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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2021-05-07 to 2022-04-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022
Report date:
2022

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
adopted 2016-07-29
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethyl benzoate
EC Number:
202-284-3
EC Name:
Ethyl benzoate
Cas Number:
93-89-0
Molecular formula:
C9H10O2
IUPAC Name:
ethyl benzoate

Method

Species / strain
Species / strain / cell type:
lymphocytes: Human
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Human lymphocytes from healthy non-smoking donors with no known illness or recent exposures to genotoxic agents.
- Suitability of cells: Human lymphocytes are the most common cells used in the micronucleus test and have been used successfully for a long time in in vitro experiments. They show stable spontaneous micronucleus frequencies at a low level. The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with PHA and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes.
- Normal cell cycle time: The cell cycle time for lymphocytes from each donor has been determined by BrdU (bromodeoxyuridine) incorporation to assess the average generation time (AGT) for the donor pool.

For lymphocytes:
- Sex, age and number of blood donors: Blood was collected from a male donor (24 years old) for experiment I, from another male donor (24 years old) for experiment II and from a female donor (30 years old) for Experiment III.
- Whether whole blood or separated lymphocytes were used: Whole blood cultures were used.
- Whether blood from different donors were pooled or not: The blood was not pooled.
- Mitogen used for lymphocytes: Human lymphocytes were stimulated for proliferation by the addition of the mitogen PHA to the culture medium for a period of 48 hours

MEDIA USED
- Type and composition of media: The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA 1.5% (v/v) as extract, 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
- CO2 concentration, humidity level, temperature: 37 °C with 5.5 % CO2 in humidified air
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- Source of S9: Phenobarbital/β-naphthoflavone induced rat liver S9
- Method of preparation of S9 mix: MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4) was mixed with an appropriate quantity of S9 supernatant.
- Concentration or volume of S9 mix and S9 in the final culture medium: The protein concentration of the S9 preparation was 33.8 mg/mL in the pre-experiment and the main experiment. Thus, the final concentration of S9 in the treatment medium was approximately 0.75%.
- Quality controls of S9: Each batch of S9 is routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2 aminoanthracene in the Ames test.
Test concentrations with justification for top dose:
Experiment I (pre-test): 9.8,17.1, 29.9, 52.3, 91.5, 160, 280, 490 858 and 1502 μg/mL (4-hour exposure, 16-hour recovery, 20-hour Cytochalasin B exposure, with and without S9). 160, 280 and 490 µg/mL were evaluated for micronuclei.
1502 μg/mL (approx. 10 mM) were applied as top concentration for treatment of the cultures in the pre-test following the recommendations from OECD guideline 487.

Experiment II: 60.7, 106, 186, 260, 364, 510, 714 and 1000 µg/mL (20-hour exposure, 20-hour Cytochalasin B exposure, without S9)

Experiment III: 62.8, 110, 192, 336, 370, 407, 448, 492, 542 and 650 µg/mL (20-hour exposure, 20-hour Cytochalasin B exposure, without S9). Concentrations of 407, 448 and 492 µg/mL were evaluated for micronuclei.

In the pre-test for toxicity, phase separation of the test item was observed at the end of treatment at 490 μg/mL and above in the absence and presence of S9 mix. No cytotoxic effects were observed in the-pre-test after 4 hours treatment in the absence and presence of S9 mix. Due to the phase separation data in Experiment I, 1000 μg/mL was chosen as top treatment concentration for Experiment II. Concentrations for Experiment III were chosen based on phase separation observed at 714 µg/mL and above in Experiment II.
Vehicle / solvent:
- Vehicles used: DMSO (test substance), deionised water (MMC, DC), 0.9 % NaCl (CPA)

- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.

- Justification for percentage of solvent in the final culture medium: The final concentration of DMSO in the culture medium was 0.5 %. This concentration is known from historical control data to produce no toxicity.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO (test substance), deionised water (MMC, DC), 0.9 % NaCl (CPA)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Demecolcine
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration Duplicate
- Number of independent experiments: 3

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

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 hours (experiment I and II) or 20 hours (experiment III)
- Harvest time after the end of treatment: 36 hours (16 hours recovery + 20 hours Cytochalasin B incubation, experiment I and II) and 20 hours (20 hours Cytochalasin B incubation, experiment III)

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Identity of cytokinesis blocking substance, its concentration, and duration and period of cell exposure: Cytochalasin B, 4 μg/mL, 20 hours
- Methods of slide preparation and staining technique used including the stain used: Cells were incubated in 1 mL of ice-cold fixative mixture of methanol and glacial acetic acid (19 parts plus 1 part, respectively). After removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide. The cells were stained with Giemsa, mounted after drying and covered with a coverslip.
- Number of cells spread and analysed per concentration: At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. To describe a cytotoxic effect the CBPI was determined in 500 cells per culture.
- Criteria for scoring micronucleated cells: The micronuclei were counted in cells showing a clearly visible cytoplasm area. The area of the micronucleus should not extend the third part of the area of the main nucleus. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976).

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Cytokinesis-block proliferation index

METHODS FOR MEASUREMENTS OF GENOTOXICIY
% micronucleated cells
Rationale for test conditions:
Test concentrations for experiment II and III were chosen based on the results of experiment I. The top dose in experiment I was chosen based on the recommendations specified in OECD guideline 487.
Evaluation criteria:
Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in all of the experimental conditions examined:
− None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− There is no concentration-related increase
− The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data (95% control limit realized as 95% confidence interval)

The test item is then considered unable to induce chromosome breaks and/or gain or loss in this test system.
Providing that all of the 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 item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− The increase is concentration-related in at least one experimental condition
− The results are outside the range of the laboratory historical solvent control data (95% control limit realized as 95% confidence interval)

When all of the criteria are met, the test item is then considered able to induce chromosome breaks and/or gain or loss in this test system.
Statistics:
Statistical significance was confirmed by the Chi Square Test (p < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control. A linear regression was performed using a validated test script of “R”, to assess a possible dose dependency in the rates of micronucleated cells. The number of micronucleated cells obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. Both, biological and statistical significance were considered together.

Results and discussion

Test results
Key result
Species / strain:
lymphocytes: Human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In Experiment III in the absence of S9 mix after continuous treatment, clear cytotoxicity was observed at the highest evaluated concentration. In all other experiment, phase precipitation was observed.
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 osmolarity and pH were determined by using a pH meter in the solvent control and the maximum concentration without metabolic activation. No relevant influence on pH was observed.
- Data on osmolality: The osmolarity and pH were determined by using an osmometer in the solvent control and the maximum concentration without metabolic activation. No relevant influence on osmolarity was observed.
- Water solubility: The test substance was soluble in DMSO
- Precipitation and time of the determination: In Experiment I, phase separation of the test item in the culture medium was observed at 490 μg/mL and above in the absence and presence of S9 mix. Phase separation occurred in Experiment II in the absence of S9 mix at 714 μg/mL and above and in Experiment III at 542 μg/mL and above at the end of treatment.
- Definition of acceptable cells for analysis: See "Details on test system and experimental conditions"

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: No concentration-dependency was observed.
- Statistical analysis: In Experiment III in the absence of S9 mix after continuous treatment, the value of 0.60% micronucleated cells was statistically significantly increased. Since the value is clearly within the 95% control limit of the historical control data (0.06 – 0.88% micronucleated cells) and no dose dependency was observed, this finding can be considered as biologically irrelevant.

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements: In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation. In Experiment III in the absence of S9 mix after continuous treatment, clear cytotoxicity was observed at the highest evaluated concentration.
o In the case of the cytokinesis-block method: CBPI or RI; distribution of mono-, bi- and multi-nucleated cells: See "Attached background material"

- Genotoxicity results
o Number of cells with micronuclei separately for each treated and control culture and defining whether from binucleated or mononucleated cells: See "Attached background material"

HISTORICAL CONTROL DATA
- Positive historical control data: The historical control data were generated in accordance with the OECD Guideline 487. For the positive controls, data range (min-max) and data distribution (standard deviation) were calculated for each experimental part of at least 20 experiments. The min-max range of the positive controls was applied for the evaluation of acceptability (See "Attached background material").
- Negative (solvent/vehicle) historical control data: The historical control data were generated in accordance with the OECD Guideline 487. For the solvent controls, data range (min-max) and data distribution (standard deviation) were calculated for each experimental part of at least 20 experiments. The calculated 95% control limit of the solvent controls (realized as 95% confidence interval) was applied for the evaluation of acceptability and interpretation of the data (See "Attached background material").

Applicant's summary and conclusion

Conclusions:
In an in vitro Micronucleus Test in Human Lymphocytes according to OECD guideline 487, under the experimental conditions reported, the test item did not induce micronuclei. Therefore, the test item is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to cytotoxic or phase separating concentrations.
Executive summary:

In an in vitro Micronucleus Test in Human Lymphocytes according to OECD guideline 487 and GLP, the test item, dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in three independent experiments. The following study design was performed:















































 



Without S9 mix



With S9 mix



 



Exp. I


 



Exp. II & III


 



Exp. I


 



Stimulation period



48 hrs



48 hrs



48 hrs



Exposure period



4 hrs



20 hrs



4 hrs



Recovery



16 hrs



-



16 hrs



Cytochalasin B exposure



20 hrs



20 hrs



20 hrs



Total culture period



88 hrs



88 hrs



88 hrs



 


In each experimental group, two parallel cultures were analysed. Per culture at least 1000 binucleated cells were evaluated for cytogenetic damage.  The highest applied concentration in this study (1502 μg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 487. Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item phase separation in accordance with OECD Guideline 487. In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation. In Experiment III in the absence of S9 mix after continuous treatment, clear cytotoxicity was observed at the highest evaluated concentration. In Experiment I and III in the absence and presence of S9 mix, no relevant increases in the number of micronucleated cells were observed after treatment with the test item. In Experiment III in the absence of S9 mix after continuous treatment, however, the value of 0.60% micronucleated cells was statistically significantly increased. Since the value is clearly within the 95% control limit of the historical control data (0.06 – 0.88% micronucleated cells) and no dose dependency was observed, this finding can be considered as biologically irrelevant.  Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.


 


In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, the test item is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to cytotoxic or phase separating concentrations.