Phenethyl benzoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation test:

The Substance was tested non-mutagenic (negative) in Salmonella Typhimurium TA98, TA100, TA 1535, TA 1537 and Escherichia Coli WP2 tester strain both in the presence and absence of liver S9 microsomal activation system. The test was performed according to OECD TG 471 and in compliance with GLP.

In vitro mammalian chromosome aberration assay:

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 Practice (GPC).

In vitro mammalian cell gene mutation assay:

In vitro gene mutation study in mammalian cells according to OECD TG 476 with the registered substance is ongoing as negative results are obtained from OECD 471 and OECD 473 studies and will be submitted later based on ECHA communication/decision number CCH-D-2114536388-40-01/F

Link to relevant study records

Reference

Reference 1

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

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

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)

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

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.

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

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

September 2002 - January 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from guideline study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

not specified

Principles of method if other than guideline:

To evaluate the mutagenic potential of test substance in bacteria by AMES assay.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine locus in the genome of Salmonella typhimurium and tryptophan locus of Escherichia coli

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

In addition to a mutation in either the histidine or tryptophan operons, the tester strains contain additional mutations that enhance their sensitivity to some mutagenic compounds. Mutation of either the uvrA gene (Escherichia coli) or the uvrB gene (Salmonella typhimurium) results in a deficient DNA excision repair system, which greatly enhances the sensitivity of these strains to some mutagens. Since the uvrB deletion extends through the bio gene, Salmonella typhimurium tester strains containing this deletion also require the vitamin biotin for growth.

Salmonella typhimurium tester strains also contain the rfa wall mutation, which results in the loss of one of the enzymes responsible for the synthesis of part of the lipopolysaccharide barrier that forms the surface of the bacterial cell wall. The resulting cell wall deficiency increases permeability to certain classes of chemicals such as those containing large ring systems (i.e., benzo[a]pyrene) that would otherwise be excluded by a normal intact cell wall.

Tester strains TA98 and TAlOO also contain the pKMlOl plasmid, which further increases the sensitivity of these strains to some mutagens. The suggested mechanism by which this plasmid increases sensitivity to mutagens is by modification of an existing bacterial DNA repair polymerase complex involved with the mismatch-repair process.

Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strains TAlOO, TA1535, and WP2uvrA are reverted from auxotrophy to prototrophy by base substitution mutagens.

Additional strain / cell type characteristics:

DNA polymerase A deficient

Metabolic activation:

with and without

Metabolic activation system:

S9 Homogenate (Aroclor) in S9Mix

Test concentrations with justification for top dose:

Salmonella tester strains (with S9 mix): 33.3, 100, 333, 1000, 3330, and 5000 ug per plate
Salmonella tester strains (without S9 mix): 3.33, 10.0, 33.3, 100, 333, 1000, 3330, and 5000 ug per plate
Escherichia coli tester strain (with and without S9 mix): 33.3, 100, 333, 1000, 3330, and 5000 ug per plate

Cytotoxicity was observed in the dose range finding study, and the highest dose level of test article used in the subsequent mutagenicity assay was a dose which gave a reduction of revertants per plate and/or a thinning or disappearance of the bacterial background lawn.

Vehicle / solvent:

The test article was .observed to form a transparent, colorless solution at a concentration of 100 mg per mL in dimethylsulfoxide (DMSO). DMSO was selected as the vehicle. At 100 mg per mL, which was the most concentrated stock dilution prepared for the mutagenicity assay, the test article was observed to form a transparent, non-viscous, colorless solution. The test article remained a solution in all succeeding dilutions prepared for the mutagenicity assay.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene - TA100, TA1535, TA1537, WP2uvrA with S9 Mix; ICR-191 - TA1537 without S9 Mix

Details on test system and experimental conditions:

Tester strains were exposed to the test article via the plate incorporation methodology originally described by Ames et al. (1975) and Maron and Ames (1983). This methodology has been shown to detect a wide range of classes of chemical mutagens. In the plate incorporation methodology, test article, tester strain, and S9 mix (when appropriate) were combined in molten agar, which was overlaid onto a minimal agar plate. Following incubation, revertant colonies were counted. All doses of test article, vehicle controls and positive controls were plated in triplicate.

Rationale for test conditions:

Experimental materials, methods and procedures are based on those described by Ames et al. (1975) and Green and Muriel (1976). The assay design is based on the OECD Guideline 471, updated and adopted 21 July 1997.

Evaluation criteria:

The condition of the bacterial background lawn was evaluated both macroscopically and microscopically (using a dissecting microscope) for indications of cytotoxicity and test article precipitate. Evidence of cytotoxicity was scored relative to the vehicle control plate and was recorded along with the revertant counts for all plates at that dose level.
Lawns were scored as normal (N), reduced (R), obscured by precipitate (0), macroscopic precipitate present (P), absent (A), or enhanced (E); contaminated plates (C) were also noted.

Revertant colonies were counted by automated colony counter or by hand.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Dose Range Finding Assay
Doses tested in the mutagenicity assay were selected based on results of the dose rangefinding assay conducted on the test article using tester strains TA100 and WP2uvrA in both the presence and absence of S9 mix with one plate per dose. Ten doses of test article, from 6.67 to 5000 ug per plate were tested.
Cytotoxicity was observed with tester strain TA100 at 333 ug per plate and above in the absence of S9 mix as evidenced by reduced background lawns and a decrease in the number of revertants per plate. No cytotoxicity was observed with tester strain TA100 in the presence of S9 mix or with tester strain WP2uvrA in the presence or absence of S9 mix.

Mutagenicity Assay
In the initial mutagenicity assay, first trial (B1), all data were acceptable, and no positive increases in the mean number of revertants per plate were observed with any of the tester strains in either the presence or absence of S9 mix.
In the confirmatory mutagenicity assay, second trial (C1), contamination was observed on many of the assay plates and several of the plates were observed to have reduced or absent bacterial background lawns. Due to the multiple technical problems observed the data generated were not used in the evaluation of the test article (the results have not been included).
The confirmatory assay was repeated in third trial (D1). In the repeat confirmatory mutagenicity assay, all data were acceptable, and no positive increases in the mean number of revertants per plate were observed with any of the tester strains in either the presence or absence of S9 mix. In this trial, a 2.7-fold increase was observed with tester strain WP2uvrA in the presence of S9 mix, however, this increase was not clearly dose-responsive and did not meet the criteria for a positive evaluation. In order to clarify this response, the test article was retested with tester strain WP2uvrA at the same doses in the presence of S9 mix in
fourth trial (D2). Also, due to variability in the vehicle control counts for tester strain TA100 in the absence of S9 mix, the test article was retested with tester strain TAI100 at the same doses in the absence of S9 mix.
In the fourth trial, all data were acceptable, and no positive increases in the mean number of revertants per plate were observed with tester strain WP2uvrA in the presence of S9 mix or with tester strain T A100 in the absence of S9 mix.

All criteria for a valid study were met.

Conclusions:

The substance was tested non-mutagenic (negative) in Salmonella Typhimurium TA98, TA100, TA 1535, TA 1537 and Escherichia Coli WP2 tester strain both in the presence and absence of liver S9 microsomal activation system. The test was performed according to OECD TG 471 and in compliance with GLP.

Executive summary:

A bacterial reverse mutation assay was performed to assess the mutagenic activitythe test chemicalin in Salmonella typhimuriumTA 98, TA100, TA 1535, TA 1537 andEscherichia colistrain WP2 uvrA tester strains bytheplate incorporation method. The doses tested in the mutagenicity assay were selected based on the results of a dose-range finding assay using tester strains TA100 and WP2uvrA and ten doses of test article ranging from 6.67 to 5000 µg per plate, both in the presence and absence of mammalian liver microsomal enzymes (S9 mix).The growth inhibitory effect (cytotoxicity)induced by the test chemicalwas determinedas a decrease in thenumber of revertant colonies per plate and/or by a thinning or disappearance of the bacterialbackground lawn.Cytotoxicity induced by the test chemical was observed with tester strain TA100 at≥333 ug per plate in the absence of S9 mix as evidenced by reduced background lawns and a decrease in the number of revertant per plate. No cytotoxicity was observed with tester strain TA100 in the presence of S9 mix or with tester strain WP2uvrA either in the presence or absence of S9 mix.The mutagenicity assay was performed using tester strains TA98, TA100, TA1535. TA1537, and WP2uvrA both in the presence and absence of S9 mix along with the appropriate vehicle (DMSO) and positive controls. The doses tested with the Salmonella tester strains were 0, 33.3, 100, 333, 1000, 3330, 5000 µg per plate in the presence of S9 mix and 0, 3.33, 10, 33.3, 100, 333, 1000, 3330 and 5000 µg per plate in the absence of S9 mix. The doses tested with tester strain WP2uvrA were 0, 33.3, 100, 333, 1000, 3330 and 5000 µg per plate both in the presence and absence of S9 mix. The results of the initial mutagenicity assay were confirmed in independentconfirmatoryexperiments. The number of revertant colonies per plate for the vehicle controls and all doses were counted manually.In the initial mutagenicity assay, no substantial increases in the mean number of revertantsper plate were observed with any of the tester strains either in the presence or absence of metabolic activation. In the confirmatory mutagenicity assay with Salmonella tester strains no substantial increase of His⁺colony numbers were seen at any doses tested either with or without metabolic activation. In E. coli WP2uvrA and in the presence of S9 mix, a 2.7-fold increase of revertant colony numbers was observed, but this increase was not clearly dose-responsive and did not meet the criteria for a positive evaluation. In order to clarify this response, the test article was retested with tester strain WP2uvrA at the same doses in the presence of S9 mix. Also, due to variability in the vehicle control counts for tester strain TA100 in the absence of S9 mix, the test article was retested with tester strain TA100 at the same doses without S9 mix. In the last trial, all data were acceptable, and no positive increases in the mean number of revertants per plate were observed with tester strain WP2uvrA in the presence of S9 mix or with tester strain T A100 in the absence of S9 mix.In conclusion,the test chemicaldid not induce gene mutations by base pair changes or frameshifts in the genome ofS. typhimurium and E. coli (WP2 uvrA) tester strainsneither in the presence nor in the absence of metabolic activation and consequently, it was considered non-mutagenic (negative) in bacterial reverse mutation assay, under the experimental conditions described.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Bacterial reverse mutation test:

A bacterial reverse mutation assay was performed to assess the mutagenic activitythe test chemicalin in Salmonella typhimuriumTA 98, TA100, TA 1535, TA 1537 andEscherichia colistrain WP2 uvrA tester strains bytheplate incorporation method. The doses tested in the mutagenicity assay were selected based on the results of a dose-range finding assay using tester strains TA100 and WP2uvrA and ten doses of test article ranging from 6.67 to 5000 µg per plate, both in the presence and absence of mammalian liver microsomal enzymes (S9 mix).The growth inhibitory effect (cytotoxicity)induced by the test chemicalwas determinedas a decrease in thenumber of revertant colonies per plate and/or by a thinning or disappearance of the bacterialbackground lawn.Cytotoxicity induced by the test chemical was observed with tester strain TA100 at≥333 ug per plate in the absence of S9 mix as evidenced by reduced background lawns and a decrease in the number of revertant per plate. No cytotoxicity was observed with tester strain TA100 in the presence of S9 mix or with tester strain WP2uvrA either in the presence or absence of S9 mix.The mutagenicity assay was performed using tester strains TA98, TA100, TA1535. TA1537, and WP2uvrA both in the presence and absence of S9 mix along with the appropriate vehicle (DMSO) and positive controls. The doses tested with the Salmonella tester strains were 0, 33.3, 100, 333, 1000, 3330, 5000 µg per plate in the presence of S9 mix and 0, 3.33, 10, 33.3, 100, 333, 1000, 3330 and 5000 µg per plate in the absence of S9 mix. The doses tested with tester strain WP2uvrA were 0, 33.3, 100, 333, 1000, 3330 and 5000 µg per plate both in the presence and absence of S9 mix. The results of the initial mutagenicity assay were confirmed in independentconfirmatoryexperiments. The number of revertant colonies per plate for the vehicle controls and all doses were counted manually.In the initial mutagenicity assay, no substantial increases in the mean number of revertantsper plate were observed with any of the tester strains either in the presence or absence of metabolic activation. In the confirmatory mutagenicity assay with Salmonella tester strains no substantial increase of His+colony numbers were seen at any doses tested either with or without metabolic activation. In E. coli WP2uvrA and in the presence of S9 mix, a 2.7-fold increase of revertant colony numbers was observed, but this increase was not clearly dose-responsive and did not meet the criteria for a positive evaluation. In order to clarify this response, the test article was retested with tester strain WP2uvrA at the same doses in the presence of S9 mix. Also, due to variability in the vehicle control counts for tester strain TA100 in the absence of S9 mix, the test article was retested with tester strain TA100 at the same doses without S9 mix. In the last trial, all data were acceptable, and no positive increases in the mean number of revertants per plate were observed with tester strain WP2uvrA in the presence of S9 mix or with tester strain T A100 in the absence of S9 mix.In conclusion,the test chemicaldid not induce gene mutations by base pair changes or frameshifts in the genome ofS. typhimurium and E. coli (WP2 uvrA) tester strainsneither in the presence nor in the absence of metabolic activation and consequently, it was considered non-mutagenic (negative) in bacterial reverse mutation assay, under the experimental conditions described.

Chromosomal Aberration study:

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.

Justification for classification or non-classification

Based on the data available the given test chemical does not exhibit gene mutation in vitro by Ames assay and In vitro mammalian chromosome aberration study.  Hence, the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.