Phenethyl benzoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

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 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Phenethyl benzoate

EC Number:

202-336-5

EC Name:

Phenethyl benzoate

Cas Number:

94-47-3

Molecular formula:

C15H14O2

IUPAC Name:

2-phenylethyl benzoate

Specific details on test material used for the study:

Appearance : Clear Colorless Liquid
Batch Number : 0017
Purity: 99.06%
Manufactured by: Otto Chemie Pvt. Ltd.,
Manufacturing date:14-Oct-2020
Expiry Date:14-Oct-2025
Storage condition: Room Temperature (20 to 30oC)

Method

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Source: NCCS, Pune, India

Metabolic activation:

with and without

Metabolic activation system:

Cofactor-supplemented liver S9 microsomal fraction was used as an exogenous metabolic activation system. The S9 fraction derived from the liver of an phenobarbitone and β-naphthoflavone-injected rat.

Composition of the cofactor mix (S9 mix);

Glucose-6-phosphate (180 mg/ml); 1ml
NADP (25 mg/ml): 1 ml
Potassium chloride (150 mM): 1 ml
S9 Fraction (ml): 2 ml
Final Volume (ml): 5 ml
S9 Mix: 40 %

Test concentrations with justification for top dose:

Test concentrations:
0.0 (NC), 0.0 (VC), 0.0625, 0.125, 0.25 mg/ml

Justification:
Test concentrations were chosen based on solubility and precipitation tests and an initial preliminary cytotoxicity test. In this pre-test, CHO cells were exposed to the Test Item at concentrations of 0.125, 0.25, 0.5, 1 and 2 mg/ml of culture medium, both in the presence (1 % v/v S9 mix) and absence of metabolic activation system along with the negative (Distilled water) and vehicle control (DMSO). Excessive cytotoxicity (defined by a decrease in Relative Increase in Cell Count [RICC] to <26% of the concurrent vehicle control) was observed for the Test Item at ≥0.5 mg/ml, both in the presence or absence of metabolic activation. Limited cytotoxicity was observed for the Test Item at 0.25 (RICC values: 48.99% without S9 mix; 47.48% with S9 mix). A concentration that causes a decrease in RICC to 45±5% of the concurrent vehicle control was selected as the highest test concentration for the chromosome aberration assay. Therefore 0.25 mg/ml was selected as the highest test item concentration.

Vehicle / solvent:

Dimethyl sulfoxide

Controls

Untreated negative controls:

yes

Remarks:

Distilled water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Single cultures were used.
- Number of independent experiments: 3 (Phase I-III)
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1 × 106 cells/flask
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk: In medium
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: NA
- Exposure duration/duration of treatment: 4hrs (Phase I-II), 24 hrs (Phase III)
- Harvest time after the end of treatment (sampling/recovery times): 24 hrs (Phase I-III)
FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure: Two hours prior to harvesting, a volume of 50 µl from 0.1 mg/ml of colchicine stock was added to culture flasks to achieve a final concentration of 1 µg/ml.
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure: NA
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Cell suspension obtained after harvesting was dropped on a clean chilled slide using the hanging drop method and kept for drying on a slide warmer. Duplicate slides were prepared from each culture. Slides were stained with freshly prepared 5 % Giemsa stain for 5 minutes and rinsed in distilled water for 2 minutes. After drying, slides were mounted using DPX mountant.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): At least 300 well-spread metaphases per concentration (single culture) were analyzed using 100x magnification for the incidence of structural aberrations.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): NA
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Cells with structural chromosomal aberration(s), including and excluding gaps, were scored. Chromatid and chromosome-type aberrations were recorded separately and classified by sub-types (breaks, exchanges). Cells that contain the number of centromeres equal to the number 2n ± 2 were scored.
- Determination of polyploidy:
- Determination of endoreplication:
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other: Cytotoxicity was measured by calculating the Relative increase in cell count (RICC) for treated and control cultures.
- Any supplementary information relevant to cytotoxicity:

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a Test Item was considered to be clearly positive if, in any of the experimental conditions examined:
• At least one of the test concentrations exhibits a significant increase compared with the concurrent negative/vehicle control,
• The increase is dose-related when evaluated with an appropriate trend test,
• Any of the results are outside of the distribution of the laboratory historical negative/vehicle control database
When all these criteria are met, the Test Item is then considered able to induce chromosomal aberrations in cultured mammalian cells in this test system.
The Test Item was considered clearly negative if, in all experimental conditions examined:
• None of the test concentrations exhibits a significant increase compared with the concurrent negative/vehicle control.
• There is no concentration-related increase when evaluated with an appropriate trend test.
• The results are inside the distribution of the laboratory historical negative control database.

Statistics:

Statistical analysis was performed to assess a possible dose-dependent increase of aberrant cell frequencies using Fisher’s Exact Test (NCSS statistics software). The percentage of aberrant cells from the Test Item treated group was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 0.25 mg/ml 51.42%, 52.68% and 50.00% cytotoxicity was observed in Phase I, Phase Ii and Phase III experiments, respectively.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Solubility and precipitation tests:
The Test Item was found to be soluble at 200 mg/ml concentration in dimethyl sulfoxide. No precipitation was observed at the concentration of 2 mg/ml. The test item did not affect the pH of medium after 4 hrs incubation.

Preliminary cytotoxicity test:
CHO cells were exposed to the Test Item at concentrations of 0.125, 0.25, 0.5, 1 and 2 mg/ml of culture medium, both in the presence (1 % v/v S9 mix) and absence of metabolic activation system along with the negative (Distilled water) and vehicle control (DMSO). Excessive cytotoxicity (defined by a reduction in Relative Increase in Cell Count [RICC] to <26% of the concurrent vehicle control data) was observed for the Test Item at ≥0.5 mg/ml, both in the presence or absence of metabolic activation. In comparison, limited cytotoxicity (defined by a reduction in Relative Increase in Cell Count [RICC] to >47% of the concurrent vehicle control data) was observed for the Test Item at 0.25 (RICC values: 48.99% without S9 mix; 47.48% with S9 mix) and 0.125 mg/ml (RICC values: 75.00% without S9 mix, 77.31% with S9 mix) either in the presence or absence of metabolic activation.

Remarks on result:

other: Non-clastogenic

Any other information on results incl. tables

Appendix 1: Relative Increase in Cell Counts – Preliminary Cytotoxicity Assay

Dose Level	Conc. (mg/ml)	Absence of Metabolic activation				Presence of Metabolic activation
		Cell count		RICC	% Cytotoxicity	Cell count		RICC	% Cytotoxicity
		Starting	Final			Starting	Final
NC	Distilled water	1000000	3520000	100.00	0.00	1000000	3485000	100.00	0.00
VC	DMSO	1000000	3480000	98.41	1.59	1000000	3380000	95.77	4.23
T1	0.125	1000000	2860000	75.00	25.00	1000000	2840000	77.31	22.69
T2	0.25	1000000	2215000	48.99	51.01	1000000	2130000	47.48	52.52
T3	0.5	1000000	1640000	25.81	74.19	1000000	1360000	15.13	84.87
T4	1	1000000	365000	-25.60	125.60	1000000	280000	-30.25	130.25
T5	2	1000000	0	-40.32	140.32	1000000	0	-42.02	142.02

Key: NC = Negative Control, VC = Vehicle Control, Conc. = Concentration, mg = milligram, ml = milliliter, RICC = Relative Increase in Cell Counts, % = percentage.

 

Appendix 2: Relative Increase in Cell Counts- Main Study

Dose Level	Conc.	Phase I -Absence of Metabolic activation
		Cell count		RICC	% Cytotoxicity
		Starting	Final
NC	Distilled water	1000000	3420000	100.00	0.00
VC	DMSO	1000000	3285000	94.42	5.58
T1	0.0625 mg/ml	1000000	2855000	81.18	18.82
T2	0.125 mg/ml	1000000	2690000	73.96	26.04
T3	0.25 mg/ml	1000000	2110000	48.58	51.42
PC	20 µg/ml	1000000	2440000	63.02	36.98

Dose Level	Conc.	Phase II -Presence of Metabolic activation
		Cell count		RICC	% Cytotoxicity
		Starting	Final
NC	Distilled water	1000000	3385000	100.00	0.00
VC	DMSO	1000000	3240000	93.92	6.08
T1	0.0625 mg/ml	1000000	2825000	81.47	18.53
T2	0.125 mg/ml	1000000	2725000	77.01	22.99
T3	0.25 mg/ml	1000000	2060000	47.32	52.68
PC	30 µg/ml	1000000	2465000	65.40	34.60

Dose Level	Conc.	Phase III -Absence of Metabolic activation
		Cell count		RICC	% Cytotoxicity
		Starting	Final
NC	Distilled water	1000000	3420000	100.00	0.00
VC	DMSO	1000000	3280000	94.21	5.79
T1	0.0625 mg/ml	1000000	2860000	81.58	18.42
T2	0.125 mg/ml	1000000	2695000	74.34	25.66
T3	0.25 mg/ml	1000000	2140000	50.00	50.00
PC	20 µg/ml	1000000	2440000	63.16	36.84

Appendix 3:Individual Data on Chromosome Aberrations- Phase I: Absence of metabolic activation (short term)

Dose level & Concentration	No. of Metaphases	Frequencies of Aberration	Total No of Aberrant cells
			with gap	without gap
NC (Distilled water)	300	-	0	0
VC (DMSO)	300	1 Fragment	1	1
T1 0.0625 mg/ml	300	1 Ctb	1	1
T2 0.125 mg/ml	300	1 Fragment	1	1
T3 0.25 mg/ml	300	1 Ctb, 1 Csb	2	2
PC 20 µg/ml	300	1 Ctg, 2 Csg, 6 Ctb, 4 deletion, 7 fragments, 6 Csb,2 ring, 1 exchange, 5 dicentric	24	23

Key: NC = Negative Control (distilled water), VC = Vehicle Control (dimethyl sulfoxide), PC = Positive Control (methyl methanesulfonate), mg = milligram, µg = microgram, ml = milliliter, T3- T1 = Test Item concentration from higher to lower, Ctg = Chromatid gap, Csg = Chromosome gap, Ctb = Chromatid break, Csb = Chromosome break, dic = dicentric.

Appendix 4: Individual Data on Chromosome Aberrations- Phase II:Presence of metabolic activation (short term)

Dose level & Concentration	No. of Metaphases	Frequencies of Aberration	Total No of Aberrant cells
			with gap	without gap
NC (Distilled water)	300	-	0	0
VC (DMSO)	300	-	0	0
T1 0.0625 mg/ml	300	1 ring	1	1
T2 0.125 mg/ml	300	-	0	0
T3 0.25 mg/ml	300	1 Ctg, 1 Csb	2	1
PC 20 µg/ml	300	2 Ctg, 6 Ctb, 6 Csb, 2 fragments,2 deletion, 4 exchange, 7 dicentic	21	20

Key:NC = Negative Control (distilled water), VC = Vehicle Control (dimethyl sulfoxide), PC = Positive Control (methyl methanesulfonate), mg = milligram, µg = microgram, ml = milliliter, T3-T1 = Test Item concentration from higher to lower, Ctg = Chromatid gap, Csg = Chromosome gap, Ctb = Chromatid break, Csb = Chromosome break, dic = dicentric.

 

Appendix 5: Individual Data on Chromosome Aberrations- Phase III: Absence of metabolic activation (Continuous)

Dose level & Concentration	No. of Metaphases	Frequencies of Aberration	Total No of Aberrant cells
			with gap	without gap
NC (Distilled water)	300	-	0	0
VC (DMSO)	300	-	0	0
T1 0.0625 mg/ml	300	2 Ctb	2	2
T2 0.125 mg/ml	300	1 Ctb	1	1
T3 0.25 mg/ml	300	-	0	0
PC 20 µg/ml	300	4 Ctg, 2 Csg, 7 Ctb, 4 Csb, 5 fragements, 2 deletions, 1 ring, 3 exchange, 1 dicentric	23	22

Appendix 6:Summary Data on Chromosome Aberrations - Phase I

Dose Level	Concentration	Absence of metabolic activation
		Total No. of Aberrant cells without gap	Percent aberrant cells
NC	Distilled water	0	0.00
VC	DMSO	1	0.33
T1	0.0625 mg/ml	1	0.33
T2	0.125 mg/ml	1	0.33
T3	0.25 mg/ml	2	0.67
PC	20 µg/ml*	23	7.67

Key: NC = Negative Control (distilled water), VC = Vehicle Control (dimethyl sulfoxide), PC = Positive Control, mg = milligram, ml = milliliter, µg = microgram, * = Statistical significant increase in % aberrant cell (p<0.05).

 

Appendix 7:Summary Data on Chromosome Aberrations - Phase II

Dose Level	Concentration	Presence of metabolic activation
		Total No. of Aberrant cells without gap	Percent aberrant cells
NC	Distilled water	0	0.00
VC	DMSO	0	0.00
T1	0.0625 mg/ml	1	0.33
T2	0.125 mg/ml	0	0.00
T3	0.25 mg/ml	1	0.33
PC	30 µg/ml*	20	6.67

 

Appendix 8: Summary Data on Chromosome Aberrations - Phase III

Dose Level	Concentration	Absence of metabolic activation
		Total No. of Aberrant cells without gap	Percent aberrant cells
NC	Distilled water	0	0.00
VC	DMSO	0	0.00
T1	0.0625 mg/ml	2	0.67
T2	0.125 mg/ml	1	0.33
T3	0.25 mg/ml	0	0.00
PC	20 µg/ml*	22	7.33

 

Historical Control Data

n=13	Percent Aberrant cells
	Phase I			Phase II			Phase III
	Absence- 4 hours			Presence- 4 hours			Absence- 24 hours
	NC	VC	PC	NC	VC	PC	NC	VC	PC
Mean	0.09	0.28	6.70	0.15	0.22	6.80	0.18	0.24	7.37
SD	0.15	0.12	0.43	0.17	0.24	0.28	0.17	0.16	0.58
Min	0.00	0.00	5.67	0.00	0.00	6.33	0.00	0.00	6.67
Max	0.33	0.33	7.33	0.33	0.67	7.33	0.33	0.33	8.33

Applicant's summary and conclusion

Conclusions:

The registered substance, Phenethyl Benzoate (CAS 94-47-3), was tested non-clastogenic (negative) in cultured Chinese Hamster Ovary (CHO) cells up to 0.25 mg/ml of culture medium, either in the presence or absence of S9 metabolic activation system. The test was performed according to OECD TG 473 and in compliance with the OECD Principles of Good Laboratory Practise (GPC).

Executive summary:

The clastogenic potential of the registered substance, Phenethyl Benzoate (CAS 94-47-3), was tested according to OECD TG 473 both in the presence and absence of S9 microsomal metabolic activation system using cultured Chinese Hamster Ovary (CHO) cells. The S9 fraction was obtained from the liver of a phenobarbitone and β-naphthoflavone-injected rat. The test substance was dissolved in dimethyl sulfoxide (DMSO).  Test concentrations were chosen based on solubility and precipitation tests and an initial cytotoxicity test. In this pre-test, CHO cells were exposed to the Test Item at concentrations of 0.125, 0.25, 0.5, 1 and 2 mg/ml of culture medium, both in the presence (1 % v/v S9 mix) and absence of metabolic activation system along with the negative (Distilled water) and vehicle control (DMSO). Excessive cytotoxicity (defined by a decrease in Relative Increase in Cell Count [RICC] to <26% of the concurrent vehicle control) was observed for the Test Item at ≥0.5 mg/ml, both in the presence or absence of metabolic activation. Limited cytotoxicity was observed for the Test Item at 0.25 (RICC values: 48.99% without S9 mix; 47.48% with S9 mix). A concentration causing a decrease in RICC to 45±5% of the concurrent vehicle control (cytotoxicity: 55±5%) was selected as the highest test concentration for the chromosome aberration assay. Based on the preliminary cytotoxicity assay results, Phase I (short-term exposure in the absence of metabolic activation), Phase II (short-term exposure in the presence of metabolic activation) and Phase III (continuous exposure in the absence of metabolic activation) experiments were conducted with the Test Item at the concentrations of 0.0625, 0.125 and 0.25 mg/ml, along with vehicle (DMSO), negative (Distilled water) and concurrent positive controls (20 µg/ml of Methyl methanesulfonate without S9 mix, 30 µg/ml of Benzo (a) pyrene with S9 mix). Results: In Phase I, cultures were exposed to Test Item, negative, vehicle and positive control for 4 hours (short-term exposure) in the absence of metabolic activation. The RICC values were 100.00 % (negative control), 94.42 % (vehicle control), 81.18 % (at 0.0625 mg/ml), 73.96 % (at 0.125 mg/ml) and 48.58 % (at 0.25 mg/ml). No significant increase in the mean percent aberrant cells at 0.0625 mg/ml (the mean % aberrant cells: 0.33%, p=0.500), 0.125 mg/ml (the mean % aberrant cells: 0.33%, p=0.500), 0.25  mg/ml (the mean % aberrant cells: 0.67%, p=0.500), was observed when compared to the vehicle control (the mean % aberrant cells 0.33%).  In Phase II, cultures were exposed to Test Item, negative, vehicle and positive control for 4 hours (short-term exposure) in the presence of metabolic activation (1 % v/v S9 mix). The RICC values were 100.00 % (negative control), 93.92 % (vehicle control), 81.47 % (at 0.0625 mg/ml), 77.01 % (at 0.125 mg/ml) and 47.32 % (at 0.25 mg/ml). No significant increase in the mean percent aberrant cells at 0.0625 mg/ml (the mean % aberrant cells: 0.33%, p=1.000), 0.125 mg/ml (the mean % aberrant cells: 0.00%, p=1.000), 0.25 mg/ml (the mean % aberrant cells: 0.33%, p=1.000), was observed when compared to the vehicle control (the mean % aberrant cells 0.00 %). In Phase III, cultures were exposed to Test Item, negative, vehicle and positive control for 24 hours (continuous exposure) in the absence of metabolic activation. The RICC values were 100.00 % (negative control), 94.21 % (vehicle control), 81.58 % (at 0.0625 mg/ml), 74.34 % (at 0.125 mg/ml) and 50.00 % (at 0.25 mg/ml). No significant increase in the mean percent aberrant cells at 0.0625 mg/ml (the mean % aberrant cells: 0.67%, p=0.500), 0.125 mg/ml (the mean % aberrant cells: 0.33%, p=1.000), 0.25 mg/ml (the mean % aberrant cells: 0.00%, p=1.000), was observed when compared to the vehicle control (the mean % aberrant cells 0.00 %). In all phases of the study, no significant reduction in RICC (cytotoxicity) and no increase in percent aberrant cells were observed in vehicle control (dimethyl sulfoxide) either in the presence or absence of metabolic activation. The positive controls (-S9: methyl methanesulfonate, +S9: Benzo[a]pyrene) used in the study produced statistically significant increases in the percent of cells with structural chromosome aberrations (Phase I: 7.67% p<0.0001, Phase II: 6.67% p=0.0070, Phase III: 7.33%, p=0.0070) indicating the sensitivity of the test system to specific mutagens and confirmed that the test conditions were appropriate and that the metabolic activation system functioned properly. Conclusion: The registered substance, Phenethyl Benzoate (CAS: 94-47-3), did not induce chromosomal aberration in cultured CHO cells up to 0.25 mg/ml of culture medium, either in the presence or absence of S9 metabolic activation system.