Benzyl benzoate

Genetic toxicity: in vivo

Administrative data

Endpoint:

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

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Published study.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

publication

Title:

Measurement of Unscheduled DNA Synthesis and S-Phase Synthesis in Rodent Hepatocytes Following In Vivo Treatment: Testing of 24 Compounds

Author:

Mirsalis JC, Tyson CK, Steinmetz KL, Loh EK, Hamilton CM, Bakke JP & Spalding JW

Year:

1989

Bibliographic source:

Environmental and Molecular Mutagenesis 14:155-164

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Induction of UDS in the rat liver in vivo

GLP compliance:

not specified

Remarks:

Data is published literature

Type of assay:

unscheduled DNA synthesis

Test material

Test material information

Constituent 1

Reference substance name:

Benzyl acetate

EC Number:

205-399-7

EC Name:

Benzyl acetate

Cas Number:

140-11-4

Molecular formula:

C9H10O2

IUPAC Name:

benzyl acetate

Details on test material:

- Name of test material (as cited in study report): Benzyl acetate

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Simonsen Laboratories (Gilroy, CA) and Hilltop Laboratory Animals (Chatsworth CA).
- Age at study initiation: no data
- Weight at study initiation: 180 - 300g
- Assigned to test groups randomly: no data
- Fasting period before study: no data
- Housing: Three rats per cage in polypropylene cages with hardwood chip bedding.
- Diet (e.g. ad libitum): Purina Rodent Chow # 5001 (Ralston Purina Co., St. Louis) ad libitum
- Water (e.g. ad libitum): Deionized 0.5um charcoal filtered tap water ad libitum.
- Acclimation period: Not documented

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not documented
- Humidity (%): Not documented
- Air changes (per hr): Not documented
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle.

IN-LIFE DATES: From: To: Not documented

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Animals were administered the test substance by oral gavage as a single bolus dissolved or suspended in corn oil or water. Doses were selected based approximately on the oral LD50 of the compound and were generally selected as 80%, 40% and 10% of the LD50. The acute LD50 was never exceeded and 1000mg/kg was selected as the highest dose if hte LD50 exceeded this value.

Duration of treatment / exposure:

Animals were exposed to a single dose of the test substance.

Frequency of treatment:

Single exposure

Post exposure period:

No information provided

Doses / concentrations

Remarks:

Doses / Concentrations:
50, 200, 1000 mg/kg bw
Basis:
no data

No. of animals per sex per dose:

3 male rats

Control animals:

not specified

Positive control(s):

corn oil

Examinations

Tissues and cell types examined:

Hepatocyte cultures prepared from perfused liver.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Doses were selected based approximately on the oral LD50 of the compound and were generally selected as 80%, 40%, and 10% of the LD50. The acuteLD50 was never exceeded, and 1000 mg/kg was selected as the highest dose if the LD50 exceeded this value.
If hepatocyte cultures showed very poor viability and attachment at nonlethal doses, the top dose was reduced to achieve acceptable hepatocyte attachment.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
A single-cell suspension of hepatocytes was obtained by combing out cells from the perfused liver into a petri dish containing 37°C collagenase solution. Cells were collected by 5 min centrifugation at 50g, resuspended in cold medium, and filtered through sterile gauze. Viability was determined using Trypan blue exclusion. In general, hepatocyte viability was not adversely affected by chemical treatment; i.e., viability generally exceeded 70%, and attachment did not vary. In cases where attachment or viability were poor, lower doses were selected for testing.
Approximately 6 x 10E5 cells were seeded into each well of a Falcon 9.6 cm2, 6-well culture plate. Each well contained a 25 mm round Thermanox coverslip in Williams' medium E (WE) supplemented with 2 mM I-glutamine, 50 ug/ml gentamycin sulfate, and 10% fetal bovine serum. After 1.5-2.0 hr incubation in a humidified atmosphere at 37°C, 5% CO2 the cultures were washed to remove nonviable cells (those not attached to the coverslips). All washes and subsequent culturing were done using serum-free WE medium.

The sampling for the UDS test was conducted at 2 and 12 hours.

DETAILS OF SLIDE PREPARATION:
Cultures were incubated in Williams medium E (WE) containing 10 uCi/ml 3H-(methyl)-thymidine (specific activity, approximately 80 Ci/mmol) for 4 hr at 37°C, 5% C02, followed by 14-18 hr in WE containing 0.25 mM unlabeled thymidine. The cultures were then washed twice with WE, followed by 10 min in 1% sodium citrate to swell cells, fixed in 1:3 glacial acetic acid:ethanol for 30 min, and washed 3 to 6 times with deionized water. The dried coverslips were mounted to glass slides using Permount. The slides were dipped in Kodak NTB-2 nuclear track emulsion diluted 1: 1 with deionized water, and exposed at -20°C for 7-14 days and then developed and stained

METHOD OF ANALYSIS:
UDS Measurement:
An area of a slide was randomly selected, and 50 morphologically unaltered cells were counted using an ARTEK Model 880 colony counter interfaced to a VAX 8800 computer. The highest of two nuclear-sized areas over the cytoplasm and adjacent to the nucleus was subtracted from the nuclear count to determine the net grains/nucleus (NG). The percentage of cells undergoing repair (%IR) was determined as the percent of those cells exhibiting 5 or more NG. Three slides \vere scored for each animal or concentration for a total of 150 cells per animal.
The test substance was considered negative if the NG of all dose groups was a negative number and the %IR was less than 10%. It was considered positive if the average NG of any dose group exceeded 0 NG. If the test compound had negative NG values, but %IR values greater than 10%, it was considered equivocal.

Evaluation criteria:

UDS:
Compounds were considered negative if the NG of all dose groups was a negative number and the %IR was less than 10%. Compounds were considered positive if the average NG of any dose group exceeded 0 NG. Compounds with negative NG values, but %IR values greater than 10% were considered equivocal.

Statistics:

No information provided.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

not specified

Additional information on results:

The test substance was considered to be negative as the net grain/nucleus was always a negative number and the percentage of cells undergoing repair was always less than 10% for each dose level tested at each timepoint examined.

Any other information on results incl. tables

Measurement of UDS in Male Rat Hepatocytes Following In Vivo Treatment:

			Male rat
	Dose (mg/kg)	Time (hr)	NG	(n)	%IR
Control / corn oil	-	2 12	-6.4±2.9 -5.6± 0.4	(2) (52)	1± 0 2±0
Benzyl Acetate	50     200     1000	2 12   2 12   2 12	-4.1± 1.4 -2.2 ±0.2   -5.3±0.3 -4.8±1.0   -4.5±1.1 -4.6±0.3	(3) (3)   (3) (3)   (3) (3)	3±1 1±0   1± 1 2 ± 1   1± 0 1 ± 1

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative
No evidence for the induction of UDS was seen in this assay

Executive summary:

The read-across substance benzyl acetate was investigated for its ability to induce unscheduled DNA synthesis (UDS) in male F344 rats following in vivo treatment. Hepatocytes, isolated by liver perfusion, were used to assess the DNA-damaging potential of benzyl acetate. Rats received a single dose of the test substance dissolved or suspended in corn oil administered by oral gavage. The dose levels used were 5, 200 and 1000 mg/kg bw. The responses were examined at the 2 and 12 hour timepoints. The results of this study indicate that benzyl acetate was negative under the conditions employed in this study as the net grain/nucleus count was always negative and the percentage of cells undergoing repair was less than 10%.