methyl 2-hydroxybut-3-enoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation in bacteria

The non-purified and non-stabilised test substance showed mutagenic effects (base pair changes in the genome of tester strain TA100) in a bacterial reverse mutation assay according to OECD guideline 471.

 

The purified but non-stabilised test substance did not cause gene mutations in a bacterial reverse mutation assay according to OECD guideline 471.

 

To test the effects of purification and addition of different stabilisers, a screening test (MiniAmes) with 6 different test substance batches was performed using two Salmonella typhimurium strains (TA98 and TA100). The purified substance stored under oxygen exclusion was found to be non-mutagenic (test item 1). A non-purified batch exposed to oxygen was mutagenic (test item 2). The purified substance exposed to oxygen but containing an appropriate stabiliser (0.5% propyl gallate or 0.5% BHT) was not mutagenic (test item 3 and test item 5). Likewise, storage of the purified substance in a nitrogen-stabilised atmosphere showed no mutagenic effect under exposure to oxygen (test item 6). The pure test substance with 0.5% vitamin E showed insufficient stabilisation and was mutagenic under the influence of oxygen (test item 4).

It can be concluded that the non-stabilised test substance forms mutagenic oxidation products if exposed to oxygen/air during storage. The properly stabilised test substance was shown not to form oxidising decomposition products even under O2 stress. The registered form of the test substance contains such a stabiliser and is therefore be considered non-mutagenic in the Bacterial Reverse Mutation Assay. 

 

In vitro gene mutation in mammalian cells

The purified but non-stabilised test substance was tested in an in vitro Mouse Lymphoma Assay according to OECD guideline 490. The test substance was mutagenic with and without metabolic activation. An increase in mainly small colonies indicates a clastogenic mechanism.

 

The purified and stabilised (0.05% BHT) test substance was tested in an in vitro Mammalian Cell Gene Mutation Test (HPRT assay) according to OECD guideline 476. The test substance was not mutagenic with and without metabolic activation. 

 

In vitro cytogenicity in mammalian cells

The purified and stabilised (0.05% BHT) test substance was tested in an in vitro Chromosome Aberration Test according to OECD guideline 473. The test substance was clastogenic without metabolic activation. 

 

Overall conclusion

The non-purified and non-stabilised test substance is mutagenic and clastogenic in all available in vitro assays. The purified and properly stabilised (e.g. 0.05-0.5% BHT) test substance as being produced and marketed shows no mutagenic properties as shown in the bacterial reverse mutation screening assays and HPRT test. In contrast, clastogenicity also occured with the purified and stabilised test item in vitro (chromosome aberration test). The fact that the purified but non-stabilised test substance was non-mutagenic in the bacterial reverse mutation assay but indicated clastogenicity in the Mouse Lymphoma Assay supports the assumption that clastogenicity is of greater concern than mutagenicity. Thus, it is concluded that purification and stabilisation reliably eliminates impurities and prevents oxidation products leading to mutagenic reactions but clastogenicity remains of concern. For that reason, further in vivo testing was conducted.

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:

weight of evidence

Study period:

2017-05-19 to 2017-09-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted with the purified but non-stabilised form of the test substance. Storage conditions were not specified.

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

2008-05-30

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 2016-07-29

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:

TK locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Mouse Lymphoma L5178Y cells (clone TK+/- -3.7.2C)
- Suitability of cells: Yes, recommended according to OECD 490 because they are heterozygous at the thymidine kinase (TK) locus, because of their high proliferation rate, good cloning efficiency and stable karyotype
- Normal cell cycle time (negative control): 10-12 h doubling time

For cell lines:
- Absence of Mycoplasma contamination: Yes. Each cell batch is routinely checked for mycoplasma infection.
- Methods for maintenance in cell culture: Thawed stock cultures are maintained in plastic culture flasks in RPMI 1640 complete medium and subcultured three times per week.
- Doubling time: 10-12 h
- Modal number of chromosomes: 40 +/- 2 chromosomes
- Periodically ‘cleansed’ of spontaneous mutants: Yes


MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature:
Complete culture medium (CCM): RPMI 1640 supplemented with 10% horse serum, 100 U/100 µg/mL penicillin/streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES and 2.5 µg/mL amphotericin B
Treatment medium: CCM, but with 5% horse serum instead of 10%
Selective medium: CCM, but with 20% horse serum instead of 10% and 5 µg/mL TFT
Temperature: 37°C
Humidity: 95%
CO2 concentration: 5%

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: Phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw) induced rats
- Method of preparation of S9 mix: The cofactor solution was prepared by mixing 8 mM MgCl2, 33 mM KCl, 5 mM Glucose-6-phosphate and 5 mM NADP in ice-cold 100 mM sodium phosphate buffer pH 7.4. An appropriate quantity of the S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL
- Concentration or volume of S9 mix and S9 in the final culture medium: Volumes were chosen to achieve a final protein concentration of 0.75 mg/mL in the cultures (approx. 2% S9 in the final culture medium)
- Quality controls of S9: Biological activity was tested in the Salmonella typhimurium assay using 2-aminoanthracene, in the mouse lymphoma assay using benzo[a]pyrene and in the chromosome aberration assay using cyclophosphamide. Sterility was also tested (not specified).

Test concentrations with justification for top dose:

Pre-experiment: 0.2, 0.5, 2.5, 5.0, 7.5, 10 mM (with and without S9 mix)
Main experiment (with S9 mix): 0.10, 0.15, 0.30, 0.35, 0.40 and 0.45 mM
Main experiment (without S9 mix): 0.10, 0.2, 0.3, 0.4, 0.5 and 0.6 mM

Vehicle / solvent:

- Vehicle used: Treatment medium (test item, EMS), 0.9% NaCl (MMS), DMSO (Benzo[a]pyrene)

- Justification for choice of solvent/vehicle: The vehcile was chosen based on the results of the solubility test.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Same as untreated negative control as treatment medium was used as solvent (test item, EMS). For MMS, 0.9% NaCl was used as solvent control and for Benzo[a]pyrene, DMSO was used

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

methylmethanesulfonate

Remarks:

Without S9:
EMS: 300 µg/mL
MMS: 10 µg/mL
With S9:
BEP: 1.5 µg/mL

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Single
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 1 x 10^7 cells in 11 mL medium were exposed with the test item. During the expression period, the cell density was determined each day and adjusted to 3 x 10^5 cells/mL. After the expression period the cloning efficiency (CE) was determined by seeding 1.6 cells/well. For selection, 2000 cells/well were seeded in 200 μL selective medium.
- Test substance added in: Medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 hours (with and without metabolic activation)
- Harvest time after the end of treatment: After 4 hours of treatment, cells were incubated for a expression and growth period of 2 days. After the expression period, cells were incubated for at least 6 days for CE determination. Additionally, cells were seeded in selective medium and incubated for approx. 12 days


FOR GENE MUTATION:
- Expression time: 2 days
- Selection time: Approx. 12 days
- Fixation time (start of exposure up to fixation or harvest of cells): Approx. 20 days
- Method used: Microwell method
- Selective agent used Trifluorothymidine (5 µg/mL, cells were treated for 12 days)
- Criteria for small (slow growing) and large (fast growing) colonies: Approx. ≤ ¼ of well diameter for small colonies and approx. > ¼ of well diameter for large colonies

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Relative total growth (RTG)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The mutant frequency was calculated by dividing the number of TFT resistant colonies by the number of cells plated for selection, corrected for the plating efficiency of cells from the same culture grown in the absence of TFT.

Rationale for test conditions:

The test concentrations were selected based on a preliminary test for toxicity.

Evaluation criteria:

The test item is considered mutagenic if the following criteria are met:
- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 10^6 cells and
- a dose-dependent increase in mutant frequency is detected

Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations. According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation of the test result. A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend of the test is negative.

Statistics:

The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative/solvent controls. Mutant frequencies of the solvent/negative controls were used as reference.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

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: The pH-value detected with the test item was within the physiological range.
- Data on osmolality: The osmolality was within the physiological range.
- Water solubility: Yes.
- Precipitation and time of the determination: No precipitation of the test item was noted in the pre-experiment and main experiment at the time of evaluation.

RANGE-FINDING/SCREENING STUDIES: The toxicity of the test item was determined in a pre-experiment up to a maximum concentration of 10 mM. Six concentrations [0.2, 0.5, 2.5, 5.0, 7.5, 10 mM] were tested without and with metabolic activation. After a 2-day growth period the relative suspension growth (RSG) of the treated cell cultures was calculated according to the method of Clive and Spector. The selection of the concentrations used in the main experiment was based on data from the pre-experiment. 0.6 mM (without metabolic activation) and 0.45 mM (with metabolic activation) were selected as the highest concentrations.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: See "Attached background material"

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship: Yes.
- Statistical analysis; p-value: Statistical significant increase in mutant frequency compared to negative controls (Mann Whitney test , p<0.05) at all concentrations without metabolic activation and at 0.30 mM and above with metabolic activation.
- Any other criteria: The mutant frequencies obtained from all experiments were compared with the Global Evaluation Factor (GEF). For the microwell method the GEF was defined to be 126. The GEF was exceeded at 0.5 mM and above without metabolic activation and at 0.35 mM and above with metabolic activation.

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: The relative total growth (RTG) was 13.1% (without metabolic activation) and 12.3% (with metabolic activation) for the highest concentration evaluated.
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency: See "Attached background material"

- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures: See "Details on test system and experimental conditions"
o Number of cells plated in selective and non-selective medium: See "Details on test system and experimental conditions"
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency: See "Attached background material"
o Colony sizing for the negative and positive controls and test chemical, related mutant frequency and GEF evaluation: Colony sizing was performed for the highest concentrations of the test item and for the negative and positive controls. An extension of the GEF by the induced mutant frequency in combination with an increased occurrence of small colonies is an indication for potential clastogenic effects and/or chromosomal aberrations. The positive controls MMS and B[a]P induced a significant increase in mutant frequency and a biologically significant increase of small colonies (≥40%), thus proving the ability of the test system to indicate potential clastogenic effects. In the main experiment without and with metabolic activation the percentage of small colonies in the negative controls was found to be lower than 40%. Due to the increased number of small colonies and corresponding mutagenicity in the two highest dose groups (without metabolic activation) and in the highest dose group (with metabolic activation), these concentrations of the test items were considered as clastogenic.

HISTORICAL CONTROL DATA
- Positive historical control data: See "Attached background material"
- Negative (solvent/vehicle) historical control data: See "Attached background material"

Conclusions:

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item is considered to be mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Executive summary:

The test item was assessed for its potential to induce mutations according to OECD guideline 490 and GLP at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The experiment without and with metabolic activation was performed as a 4 h short-term exposure assay. The selection of the concentrations used in the main experiment was based on data from the pre- experiment. The test item was investigated at the following concentrations:

 

 

Without metabolic activation: 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 mM

 

and with metabolic activation: 0.10, 0.15, 0.30, 0.35, 0.40 and 0.45 mM

 

No precipitation of the test item was noted in the experiment. Growth inhibition was observed in the main experiment without and with metabolic activation: The relative total growth (RTG) was 13.1% (without metabolic activation) and 12.3% (with metabolic activation) for the highest concentration evaluated.

Biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). The Global Evaluation Factor (GEF; defined as the mean of the negative/vehicle mutant frequency plus one standard deviation; data gathered from ten laboratories) was exceeded by the induced mutant frequency at concentrations of 0.5 mM and higher (without metabolic activation) and at concentrations of 0.35 mM and higher (with metabolic activation). Moreover, a dose-response relationship was observed. Additionally, colony sizing showed clastogenic effects induced by the test item under the experimental conditions (without and with metabolic activation). EMS, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.

 

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item is considered to be mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2021-06-09 to 2021-07-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted with the purified and stabilised form of the test substance. The test item was stored under inert atmosphere (nitrogen).

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

2016-07-29

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Sub-line (K1) of Chinese hamster ovary cell line CHO
- Suitability of cells: Yes, CHO cells have a demonstrated sensitivity to chemical mutagens, a high cloning efficiency, a stable karyotype, and a stable spontaneous mutant frequency.
- Normal cell cycle time (negative control): Approx. 22 hours doubling time

For cell lines:
- Absence of Mycoplasma contamination: Yes (Each batch of frozen cells was tested for mycoplasma infections)
- Methods for maintenance in cell culture: Growing cells were subcultured in an appropriate number of flasks.
- Doubling time: Approx. 22 hours
- Periodically checked for karyotype stability: Not specified
- Periodically ‘cleansed’ of spontaneous mutants: Yes (each batch of frozen cells was purged of HPRT mutants)

MEDIA USED
Solvent: Ham's F12 medium supplemented with 1 % Antibiotic-antimycotic solution (containing 10000 U/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B), 0.0146 mg/mL L-Glutamine
Growth medium: Ham's F12 medium (F12-10) supplemented with 1 % Antibiotic-antimycotic solution (containing 10000 U/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B), 0.0146 mg/mL L-Glutamine and heat-inactivated bovine serum (final concentration 10 %)
Treatment medium: Ham's F12 medium (F12-10) supplemented with 1 % Antibiotic-antimycotic solution (containing 10000 U/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B), 0.0146 mg/mL L-Glutamine and heat-inactivated bovine serum (final concentration 5 %)
Selection medium: Ham's F12*-SEL (EX-CELL® CD CHO Serum-Free Medium for CHO Cells F12 medium without hypoxanthine, without thymidine) supplemented with 1 % Antibiotic-antimycotic solution (containing 10000 U/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B), 0.03 mg/mL L-Glutamine and heat-inactivated bovine serum (final concentration 10 %) and 3.4 μg/mL 6-thioguanine (6-TG)
CO2 concentration: 5 %
Humidity: Humidified atmosphere (approx. 95%)
Temperature: 37 °C

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: phenobarbital (PB) and β-naphthoflavone (BNF) induced rats
- Method of preparation of S9 mix: 0.2 mg/mL of 20 mM HEPES, 0.1 mg/mL 330 mM KCl, 0.1 mg/mL 50 mM MgCl2, 0.1 mg/mL 40 mM NADP, 0.1 mg/mL 50 mM D-Glucose-6-phosphate, 0.1 mg/mL Ham’s F12 medium and 0.3 mL/mL S9 were mixed and kept in an ice bath.
- Concentration or volume of S9 mix and S9 in the final culture medium: 600 µL S9 mix containing approx 33% S9 in a final culture medium of 15 mL
- Quality controls of S9: A certificate of analysis is available from the S9 supplier

Test concentrations with justification for top dose:

Preliminary experiment with S9-mix: 1, 2, 4, 7.9, 15.7, 31.3 and 62.5 µg/mL;

Preliminary experiment without S9-mix: 17.9, 15.7, 31.3 and 62.5, 125, 250 and 300 µg/mL;

Main experiment, 5-hour treatment period without S9-mix: 25, 50, 100, 170, 230 and 260 μg/mL;

Main experiment, 5-hour treatment period with S9-mix: 1, 2, 4, 8, 12, and 16 μg/mL

Vehicle / solvent:

- Vehicle used: Ham's F12 medium (test item, EMS), DMSO (DMBA)

- Justification for choice of solvent/vehicle: Solubility was tested in a preliminary solubility test. The test item was soluble in Ham's F12 medium up to a concentration of 100 mg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ham's F12 medium (test item, EMS), DMSO (DMBA)

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Remarks:

Without S9:
EMS: 1.0 μL/mL
With S9:
DMBA: 20 μg/mL

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate
- Number of independent experiments: 1 (one experiment with and one without metabolic activation)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 10 x10^6 cells/dish
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 5 hours
- Harvest time after the end of treatment: 6 days (for determination of survival), 13-15 days for determination of viability (7-9 days expression period + 6 days for colony growing), 15-17 days for determination of mutagenicity (7-9 days expression period + 8 days selection period)

FOR GENE MUTATION:
- Expression time: 7-9 days
- Selection time: 8 days
- Fixation time: 15-17 days
- Selective agent: 6-thioguanine (3.4 μg/mL), 8 days
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 10 x10^6 cells/dish for treatment, 10^5 cells/mL for expression period (during this period, cells were regularly sub-cultured to maintain them in exponential growth. 2x10^6 cells/dish), 200 cells/dish for survival, 2x10^5 cells / dish for selection, 10^5 cells/mL for determination of viability. After the selection period, the colonies were fixed with methanol for five minutes, stained with Giemsa and counted for either mutant selection or cloning efficiency determination. For viability, colonies were fixed with methanol, stained with Giemsa and counted. Cloning Efficiency was assessed
(Number of colonies/Number of cells plated).

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Relative Survival / Cloning efficiency

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The mutation frequency was calculated by dividing the total number of mutant colonies by the number of cells selected (2x10^6 cells: 2 x 5 dishes at 2x10^5 cells/dish), corrected for the cloning efficiency of cells prior to mutant selection (viability), and was expressed as 6-TG resistant mutants per 10^6 clonable cells.

Rationale for test conditions:

Concentration were selected based on a preliminary cytotoxicity test

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
• at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
• any of the results are outside the distribution of the laboratory historical negative control data (based 95% control limit),
• the increase of mutant frequency is concentration-related when evaluated with an appropriate trend test.

Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if, in all experimental conditions examined:
• none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
• there is no concentration-related increase when evaluated with an appropriate trend test, all results are inside the distribution of the historical negative control data (based 95% control limit).

Statistics:

Statistical Analysis was performed with SPSS PC+ software for the following data:
• mutant frequency between the negative (solvent) control group and the test item or positive control item treated groups.
• mutant frequency between the laboratory historical negative (solvent) control group and concurrent negative (solvent) control, the test item or positive control item treated groups.
• The data were checked for a linear trend in mutant frequency with treatment dose using the adequate regression analysis by Microsoft Excel software.

The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis one-way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using the Mann Whitney U-test.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

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: No biologically relevant changes in pH of the test system were noted at the different concentration levels tested.
- Data on osmolality: No biologically relevant changes in osmolality of the test system were noted at the different concentration levels tested.
- Water solubility: The test item was soluble in Ham's F12 medium
- Precipitation and time of the determination: For examined test item concentrations, no precipitation in the medium was noted at the beginning and end of treatment.

RANGE-FINDING/SCREENING STUDIES: A Pre-test on Toxicity was performed to establish an appropriate concentration range for the main Mutation Assay, both in the absence and in the presence of metabolic activation (rodent S9-mix). Cells were treated for 5 hours and then sub-cultured for days. Cytotoxicity was determined by RS (relative survival), where the cloning efficiency (CE) of cells plated immediately after treatment adjusted by any loss of cells during treatment as compared with adjusted cloning efficiency in negative controls (assigned a survival of 100%).

STUDY RESULTS
- Concurrent vehicle negative and positive control data: See "Attached background material"

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: See "Attached background material"
o Relative survival (RS) and cloning efficiency: See "Attached background material"

- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures: See "Details on test system and experimental conditions"
o Number of cells plated in selective and non-selective medium: See "Details on test system and experimental conditions"
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency: See "Attached background material"

HISTORICAL CONTROL DATA
- Positive historical control data: See "Attached background material"
- Negative (solvent/vehicle) historical control data: See "Attached background material"

Conclusions:

In an in vitro Mammalian Cell Gene Mutation Test accoding to OECD guideline 476, the test item tested up to the marked cytotoxic concentration without and with metabolic activation system over a 5-hour treatment period did not induce statistically significant or biologically relevant increases in mutant frequency compared to the solvent control and the historical control data. Thus, it is concluded that the test item was not mutagenic in this In Vitro Mammalian Cell Gene Mutation Test performed with Chinese hamster ovary cells.

Executive summary:

The test item was tested in a Mammalian Cell Gene Mutation Test (HPRT test) in CHO-K1 cells according to OECD guideline 476 and GLP. The purpose of this study was to determine whether the test item or its metabolites can induce forward mutation at the hypoxanthineguanine phosphoribosyl transferase enzyme locus (hprt) in cultured Chinese hamster cells. The test item was dissolved in Ham's F12 medium and the concentrations of the main test were selected on the basis of cytotoxicity investigations made in a preliminary study without and with metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver. In the performed Mutation Assay the concentration levels were chosen based on the cytotoxicity noted in the preliminary study. The Mutation Assay was performed at the concentrations and treatment intervals given below:

 

Mutation Assay 5-hour treatment period without S9-mix: 25, 50, 100, 170, 230 and 260 μg/mL

Mutation Assay 5-hour treatment period with S9-mix: 1, 2, 4, 8, 12, and 16 μg/mL

 

Following treatment, phenotypic expression was allowed for 8 days followed by a mutant selection period for another 8 days. In the absence of metabolic activation, clear cytotoxicity (survival 12 %) of the test item was observed at the highest concentration applied (260 μg/mL). In the presence of metabolic activation, clear cytotoxicity (survival 12 %) of the test item was observed at the highest concentration applied (16 μg/mL). Thus, the requirements for the highest test concentrations with regard to cytotoxicity as required in the guideline were met. In this In Vitro Mammalian Cell Gene Mutation Test, the frequency of cells with mutations did not show any biologically relevant or statistically significant increases compared to the concurrent and historical controls when the test item was tested in the absence and in the presence of metabolic activation even at test concentrations causing massive cytotoxicity. All values were within the range of the laboratory historical control data and no dose-response relationship was noted.

 

There was no precipitation of the test item at any concentration tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different concentration levels tested.

The mutation frequency found in the negative controls (-S9: 5.94-6.93 and +S9: 5.94-7) were well within the 95% control limits of historical laboratory control data (-S9: 5.36 - 7.81 and +S9: 5.19 - 8.71). The concurrent positive controls Ethyl methanesulfonate (1.0 μL/mL) and 7, 12-Dimethyl benzanthracene (20 μg/mL) caused the expected biologically relevant increase of cells with mutation frequency (1460.32-1478.69 and 706.33-714.29, respectively) as compared to solvent controls and were within the historical positive control data (1432.61-1542.21 and 700.49-769.66). Thus, the study is considered valid.

 

In conclusion, the test item tested up to the marked cytotoxic concentration without and with metabolic activation system over a 5-hour treatment period did not induce statistically significant or biologically relevant increases in mutant frequency compared to the solvent control and the historical control data. Thus, it is concluded that the test item was not mutagenic in this In Vitro Mammalian Cell Gene Mutation Test performed with Chinese hamster ovary cells.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2019-08-22 to 2019-10-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted with the purified and stabilised form of the test substance. The test item was stored under inert atmosphere (nitrogen).

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2017-02-14

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

2016-07-29

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: V79 Chinese hamster lung fibroblasts (male)
- Suitability of cells: The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes it suitable for gene toxicity assays with low background aberrations. These cells were chosen because of their small number of chromosomes (diploid number, 2n = 22) and because of the high proliferation rates (doubling time 12-14 h).
- Normal cell cycle time: 12-14 hours doubling time

For cell lines:
- Absence of Mycoplasma contamination: Yes, regularly checked
- Methods for maintenance in cell culture: The laboratory cultures were maintained in 75 cm^2 plastic flasks at 37 +/- 0.5 °C in an incubator with a humidified atmosphere, set at 5 % CO2.
- Doubling time: 12-14 hours
- Modal number of chromosomes: 22 (diploid)
- Periodically checked for karyotype stability: Not specified
- Periodically ‘cleansed’ of spontaneous mutants: Not specified

MEDIA USED
Solvent: Dulbecco’s Modified Eagle’s medium (DMEM)
Growth medium: DMEM supplemented with L-glutamine (2 mM) and 1% of Antibiotic-antimycotic solution (containing 10000 units/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphoptericin-B) and heat-inactivated bovine serum (final concentration 10%).
Treatment medium: DMEM supplemented with L-glutamine (2 mM) and 1% of Antibiotic-antimycotic solution (containing 10000 units/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphoptericin-B) and heat-inactivated bovine serum (final concentration 5%).
CO2 concentration: 5%
Humidity: Humidified atmosphere
Temperature: 37 +/- 0.5°C

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: Phenobarbital (PB) and β-naphthoflavone (BNF) induced rats
- Method of preparation of S9 mix: 2 mL of 20 mM HEPES, 1 mL 330 mM KCl, 1 mL 50 mM MgCl2, 1 mL 40 mM NADP, 1 mL 50 mM D-Glucose-6-phosphate, 1mL DMEM and 3 mL S9 were mixed and kept in an ice bath.
- Concentration or volume of S9 mix and S9 in the final culture medium: 250 µL S9 mix containing approx 33% S9 in a final culture medium of 5 mL
- Quality controls of S9: A certificate of analysis is available from the S9 supplier

Test concentrations with justification for top dose:

Experiment A with 3/20 h treatment/sampling time
Without S9 mix: 18, 36, 72, 144 and 2161 μg/mL test item
With S9 mix: 3, 9, 12 and 151 μg/mL test item;

Experiment B with 20/20 h treatment/sampling time
without S9 mix: 9, 18 and 36 μg/mL test item;

Experiment B with 20/28 h treatment/sampling time
without S9 mix: 9, 18 and 36 μg/mL test item;

Experiment B with 3/28 h treatment/sampling time
with S9 mix: 3, 6, 12 and 151 μg/mL test item

Vehicle / solvent:

- Vehicle used: DMEM (test item and positive conrols)

- Justification for choice of solvent/vehicle: This vehicle is compatible with the survival of the V79 cells and the S9 activity and was chosen based on the results of the preliminary solubility test, and its suitability is confirmed with the available laboratory’s historical database.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dulbecco’s Modified Eagle’s medium (test item, EMS, CP)

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

Without S9
EMS: 0.4 and 1.0 μL/mL

With S9
CP: 5.0 μL/mL

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 5 x 10^5 cells
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 hours (experiment A, with and without metabolic activation), 20 hours (experiment B, without metabolic activation), 3 hours (experiment B, with metabolic activation)
- Harvest time: 20 hours after treatment start (experiment A, with and without metabolic activation), 20 hours after treatment start (experiment B, without metabolic activation), 28 hours after treatment start (experiment B, with and without metabolic activation)

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor: Colchicine (0.2 µg/mL), addition 2.5 hours prior to harvesting
- Methods of slide preparation and staining technique used including the stain used: Following the selection time, cells were swollen with 0.075 M KCl hypotonic solution, then washed in fixative (approx. 10 min. in 3:1 mixture of methanol: acetic-acid until the preparation becomes plasma free) and dropped onto slides and air-dried. The preparation was stained with 5 % Giemsa for subsequent scoring of chromosome aberration frequencies.
- Number of cells spread and analysed per concentration: 300 well-spread metaphase cells containing 22 ± 2 chromosomes were scored per test item concentration as well as the negative and positive controls and were equally divided among the duplicates (150 metaphases/slide).
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): The nomenclature and classification of chromosome aberrations were given based upon ISCN, 1985, and Savage, 1976, 1983. Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately.
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Relative Increase in Cell Counts (RICC)

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Increase in numbers of chromosome aberrations

Rationale for test conditions:

Test concentrations were based on a preliminary toxicity test. Sampling times were chosen to cover approx. 1.5 cell cycles (20 hours, without S9 mix only) and approx. 2 normal cell cycles (28 hours, without and with S9 mix) from the beginning of treatment to cover a potential mitotic delay.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if:
– at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– the increase is dose-related when evaluated with an appropriate trend test,
– any of the results are outside the distribution of the laboratory historical negative control data.

Providing that all acceptability criteria are fulfilled, the test item is considered clearly negative if, in all experimental conditions examined:
– none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– there is no concentration-related increase when evaluated with an appropriate trend test,
– all results are inside the distribution of the laboratory historical negative control data.

Both biological and statistical significance should be considered together. There is no requirement according to the respective OECD guideline for verification of a clearly positive or negative response.

Statistics:

For statistical analysis, the CHI^2 test was utilized. The parameters evaluated for statistical analysis were the number of aberrations (with and without gaps) and number of cells with aberrations (with and without gaps). The number of aberrations in the treatment and positive control groups were compared to the concurrent negative control. The concurrent negative and positive controls and the treatment groups were compared to the laboratory historical controls, too. The lower and upper 95% confidence intervals of historical control data were calculated with C-chart. The data were checked for a linear trend in number of cells with aberrations (without gaps). with treatment concentrations using the adequate regression analysis by Microsoft Excel software.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

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

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

positive

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: There were no relevant changes in pH after treatment with the test item.
- Data on osmolality: There were no relevant changes in osmolality after treatment with the test item.
- Water solubility: The test item was soluble in DMEM
- Precipitation and time of the determination: There was no precipitation in the medium at any concentration tested.
- Definition of acceptable cells for analysis: Metaphase cells containing 22 ± 2 chromosomes

RANGE-FINDING/SCREENING STUDIES: In order to determine the treatment concentrations of test item in the cytogenetic study a dose selection (cytotoxicity assay) was performed. During the cytotoxicity assay the cells were seeded at 5 x 10^5 cells each and were incubated for 24 hours in 10 mL of DMEM containing 10 % foetal bovine serum. After 24 hours the cells were treated using increasing concentrations of test item in the absence or presence of S9 mix (50 mg/mL) and were incubated at 37 °C for 3 hours. Cell counts were performed after 20 hours. Additional groups of cells were treated for 20 hours without metabolic and for 3 hours with metabolic activation, with cell counts conducted after 20 hours (without S9 mix only) and 28 hours (without and with S9 mix). Additionally, 4 cultures were set up for determining the initial cell count. Based on the cell counts Relative Increase in Cell Counts (RICC) was calculated, which is an indicator of cytotoxicity. For the 3-hour treatment and 20 hours sampling time, cytotoxicity increased from 3.06% at 15.7 µg/mL test item up to 51.59% at 250 µg/mL test item without S9 mix. With S9 mix, strong cytotoxicity (100%) was noted at 62.5 µg/mL test item and above. For the 20-hour treatment without S9 mix and 20 hours sampling time, strong cytotoxicity (89.21-100%) was noted at 62.5 µg/mL test item and above. For the 20-hour treatment without S9 mix and 28 hours sampling time, strong cytotoxicity (94.86-100%) was noted at 62.5 µg/mL test item and above. For the 3-hour treatment with S9 mix and 28 hours sampling time, strong cytotoxicity (83.99-100%) was noted at 15.7 µg/mL test item and above.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: See "Attached background material"

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship: Yes. In the Experiment B, 20-hour treatment at the doses of 18 and 36 μg/mL in the absence of S9 mix with 20 and 28-hour harvest from the beginning of treatment caused a dose associated and biologically relevant increases in the number of cells with structural chromosome aberrations. Statistically significant difference were observed at the dose of 36 μg/mL.
- Statistical analysis; p-value if any: p < 0.01 to the concurrent negative control and to the historical control at the highest tested concentration (experiment B, 20-hour treatment, 20- and 28- hour sampling time, without metabolic activation)

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements: See "Attached background material"
o For cell lines: relative population doubling (RPD), relative Increase in cell count (RICC), number of cells treated and cells harvested for each culture, information on cell cycle length, doubling time or proliferation index: See "Attached background material" and "Details on mammalian cell type"
- Genotoxicity results: See "Attached background material"
o Definition for chromosome aberrations, including gaps: According to the nomenclature and classification of chromosome aberrations given in ISCN, 1985, and Savage, 1976, 1983.
o Number of cells scored for each culture and concentration, number of cells with chromosomal aberrations and type given separately for each treated and control culture, including and excludling gaps: 300 well-spread metaphase cells containing 22 ± 2 chromosomes were scored per test item concentration as well as the negative and positive controls and were equally divided among the duplicates (150 metaphases/slide). Gaps were scored but not included in the evaluation. Results are given under "Attached background material"
o Changes in ploidy (polyploidy cells and cells with endoreduplicated chromosomes): No increase in the rate of polyploid and endoreduplicated metaphases were found after treatment with the different concentrations.

HISTORICAL CONTROL DATA
- Positive historical control data: See "Attached background material"
- Negative (solvent/vehicle) historical control data: See "Attached background material"

Conclusions:

In an in vitro Mammalian Chromosome Aberration Test according to OECD guideline 473, the test item is considered clastogenic without metabolic activation.

Executive summary:

The test item, dissolved in DME (Dulbecco’s Modified Eagle’s) medium, was tested in a chromosome aberration assay in V79 cells according to OECD guideline 473 and GLP in two independent experiments. For the cytogenetic experiments, the following concentrations were selected on the basis of a pre-test on cytotoxicity (without and with metabolic activation using rodent S9 mix) in accordance with the current OECD Guideline 473:

 

Experiment A with 3/20 h treatment/sampling time

Without S9 mix: 18, 36, 72, 144 and 2161 μg/mL test item

with S9 mix: 3, 9, 12 and 151 μg/mL test item

 

Experiment B with 20/20 h treatment/sampling time

without S9 mix: 9, 18 and 36 μg/mL test item

 

Experiment B with 20/28 h treatment/sampling time

without S9 mix: 9, 18 and 36 μg/mL test item

 

Experiment B with 3/28 h treatment/sampling time

with S9 mix: 3, 6, 12 and 151 μg/mL test item

 

Following treatment and recovery, the cells were exposed to the spindle inhibitor colchicine (0.2 μg/mL) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained. In each experimental group, duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture). No precipitation of the test item was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality due to the test item. Clear cytotoxicity (52-60 %) in line with the OECD test guideline was observed after test item treatment in all experimental parts for the highest test concentrations.

 

In Experiment A, in the absence of metabolic activation, no relevant increases in cells carrying structural chromosomal aberrations were observed. In experiment A and in the Experiment B in the presence of metabolic activation, the frequency of the cells with structural chromosome aberrations without gaps at concentration of 12 μg/mL was little above (6/150 cells) the 95% confidence interval of the control values. In Experiment A, this value was in the range of the historical control. In addition, no statistical significant differences were observed after test item treatment when compared to the concurrent solvent control as well as the historical control groups in any of these experiments. Thus, the substance was evaluated not to be clastogenic in the presence of a metabolic system.

 

In the Experiment B, 20-hour treatment at the doses of 18 and 36 μg/mL in the absence of S9 mix with 20 and 28-hour harvest from the beginning of treatment caused a dose associated and biologically relevant increases in the number of cells with structural chromosome aberrations. Statistically significant difference were observed at the dose of 36 μg/mL causing 60 and 59 % cytotoxicity respectively when compared to the concurrent solvent as well as the historical control groups in both experiments. There were no polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation. The number of aberrations found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 μL/mL) and cyclophosphamide (5 μg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid. The test item without metabolic activation induced at clear cytotoxic concentrations a statistically significant and biological relevant increase of structural chromosome aberrations in Chinese Hamster lung cells at the prolonged exposure interval only.

 

Based on this result, the test item without metabolic activation system is considered clastogenic in this system.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-05-19 to 2017-08-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted with the purified but non-stabilised form of the test substance. Storage conditions were not specified.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998-08

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008-05-30

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997-07-21

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA1535, TA100, TA102) and frameshift (TA1537, TA98) mutations.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: Phenobarbital/ß-naphthoflavone-induced rats
- Method of preparation of S9 mix: 100 mM of ice-cold sodium-ortho-phosphate buffer pH 7.4 was mixed with 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate and 4 mM NADP. 9.5 parts of the cofactor solution was then mixed with 0.5 parts of S9.
- Concentration or volume of S9 mix and S9 in the final culture medium: 500 µL of 5% S9 mix (containing 25 µL S9) in a final culture medium of 2700 µL
- Quality controls of S9: Biological activity was tested in the Salmonella typhimurium assay using 2-AA and Benzo[a]pyrene. Sterility was also tested (not further described). Furthermore, alkoxyresorufin-0-dealkylase activities and a test for the presence of adventitious agents was performed by the supplier of S9.

Test concentrations with justification for top dose:

±S9: 0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate (pre-experiment in TA98 and TA100);
±S9: 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate (main experiment I+II, all strains);

A maximum concentration of 5 µL/plate was selected based on a preliminary experiment on toxicity, solubility testing and as the maximum recommended concentration according to current regulatory guidelines (OECD, 1997).

Vehicle / solvent:

- Vehicle used: DMSO (4-NOPD, 2-AA), Distilled water (SAZ, MMS)

- Justification for choice of solvent/vehicle: Solubility properties

- Justification for percentage of solvent in the final culture medium: The percentage of vehicle is compatible with the survival of the bacteria and S9 activity

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (4-NOPD, 2-AA), Distilled water (test item, SAZ, MMS)

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other:

Remarks:

Without S9:
SAZ: 10 µg/plate (TA100 and TA1535)
4-NOPD: 10 µg/plate (TA98), 40 µg/plate (TA1537)
MMS: 1 µL/plate (TA102)
With S9:
2AA: 2.5 µg/plate (TA98, TA100, TA1535, TA1537), 10 µg/plate (TA102)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE
Experiment I: Plate incorporation method
Experiment II: Pre-incubation method

TREATMENT AND HARVEST SCHEDULE
- Preincubation period: 60 min
- Exposure duration: 48 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Diminuation of background lawn/ reduction in the number of revertants down to a mutation factor of approx. ≤0.5 in relation to the solvent

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Mean revertant numbers

Evaluation criteria:

A test item is considered mutagenic if:
- a clear dose-dependent increase in the number of revertants occur and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and TA102 the number of reversions is at least twice as high
- if in tester strains TA1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control

A test item producing neither a dose-dependent increase in the number of revertants nor a reproducible biologically relevant positive response in any of the dose groups is considered to be non-mutagenic.

Statistics:

Not required according to OECD guideline 471

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

non-cytotoxic in experiment I, cytotoxic in experiment II

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

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

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

non-cytotoxic in experiment I, cytotoxic in experiment II

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

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

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

non-cytotoxic in experiment I, cytotoxic in experiment II

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

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

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

non-cytotoxic in experiment I, cytotoxic in experiment II

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

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

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

non-cytotoxic in experiment I, cytotoxic in experiment II

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

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
- Water solubility: The test item was soluble in water at all concentrations
- Precipitation and time of the determination: At the time of evaluation, no precipitation was observed for all test items at any concentration both in experiment I and II

STUDY RESULTS
- Concurrent vehicle negative and positive control data: See "Attached background material"

Ames test:
- Signs of toxicity: Yes, see "Attached background material"
- Individual plate counts: See "Attached background material"
- Mean number of revertant colonies per plate and standard deviation: See "Attached background material"

HISTORICAL CONTROL DATA
- Positive historical control data: See "Attached background material"
- Negative (solvent/vehicle) historical control data: See "Any other information on materials and methods incl. tables"

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

In order to investigate the potential of the test item for its ability to induce gene mutations, the plate incorporation test (experiment I) and the pre-incubation test (experiment II) were performed with the Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA102 according to OECD guideline 471 and GLP. In two independent experiments, several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:

 

0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5 µL/plate

 

No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation). Toxic effects of the test item were noted in all tester strains used in experiment I and II:

• In experiment I, toxic effects of the test item were observed at concentrations of 1.0 µL/plate and higher (with metabolic activation) depending on the respective tester strain


• In experiment II, toxic effects of the test item were observed at concentrations of 2.5 µL/plate and higher (with and without metabolic activation) depending on the respective tester strain

 

No biologically relevant increases in revertant colony numbers were noted in any of the five tester strains following treatment with the test item at any concentration level, neither in the presence or absence of metabolic activation in experiment I and II. All criteria for validity were met.

 

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2019-02-05 to 2019-04-08

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Screening assay with different batches (different purities, stabilisators and storage conditions), only two strains tested

Qualifier:

according to guideline

Guideline:

other: ICH Guidance S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use

Version / remarks:

2012-06

Deviations:

yes

Remarks:

Screening assay, only 2 strains tested

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008-08

Deviations:

yes

Remarks:

Screening assay, only 2 strains tested

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008-05-30

Deviations:

yes

Remarks:

Screening assay, only 2 strains tested

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997-07-21

Deviations:

yes

Remarks:

Screening assay, only 2 strains tested

Principles of method if other than guideline:

The purpose of this study was to screen different batches of the test item (with different purities and/or different stabilizers added, with or without oxygen exclusion) for mutagenic potential. For this reason, the test was performed only in 2 strains (TA98 and TA100) in the presence and absence of activated rat liver S9.

GLP compliance:

no

Remarks:

The study was not performed in compliance with the principles of GLP; however, all procedures were performed according to the laboratory's SOPs.

Type of assay:

bacterial reverse mutation assay

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA100) and frameshift (TA98) mutations.

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 98

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: Phenobarbital/ß-naphthoflavone-induced rats
- Method of preparation of S9 mix: The complete S9 mix was freshly prepared containing 500 mL of ice-cold 0.2 M sodium phosphate buffer pH 7.4, 100 mL S9 and 400 mL salt solution for S9 mix
- Concentration or volume of S9 mix and S9 in the final culture medium: 500 µL of 10% S9 mix (containing 50 µL S9) in a final culture medium of 2650 - 2800 µL (depending on the concentration of dosing solutions)
- Quality controls of S9: Each batch of S9 is checked for protein content, alkoxyresorufin-o dealkylase activity, for presence of adventitious agents, and promutagen activation (ethidium bromide, cyclophosphamide, benzo(a)pyrene and 2-aminoanthracene) by the supplier.

Test concentrations with justification for top dose:

±S9: 5, 4.2, 3.4, 2.8, 2, 1.2, 0.6, 0.3, 0.15 and 0.075 μL/plate;

A maximum concentration of 5 µL/plate was selected based on preliminary solubility testing and as the maximum recommended concentration according to current regulatory guidelines (OECD, 1997).

Vehicle / solvent:

- Vehicle used: DMSO (test item, NPD, 2AA), ultrapure water (SAZ)

- Justification for choice of solvent/vehicle: Solubility properties. The test item was soluble in DMSO at 50 µL/mL

- Justification for percentage of solvent in the final culture medium: The percentage of vehicle is compatible with the survival of the bacteria and the S9 activity as determined in a preliminary solubility test.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (test item 2AA, NPD) and ultrapure water (SAZ)

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other:

Remarks:

With S9:
2AA: 2 µg/plate (TA98 and TA100)
Without S9:
NPD: 4 µg/plate (TA98)
SAZ: 2 µg/plate (TA100)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

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

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Mean revertant numbers

Evaluation criteria:

A test item is considered mutagenic if:
- A dose- related increase in the number of revertants occur and/or
- A biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation
An increase is considered biologically relevant if:
- In strain TA100, the number of reversions is at least twice as high as the reversion rate of the vehicle control
- In strain TA98, the number of reversions is at least three times higher than the reversion rate of the vehicle control

A test item is considered non-mutagenic if:
A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation

Statistics:

Not required according to OECD guideline 471

Key result

Species / strain:

E. coli WP2 uvr A

Remarks:

Not tested, screening assay only in TA98 and TA100.

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

Not tested, screening assay only in TA98 and TA100.

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

Not tested, screening assay only in TA98 and TA100.

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Tested with test item 4

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 examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

Tested with test item 4

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

Tested with test item 6

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Tested with test item 5

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 examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

Tested with test item 5

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 examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Tested with test item 4

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

Tested with test item 4

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

A clear concentration-response effect was noted exceeding the factor of 2 but not the trigger value of 3. This also indicates a potential weak mutagenic effect in this tester strain.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Tested with test item 3

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 examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

Tested with test item 3

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 examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Tested with test item 2

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

Tested with test item 2

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

A clear concentration-response effect was noted exceeding the factor of 2 but not the trigger value of 3. This also indicates a potential weak mutagenic effect in this tester strain.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

Tested with test item 1

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 examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

Tested with test item 1

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

Tested with test item 1

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The behavior of the test items was investigated also in ultrapure water. While test item 1, 3 and 6 were miscible at the concentration of 50 μL/mL, test item 5 produced a slightly opalescent suspension at 50 μL/mL. Test item 2 and 4 were not miscible with water even by ultrasonication or lower practical concentration (e.g.: 25 μL/mL).
- Precipitation and time of the determination: No precipitation was observed for all test items at any concentration

STUDY RESULTS
- Concurrent vehicle negative and positive control data: See "Attached background material"

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship: For all positive results, a concentration-response relationship was observed (see "Attached background material")

Ames test:
- Signs of toxicity: Yes, see "Attached background material"
- Individual plate counts: See "Attached background material"
- Mean number of revertant colonies per plate and standard deviation: See "Attached background material"

HISTORICAL CONTROL DATA
- Positive historical control data: See "Attached background material"
- Negative (solvent/vehicle) historical control data: See "Attached background material"

Remarks on result:

other: Screening assay. Only two strains were tested, thus, a final conclusion cannot be drawn.

Conclusions:

In this Bacterial Reverse Mutation Screening Assay which followed OECD guideline 471, 6 batches of the test item with different purities were tested in Salmonella typhimurium strains TA98 and TA100. Test item 2 and 4 showed mutagenic activity with and without metabolic activation. Test item 1, 3, 5 and 6 showed no mutagenic activity under the conditions of the test with and without metabolic activation.

Executive summary:

In a non-GLP Bacterial Reverse Mutation Assay which followed OECD guideline 471, the two Salmonella typhimurium bacterial strains TA98 and TA100 were used to screen for the mutagenic potential of 6 batches of the test item with different purities with or without the addition of stabilizers and stored with or without oxgen exclusion in a single plate incorporation test:

 

Test item 1: Purity: 98%, stored under oxygen exclusion, not exposed to oxidative stress

Test item 2: Purity: 69%, no stabilization and exposed to oxidative stress

Test item 3: Purity: 98%, stabilized (0.5 % propyl gallate) and exposed to oxidative stress

Test item 4: Purity: 98%, stabilized (0.5 % Vitamin E) and exposed to oxidative stress

Test item 5: Purity: 97%, stabilized (0.5 % BHT) and exposed to oxidative stress

Test item 6: Purity: 97%, stabilized (nitrogen atmosphere) and exposed to oxidative stress

 

The test items were dissolved in DMSO and the following concentrations were investigated in all assays:  initial and confirmatory mutation tests: ±S9: 5, 4.2, 3.4, 2.8, 2, 1.2, 0.6, 0.3, 0.15 and 0.075 μL/plate.

Each assay was conducted with and without metabolic activation (±S9). The concentrations, including the controls, were tested in triplicate. In the performed experiments positive and negative (vehicle) controls were run concurrently.

In the performed experiments, all of the validity criteria, regarding negative (vehicle) and positive controls, S9 activity and number of investigated analysable concentration levels were fulfilled.

 

Test item 1

Clear toxic (inhibitory) effect of the test item was seen in both strains. In S. typhimurium TA98 at 5 μL/plate and 4.2 μL/plate in the presence of metabolic activation (+S9); in TA100 down to and including the concentration of 3.4 μL/plate in the absence (-S9) and down to and including the concentration of 1.2 μL/plate in the presence of metabolic activation (+S9). The inhibitory effect of the test item was indicated by affected background lawn development (reduced or slightly reduced background lawn and/or affected colony development (absent colonies, small pinpoint colonies, revertant colony numbers below the actual vehicle control and historical control data ranges). No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the experiment. All of the noticed slight revertant colony number increases remained within the biological variability range of the applied test system.

In conclusion, test item 1  has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

 

Test item 2

Clear toxic (inhibitory) effect of the test item was noticed in S. typhimurium TA98 in the concentration range of 5-2 μL/plate in the absence (-S9) and at the highest examined concentration of 5 μL/plate in the presence of exogenous metabolic activation (+S9); furthermore, in TA100 in the concentration range of 5-2.8 μL μL/plate (±S9). The inhibitory effect of the test item was indicated by affected colony development (absent colonies, small pinpoint colonies, revertant colony numbers below the actual vehicle control and historical control data ranges) and/or affected background lawn development (absent, reduced or slightly reduced background lawn. No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the experiment. Dose-related changed revertant colony number increases, revertant colony numbers above the corresponding historical control data ranges and above the thresholds for being positive were noticed in both strains in the absence and presence of exogenous metabolic activation (±S9). The increases were biologically relevant, above the threshold for being positive in Salmonella typhimurium TA100 strain following treatment with test item 2 at the concentrations of 2 and 1.2 μL/plate (-S9) and borderline positive results were obtained in TA100 at 2 μL/plate (+S9). In addition a clear concentration response effect was noted in in Salmonella typhimurium TA98 exceeding the factor of 2 but not the trigger value of 3. This also indicates a potential weak mutagenic effect in this tester strain.

In conclusion, test item 2 showed mutagenic activity on the applied Salmonella typhimurium TA100 tester strains in the absence and presence of exogenous metabolic activation, under the test conditions used in this study. In addition also indications for weak borderline mutagenic potential in TA98 was noted, however a final evaluation is not possible based on this screening assay.

 

Test item 3

Clear toxic (inhibitory) effect of the test item was seen in both strains. In S. typhimurium TA98 at 5 μL/plate in the absence and presence of metabolic activation (±S9); in TA100 down to and including the concentration of 3.4 μL/plate in the absence (-S9) and down to and including the concentration of 2 μL/plate in the presence of metabolic activation (+S9). The inhibitory effect of the test item was indicated by affected background lawn development (reduced or slightly reduced background lawn) and/or affected colony development (absent colonies small pinpoint colonies, revertant colony numbers below the actual vehicle control and historical control data ranges). No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the experiment. All of the noticed slight revertant colony number increases remained within the biological variability range of the applied test system.

In conclusion, test item 3 has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

 

Test item 4

Clear toxic (inhibitory) effect of the test item was noticed in S. typhimurium TA98 in the concentration range of 5-2.8 μL/plate in the absence (-S9) and at the highest examined concentration of 5 μL/plate in the presence of exogenous metabolic activation (+S9); furthermore, in TA100 in the concentration range of 5-2.8 μL μL/plate (±S9). The inhibitory effect of the test item was indicated by affected background lawn development (absent, reduced or slightly reduced background lawn) and/or affected colony development (absent colonies, small pinpoint colonies, revertant colony numbers below the actual vehicle control and historical control data ranges). No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the experiment. Dose-related changed revertant colony number increases, revertant colony numbers above the corresponding historical control data ranges and above the thresholds for being positive were noticed in both strains in the absence and presence of exogenous metabolic activation (±S9). The increases were biologically relevant, above the threshold for being positive in Salmonella typhimurium TA100 strain following treatment with test item 4 at the concentration of 1.2 μL/plate (-S9) and at 2 and 1.2 μL/plate (+S9). In addition, a clear concentration response effect was noted in in Salmonella typhimurium TA98 exceeding the factor of 2 but not the trigger value of 3. This also indicates a potential weak mutagenic effect in this tester strain.

In conclusion, test item 4 showed mutagenic activity on the applied Salmonella typhimurium TA100 tester strains in the absence and presence of exogenous metabolic activation, under the test conditions used in this study. In addition also indications for weak borderline mutagenic potential in TA98 was noted, however a final evaluation is not possible based on this screening assay.

 

Test item 5

Clear toxic (inhibitory) effect of the test item was seen in both strains. In S. typhimurium TA98 at 5 μL/plate in the absence and presence of metabolic activation (±S9); in TA100 down to and including the concentration of 2.8 μL/plate in the absence (-S9) and down to and including the concentration of 2 μL/plate in the presence of metabolic activation (+S9). The inhibitory effect of the test item was indicated by affected background lawn development (reduced or slightly reduced background lawn and/or affected colony development (absent colonies, small pinpoint colonies, revertant colony numbers below the actual vehicle control and historical control data ranges). No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the experiment. All of the noticed slight revertant colony number increases remained within the biological variability range of the applied test system.

In conclusion, test item 6 has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

 

Test item 6

Clear toxic (inhibitory) effect of the test item was seen in both strains. In S. typhimurium TA98 at 5 and 4.2 μL/plate in the presence of metabolic activation (+S9); in TA100 down to and including the concentration of 3.4 μL/plate in the absence (-S9) and down to and including the concentration of 2 μL/plate in the presence of metabolic activation (+S9). The inhibitory effect of the test item was indicated by affected background lawn development (reduced or slightly reduced background lawn and/or affected colony development (absent colonies, small pinpoint colonies, revertant colony numbers below the actual vehicle control and historical control data ranges). No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the experiment. All of the noticed slight revertant colony number increases remained within the biological variability range of the applied test system.

In conclusion, test item 6 has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

 

Overall conclusion

In this Bacterial Reverse Mutation Screening Assay which followed OECD guideline 471, 6 batches of the test item with different purities were tested in Salmonella typhimurium strains TA98 and TA100. Test item 2 and 4 showed mutagenic activity with and without metabolic activation. Test item 1, 3, 5 and 6 showed no mutagenic activity under the conditions of the test with and without metabolic activation.

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016-12-23 to 2017-03-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted with the non-purified and non-stabilised form of the test substance. Storage conditions were not specified.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998-08

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008-05-30

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997-07-21

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA1535, TA100, TA102) and frameshift (TA1537, TA98) mutations.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: Phenobarbital/ß-naphthoflavone-induced rats
- Method of preparation of S9 mix: 100 mM of ice-cold sodium-ortho-phosphate buffer pH 7.4 was mixed with 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate and 4 mM NADP. 9.5 parts of the cofactor solution was then mixed with 0.5 parts of S9.
- Concentration or volume of S9 mix and S9 in the final culture medium: 500 µL of 5% S9 mix (containing 25 µL S9) in a final culture medium of 2700 µL
- Quality controls of S9: Biological activity was tested in the Salmonella typhimurium assay using 2-AA and Benzo[a]pyrene. Sterility was also tested (not further described). Furthermore, alkoxyresorufin-0-dealkylase activities and a test for the presence of adventitious agents was performed by the supplier of S9.

Test concentrations with justification for top dose:

±S9: 0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate (pre-experiment in TA98 and TA100);
±S9: 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate (main experiment, all strains);

A maximum concentration of 5 µL/plate was selected based on a preliminary experiment on toxicity, solubility testing and as the maximum recommended concentration according to current regulatory guidelines (OECD, 1997).

Vehicle / solvent:

- Vehicle used: DMSO (4-NOPD, 2-AA), Distilled water (SAZ, MMS)

- Justification for choice of solvent/vehicle: Solubility properties

- Justification for percentage of solvent in the final culture medium: The percentage of vehicle is compatible with the survival of the bacteria and S9 activity

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (4-NOPD, 2-AA), Distilled water (test item, SAZ, MMS)

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other:

Remarks:

Without S9:
SAZ: 10 µg/plate (TA100 and TA1535)
4-NOPD: 10 µg/plate (TA98), 40 µg/plate (TA1537)
MMS: 1 µL/plate (TA102)
With S9:
2AA: 2.5 µg/plate (TA98, TA100, TA1535, TA1537), 10 µg/plate (TA102)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments: 1 (due to the positive results in the first experiment, no independent repetition was performed)

METHOD OF TREATMENT/ EXPOSURE
Plate incorporation method

TREATMENT AND HARVEST SCHEDULE
- Exposure duration: 48 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Mean revertant numbers

Evaluation criteria:

A test item is considered mutagenic if:
- a clear dose-dependent increase in the number of revertants occur and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and TA102 the number of reversions is at least twice as high
- if in tester strains TA1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control

A test item producing neither a dose-dependent increase in the number of revertants nor a reproducible biologically relevant positive response in any of the dose groups is considered to be non-mutagenic.

Statistics:

Not required according to OECD guideline 471

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

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

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

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

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was soluble in water at all concentrations
- Precipitation and time of the determination: At the time of evaluation, no precipitation was observed for all test items at any concentration

STUDY RESULTS
- Concurrent vehicle negative and positive control data: See "Attached background material"

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship: For the positive result, a concentration-response relationship was observed (see "Attached background material")

Ames test:
- Signs of toxicity: Yes, see "Attached background material"
- Individual plate counts: See "Attached background material"
- Mean number of revertant colonies per plate and standard deviation: See "Attached background material"

HISTORICAL CONTROL DATA
- Positive historical control data: See "Attached background material"
- Negative (solvent/vehicle) historical control data: See "Any other information on materials and methods incl. tables"

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item caused gene mutations by base pair changes in the genome of tester strain TA100. Therefore, the test item is considered to be mutagenic in this bacterial reverse mutation assay.

Executive summary:

In order to investigate the potential of the test item for its ability to induce gene mutations, the plate incorporation test was performed with the Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA102 according to OECD guideline 471 and GLP. In the experiment, several concentrations of the test item were used. The assay was conducted with and without metabolic activation. The concentrations, including controls, were tested in triplicate. The following concentrations of the test item were prepared:

 

0.0316, 0.100, 0.316, 1.0, 2.5 and 5 µL/plate

 

No precipitation of the test item was observed in any tester strain used (with and without metabolic activation). Toxic effects of the test item were noted in several tester strains used at a concentration of 5 µL/plate (without metabolic activation) and at concentrations of 2.5 µL/plate and higher (with metabolic activation), depending on the respective tester strain.

 

No biologically relevant increases in revertant colony numbers were noted in tester strains TA98, TA1535, TA1537 and TA102. Biologically relevant increases of revertant colony numbers were observed in tester strain TA100 at a concentration of 5 µL/plate (without metabolic activation). The threshold value of 2.0 was exceeded and a maximum mutation factor of 2.3 was reached at a concentration of 5.0 µL/plate (without metabolic activation). Moreover, a dose-response relationship was found in this tester strain. All criteria for validity were met.

 

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item caused gene mutations by base pair changes in the genome of tester strain TA100. Therefore, the test item is considered to be mutagenic in this bacterial reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In an in vivo Mammalian Alkaline Comet Assay according to OECD guideline 489, the purified and stabilised test item was administered twice via oral gavage at the dose levels 220, 110 and 55 mg/kg body weight/day to male Wistar rats. Sampling was performed about 3 to 4 hours after the second treatment. Under the experimental conditions presented in this report, the test item induced sporadic, occasional statistically significant increases in DNA strand breaks at the investigated dose levels in liver, stomach and kidney cells compared to the concurrent control. However, a detailed analysis considering lack of dose response, and historical control data, the noticed statistical significances were proven as not indicating a mutagenic potential of the test item. In consequence, in was concluded that the investigated test item did not show genotoxic activity in the examined tissues in this Comet assay.

 

To verify the result from the Comet Assay, an in vivo Mammalian Eryhthrocyte Micronucleus Test according to OECD guideline 474 was conducted in male NMRI mice. No biologically relevant or statistically significant increases in the frequency of micronucleated polychromatic erythrocytes were seen in the groups of mice treated with the test item compared to the vehicle and historical control groups. Statistically reduced PCEs at the mid and high dose demonstrated that the bone marrow was reached by the test item did not show any genotoxic activity in this Mouse Micronucleus Test.

 

Overall conclusion

Both in vivo tests conducted with the purified and stabilised test item showed no mutagenicity or clastogenicity in vivo. Thus, the registered form of the test substance (purified and stabilised with ≥0 < 0.1% BHT) is considered to be non-mutagenic and non-clastogenic in vivo.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-09-01 to 2021-11-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted with the purified and stabilised form of the test substance. The test item was stored under inert atmosphere (nitrogen).

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

1998-08

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2016-07-29

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

NMRI

Remarks:

Win: NMRI mice

Details on species / strain selection:

The NMRI mouse is one of the standard animals used internationally in this type of mutagenicity testing.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt.
- Age at study initiation: 8 weeks
- Weight at study initiation: 34.2-38.7 g
- Assigned to test groups randomly: Yes, using a randomization scheme. The randomization was checked according to the actual body weights verifying the homogeneity and deviations between the groups.
- Fasting period before study: No
- Housing: 2 animal/cage in the pretest and in the high dose group of main test and 5 animals/cage in the other groups of the main test in type I polypropylene/polycarbonate cages with laboratory bedding
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 40 - 70
- Air changes (per hr): Not indicated
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From day 0 to day 2

Route of administration:

oral: gavage

Vehicle:

- Vehicle: Aqua Purificata
- Justification for choice of solvent/vehicle: The suitability of this vehicle was confirmed in a GLP compliant validation study (Analytical report 941-100-5436) Furthermore, suitability of the vehicle is confirmed with the laboratory’s historical control database.
- Concentration of test material in vehicle: The test item was used for treatment in concentrations of 15 mg/mL, 30 mg/mL and 60 mg/mL prepared with the Aqua Purificata.
- Amount of vehicle: 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The necessary amount of test item was weighed into a calibrated volumetric flask. A partial volume of Aqua Purificata was added and the formulation was stirred until homogeneity was reached. The formulations were prepared fresh on day of dosing and used within 2 hours.

Duration of treatment / exposure:

48 hours

Frequency of treatment:

Twice at a 24-hour interval (test item concentrations and negative control), once (positive control)

Post exposure period:

24 hours (test groups, negative and positive control)

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

600 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 male animals/group (7 male animals in the high dose group), 5 groups

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Justification for choice of positive control: Commonly used mutagen, good laboratory's HCD available
- Route of administration: Intraperitoneally
- Doses / concentrations: 60 mg/kg bw

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: A non GLP Preliminary toxicity test was performed to identify the appropriate maximum dose level for the main test as no toxicity data in mice was available. The preliminary toxicity test determined the MTD and based on death, clinical signs of test item related toxicity and whether there are differences in toxicity between the sexes or not.

DETAILS OF SLIDE PREPARATION: Smears of the vortexed and centriguged bone marrow from two femurs cell were made on standard microscope slides. Slides were then dried at room temperature. They were fixed for a minimum of 5 minutes in methanol and allowed to air-dry and then stained with Giemsa (10%) solution for 25 minutes. Afterwards, slides were rinsed in distilled water. They were dryed at room temperature (at least 12 hours) and coated with EZ-mount.

METHOD OF ANALYSIS: Four thousand mature, polychromatic erythrocytes (PCEs) were scored per animal to assess the micronucleated cells. The frequency of micronucleated cells was expressed as percent of micronucleated cells based on the first 4000 PCEs counted in the optic field. The proportion of immature among total (immature + mature) erythrocytes was determined for each animal by counting a total of at least 500 erythrocytes.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, the test item is considered clearly positive if:
• At least one of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control,
• This increase is dose-related at least at one sampling time when evaluated with an appropriate test, and
• Any of these results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits)

Providing that all acceptability criteria are fulfilled, the test item is considered clearly negative if the following criteria had been met:
• None of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control,
• There is no dose-related increase at any sampling time when evaluated by an appropriate test,
• All results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits),
• Bone marrow exposure to the test item occurred.

Statistics:

Statistical analysis was done with SPSS PC+ software for the following data:
- The frequencies of micronucleated polychromatic erythrocytes in animals in the test and positive control groups were compared to the values found in the corresponding negative (vehicle) and historical control groups.
- The proportion of immature among total (immature + mature) erythrocytes in animals in the test and positive control groups were compared to the values found in the corresponding negative (vehicle) and historical control groups.
- The data was checked for a linear trend in mutant frequency with treatment dose using the adequate regression analysis by Microsoft Excel software.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 125 - 1500 mg/kg bw
- Solubility: A clear solution was obtained up to a concentration of 200 mg/mL.
- Clinical signs of toxicity in test animals: Groups of two males and females mice were treated two times at 24-hour intervals by oral gavage. Clinical signs like decreased activity, narrow palpebra, piloerection, incoordination and decreased grip strength were observed with dose-dependent increase in severity and decreasing onset. At 750 mg/kg bw/day, one female animal died after the first treatment. Tonic and clonic convulsions were observed before her death. At 1000 mg/kg bw/day, one male animal died half an hour after the first treatment. Tonic and clonic convulsions were observed before death. At 1000 mg/kg bw/day, all animals died between 1-4 hours after treatment.
- Evidence of cytotoxicity in tissue analysed: In two females of the 1000 mg/kg bw/day group, red and haemorrhaged mucous membrane was observed in the stomach.
- Rationale for exposure: The doses were based on the effects (massive local tissue damage in the stomach) noted in acute rat studies.
- Harvest times: Animals were treated twice in 24-hour intervals. The pre-test was finished 24 hours after the last treatment. No bone marrow smears were prepared.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: The frequencies of micronucleated polychromatic erythrocytes (MPCEs) in the treated mice in all dose groups were within acceptable ranges and compatible with the historical control data for this laboratory.
- Ratio of PCE/NCE: In the dose groups of 300 and 600 mg/kg body weight a statistically significant decreased number of PCEs was observed compared to the negative and historical control groups. This effect demonstrated exposure of the test item to the bone marrow (see “Attached background material”)
- Appropriateness of dose levels and route: The dose levels were chosen based on the results of a preliminary range-finding test. In this pre-test, mortality was observed at 750 mg/kg bw/day and higher in male and female mice. Clear signs of toxicity (strong decreased activity, piloerection and grip strength, incoordination and narrow palpebra after the first and second treatment) were observed at 500 mg/kg bw /day. Thus, the maximum tolerated dose was chosen as a dose between 500 and 750 mg/kg bw/day /600 mg/kg bw/day). The dose groups were separated by a factor of 2 as suggested in OECD guideline 474. Oral administration was shown to be appropriate as it was shown that the test substance reached the bone marrow (decreased number of PCEs in the middle and high dose groups).
- Statistical evaluation: No statistical significance was observed in the frequency of MPCEs in male mice at 24 hours after the second treatment compared to the concurrent negative (vehicle) control in any of the examined dose groups (see also "Attached background material").

Conclusions:

In a Mammalian Erythrocyte Micronucleus Test according to OECD guideline 474, no biologically relevant or statistically significant increases in the frequency of MPCEs were seen in the groups of mice treated with the test item compared to the vehicle and historical control groups. Statistically reduced PCEs at the mid and high dose demonstrated that the bone marrow was reached by the test item did not show any genotoxic activity in this Mouse Micronucleus Test.

Executive summary:

The potential mutagenic activity of the test item was examined in bone marrow of male NMRI mice according to OECD guideline 474 and GLP. The doses of the test item for the Micronucleus Test were determined according to a preliminary oral toxicity study. The doses selected were 150, 300 and 600 mg test item per kg body weight.

Study Design

Negative (vehicle) control and a positive control group were included. Treatment was carried in Aqua Purificata with a constant treatment volume (10 mL/kg body weight). The test item and negative (vehicle) control item were administered by gavage two times at 24-hour intervals. Cyclophosphamide dissolved in Aqua ad injectabilia (positive control) was administered once, intraperitoneally with a treatment volume of 10 mL/kg body weight. In the low, mid and high dose groups and vehicle control group the sampling was made once at 24 hours after the second treatment. In animals treated with Cyclophosphamide (60 mg/kg bw), the sampling was performed at 24 hours post-treatment of the single administration. Five animals per dose group were used. Four thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the micronucleated cells. The suitability of the chosen vehicle for the test item was analytically verified. A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. Measured concentrations of formulations applied in the study varied in the acceptable range (between of 96-103% of the nominal concentrations) and all formulations were homogenous, thereby confirming proper dosing.

Results

The two times oral administration of 150 mg/kg body weight, 300 mg/kg body weight and 600 mg/kg body weight of Methyl vinyl glycolate (MVG) did not induce increases in the frequency of micronucleated polychromatic erythrocytes (MPCEs) in male mice 24 hours after the second treatment compared to the negative and historical control groups. The proportion of immature among total (immature + mature) erythrocytes was determined for each animal by counting a total of at least 500 erythrocytes. Compared to the negative and historical control groups the number of polychromatic erythrocytes (PCEs) at 24 hours after the second treatment in the dose group of 150 mg/kg body weight was not affected. In the dose groups of 300 and 600 mg/kg body weight a statistically significant decreased number of PCEs was observed compared to the negative and historical control groups. This effect demonstrated exposure of the test item to the bone marrow. The frequencies of MPCEs for the negative and positive control mice were within acceptable ranges and in line with the historical control data for this laboratory. Cyclophosphamide treated mice (60 mg/kg body weight) showed a large, statistically significant increase in the MPCE number compared to the negative and historical controls. Thus, the study is considered to be fully valid.

Conclusion

No biologically relevant or statistically significant increases in the frequency of MPCEs were seen in the groups of mice treated with Methyl vinyl glycolate (MVG) compared to the vehicle and historical control groups. Statistically reduced PCEs at the mid and high dose demonstrated that the bone marrow was reached by the test item did not show any genotoxic activity in this Mouse Micronucleus Test.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-05-19 to 2020-10-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted with the purified and stabilised form of the test substance. The test item was stored under inert atmosphere (nitrogen).

Qualifier:

according to guideline

Guideline:

other: Council Regulation (EU) No 2017/735, Annex Part B, B.62: In vivo Mammalian Alkaline Comet Assay

Version / remarks:

2017-02-14

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

2016-07-29

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Species:

rat

Strain:

Wistar

Remarks:

HAN:WIST of Wistar origin

Details on species / strain selection:

The Wistar rat was selected due to a wide range of experience with this strain of rat in corresponding toxicity studies and historical control data at the laboratory.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt.
- Age at study initiation: 63-68 days
- Weight at study initiation: 297-315 g, the weight variation in animals involved at the start of the study did not exceed ± 20%.
- Assigned to test groups randomly: Yes, all animals were sorted according to body weight by computer and grouped according to weight ranges.
- Housing: 3 animals/cage and for the positive control group 2 animals/cages in type III polypropylene/polycarbonate cages with certified laboratory wood bedding.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1-23.9
- Humidity (%): 39-67
- Air changes (per hr): More than 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From day 0 to day 1

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: Ultrapure water (ASTM Type I)
- Justification for choice of solvent: Water was also used in a previous acute oral toxicity test and dose-range finding study. At the chosen target tissues, the suitability of ultrapure water vehicle in the in vivo Comet assay was evaluated in separate validation, reliability studies (Study codes: 392-489-1283, 392-489-5501) performed in the testing laboratory under the same conditions as the present study; furthermore it suitability is confirmed with an available own laboratory’s historical
control database: that is based on seven experiments in the case of liver, on four experiments
in the case of stomach, on six experiments in the case of duodenum and on two experiments
in the case of kidney.
- Concentration of test material in vehicle: The test item was formulated in ultrapure water (ASTM Type I) at the concentrations of 22, 11 and 5.5 mg/mL.
- Amount of vehicle: A constant treatment volume of 10 mL dose preparation/kg body weight was administered in all groups.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was formulated in ultrapure water (ASTM Type I) at the concentrations of 22, 11 and 5.5 mg/mL. The formulations were prepared in the laboratory of the Test Facility just before each treatment.

Duration of treatment / exposure:

Approx. 27-28 hours

Frequency of treatment:

Twice for the treatment groups and negative control (once on the day 0 and 24 hours thereafter), the
positive control animals were treated once on day 1.

Post exposure period:

Sampling was performed 3-4 hours after the second treatment (treatment groups and negative control) or after the single treatment (positive control group).

Dose / conc.:

220 mg/kg bw/day (nominal)

Dose / conc.:

110 mg/kg bw/day (nominal)

Dose / conc.:

55 mg/kg bw/day (nominal)

Dose / conc.:

0 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6 male animals per dose group and negative conttrol, 4 male animals for the positive control

Control animals:

yes, concurrent vehicle

Positive control(s):

Ethyl methanesulfonate (EMS)
- Justification for choice of positive control: EMS is a known mutagen. A good historical control database exisits for this mutagen at the laboratory.
- Route of administration: Oral gavage
- Doses / concentrations: 200 mg/kg bw

Tissues and cell types examined:

Glandular stomach (stomach epithelia), liver (mixed cell types), duodenum (surface epithelia), kidney (mixed cell types)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The dose levels investigated in the main test were chosen based on the results of earlier performed acute oral toxicity test (941-423-5434) and the additional preliminary dose range finding tests simulating the dosing regimen in the Comet assay (two subsequent administrations within 2 days). The dose range-finding test was started with 500 mg/kg body weight/day, using 2 animals. Because of the lethal effect of this test item dose (one animal died before the second treatment, one animal died within one hour after the second treatment), further 2 animals were exposed to 250 mg/kg body weight/day dose. Based on this information the maximum dose of 220 mg/kg body weight/day (as maximum tolerated dose, MTD) and in addition two additional lower doses: 110 and 55 mg/kg body weight/day were selected.

TREATMENT AND SAMPLING TIMES: The sampling time is a critical variable because it is determined by the period needed for the test chemicals to reach maximum concentration in the target tissue and for DNA strand breaks to be induced but before those breaks are removed, repaired or lead to cell death. A suitable compromise for the measurement of genotoxicity is to sample at 2-6 hour after the last treatment. In this particular test and based on the experience of the laboratory over the last years the sampling was performed 3-4 hours after the second treatment.

DETAILS OF SLIDE PREPARATION: The supernatant of the tissue preparations was used to prepare the comet slides. The slide preparation was done within one hour after single cell preparation. Four slides were prepared for each animal for each tissue sample, with six animals per dose group and vehicle control and four animals for the positive control groups. In summary 24 slides per treatment per tissue for the treatment groups and vehicle control and 16 slides per tissue for the positive controls. According to general procedure the conventional (superfrost) slides were dipped in hot 0.5 % normal melting point agarose in water. After gently remove the underside of the slides were wiped in order to remove the excess of agarose. The slides were then laid on a flat surface and let allow drying. A volume of 130 μL of 0.5 % normal melting point agarose (NMA) was added on a microscope slide pre-layered with 0.5 % NMA (see above) and covered with a glass coverslip. The slides were placed on a tray until the agarose hardens (~ 5 minutes). After the cell isolations each cell suspension was mixed with 0.5 % or 1.0 % Low Melting Point Agarose. Thereafter 85 or 165 μL (~1-9 x 10^4 cells) of this mixture was added on the microscope slide after gentle slide off the coverslip. The microscope slides were covered with a new coverslip. After the LMPA-cell mixture hardens an additional 70 μL of NMA was dropped on the microscope slide after a gentlenslide off the (second) coverslip and an additional new coverslip was laid on the slide. After the repeated NMA layer hardens the coverslip was removed. After the top layer of agarose solidifies and the last glass coverslip was removed the slides were immersed in chilled lysing solution for overnight 2-8°C (in refrigerator) in the dark. After the incubation period, the slides were rinsed to remove residual detergents and salts prior to the alkali unwinding step. This latter procedure was performed in electrophoresis buffer. The slides were removed from the lysing solution and randomly placed on a horizontal gel electrophoresis unit. The unit was filled up with freshly prepared electrophoresis solution until the surfaces of the slides were completely covered with the solution (to about 1-2 mm above the slides). During the unwinding and electrophoresis, a balanced design was used to place slides in the electrophoresis tank to mitigate the effects of any trends or edge effect within the tank and to minimize batch to batch variability. The slides were left for 30 min. for the DNA to unwind. Thereafter the electrophoresis was conducted for 30 min. by applying a constant voltage of 0.7 V/cm and an electric current of about 300 mA (actual values: 283-302 mA). The current was recorded at the start and end of each electrophoresis period. All of these steps were sheltered from the daylight to prevent the occurrence of additional DNA damage. The temperature of the electrophoresis solution through unwinding and electrophoresis was maintained at a low temperature, at 5°C using a special cooler designed for Comet electrophoresis tank and recorded. The temperature of the electrophoresis solution before the unwinding, during the unwinding and electrophoresis was kept at 5°C, and recorded once during the procedure. After electrophoresis, the slides were removed from the electrophoresis unit, were covered with neutralization solution, allowed to stand covered for about 5 minutes, thereafter blotted and covered again with neutralization solution. This procedure was repeated twice. Subsequently the slides were exposed for additional 5 minutes to absolute ethanol in order to preserve all of the slides. The slides were air dried and then stored at room temperature until they were scored for comets. Just prior the scoring the DNA, the slides were stained using 2 μg/mL Ethidium bromide.

METHOD OF ANALYSIS: The slides were examined with an appropriate magnification (200x) using fluorescent microscope equipped with an appropriate excitation filter (TRITC). For image analysis the Andor Kinetic Imaging Komet 6.0 (Andor Technology) was used. For stomach liver and duodenum samples fifty cells per slide were randomly scored i.e. 150 cells per animal (750 analysed cells per test item treatment, per vehicle control and 450 per positive control). For kidney samples fifty cells per slide were randomly scored i.e. 200 cells per animal (1200 analysed cells per test item treatment and per vehicle control; furthermore and 450 per positive control, see above). DNA strand breaks in the comet assay were measured by independent endpoints such as % tail DNA, olive tail moment (OTM) and tail length. The tail % DNA (also known as tail intensity) was applied as a key parameter for the evaluation and interpretation of the results and determined by the DNA fragment intensity in the tail expressed as a percentage of the cell’s total intensity. The OTM is expressed in arbitrary units and is calculated by multiplying the percentage of DNA (fluorescence) in the tail by the length of the tail in μm. The tail length is measured between the center of the comet head and the end of the comet tail. In addition, each slide was examined for presence of ghost cells (possible indicator of toxicity and/or apoptosis). Ghost cells results from a total migration of the DNA from the nucleus into the comet tail, reducing the size of the head to a minimum. Ghost cells, also known as clouds or hedgehogs, are morphological indicative of highly damaged cells and their presence is often associated with severe genotoxicity, necrosis and apoptosis. Ghost cells were excluded from the image analysis data collection, however determining of their frequency is useful for the data interpretation. The ghost cells were recorded for each slide per animal, per treatment and per tissue.

Evaluation criteria:

Providing that all validity criteria are fulfilled, the test chemical is clearly negative if:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control;
- there is no concentration-related increase when evaluated with an appropriate trend test;
- all results are inside the distribution of the historical negative control data for given species, vehicle, route, tissue and number of administration;
- direct or indirect evidence supportive of exposure of, or toxicity to, the target tissue(s) is demonstrated.

Providing that all validity criteria are fulfilled, the test chemical is clearly positive if:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control;
- the increase is dose-related when evaluated with an appropriate trend test;
- any of the results are outside the distribution of the historical negative control data for given species, vehicle, route, tissue and number of administration

There is no requirement for verification of the clearly negative or positive response.

Statistics:

The heterogeneity of the obtained data was tested. The statistical significance of % tail DNA values, tail length and OTM values; furthermore, the number of ghost cells was carried out using the appropriate statistical method, using SPSS software. The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. In case of a positive analysis, Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. If the data were not normal distributed, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. In case of a positive analysis result, the inter-group comparisons were performed using Mann-Whitney U-test. Additionally, % tail DNA and tail length values of liver and stomach samples were compared statistically (with 2-Sample T-test (α=0.05), SPSS software) with the corresponding whole historical control database.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 250-500 mg/kg bw/day
- Solubility: Yes
- Clinical signs of toxicity in test animals: Lethal effects at 500 mg/kg bw/day (one animal died before the second treatment, one animal died within one hour after the second treatment). For the at 250 mg/kg bw/day treatment, reddish excoriation (hyperaemia) at the mouth was observed 0.5 hours after the second treatment in both animals, bedding digging and eating was observed in one animal and depression was observed in the other animal 1 hour after the second treatment, piloerection, bluish discoloration of mouth periphery was observed 3 hours after the second treatment in both animals.
- Evidence of cytotoxicity in tissue analysed: Hyperaemia, haemorrhage was noticed at the throat, stomach, and intestine, at the gastric mucosa layer and liver at 500 mg/kg bw/day. General hyperaemia was noticed at the opening of abdominal cavity, ulcers (blistered, characteristic) and tympanites were observed in the (fore)stomach and intestine.
- Harvest times: Three hours after the second treatment

RESULTS OF DEFINITIVE STUDY
- Appropriateness of dose levels and route: Dose levels were based on the maximum tolerated dose determined in a range-finding study. As animals were in bad condition at scheduled termination at 250 mg/kg bw/day in the range-finding study, the dose was slightly lowered for the main study. At the highest dose chosen for the main study (220 mg/kg bw/day), signs of toxicity were observed after the second treatment (continuous head grooming, bedding digging and eating, increased nervous activity, piloerection, decreased activity, weight decrease), showing that the maximum tolerated dose was reached. Oral application was shown to be appropriate as it was shown that the test substance reached the target tissues (signs of toxicity was observed in the target organs stomach, intestine and liver).
- Statistical evaluation: See "Attached background material"

Conclusions:

The test item was investigated by the means of the in vivo comet assay according to OECD guideline 489 and GLP on isolated liver, stomach, duodenum and kidney cells under alkaline conditions in the male HAN:WIST rats. The test item was administered twice via oral gavage at the dose levels 220, 110 and 55 mg/kg body weight/day. Sampling was performed about 3 to 4 hours after the second treatment. Under the experimental conditions presented in this report, the test item induced sporadic, occasional statistically significant increases in DNA strand breaks at the investigated dose levels in liver, stomach and kidney cells compared to the concurrent control. However, a detailed analysis considering lack of dose response, and historical control data, the noticed statistical significances were proven as not indicating a mutagenic potential of the test item. In consequence, the investigated test item did not show genotoxic activity in the examined tissues in this Comet assay.

Executive summary:

In this In Vivo Alkaline Comet Assay on Different Rat Tissues with the test item according to OECD guideline 489 and GLP, the mutagenic potential of the test item in vivo was evaluated by measuring its ability to induce DNA damage in the target organ tissues (liver, stomach, duodenum, and kidney).

Methodology

6 male Wistar rats in the dose groups and negative control group and  4 animals in the positive control group were treated with 55, 110 and 220 mg/kg body weight/day. Ultrapure water was used as vehicle and negative control and ethyl methanesulfonate at 200 mg/kg bw was used as positive control. Formulations were prepared before each treatment. The test item was formulated in the vehicle in nominal concentrations of 22, 11 and 5.5 mg/mL. In the analytical measurements, the measured concentration values remained within the ± 10% of nominal range in the test item solutions at all concentrations prepared. The test item concentrations in the samples varied between 97 and 106 % in comparison to the nominal values; therefore, the nominal concentration values 22, 11 and 5.5 mg/mL (corresponding dose levels: 220, 110 and 55 mg/kg body weight/day) were applied and referred throughout the study. The test doses and negative control were administered twice by oral gavage (on day 0 and 24 hours thereafter). The positive control animals were treated by oral gavage once during the experiment on the day 1. The treatment volume was 10 mL/kg bw at all treatments (vehicle control: ultrapure water, test item doses and at the positive control: EMS). 3-4 hours after the second treatment (doses and vehicle control) and 3-4 hours after the treatment (positive control) the animals were euthanized and the cells of the target tissues were isolated. Cytotoxicity was determined (as a first screening) on a small sample of each isolated cell suspension following the Trypan blue dye exclusion technique, directly after sampling.

Comet Assay steps

Cells were embedded, lysis was performed at pH=10 and unwinding was performed at pH>13 for 30 min. Electrophoresis (pH>13) was performed for 30 min at 0.7 V/cm and about 300 mA. After neutralization (pH=7.5; 3 times for 5 min), slides were preserved in abs. ethanol for 5 min and air dried.

Evaluation

For scoring, the DNA was stained with 2 μg/mL ethidium bromide. The comets were measured via a digital camera linked to an image analyzer system using a fluorescence microscope equipped with an appropriate excitation filter at a magnification of 200x. For image analysis, the Komet 6.0 F (Andor Technology) was used. In addition, each slide was examined for presence of ghost cells (possible indicator of toxicity and/or apoptosis). Ghost cells were excluded from the image analysis data collection.

Liver, stomach and duodenum samples of 5 animals for all dose groups and negative control group; respectively and 3 animals for the positive control group were analysed. Fifty cells per slide were randomly scored i.e. 150 cells (three slides) per animal (750 analyzed cells per test item treatment and per vehicle control; 450 per positive control).

Kidney samples were evaluated in two steps. At the first step the same procedure was followed as applied for the other tissue samples; however, to allow a better interpretation of the findings further analysis was considered necessary. Thus, kidney samples of 6 animals for all dose groups and negative control group; respectively and 3 animals in the positive control group were analysed. In addition, fifty cells per slide were randomly scored i.e. 200 cells (four slides) per animal (1200 analyzed cells per test item treatment and per vehicle control; 450 per positive control) for kidney samples.

Results and discussion

All of the validity criteria regarding the negative and positive control treatments as well as the number of analysed cells, and the investigated dose levels were met. For dose selection, the criteria of the referred guideline (OECD 489), the information obtained from a previously performed acute oral toxicity study of the test item Methyl vinyl glycolate (MVG) in the same rat strain using the same vehicle; and the results of the performed preliminary dose range finding test (simulating the treatment regimen of the Comet Assay) were taken into consideration. No mortality was observed during the treatments and expression period in any dose group up to the highest dose of 220 mg/kg body weight/day and in the controls. During the treatment period, after the second treatment unequivocal signs of test item toxicity, mostly behavioral changes that appeared in dose-related manner were noticed. Eating and digging of bedding and/or an increased nervous activity followed by decreased activity (as a kind of fatigue) was observed at the test item treated groups (it was most obvious at the highest dose of 220 mg/kg body weight/day). At the tissue isolation normal appearance and anatomy of liver, stomach, duodenum and kidney was noticed at the vehicle control animals and at two animals of the positive control. The stomach of two positive control animals contained bedding material (obviously different amounts). The further noticed symptoms were different: hyperaemia (at one animal generally appeared at the insides, at the other at the liver, only) at one animal inflammation and ulcer on duodenum was observed, but normal appearance of duodenum at the other. Based on their occasional appearance, these symptoms were considered rather as signs of individual sensitivity than characteristic positive control material effect.

Unequivocal signs of toxicity evident as local tissue effects (e.g.: tympanites in the gastrointestinal tract, ulcer, inflammation on mucous membranes, hyperaemia at insides, bedding material in the stomach) were noticed at all test item doses. The intensity and degree of these observations showed a dose dependent increase. Furthermore, at the highest dose of 220 mg/kg body weight/day at five animals mosaic pattern on the liver, at one animal additionally mosaic pattern on the kidney was noticed, and in general at this dose characteristic stomach content: bedding material and significant volumes of liquid (assumed: water) was observed. The average body weights slightly increased (by 0.43-1.39 %) in the negative and positive controls and in the test item treated dose group of 55 mg/kg body weight/day when comparing the weight values measured on day 0 and just before the sacrifice, which is in the range of the expected increase for such exposure times. At the dose group of 110 mg/kg body weight/day in average 0.98 % weight reduction, at the dose group of 220 mg/kg body weight/day in average 2.75 % weight reduction was noticed, also indicating a systemic toxic effect and being in line with the local effects noted in the stomach.

At the cytotoxicity screening measurements (using Trypan blue dye exclusion method) no cytotoxicity was noticed in any test item and control item treatments at any target tissue. Each slide was examined for presence of ghost cells (possible indicator of cytotoxicity and/or apoptosis, however there is no consensus, how the ghost cells percentage should be concluded). The ghost cells percentages calculated for each tissue at each test item dose and positive control were compared to that of the vehicle control and laboratory’s historical control data, In the case of liver and duodenum samples the percentages of ghost cells at the test item treated doses did not differ statistically significantly from that of the vehicle control in any case and the actual values fitted well into the laboratory’s minimum-maximum ranges. In the case of stomach and kidney samples the ghost cell percentages differed statistically significantly from that of the vehicle control, additionally were above the actual laboratory’s range at one (stomach) or more (kidney) dose levels. The relatively higher number of ghost cells in these cases are likely be due to toxic effects attributable to the test item, which was observed during macroscopic inspection of the tissue prior to cell isolation. Statistically significant increase of ghost cells was noticed at the EMS treatments in the liver, stomach, duodenum and kidney samples too. This is a common finding according to historical control data and related to the cellular toxicity of EMS. The observed relatively higher percentage of ghost cells at the EMS treatments (liver 8 %, stomach 30 %, duodenum 27 % and kidney 21 %) were within the testing laboratory’s range for EMS positive control in all tissue preparations. The tail % DNA was applied as the main parameter for the evaluation and interpretation of the results; however further parameters: the tail length and Olive Tail Moment (OTM) values were also collected, evaluated and discussed. In the case of liver samples, most of the mean median % tail DNA values were within the corresponding historical control ranges, 95 % confidence intervals of C-charts, and the slightly higher value (above the historical control data range) noticed at 110 mg/kg body weight/day dose was neither an extreme outlier nor did it differ statistically significantly from that of the vehicle control The linear trend analyses did not show significance, either. In addition, at the higher test dose of 220 mg/kg body weight/day, the corresponding value was within the historical control range, confirms the accidental character of the findings.

At the data analysis performed on the mean median % tail DNA, tail length and OTM values of these samples sporadically occurred statistically significant differences were obtained; however, the linear trend analyses did not show dose dependent changes in any cases and the targeted historical control data comparisons did not show statistically significant differences either. The calculated low effect ratio values of 1.2-1.4 (ratio indicated an increase of means of % tail DNA of each dose over the vehicle control value) confirmed that the slight variations noted are accidental findings and are no indication for a mutagenic effect. In the case of stomach samples, the mean median % tail DNA values, as well as the further parameters (tail length and OTM values) at each dose remained well within the historical control ranges. The results of statistical analysis of stomach samples showed sporadically occurred significant differences; however, the further analysis of these significances (linear trend analysis, statistical comparison with corresponding historical control database) confirmed their irrelevance in mutagenicity point of view. In the case of duodenum samples, the mean median % tail DNA values, as well as the further parameters (tail length and OTM values) at each dose remained well within the historical control ranges, and the statistical evaluation of duodenum sample data did not show statistically significant differences from that of the vehicle control in any case.

For a better and more robust interpretation of the findings in the kidney, these samples were evaluated in two steps (see above). In the case of kidney samples, the mean median % tail DNA values of each dose; furthermore, the mean median % tail DNA values of each animal remained within the same range, well within the established historical control ranges (95 % confidence intervals of C-charts), and the linear trend analysis did not show a dose related increase in % tail DNA values confirming a dose response relationship. However, the noted values differed statistically significantly from that of the concurrent vehicle control. The obtained % tail DNA data results were further evaluated in context of published literature with focusing of test item induced toxicity and its potential effects. The obtained reduced body weight gain data, the obtained higher ghost cell percentages, the noticed clinical signs and macroscopic observations adequately demonstrated that the test item caused clear toxicity in the kidney. In the case of kidney samples all of the mean median tail length and OTM values remained well within the corresponding 95 % confidence intervals of C-charts. The OTM values differed however statistically significantly from that of the concurrent vehicle control at all test item doses without revealing any dose response relationship.

While the tail length values of 55 and 110 mg/kg body weight doses differed statistically significantly from that of the concurrent control, no statistically significant differences were noticed at the top dose of 220 mg/kg body weight. The linear trend analysis did not show significance at these parameters either. The missing dose dependency of tail length values of kidney samples and the results of their statistical analysis (no significance at 220 mg/kg body weight/day dose) further confirmed the data interpretation that the variations noted reflect the biological variability of the applied test system and/or signs of excessive toxic effect of the test item.

Conclusion

The test item was investigated by the means of the in vivo comet assay according to OECD guideline 489 and GLP on isolated liver, stomach, duodenum and kidney cells under alkaline conditions in the male HAN:WIST rats. The test item was administered twice via oral gavage at the dose levels 220, 110 and 55 mg/kg body weight/day. Sampling was performed about 3 to 4 hours after the second treatment. Under the experimental conditions presented in this report, the test item induced sporadic, occasional statistically significant increases in DNA strand breaks at the investigated dose levels in liver, stomach and kidney cells compared to the concurrent control. However, a detailed analysis considering lack of dose response, and historical control data, the noticed statistical significances were proven as not indicating a mutagenic potential of the test item. In consequence, the investigated test item did not show genotoxic activity in the examined tissues in this Comet assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

WoE, Bacterial Reverse Mutation Assay, RL1 (Non-purified test substance, non-stabilised)

In order to investigate the potential of the test item for its ability to induce gene mutations, the plate incorporation test was performed with the Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA102 according to OECD guideline 471 and GLP. In the experiment, several concentrations of the test item were used. The assay was conducted with and without metabolic activation. The concentrations, including controls, were tested in triplicate. The following concentrations of the test item were prepared:

 

0.0316, 0.100, 0.316, 1.0, 2.5 and 5 µL/plate

 

No precipitation of the test item was observed in any tester strain used (with and without metabolic activation). Toxic effects of the test item were noted in several tester strains used at a concentration of 5 µL/plate (without metabolic activation) and at concentrations of 2.5 µL/plate and higher (with metabolic activation), depending on the respective tester strain.

 

No biologically relevant increases in revertant colony numbers were noted in tester strains TA98, TA1535, TA1537 and TA102. Biologically relevant increases of revertant colony numbers were observed in tester strain TA100 at a concentration of 5 µL/plate (without metabolic activation). The threshold value of 2.0 was exceeded and a maximum mutation factor of 2.3 was reached at a concentration of 5.0 µL/plate (without metabolic activation). Moreover, a dose-response relationship was found in this tester strain. All criteria for validity were met.

 

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item caused gene mutations by base pair changes in the genome of tester strain TA100. Therefore, the test item is considered to be mutagenic in this bacterial reverse mutation assay.

 

 

WoE, Bacterial Reverse Mutation Assay, RL1 (Purified test substance, non-stabilised)

In order to investigate the potential of the test item for its ability to induce gene mutations, the plate incorporation test (experiment I) and the pre-incubation test (experiment II) were performed with the Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA102 according to OECD guideline 471 and GLP. In two independent experiments, several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:

 

0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5 µL/plate

 

No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation). Toxic effects of the test item were noted in all tester strains used in experiment I and II:

• In experiment I, toxic effects of the test item were observed at concentrations of 1.0 µL/plate and higher (with metabolic activation) depending on the respective tester strain


• In experiment II, toxic effects of the test item were observed at concentrations of 2.5 µL/plate and higher (with and without metabolic activation) depending on the respective tester strain

 

No biologically relevant increases in revertant colony numbers were noted in any of the five tester strains following treatment with the test item at any concentration level, neither in the presence or absence of metabolic activation in experiment I and II. All criteria for validity were met.

 

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.

 

 

WoE, Bacterial Reverse Mutation Assay (Screening Assay), RL2 (Different batches with different purification and stabilisation levels, different storage conditions)

In a non-GLP Bacterial Reverse Mutation Assay which followed OECD guideline 471, the two Salmonella typhimurium bacterial strains TA98 and TA100 were used to screen for the mutagenic potential of 6 batches of the test item with different purities with or without the addition of stabilizers and stored with or without oxgen exclusion in a single plate incorporation test:

 

Test item 1: Purity: 98%, stored under oxygen exclusion, not exposed to oxidative stress

Test item 2: Purity: 69%, no stabilization and exposed to oxidative stress

Test item 3: Purity: 98%, stabilized (0.5 % propyl gallate) and exposed to oxidative stress

Test item 4: Purity: 98%, stabilized (0.5 % Vitamin E) and exposed to oxidative stress

Test item 5: Purity: 97%, stabilized (0.5 % BHT) and exposed to oxidative stress

Test item 6: Purity: 97%, stabilized (nitrogen atmosphere) and exposed to oxidative stress

 

The test items were dissolved in DMSO and the following concentrations were investigated in all assays:  initial and confirmatory mutation tests: ±S9: 5, 4.2, 3.4, 2.8, 2, 1.2, 0.6, 0.3, 0.15 and 0.075 μL/plate.

Each assay was conducted with and without metabolic activation (±S9). The concentrations, including the controls, were tested in triplicate. In the performed experiments positive and negative (vehicle) controls were run concurrently.

In the performed experiments, all of the validity criteria, regarding negative (vehicle) and positive controls, S9 activity and number of investigated analysable concentration levels were fulfilled.

 

Test item 1

Clear toxic (inhibitory) effect of the test item was seen in both strains. In S. typhimurium TA98 at 5 μL/plate and 4.2 μL/plate in the presence of metabolic activation (+S9); in TA100 down to and including the concentration of 3.4 μL/plate in the absence (-S9) and down to and including the concentration of 1.2 μL/plate in the presence of metabolic activation (+S9). The inhibitory effect of the test item was indicated by affected background lawn development (reduced or slightly reduced background lawn and/or affected colony development (absent colonies, small pinpoint colonies, revertant colony numbers below the actual vehicle control and historical control data ranges). No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the experiment. All of the noticed slight revertant colony number increases remained within the biological variability range of the applied test system.

In conclusion, test item 1  has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

 

Test item 2

Clear toxic (inhibitory) effect of the test item was noticed in S. typhimurium TA98 in the concentration range of 5-2 μL/plate in the absence (-S9) and at the highest examined concentration of 5 μL/plate in the presence of exogenous metabolic activation (+S9); furthermore, in TA100 in the concentration range of 5-2.8 μL μL/plate (±S9). The inhibitory effect of the test item was indicated by affected colony development (absent colonies, small pinpoint colonies, revertant colony numbers below the actual vehicle control and historical control data ranges) and/or affected background lawn development (absent, reduced or slightly reduced background lawn. No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the experiment. Dose-related changed revertant colony number increases, revertant colony numbers above the corresponding historical control data ranges and above the thresholds for being positive were noticed in both strains in the absence and presence of exogenous metabolic activation (±S9). The increases were biologically relevant, above the threshold for being positive in Salmonella typhimurium TA100 strain following treatment with test item 2 at the concentrations of 2 and 1.2 μL/plate (-S9) and borderline positive results were obtained in TA100 at 2 μL/plate (+S9). In addition a clear concentration response effect was noted in in Salmonella typhimurium TA98 exceeding the factor of 2 but not the trigger value of 3. This also indicates a potential weak mutagenic effect in this tester strain.

In conclusion, test item 2 showed mutagenic activity on the applied Salmonella typhimurium TA100 tester strains in the absence and presence of exogenous metabolic activation, under the test conditions used in this study. In addition also indications for weak borderline mutagenic potential in TA98 was noted, however a final evaluation is not possible based on this screening assay.

 

Test item 3

Clear toxic (inhibitory) effect of the test item was seen in both strains. In S. typhimurium TA98 at 5 μL/plate in the absence and presence of metabolic activation (±S9); in TA100 down to and including the concentration of 3.4 μL/plate in the absence (-S9) and down to and including the concentration of 2 μL/plate in the presence of metabolic activation (+S9). The inhibitory effect of the test item was indicated by affected background lawn development (reduced or slightly reduced background lawn) and/or affected colony development (absent colonies small pinpoint colonies, revertant colony numbers below the actual vehicle control and historical control data ranges). No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the experiment. All of the noticed slight revertant colony number increases remained within the biological variability range of the applied test system.

In conclusion, test item 3 has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

 

Test item 4

Clear toxic (inhibitory) effect of the test item was noticed in S. typhimurium TA98 in the concentration range of 5-2.8 μL/plate in the absence (-S9) and at the highest examined concentration of 5 μL/plate in the presence of exogenous metabolic activation (+S9); furthermore, in TA100 in the concentration range of 5-2.8 μL μL/plate (±S9). The inhibitory effect of the test item was indicated by affected background lawn development (absent, reduced or slightly reduced background lawn) and/or affected colony development (absent colonies, small pinpoint colonies, revertant colony numbers below the actual vehicle control and historical control data ranges). No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the experiment. Dose-related changed revertant colony number increases, revertant colony numbers above the corresponding historical control data ranges and above the thresholds for being positive were noticed in both strains in the absence and presence of exogenous metabolic activation (±S9). The increases were biologically relevant, above the threshold for being positive in Salmonella typhimurium TA100 strain following treatment with test item 4 at the concentration of 1.2 μL/plate (-S9) and at 2 and 1.2 μL/plate (+S9). In addition, a clear concentration response effect was noted in in Salmonella typhimurium TA98 exceeding the factor of 2 but not the trigger value of 3. This also indicates a potential weak mutagenic effect in this tester strain.

In conclusion, test item 4 showed mutagenic activity on the applied Salmonella typhimurium TA100 tester strains in the absence and presence of exogenous metabolic activation, under the test conditions used in this study. In addition also indications for weak borderline mutagenic potential in TA98 was noted, however a final evaluation is not possible based on this screening assay.

 

Test item 5

Clear toxic (inhibitory) effect of the test item was seen in both strains. In S. typhimurium TA98 at 5 μL/plate in the absence and presence of metabolic activation (±S9); in TA100 down to and including the concentration of 2.8 μL/plate in the absence (-S9) and down to and including the concentration of 2 μL/plate in the presence of metabolic activation (+S9). The inhibitory effect of the test item was indicated by affected background lawn development (reduced or slightly reduced background lawn and/or affected colony development (absent colonies, small pinpoint colonies, revertant colony numbers below the actual vehicle control and historical control data ranges). No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the experiment. All of the noticed slight revertant colony number increases remained within the biological variability range of the applied test system.

In conclusion, test item 6 has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

 

Test item 6

Clear toxic (inhibitory) effect of the test item was seen in both strains. In S. typhimurium TA98 at 5 and 4.2 μL/plate in the presence of metabolic activation (+S9); in TA100 down to and including the concentration of 3.4 μL/plate in the absence (-S9) and down to and including the concentration of 2 μL/plate in the presence of metabolic activation (+S9). The inhibitory effect of the test item was indicated by affected background lawn development (reduced or slightly reduced background lawn and/or affected colony development (absent colonies, small pinpoint colonies, revertant colony numbers below the actual vehicle control and historical control data ranges). No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the experiment. All of the noticed slight revertant colony number increases remained within the biological variability range of the applied test system.

In conclusion, test item 6 has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

 

Overall conclusion

In this Bacterial Reverse Mutation Screening Assay which followed OECD guideline 471, 6 batches of the test item with different purities were tested in Salmonella typhimurium strains TA98 and TA100. Test item 2 and 4 showed mutagenic activity with and without metabolic activation. Test item 1, 3, 5 and 6 showed no mutagenic activity under the conditions of the test with and without metabolic activation.

 

 

WoE, Mouse Lymphoma Assay, RL1 (Purified test substance, non-stabilised)

 

The test item was assessed for its potential to induce mutations according to OECD guideline 490 and GLP at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The experiment without and with metabolic activation was performed as a 4 h short-term exposure assay. The selection of the concentrations used in the main experiment was based on data from the pre- experiment. The test item was investigated at the following concentrations:

 

 

Without metabolic activation: 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6 mM

 

and with metabolic activation: 0.10, 0.15, 0.30, 0.35, 0.40 and 0.45 mM

 

No precipitation of the test item was noted in the experiment. Growth inhibition was observed in the main experiment without and with metabolic activation: The relative total growth (RTG) was 13.1% (without metabolic activation) and 12.3% (with metabolic activation) for the highest concentration evaluated.

Biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). The Global Evaluation Factor (GEF; defined as the mean of the negative/vehicle mutant frequency plus one standard deviation; data gathered from ten laboratories) was exceeded by the induced mutant frequency at concentrations of 0.5 mM and higher (without metabolic activation) and at concentrations of 0.35 mM and higher (with metabolic activation). Moreover, a dose-response relationship was observed. Additionally, colony sizing showed clastogenic effects induced by the test item under the experimental conditions (without and with metabolic activation). EMS, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.

 

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item is considered to be mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

 

 

WoE, In vitro Mammalian Cell Gene Mutation Test, RL1 (Purified and stabilised test substance)

The test item was tested in a Mammalian Cell Gene Mutation Test (HPRT test) in CHO-K1 cells according to OECD guideline 476 and GLP. The purpose of this study was to determine whether the test item or its metabolites can induce forward mutation at the hypoxanthineguanine phosphoribosyl transferase enzyme locus (hprt) in cultured Chinese hamster cells. The test item was dissolved in Ham's F12 medium and the concentrations of the main test were selected on the basis of cytotoxicity investigations made in a preliminary study without and with metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver. In the performed Mutation Assay the concentration levels were chosen based on the cytotoxicity noted in the preliminary study. The Mutation Assay was performed at the concentrations and treatment intervals given below:

 

Mutation Assay 5-hour treatment period without S9-mix: 25, 50, 100, 170, 230 and 260 μg/mL

Mutation Assay 5-hour treatment period with S9-mix: 1, 2, 4, 8, 12, and 16 μg/mL

 

Following treatment, phenotypic expression was allowed for 8 days followed by a mutant selection period for another 8 days. In the absence of metabolic activation, clear cytotoxicity (survival 12 %) of the test item was observed at the highest concentration applied (260 μg/mL). In the presence of metabolic activation, clear cytotoxicity (survival 12 %) of the test item was observed at the highest concentration applied (16 μg/mL). Thus, the requirements for the highest test concentrations with regard to cytotoxicity as required in the guideline were met. In this In Vitro Mammalian Cell Gene Mutation Test, the frequency of cells with mutations did not show any biologically relevant or statistically significant increases compared to the concurrent and historical controls when the test item was tested in the absence and in the presence of metabolic activation even at test concentrations causing massive cytotoxicity. All values were within the range of the laboratory historical control data and no dose-response relationship was noted.

 

There was no precipitation of the test item at any concentration tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different concentration levels tested.

The mutation frequency found in the negative controls (-S9: 5.94-6.93 and +S9: 5.94-7) were well within the 95% control limits of historical laboratory control data (-S9: 5.36 - 7.81 and +S9: 5.19 - 8.71). The concurrent positive controls Ethyl methanesulfonate (1.0 μL/mL) and 7, 12-Dimethyl benzanthracene (20 μg/mL) caused the expected biologically relevant increase of cells with mutation frequency (1460.32-1478.69 and 706.33-714.29, respectively) as compared to solvent controls and were within the historical positive control data (1432.61-1542.21 and 700.49-769.66). Thus, the study is considered valid.

 

In conclusion, the test item tested up to the marked cytotoxic concentration without and with metabolic activation system over a 5-hour treatment period did not induce statistically significant or biologically relevant increases in mutant frequency compared to the solvent control and the historical control data. Thus, it is concluded that the test item was not mutagenic in this In Vitro Mammalian Cell Gene Mutation Test performed with Chinese hamster ovary cells.

 

 

WoE, In vitro Chromosome Aberration Test , RL1 (Purified and stabilised test substance)

The test item, dissolved in DME (Dulbecco’s Modified Eagle’s) medium, was tested in a chromosome aberration assay in V79 cells according to OECD guideline 473 and GLP in two independent experiments. For the cytogenetic experiments, the following concentrations were selected on the basis of a pre-test on cytotoxicity (without and with metabolic activation using rodent S9 mix) in accordance with the current OECD Guideline 473:

 

Experiment A with 3/20 h treatment/sampling time

Without S9 mix: 18, 36, 72, 144 and 2161 μg/mL test item

with S9 mix: 3, 9, 12 and 151 μg/mL test item

 

Experiment B with 20/20 h treatment/sampling time

without S9 mix: 9, 18 and 36 μg/mL test item

 

Experiment B with 20/28 h treatment/sampling time

without S9 mix: 9, 18 and 36 μg/mL test item

 

Experiment B with 3/28 h treatment/sampling time

with S9 mix: 3, 6, 12 and 151 μg/mL test item

 

Following treatment and recovery, the cells were exposed to the spindle inhibitor colchicine (0.2 μg/mL) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained. In each experimental group, duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture). No precipitation of the test item was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality due to the test item. Clear cytotoxicity (52-60 %) in line with the OECD test guideline was observed after test item treatment in all experimental parts for the highest test concentrations.

 

In Experiment A, in the absence of metabolic activation, no relevant increases in cells carrying structural chromosomal aberrations were observed. In experiment A and in the Experiment B in the presence of metabolic activation, the frequency of the cells with structural chromosome aberrations without gaps at concentration of 12 μg/mL was little above (6/150 cells) the 95% confidence interval of the control values. In Experiment A, this value was in the range of the historical control. In addition, no statistical significant differences were observed after test item treatment when compared to the concurrent solvent control as well as the historical control groups in any of these experiments. Thus, the substance was evaluated not to be clastogenic in the presence of a metabolic system.

 

In the Experiment B, 20-hour treatment at the doses of 18 and 36 μg/mL in the absence of S9 mix with 20 and 28-hour harvest from the beginning of treatment caused a dose associated and biologically relevant increases in the number of cells with structural chromosome aberrations. Statistically significant difference were observed at the dose of 36 μg/mL causing 60 and 59 % cytotoxicity respectively when compared to the concurrent solvent as well as the historical control groups in both experiments. There were no polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation. The number of aberrations found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 μL/mL) and cyclophosphamide (5 μg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid. The test item without metabolic activation induced at clear cytotoxic concentrations a statistically significant and biological relevant increase of structural chromosome aberrations in Chinese Hamster lung cells at the prolonged exposure interval only.

 

Based on this result, the test item without metabolic activation system is considered clastogenic in this system.

 

 

Key, In vivo Mammalian Alkaline Comet Assay, rat, RL1 (Purified and stabilised test substance)

In this In Vivo Alkaline Comet Assay on Different Rat Tissues with the test item according to OECD guideline 489 and GLP, the mutagenic potential of the test item in vivo was evaluated by measuring its ability to induce DNA damage in the target organ tissues (liver, stomach, duodenum, and kidney).

Methodology

6 male Wistar rats in the dose groups and negative control group and  4 animals in the positive control group were treated with 55, 110 and 220 mg/kg body weight/day. Ultrapure water was used as vehicle and negative control and ethyl methanesulfonate at 200 mg/kg bw was used as positive control. Formulations were prepared before each treatment. The test item was formulated in the vehicle in nominal concentrations of 22, 11 and 5.5 mg/mL. In the analytical measurements, the measured concentration values remained within the ± 10% of nominal range in the test item solutions at all concentrations prepared. The test item concentrations in the samples varied between 97 and 106 % in comparison to the nominal values; therefore, the nominal concentration values 22, 11 and 5.5 mg/mL (corresponding dose levels: 220, 110 and 55 mg/kg body weight/day) were applied and referred throughout the study. The test doses and negative control were administered twice by oral gavage (on day 0 and 24 hours thereafter). The positive control animals were treated by oral gavage once during the experiment on the day 1. The treatment volume was 10 mL/kg bw at all treatments (vehicle control: ultrapure water, test item doses and at the positive control: EMS). 3-4 hours after the second treatment (doses and vehicle control) and 3-4 hours after the treatment (positive control) the animals were euthanized and the cells of the target tissues were isolated. Cytotoxicity was determined (as a first screening) on a small sample of each isolated cell suspension following the Trypan blue dye exclusion technique, directly after sampling.

Comet Assay steps

Cells were embedded, lysis was performed at pH=10 and unwinding was performed at pH>13 for 30 min. Electrophoresis (pH>13) was performed for 30 min at 0.7 V/cm and about 300 mA. After neutralization (pH=7.5; 3 times for 5 min), slides were preserved in abs. ethanol for 5 min and air dried.

Evaluation

For scoring, the DNA was stained with 2 μg/mL ethidium bromide. The comets were measured via a digital camera linked to an image analyzer system using a fluorescence microscope equipped with an appropriate excitation filter at a magnification of 200x. For image analysis, the Komet 6.0 F (Andor Technology) was used. In addition, each slide was examined for presence of ghost cells (possible indicator of toxicity and/or apoptosis). Ghost cells were excluded from the image analysis data collection.

Liver, stomach and duodenum samples of 5 animals for all dose groups and negative control group; respectively and 3 animals for the positive control group were analysed. Fifty cells per slide were randomly scored i.e. 150 cells (three slides) per animal (750 analyzed cells per test item treatment and per vehicle control; 450 per positive control).

Kidney samples were evaluated in two steps. At the first step the same procedure was followed as applied for the other tissue samples; however, to allow a better interpretation of the findings further analysis was considered necessary. Thus, kidney samples of 6 animals for all dose groups and negative control group; respectively and 3 animals in the positive control group were analysed. In addition, fifty cells per slide were randomly scored i.e. 200 cells (four slides) per animal (1200 analyzed cells per test item treatment and per vehicle control; 450 per positive control) for kidney samples.

Results and discussion

All of the validity criteria regarding the negative and positive control treatments as well as the number of analysed cells, and the investigated dose levels were met. For dose selection, the criteria of the referred guideline (OECD 489), the information obtained from a previously performed acute oral toxicity study of the test item Methyl vinyl glycolate (MVG) in the same rat strain using the same vehicle; and the results of the performed preliminary dose range finding test (simulating the treatment regimen of the Comet Assay) were taken into consideration. No mortality was observed during the treatments and expression period in any dose group up to the highest dose of 220 mg/kg body weight/day and in the controls. During the treatment period, after the second treatment unequivocal signs of test item toxicity, mostly behavioral changes that appeared in dose-related manner were noticed. Eating and digging of bedding and/or an increased nervous activity followed by decreased activity (as a kind of fatigue) was observed at the test item treated groups (it was most obvious at the highest dose of 220 mg/kg body weight/day). At the tissue isolation normal appearance and anatomy of liver, stomach, duodenum and kidney was noticed at the vehicle control animals and at two animals of the positive control. The stomach of two positive control animals contained bedding material (obviously different amounts). The further noticed symptoms were different: hyperaemia (at one animal generally appeared at the insides, at the other at the liver, only) at one animal inflammation and ulcer on duodenum was observed, but normal appearance of duodenum at the other. Based on their occasional appearance, these symptoms were considered rather as signs of individual sensitivity than characteristic positive control material effect.

Unequivocal signs of toxicity evident as local tissue effects (e.g.: tympanites in the gastrointestinal tract, ulcer, inflammation on mucous membranes, hyperaemia at insides, bedding material in the stomach) were noticed at all test item doses. The intensity and degree of these observations showed a dose dependent increase. Furthermore, at the highest dose of 220 mg/kg body weight/day at five animals mosaic pattern on the liver, at one animal additionally mosaic pattern on the kidney was noticed, and in general at this dose characteristic stomach content: bedding material and significant volumes of liquid (assumed: water) was observed. The average body weights slightly increased (by 0.43-1.39 %) in the negative and positive controls and in the test item treated dose group of 55 mg/kg body weight/day when comparing the weight values measured on day 0 and just before the sacrifice, which is in the range of the expected increase for such exposure times. At the dose group of 110 mg/kg body weight/day in average 0.98 % weight reduction, at the dose group of 220 mg/kg body weight/day in average 2.75 % weight reduction was noticed, also indicating a systemic toxic effect and being in line with the local effects noted in the stomach.

At the cytotoxicity screening measurements (using Trypan blue dye exclusion method) no cytotoxicity was noticed in any test item and control item treatments at any target tissue. Each slide was examined for presence of ghost cells (possible indicator of cytotoxicity and/or apoptosis, however there is no consensus, how the ghost cells percentage should be concluded). The ghost cells percentages calculated for each tissue at each test item dose and positive control were compared to that of the vehicle control and laboratory’s historical control data, In the case of liver and duodenum samples the percentages of ghost cells at the test item treated doses did not differ statistically significantly from that of the vehicle control in any case and the actual values fitted well into the laboratory’s minimum-maximum ranges. In the case of stomach and kidney samples the ghost cell percentages differed statistically significantly from that of the vehicle control, additionally were above the actual laboratory’s range at one (stomach) or more (kidney) dose levels. The relatively higher number of ghost cells in these cases are likely be due to toxic effects attributable to the test item, which was observed during macroscopic inspection of the tissue prior to cell isolation. Statistically significant increase of ghost cells was noticed at the EMS treatments in the liver, stomach, duodenum and kidney samples too. This is a common finding according to historical control data and related to the cellular toxicity of EMS. The observed relatively higher percentage of ghost cells at the EMS treatments (liver 8 %, stomach 30 %, duodenum 27 % and kidney 21 %) were within the testing laboratory’s range for EMS positive control in all tissue preparations. The tail % DNA was applied as the main parameter for the evaluation and interpretation of the results; however further parameters: the tail length and Olive Tail Moment (OTM) values were also collected, evaluated and discussed. In the case of liver samples, most of the mean median % tail DNA values were within the corresponding historical control ranges, 95 % confidence intervals of C-charts, and the slightly higher value (above the historical control data range) noticed at 110 mg/kg body weight/day dose was neither an extreme outlier nor did it differ statistically significantly from that of the vehicle control The linear trend analyses did not show significance, either. In addition, at the higher test dose of 220 mg/kg body weight/day, the corresponding value was within the historical control range, confirms the accidental character of the findings.

At the data analysis performed on the mean median % tail DNA, tail length and OTM values of these samples sporadically occurred statistically significant differences were obtained; however, the linear trend analyses did not show dose dependent changes in any cases and the targeted historical control data comparisons did not show statistically significant differences either. The calculated low effect ratio values of 1.2-1.4 (ratio indicated an increase of means of % tail DNA of each dose over the vehicle control value) confirmed that the slight variations noted are accidental findings and are no indication for a mutagenic effect. In the case of stomach samples, the mean median % tail DNA values, as well as the further parameters (tail length and OTM values) at each dose remained well within the historical control ranges. The results of statistical analysis of stomach samples showed sporadically occurred significant differences; however, the further analysis of these significances (linear trend analysis, statistical comparison with corresponding historical control database) confirmed their irrelevance in mutagenicity point of view. In the case of duodenum samples, the mean median % tail DNA values, as well as the further parameters (tail length and OTM values) at each dose remained well within the historical control ranges, and the statistical evaluation of duodenum sample data did not show statistically significant differences from that of the vehicle control in any case.

For a better and more robust interpretation of the findings in the kidney, these samples were evaluated in two steps (see above). In the case of kidney samples, the mean median % tail DNA values of each dose; furthermore, the mean median % tail DNA values of each animal remained within the same range, well within the established historical control ranges (95 % confidence intervals of C-charts), and the linear trend analysis did not show a dose related increase in % tail DNA values confirming a dose response relationship. However, the noted values differed statistically significantly from that of the concurrent vehicle control. The obtained % tail DNA data results were further evaluated in context of published literature with focusing of test item induced toxicity and its potential effects. The obtained reduced body weight gain data, the obtained higher ghost cell percentages, the noticed clinical signs and macroscopic observations adequately demonstrated that the test item caused clear toxicity in the kidney. In the case of kidney samples all of the mean median tail length and OTM values remained well within the corresponding 95 % confidence intervals of C-charts. The OTM values differed however statistically significantly from that of the concurrent vehicle control at all test item doses without revealing any dose response relationship.

While the tail length values of 55 and 110 mg/kg body weight doses differed statistically significantly from that of the concurrent control, no statistically significant differences were noticed at the top dose of 220 mg/kg body weight. The linear trend analysis did not show significance at these parameters either. The missing dose dependency of tail length values of kidney samples and the results of their statistical analysis (no significance at 220 mg/kg body weight/day dose) further confirmed the data interpretation that the variations noted reflect the biological variability of the applied test system and/or signs of excessive toxic effect of the test item.

Conclusion

The test item was investigated by the means of the in vivo comet assay according to OECD guideline 489 and GLP on isolated liver, stomach, duodenum and kidney cells under alkaline conditions in the male HAN:WIST rats. The test item was administered twice via oral gavage at the dose levels 220, 110 and 55 mg/kg body weight/day. Sampling was performed about 3 to 4 hours after the second treatment. Under the experimental conditions presented in this report, the test item induced sporadic, occasional statistically significant increases in DNA strand breaks at the investigated dose levels in liver, stomach and kidney cells compared to the concurrent control. However, a detailed analysis considering lack of dose response, and historical control data, the noticed statistical significances were proven as not indicating a mutagenic potential of the test item. In consequence, the investigated test item did not show genotoxic activity in the examined tissues in this Comet assay.

 

 

Key, In vivo Mammalian Eryhthrocyte Micronucleus Test, mouse, RL1 (Purified and stabilised test substance)

 

The potential mutagenic activity of the test item was examined in bone marrow of male NMRI mice according to OECD guideline 474 and GLP. The doses of the test item for the Micronucleus Test were determined according to a preliminary oral toxicity study. The doses selected were 150, 300 and 600 mg test item per kg body weight.

Study Design

Negative (vehicle) control and a positive control group were included. Treatment was carried in Aqua Purificata with a constant treatment volume (10 mL/kg body weight). The test item and negative (vehicle) control item were administered by gavage two times at 24-hour intervals. Cyclophosphamide dissolved in Aqua ad injectabilia (positive control) was administered once, intraperitoneally with a treatment volume of 10 mL/kg body weight. In the low, mid and high dose groups and vehicle control group the sampling was made once at 24 hours after the second treatment. In animals treated with Cyclophosphamide (60 mg/kg bw), the sampling was performed at 24 hours post-treatment of the single administration. Five animals per dose group were used. Four thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the micronucleated cells. The suitability of the chosen vehicle for the test item was analytically verified. A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. Measured concentrations of formulations applied in the study varied in the acceptable range (between of 96-103% of the nominal concentrations) and all formulations were homogenous, thereby confirming proper dosing.

Results

The two times oral administration of 150 mg/kg body weight, 300 mg/kg body weight and 600 mg/kg body weight of Methyl vinyl glycolate (MVG) did not induce increases in the frequency of micronucleated polychromatic erythrocytes (MPCEs) in male mice 24 hours after the second treatment compared to the negative and historical control groups. The proportion of immature among total (immature + mature) erythrocytes was determined for each animal by counting a total of at least 500 erythrocytes. Compared to the negative and historical control groups the number of polychromatic erythrocytes (PCEs) at 24 hours after the second treatment in the dose group of 150 mg/kg body weight was not affected. In the dose groups of 300 and 600 mg/kg body weight a statistically significant decreased number of PCEs was observed compared to the negative and historical control groups. This effect demonstrated exposure of the test item to the bone marrow. The frequencies of MPCEs for the negative and positive control mice were within acceptable ranges and in line with the historical control data for this laboratory. Cyclophosphamide treated mice (60 mg/kg body weight) showed a large, statistically significant increase in the MPCE number compared to the negative and historical controls. Thus, the study is considered to be fully valid.

Conclusion

No biologically relevant or statistically significant increases in the frequency of MPCEs were seen in the groups of mice treated with Methyl vinyl glycolate (MVG) compared to the vehicle and historical control groups. Statistically reduced PCEs at the mid and high dose demonstrated that the bone marrow was reached by the test item did not show any genotoxic activity in this Mouse Micronucleus Test.

 

 

Overall conclusion

The non-purified and non-stabilised test substance is mutagenic and clastogenic in all available in vitro assays. The purified and properly stabilised (e.g. 0.05-0.5% BHT) test substance shows no mutagenic properties in vitro, as shown in the bacterial reverse mutation screening assays and HPRT test. In contrast, clastogenicity also occured with the purified and stabilised test item in vitro (chromosome aberration test). Thus, the test substance was tested in vivo. Both the in vivo Comet Assay and Micronucleus Test conducted with the purified and stabilised test substance showed no mutagenic or clastogenic potential of the test substance in vivo. Thus, the registered form of the test substance (purified and stabilised with ≥0 < 0.1% BHT) is considered to be non-mutagenic and non-clastogenic.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item is not classified and labelled according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighteenth time in Regulation (EU) 2022/692.