Ethyl benzoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a Bacterial Reverse Mutation Assay according to OECD guideline 471, the results indicate that the test item, under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolizing system.

 

In an in vitro Gene Mutation Assay in Chinese Hamster V79 Cells according to OECD guideline 476, under the experimental conditions reported, the test substance did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.

 

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.

 

Overall conclusion: The test substance is neither mutagenic nor clastogenic in the three in vitro genotoxicity tests.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-05-07 to 2022-04-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

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.

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.

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").

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000-12-20 to 2001-01-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP and OECD Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella typhimurium: histidine operon

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:

rat liver S9 mix, Aroclor 1254 induced

Test concentrations with justification for top dose:

without metabolic activation: 0, 15, 50, 150, 500, 1500, 5000 µg/plate
with metabolic activation: 0, 50, 150, 500, 1500, 5000 µg/plate

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (all strains, with and wothout metabolic activation), Mitomycin C (TA102 without MA), Sodium azide (TA100, TA1535 with and without MA), 2-Nitrofluorene (TA98, with and without MA), 9-aminoacridine (TA1537, with and without MA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 to 72 h at 37°C

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
A reduction in the number of revertant colonies and/or a diminution of the background lawn was taken as an indication of bacteriotoxicity.

Evaluation criteria:

The test substance is evaluated as mutagenic if it significantly increases the mutation frequency of the tester strains.

Statistics:

For estimation of the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each dosage level a X2-test was used.

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity observed with metablic activation at 1500 µg/plate and without metabolic activation at 500 μg/plate

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

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity observed with and without metablic activation at 5000 µg/plate

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

Metabolic activation:

with and without

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity observed with metablic activation at 5000 µg/plate and without metabolic activation at 1500 μg/plate

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 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity observed without metabolic activation at 5000 μg/plate

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 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity observed with and without metablic activation at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Tables 1-4 show mean numbers of revertant colonies per plate from two independent experiments, with and without metabolizing system.

 

Table 1:Induction of revertants in S. typhimurium in the absence of a metabolizing system (Experiment 1)

substance	Concentration µg/plate	Number of revertants per plate (mean of three plates)
		TA98	TA100	TA102	TA1535	TA1537
Control	0	28	141	288	23	10
Solvent control	0	17	129	247	19	8
Ethylbenzoate	15			243
	50	19	108	243	23	7
	150	20	125	233	20	9
	500	16	124	187T	23	7
	1500	8T	110	135T	24	8
	5000	0T	83T		13T	4T
NaN3	0.7		535		804
2-nitrofluorene	2.5	547
9-aminoacridine	50					168
Mitomycin C	0.15			604

T: bacteriotoxic

 

Table 2:Induction of revertants in S. typhimurium in the presence of a metabolizing system (Experiment 1)

substance	Concentration µg/plate	Number of revertants per plate (mean of three plates)
		TA98	TA100	TA102	TA1535	TA1537
Control	0	28	129	340	11	20
Solvent control	0	27	117	320	8	14
Ethylbenzoate	50	22	109	277	6	13
	150	25	100	183	8	16
	500	25	111	248	8	12
	1500	23	101	234	6	14
	5000	18T	81T	147T	4T	12
2-aminoanthra cene	0.8	497	817	431	103
	1.7					154

T: bacteriotoxic

 

Table 3:Induction of revertants in S. typhimurium in the absence of a metabolizing system (Experiment 2)

substance	Concentration µg/plate	Number of revertants per plate (mean of three plates)
		TA98	TA100	TA102	TA1535	TA1537
Control	0	32	107	310	18	10
Solvent control	0	35	103	296	23	12
Ethylbenzoate	15	36	102	293		12
	50	37	101	289	18	13
	150	34	89	267	23	13
	500	31	87	244T	19	12
	1500		90		19	12
	5000				14T
NaN3	0.7		366		798
2-nitrofluorene	2.5	402
9-aminoacridine	50					300
Mitomycin C	0.15			813

T: bacteriotoxic

 

Table 4:Induction of revertants in S. typhimurium in the presence of a metabolizing system (Experiment 2)

substance	Concentration µg/plate	Number of revertants per plate (mean of three plates)
		TA98	TA100	TA102	TA1535	TA1537
Control	0	22	127	407	14	14
Solvent control	0	20	109	366	10	15
Ethylbenzoate	15	20	120	348	8
	50	18	124	321	10	14
	150	23	105	320	10	14
	500	20	103	304	6	17
	1500	19	110	271T	12	17
	5000					15
2-aminoanthra cene	0.8	294	719	709	130
	1.7					224

T: bacteriotoxic

 

 

 

 

 

 

 

Conclusions:

The results indicate that ethylbenzoate under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolizing system.

Executive summary:

To perform a bacterial reverse mutation assay, ethylbenzoate was dissolved in DMSO. The substance was tested in concentrations of 15 to 5000 µg per plate in the presence and of 5 to 5000 µg per plate in the absence of S9. In the absence of S9-mix the substance was found to be bacteriotoxic towards the strain TA102 at 500 μg/plate, towards the strain TA98 at 1500 μg/plate, and towards the strains TA100, TA1535, and TA1537 at 5000 μg/plate. In the presence of S9-mix ethylbenzoate was bacteriotoxic towards the strain TA102 at 1500 µg/plate and towards the strains TA98, TA100, and TA1535 at 5000 µg/plate. Precipitation of the test compound and the plates was not observed. The test compound failed to induce a significant increase in the mutation frequency of the tester strains in the absence and presence of a metabolic activation system. In conclusion, these results indicate that ethylbezoate under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolizing system.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-05-06 to 2022-01-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Version / remarks:

adopted 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

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: V79 cells, supplied by the Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50%) both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22.
- Normal cell cycle time: Doubling time 12-16 hours in stock cultures

For cell lines:
- Absence of Mycoplasma contamination: Yes, each master cell stock is screened for mycoplasm contamination.
- Methods for maintenance in cell culture: Thawed stock cultures were propagated at 37 °C in 75 cm^2 plastic flasks. About 2-3×10^6 cells were seeded into each flask with 15 mL of MEM (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1%). The cells were sub-cultured once or twice weekly.
- Doubling time: 12-16 hours
- Modal number of chromosomes: 22
- Periodically checked for karyotype stability: Yes
- Periodically ‘cleansed’ of spontaneous mutants: Yes

MEDIA USED
- Type and composition of media:
Maintainance medium: MEM containing Hank’s salts supplemented with 10% FBS, neomycin (5 μg/mL) and amphotericin B (1%).
Treatment medium: Maintainance medium without FBS
Selective medium: Maintainance medium with 11 μg/mL 6-thioguanine
- CO2 concentration, humidity level, temperature: All incubations were done at 37°C with 1.5% carbon dioxide (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 was used as the metabolic activation system. The S9 was prepared from male Wistar rats induced by peroral administration of 80 mg/kg b.w. phenobarbital and by peroral administrations of ß-naphthoflavone each, on three consecutive days. The livers were prepared 24 h after the last treatment. The S9 fractions were produced by dilution of the liver homogenate with a KCl solution (1+3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant were frozen and stored in ampoules at –80 °C.
- 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 2.2%.
- 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:

Pre-test: Concentrations between 11.7 - 1502.0 μg/mL (with and without S9) were tested. The highest concentration corresponding to 10 mM, with respect to the OECD guideline 476.

Main experiment: 23.5, 46.9, 93.9, 187.8, 281.7, 375.5, 469.4, 563.3 and 657.1 µg/mL (without S9) and 23.5, 46.9, 93.9, 187.8, 375.5, 751 and 1502 (with S9)

Concentrations chosen for the mutation rate analysis were 46.9, 93.9, 187.8, 281.7 and 375.5 (without S9) and 23.5, 46.9, 93.9, 187.8 and 375.5 (with S9)

The top dose was chosen based on OECD guideline 476. The dose range of the main experiment was set according to data generated in the pre-experiment. In the pre-experiment, phase separation occurred at 751.0 μg/mL and above with and without metabolic activation. A relevant cytotoxic effect, indicated by a relative cloning efficiency of 50% or below was observed at 751.0 μg/mL and above without metabolic activation and at 1502 μg/mL with metabolic activation. In the main experiment, the individual concentrations were spaced by a factor of 2.0. Narrower spacing in the higher concentrations in the absence of S9 mix was used to cover the cytotoxic range more closely. To overcome problems with possible deviations in toxicity the main experiment was started with more than four concentrations.

Vehicle / solvent:

- Vehicles used: DMSO (test substance, DMBA), culture medium (EMS)

- 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: A final DMSO concentration of 0.5% was used throughout the study. This concentration is known from historical control data to produce no toxicity.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (test substance, DMBA), culture medium (EMS)

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate (pre-experiment and main experiment)
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 0.7 to 1.2×10^7 cells
- Test substance added: In medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 hours (with and without S9)
- Harvest time after the end of treatment: 8 ± 2 days (for determination of Relative Survival), approx. 15 days (7 days expression time + 8 days in non-selective for determination of viability), approx. 14-18 days (7 days expression time + 9 ± 2 days in selective medium for mutation analysis)

FOR GENE MUTATION:
- Expression time: Approx. 7 days
- Selection time: 9 ± 2 days
- Fixation time: 14-18 days
- Selective agent used: 6-thioguanine, 11 μg/mL, 9 ± 2 days exposure
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: At least 2.0×10^6 cells for sub-cultivation for expression, 500 cells to determine the relative survival, 4 - 5×10^5 cells for selection, 500 cells to determine viability. Colonies were stained with 10% methylene blue in 0.01% KOH solution. Colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Relative survival (RS), viability

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Numbers of mutant colonies

Rationale for test conditions:

Test concentrations for the main experiment were chosen based on the results of the pre-experiment.

The cultures at the lowest concentration without metabolic activation were not continued as a minimum of only four analysable concentrations is required by the guidelines. The cultures at the three highest concentrations without metabolic activation were not continued due to strong cytotoxic effects. The cultures at the concentration of 751 μg/mL and above were not continued to avoid analysis of too many concentrations showing phase separation.

Evaluation criteria:

A test substance is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g., Poisson-based 95% control limits).

A test substance is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (e.g., Poisson-based 95% control limits).

Statistics:

The statistical analysis was performed on the mean values of culture I and II for the main experiment. A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test substance were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. A t-test was performed using a validated test script of “R”, a language and environment for statistical computing and graphics, to evaluate a significant increase of the mutation frequency at test points exceeding the 95% confidence interval. Again, a t-test is judged as significant if the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together. A t-test was performed only for the positive controls since all mean mutant frequencies of the groups treated with the test substance were well within the 95% confidence interval of our laboratory’s historical negative control data.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Strong cytotoxicity occured at the three highest concentrations (469.4, 563.3 and 657.1 µg/mL) without metabolic activation (not evaluated for genotoxicity).

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 was determined in culture medium of the solvent control and of the highest concentration in the pre-experiment without metabolic activation. There was no relevant shift in the pH of the medium even at the maximum concentration of the test substance measured in the pre-experiment.
- Data on osmolality: The osmolarity was determined in culture medium of the solvent control and of the highest concentration in the pre-experiment without metabolic activation. There was no relevant shift of osmolarity of the medium even at the maximum concentration of the test substance measured in the pre-experiment.
- Water solubility: The test substance was soluble in DMSO
- Precipitation and time of the determination: Phase separation occured at the end of treatment at 469.4 µg/mL and above (without metabolic activation) and at 475.5 µg/mL and above (with metabolic activation)

RANGE-FINDING/SCREENING STUDIES: Test substance concentrations between 11.7 μg/mL and 1502.0 μg/mL were used in the pre-experiment with and without metabolic activation following 4 hours treatment. The highest concentration corresponding to 10 mM, with respect to the OECD guideline 476. The test medium was checked for phase separation and precipitation at the end of the treatment period (4 hours) before the test substance was removed. Phase separation occurred at 751.0 μg/mL and above with and without metabolic activation. A relevant cytotoxic effect, indicated by a relative cloning efficiency of 50% or below was observed at 751.0 μg/mL and above without metabolic activation and at 1502 μg/mL with metabolic activation. There was no relevant shift of osmolarity and pH of the medium even at the maximum concentration of the test substance measured in the pre-experiment.

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 where possible: No concentration-response relationship was observed.
- Statistical analysis: Linear regression analysis showed no statistically significant trend.


Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: No cytotoxicity was observed up to the highest evaluated concentration without metabolic activation. The next higher tested concentration, however, which was separated by a factor smaller than recommended by the guideline was outside the recommended range of cytotoxicity and therefore not evaluated for mutagenicity. No cytotoxic effects indicated by a relative adjusted cloning efficiency I (survival) below 50% (mean value of both parallel cultures) were noted with metabolic activation.
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"

HISTORICAL CONTROL DATA
- Positive historical control data: For the positive controls data range (min-max) and data distribution (standard deviation) are calculated for each experimental part of at least 20 experiments. The min-max range of the positive controls are applied for the evaluation of acceptability (see "Attached background materials").
- Negative (solvent/vehicle) historical control data: The historical control data were generated in accordance with the OECD Guideline 476. For the solvent controls data range (min-max) and data distribution (standard deviation) are calculated for each experimental part of at least 20 experiments. The calculated 95% control limit of the solvent controls (realized as 95% confidence interval) are applied for the evaluation of acceptability and interpretation of the data (see "Attached background materials")

Conclusions:

In an in vitro Gene Mutation Assay in Chinese Hamster V79 Cells according to OECD guideline 476, under the experimental conditions reported, the test substance did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.

Executive summary:

This in vitro Gene Mutation Assay in Chinese Hamster V79 Cells according to OECD guideline 476 and GLP was performed to investigate the potential of the test substance dissolved in DMSO to induce gene mutations at the Hprt locus in V79 cells of the Chinese hamster. The treatment period was 4 hours with and without metabolic activation. The maximum test substance concentration of the pre-experiment (1502 μg/mL) was equal to a molar concentration of about 10 mM with respect to the OECD guideline 476. The concentration range of the main experiment was chosen based on the observed phase separation. In the main experiment in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation in the presence of S9 mix. In the absence of S9 mix the next higher tested concentration, however, which was separated by a factor smaller than requested by the guideline was outside the recommended range of cytotoxicity and therefore not evaluated for mutagenicity. No substantial and dose dependent increase of the mutant frequency was observed in the main experiment. The tested concentrations ranged from 23.5 µg/L to 657.1 µg/mL in the 4-hour experiment without S9 mix and from 23.5 µg/L to 1502 µg/mL in the 4-hour experiment with S9 mix. The evaluated experimental points and the results were 46.9, 93.9, 187.8, 281.7 and 375.5 µg/L (without S9 mix) and 23.5, 46.9, 93.9, 187.8 and 375.5 µg/L (with S9 mix). Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

 

In conclusion it can be stated that under the experimental conditions reported the test substance did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial reverse mutation assay, RL1

A bacterial reverse mutation assay according to OECD guideline 471 and GLP was performed to assess the mutagenicity of the test item. To perform the bacterial reverse mutation assay, ethyl benzoate was dissolved in DMSO. The substance was tested in concentrations of 15 to 5000 µg per plate in the presence and of 5 to 5000 µg per plate in the absence of S9. In the absence of S9-mix the substance was found to be bacteriotoxic towards the strain TA102 at 500 μg/plate, towards the strain TA98 at 1500 μg/plate, and towards the strains TA100, TA1535, and TA1537 at 5000 μg/plate. In the presence of S9-mix ethyl benzoate was bacteriotoxic towards the strain TA102 at 1500 µg/plate and towards the strains TA98, TA100, and TA1535 at 5000 µg/plate. Precipitation of the test compound and the plates was not observed. The test compound failed to induce a significant increase in the mutation frequency of the tester strains in the absence and presence of a metabolic activation system. In conclusion, these results indicate that ethyl benzoate under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolizing system.

 

In vitro Gene Mutation Assay in Mammalian Cells, RL1

This in vitro Gene Mutation Assay in Chinese Hamster V79 Cells according to OECD guideline 476 and GLP was performed to investigate the potential of the test substance dissolved in DMSO to induce gene mutations at the Hprt locus in V79 cells of the Chinese hamster. The treatment period was 4 hours with and without metabolic activation. The maximum test substance concentration of the pre-experiment (1502 μg/mL) was equal to a molar concentration of about 10 mM with respect to the OECD guideline 476. The concentration range of the main experiment was chosen based on the observed phase separation. In the main experiment in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation in the presence of S9 mix. In the absence of S9 mix the next higher tested concentration, however, which was separated by a factor smaller than requested by the guideline was outside the recommended range of cytotoxicity and therefore not evaluated for mutagenicity. No substantial and dose dependent increase of the mutant frequency was observed in the main experiment. The tested concentrations ranged from 23.5 µg/L to 657.1 µg/mL in the 4-hour experiment without S9 mix and from 23.5 µg/L to 1502 µg/mL in the 4-hour experiment with S9 mix. The evaluated experimental points and the results were 46.9, 93.9, 187.8, 281.7 and 375.5 µg/L (without S9 mix) and 23.5, 46.9, 93.9, 187.8 and 375.5 µg/L (with S9 mix). Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions reported the test substance did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.

 

In vitro Micronucleus Test in Human Lymphocytes, RL1

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.

 

Overall conclusion: The test substance is neither mutagenic nor clastogenic in the three in vitro genotoxicity tests.

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 genotoxicity, 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.