dose-range finding test, without S9 (24h): 1, 5, 10, 25, 75 and 125 μg/mL
- Experiment 1, with S9 (3h) and without S9 (3h): 0.01, 1.0, 5.0, 10, 25, 50, 75, 125, 150, 200, 250, 300 and 400 μg/mL exposure medium.
The dose levels selected to measure mutant frequencies at the TK-locus were:
Without and with S9-mix: 0.01, 1.0, 5.0, 10, 25, 50, 75 and 125 μg/mL
- Experiment 2, without S9 (24h): 0.5, 1.0, 2.5, 5.0, 10, 25, 35, 45, 55, 65, 75, 100 and 125 µg/mL exposure medium.
The dose levels selected to measure mutant frequencies at the TK-locus were:
0.5, 1.0, 2.5, 5.0, 10, 25 and 35 µg/mL exposure medium.

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle: A solubility test was performed based on visual assessment. The test item formed a clear (light) yellow solution in dimethyl sulfoxide (DMSO, Merck, armstadt, Germany).
Test item concentrations were used within 1 hour after preparation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): test concentrations: 1 per test concentration; positive control: 1; solvent control: 2
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): below 1 x 10^6 cells/mL
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
Short term treatment: 3 hours (with and without S9-mix)
Prolonged treatment: 24 hours (without S9-mix)

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 days in which at least 4 x 10^6 cells were subcultured every day.
- Selection time (if incubation with a selective agent): 11 or 12 days
- Method used: microwell plates
- Selective agent is used: 5 μg/mL trifluorothymidine (TFT)
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-wel
l dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective m
edium.
For determination of the mutation frequency (MF) a total number of 9.6 x 105 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 105 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 1.5-2 hours, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.
- Criteria for small (slow growing) and large (fast growing) colonies: The small colonies are morphologically dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphologically less dense colonies with a hazy contour and with a diameter larger than a quarter of a well. A well containing more than on e small colony is classified as one small colony. A well containing more than one large colony is classified as one large colony. A well containing one small and one large colony is classified as one large colony.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: - Method: relative total growth (see 'Any other information on materials and methods incl. tables' for details on calculations)

Evaluation criteria:

The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/
solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for
positive or negative result can be made after an additional confirmation study.
A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested
concentrations reaches a mutation frequency of MF(controls) + 126.



ACCEPTABILITY CRITERIA
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analyzed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The suspension growth (SG) over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutation frequency,
that is, an increase above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10^-6. At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control has an increase in the small colony MF of at least 150 x 10^-6 above that seen in the concurrent solvent control (a small colony IMF of 150 x 10^-6).

Statistics:

Not performed.

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH and osmolarity: The pH and osmolarity at a concentration of 75 μg/mL were 7.28 and 0.465 Osm/kg respectively (compared to 7.29 and 0.464 Osm/kg in the solvent control).
- Precipitation and time of the determination: The test item precipitated directly and after 3 hours in the test system at concentrations of 125 μg/mL and above. After 24 hours the test item precipitated in the test system at concentrations of 75 μg/mL and above.

RANGE-FINDING/SCREENING STUDIES:
In the absence of S9-mix (3 hours treatment), the relative suspension growth was 59% at the test item concentration of 125 μg/mL compared to the relative suspension growth of the solvent control.
In the presence of S9-mix (3 hours treatment), the relative suspension growth was 68% at the test item concentration of 125 μg/mL compared to the relative suspension growth of the solvent control.
The relative suspension growth was 58% at the test item concentration of 25 μg/mL compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at the test item concentration of 75 and 125 μg/mL after the 24 hours subculture period without S9-mix.

STUDY RESULTS
- Concurrent vehicle negative and positive control data:
The mutant frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.
Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutant frequency. In addition, the mutant frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
Experiment 1:
In the absence of S9-mix, the relative total growth of the highest test item concentration was 77% compared to the total growth of the solvent controls. In the presence of S9-mix, the relative total growth of the highest test item concentration was 39% compared to the total growth of the solvent controls.
Experiment 2:
The relative total growth of the highest test item was 8% compared to the total growth of the solvent controls.


- Genotoxicity results:
No biologically relevant increase in the mutant frequency at the TK locus was observed after treatment with the test item in the absence and presence of S9-mix. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data:
Mutant frequency per 10^6 survivors
-S9 Mix +S9 mix
3 hour treatment 24 hour treatment 3 hour treatment
Mean 961 776 1154
SD 408 236 648
n 94 89 93
Lower Control Limit (95% Control Limits) 162 313 -116
Upper Control Limit (95% Control Limits) 1761 1239 2425
SD = Standard deviation
n = Number of observations
Distribution historical positive control data from experiments performed between May 2018 and May 2021.

- Negative (solvent/vehicle) historical control data:
Mutant frequency per 10^6 survivors
-S9 Mix +S9 mix
3 hour treatment 24 hour treatment 3 hour treatment
Mean 100 99 100
SD 28 28 28
n 96 87 93
Lower Control Limit (95% Control Limits) 45 44 46
Upper Control Limit (95% Control Limits) 154 153 154
SD = Standard deviation
n = Number of observations
Distribution historical negative control data from experiments performed between May 2018 and May 2021.

Conclusions:

The substance is not mutagenic in the TK mutation test system.

Executive summary:

A gene mutation test in mammalian cells was performed according to OECD guideline 490 and GLP principles, to assess the potential of the substance to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells.
In the first experiment, the test item was tested up to concentrations of 125 μg/mL in the absence and presence S9-mix, respectively. The incubation time was 3 hours. The RTG was reduced to 77% and 39% for the concentration of 125 μg/mL in the absence and presence of S9-mix, respectively. The test item precipitated in the culture medium at this dose level.
In the second experiment, the test item was tested up to concentrations of 35 μg/mL in the absence of S9-mix. The incubation time was 24 hours. The RTG was reduced to 8% and 38% for concentrations of 35 and 25 μg/mL, respectively. Reliable negative and positive controls were included in both experiments.
In the absence and presence of S9 -mix, the substance did not induce a biologically relevant increase in the mutation frequency. In conclusion, the substance is not mutagenic in the mouse lymphoma L5178Y test system.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 February 2021 - 12 April 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: Cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Sex, age and number of blood donors: Blood was collected from healthy adult, non-smoking volunteers (approximately 18 to 35 years of age). The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2020) are presented below:
Dose-range finding study/ First cytogenetic assay: age 24, AGT = 12.9 h
Second cytogenetic assay: age 29, AGT = 14.0 h
- Whether whole blood or separated lymphocytes were used: Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin (Vacuette, Greiner Bio-One, Alphen aan den Rijn, The Netherlands). Immediately after blood collection lymphocyte cultures were started.
- Mitogen used for lymphocytes: Phytohaemagglutinin

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Culture medium consisted of RPMI 1640 medium (Life technologies), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life technologies), L-glutamine (2 mM) (Life technologies), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively) (Life technologies) and 30 U/mL heparin (Sigma, Zwijndrecht, The Netherlands).
All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 39 - 100%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.1 - 39.6°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Rat S9 homogenate was obtained from Trinova Biochem GmbH, Giessen, Germany and is prepared from male Sprague Dawley rats that have been dosed orally with a suspension of
phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix components
contained per mL physiological saline: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom); 4 μmol HEPES (Life technologies).
The above solution was filter (0.22 μm)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
- concentration or volume of S9 mix and S9 in the final culture medium: The concentration of the S9-fraction in the exposure medium was 1.8% (v/v).

Test concentrations with justification for top dose:

Dose range finding test:
Without S9-mix, 24hr exposure; 24hr fixation: 10, 20, 39, 78 and 156 μg/mL
First cytogenetic test:
Without and with S9-mix, 3hr exposure time, 24hr fixation time: 39, 78 and 156 μg/mL
All doses were selected for scoring of chromosome aberrations.
Second cytogenetic test:
Without S9-mix, 24hr exposure; 24hr fixation: 10, 20, 30, 40, 50 and 60 μg/mL
The following doses were selected for scoring of chromosome aberrations:
Without S9-mix, 24hr exposure; 24hr fixation: 30, 40 and 50 μg/mL

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: A solubility test was performed based on visual assessment. The test item formed a clear solution in dimethyl sulfoxide (DMSO, SeccoSolv, Merck, Darmstadt, Germany) at concentrations of 250 mg/ml and below. In the solubility test the stock solution was treated with ultrasonic until the test item had completely dissolved.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): duplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 48 hours
- Exposure duration/duration of treatment:
- Harvest time after the end of treatment (sampling/recovery times): 3 hr (with and without S9-mix), 24 hr (without S9-mix)

FOR CHROMOSOME ABERRATION:
- Spindle inhibitor (cytogenetic assays): During the last 2.5 - 3 h of the culture period, cell division was arrested by the addition of the spindle inhibitor colchicine (0.5 μg/mL medium).
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. The slides were marked with the Charles River Den Bosch study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 6.7% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper (ClearVue Coverslipper, Thermo Fisher Scientific, Breda, The Netherlands).
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): One hundred and fifty metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Only metaphases containing 46 ± 2 centromeres (chromosomes) were analyzed.
- Determination of polyploidy: yes
- Determination of endoreplication: yes

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: The mitotic index of each culture was determined by counting the number of metaphases from at least 1000 cells (with a maximum deviation of 5%).

Evaluation criteria:

A test item is considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact
test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, onesided,
p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

Statistics:

Graphpad Prism version 8.4 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.

Key result

Species / strain:

lymphocytes: Cultured peripheral human lymphocytes

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

24 hours exposure time

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: Cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

3 hours exposure time

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH and on osmolarity: The pH and osmolarity of a concentration of 78 μg/mL were 7.7 and 436 mOsm/kg respectively (compared to 7.6 and 433 mOsm/kg in the solvent control).
- Precipitation and time of the determination: The test item precipitated at concentrations of 156 μg/mL and upwards.

RANGE-FINDING/SCREENING STUDIES: Cytotoxicity was observed at the concentrations of 39, 78 and 156 μg/mL at the 24hr treatment time without S9-mix.

STUDY RESULTS
- Concurrent vehicle negative and positive control data:
The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database, except in the absence of S9-mix at the 3 h exposure time, the response of MMC-C was above the upper control limits, these limits are 95% control limits and a higher response is within the expected response ranges. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
No cytotoxicity was observed in the duplicate cultures of the 3 hr exposure time with and without
S9-mix. Cytotoxicity was observed at the concentrations of 40 and 50 μg/mL at the 24hr
treatment time without S9-mix.
- Genotoxicity results (for both cell lines and lymphocytes)
Both in the absence and presence of S9-mix the test item did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.
No effects of the test item on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore, it can be
concluded that the test item does not disturb mitotic processes and cell cycle progression and
does not induce numerical chromosome aberrations.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data:
3 hours exposure 24 hours exposure
Gaps included Gaps excluded Gaps included Gaps excluded
-S9 mix +S9 mix -S9 mix +S9 mix -S9 mix -S9 mix
Number of aberrant cells (per 300 cells) 62.3 55.1 61.0 53.4 80.2 78.6
SD 25.5 20.6 25.6 20.7 38.0 38.5
n 67 66 67 66 63 63
Lower Control Limit (95% Control Limits)13 15 11 13 6 4
Upper Control Limit (95% Control Limits)112 96 111 94 155 154
SD = Standard deviation
n = Number of observations
Distribution historical positive control data from experiments performed between November 2017 and November 2020.

- Negative (solvent/vehicle) historical control data:
3 hours exposure 24 hours exposure
Gaps included Gaps excluded Gaps included Gaps excluded
-S9 mix +S9 mix -S9 mix +S9 mix -S9 mix -S9 mix
Number of aberrant cells (per 300 cells) 1.3 1.0 1.0 0.9 1.0 0.8
SD 1.5 1.4 1.3 1.2 1.4 1.3
n 66 66 66 66 63 63
Lower Control Limit (95% Control Limits) -2 -2 -2 -2 -2 -2
Upper Control Limit (95% Control Limits) 4 4 4 3 4 3
SD = Standard deviation
n = Number of observations
Distribution historical negative control data from experiments performed between November 2017 and November 2020.

Conclusions:

A chromosome aberration study with the substance was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. It is concluded that the substance is not clastogenic in human lymphocytes.

Executive summary:

A chromosome aberration study with the substance was performed according to OECD guideline 473 and GLP principles, in cultured peripheral human lymphocytes in two independent experiments.

The test item precipitated at the concentration of 156 μg/mL. No cytotoxicity was observed in the duplicate cultures of the 3 hr exposure time with and without S9-mix. Cytotoxicity was observed at the concentrations of 40 and 50 μg/mL at the 24hr treatment time without S9-mix. Reliable positive and negative controls were included.

Both in the absence and presence of S9-mix the test item did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.
No effects of the test item on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore, it can be concluded that the test item does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations.

Based on the results it can be concluded that Pergafast 425 is not clastogenic in human lymphocytes.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 July 2020 - 27 July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

31 may 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guideline stipulated by the Japanese Ministry of Health, Labour and Welfare, Ministry of Economy, Trade and Industry and Ministry of the Environment

Version / remarks:

revised March 31st, 2011

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Rat liver microsomal enzymes (S9 homogenate) was obtained from Trinova Biochem GmbH, Giessen, Germany and was prepared from male Sprague Dawley rats that had been injected intraperitoneal with Aroclor 1254 (500 mg/kg body weight).
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix contained per 10 mL: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg
glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 mL or 5.0 mL Milli-Q water (first or second experiment respectively) (Millipore Corp., Bedford, MA., USA); 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution (Merck); 1 mL 0.33 M KCl solution (Merck). The above solution was filter (0.22 μm)-sterilized.
- concentration or volume of S9 mix and S9 in the final culture medium: S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix in the first experiment and to 9.0 mL of S9-mix components 1.0 mL S9-fraction was added (10% (v/v) S9-fraction) to complete the S9-mix in the second experiment.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Each S9 batch is characterized with the mutagens Benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively.

Test concentrations with justification for top dose:

Experiment 1:
All five strains, without and with S9: 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate
Experiment 2:
All five strains, without and with S9: 275, 492, 878, 1568, 2800 and 5000 μg/plate

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: A solubility test was performed based on visual assessment. The test item formed a clear yellow solution in dimethyl sulfoxide (DMSO).
Test item concentrations were used within 2 hours after preparation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191, 2.5 μg in DMSO for TA1537

Remarks:

Without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA) in DMSO, 1 μg for TA98 and TA100, 2.5 μg for TA1535 and TA1537 (5% S9), 5 μg for TA1537 (10% S9), 15 μg for WP2uvrA

Remarks:

With S9

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

METHOD OF TREATMENT/ EXPOSURE:
in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Amount of revertant colonies.

OTHER:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent vehicle control.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent vehicle control.
b) In case a positive response will be repeated, the positive response should be reproducible in at least one independently repeated experiment.

Statistics:

Not performed.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

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:

cytotoxicity

Remarks:

at the highest tested concentration in experiment 2.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination:
Experiment 1: The test item precipitated on the plates at dose levels of 1600 μg/plate and upwards. Since the test item precipitated heavily on the plates at the test item concentration of 5000 μg/plate, the number of revertants of these dose level could not be determined.
Experiment 2:
The test item precipitated on the plates at dose levels of 1568 μg/plate and higher. Since the test item precipitated heavily on the plates at the test item concentration of 5000 μg/plate, the number of revertants of these dose level could not be determined.

STUDY RESULTS
- Concurrent vehicle negative and positive control data :
The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Ames test:
In the first mutation assay, the test item was tested up to concentrations of 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix. The test item precipitated on the plates at dose levels of 1600 μg/plate and upwards. Since the test item precipitated heavily on the plates at the test item concentration of 5000 μg/plate, the number of revertants of these dose level could not be determined. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.
In the second mutation assay, the test item was tested up to concentrations of 5000 µg/plate in the absence and presence of 10% (v/v) S9-mix. The test item precipitated on the plates at dose levels of 1568 μg/plate and higher. Since the test item precipitated heavily on the plates at the test item concentration of 5000 μg/plate, the number of revertants of these dose level could not be determined. Cytotoxicity, as evidenced by a decrease in the number of revertants, was only observed in tester strain WP2uvrA in the absence of S9-mix at the highest tested concentration.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data:
TA1535 TA1537 TA98
S9-mix - + - + - +
Range 107 – 1530 78 – 1481 58 – 1467 48 – 1239 365 – 2118 258 – 2033
Mean 955 282 811 290 1220 903
SD 174 114 372 138 221 367
n 2598 2587 2223 2630 2670 2621

TA100 WP2uvrA
S9-mix - + - +
Range 439 – 1993 397 – 2666 93 – 1999 109 - 1968
Mean 879 1371 1167 421
SD 170 412 527 169
n 2654 2634 2516 2528

SD = Standard deviation
n = Number of observations
Historical control data from experiments performed between Jun 2017 and Jun 2020.

- Negative (solvent/vehicle) historical control data:
TA1535 TA1537 TA98 TA100 WP2uvrA
S9-mix - + - + - + - + - +
Range 3 – 29 4 – 25 2 – 24 2 – 18 5 – 61 8 – 60 61 – 188 55 - 176 11 – 61 12 - 68
Mean 10 11 5 6 14 19 110 103 25 31
SD 3 3 2 2 5 6 18 21 8 10
n 2733 2694 2707 2712 2717 2754 2806 2724 2622 2596

SD = Standard deviation
n = Number of observations
Historical control data from experiments performed between Jun 2017 and Jun 2020.

Conclusions:

In an Ames test, performed according to OECD 471 guideline and GLP principles, the substance was found not to be mutagenic with or without metabolic activation.

Executive summary:

An Ames test was performed according to OECD 471 guideline and GLP principles.

In the first mutation assay, the test item was tested up to concentrations of 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix. The test item precipitated on the plates at dose levels of 1600 μg/plate and upwards. Since the test item precipitated heavily on the plates at the test item concentration of 5000 μg/plate, the number of revertants of these dose level could not be determined. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the second mutation assay, the test item was tested up to concentrations of 5000 µg/plate in the absence and presence of 10% (v/v) S9-mix. The test item precipitated on the plates at dose levels of 1568 μg/plate and higher. Since the test item precipitated heavily on the plates at the test item concentration of 5000 μg/plate, the number of revertants of these dose level could not be determined. Cytotoxicity, as evidenced by a decrease in the number of revertants, was only observed in tester strain WP₂uvrA in the absence of S9-mix at the highest tested concentration.

The test item did not induce a dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP₂uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

In conclusion, based on the results of this study it is concluded that Pergafast 425 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test:

An Ames test was performed according to OECD 471 guideline and GLP principles.

In the first mutation assay, the test item was tested up to concentrations of 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix. The test item precipitated on the plates at dose levels of 1600 μg/plate and upwards. Since the test item precipitated heavily on the plates at the test item concentration of 5000 μg/plate, the number of revertants of these dose level could not be determined. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the second mutation assay, the test item was tested up to concentrations of 5000 µg/plate in the absence and presence of 10% (v/v) S9-mix. The test item precipitated on the plates at dose levels of 1568 μg/plate and higher. Since the test item precipitated heavily on the plates at the test item concentration of 5000 μg/plate, the number of revertants of these dose level could not be determined. Cytotoxicity, as evidenced by a decrease in the number of revertants, was only observed in tester strain WP₂uvrA in the absence of S9-mix at the highest tested concentration.

The test item did not induce a dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP₂uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

In conclusion, based on the results of this study it is concluded that Pergafast 425 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

 

Chromosome aberration test:

A chromosome aberration study with the substance was performed according to OECD guideline 473 and GLP principles, in cultured peripheral human lymphocytes in two independent experiments.

The test item precipitated at the concentration of 156 μg/mL. No cytotoxicity was observed in the duplicate cultures of the 3 hr exposure time with and without S9-mix. Cytotoxicity was observed at the concentrations of 40 and 50 μg/mL at the 24hr treatment time without S9-mix. Reliable positive and negative controls were included.

Both in the absence and presence of S9-mix the test item did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.
No effects of the test item on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore, it can be concluded that the test item does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations.

Based on the results it can be concluded that Pergafast 425 is not clastogenic in human lymphocytes.

 

Mouse lymphoma test:

A gene mutation test in mammalian cells was performed according to OECD guideline 490 and GLP principles, to assess the potential of the substance to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells.
In the first experiment, the test item was tested up to concentrations of 125 μg/mL in the absence and presence S9-mix, respectively. The incubation time was 3 hours. The RTG was reduced to 77% and 39% for the concentration of 125 μg/mL in the absence and presence of S9-mix, respectively. The test item precipitated in the culture medium at this dose level.
In the second experiment, the test item was tested up to concentrations of 35 μg/mL in the absence of S9-mix. The incubation time was 24 hours. The RTG was reduced to 8% and 38% for concentrations of 35 and 25 μg/mL, respectively.  Reliable negative and positive controls were included in both experiments.
In the absence and presence of S9 -mix, the substance did not induce a biologically relevant increase in the mutation frequency. In conclusion, the substance is not mutagenic in the mouse lymphoma L5178Y test system.

Justification for classification or non-classification

Based on the results of the Ames test, in vitro chromosome aberration and mouse lymphoma studies, the substance does not have to be classified for genotoxicity in accordance with Regulation (EC) No 1272/2008 and its amendments.