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Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
other: absorption and distribution
Qualifier:
no guideline followed
Principles of method if other than guideline:
Toxicokinetic investigations using percutaneous absorption in the rat, in vivo, with radiolabelled test material
GLP compliance:
not specified
Specific details on test material used for the study:
- Name of test material (as cited in study report): Benzyl Acetate
- Radiochemical purity (if radiolabelling): >96%
- Specific activity (if radiolabelling): 53mCi/mmol
Radiolabelling:
yes
Remarks:
>96% radiochemical purity
Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Oxford Laboratory Animal Co. (Oxford)
- Age at study initiation: Not documented
- Weight at study initiation: 200g
- Fasting period before study: The animals were not fasted - they were allowed access to food and water ad libitum throughout the experiments.
- Housing: Not documented
- Individual metabolism cages: Not documented
- Diet (e.g. ad libitum): Oxoid 41B Pellets ad libitum
- Water (e.g. ad libitum): 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): Not documented

IN-LIFE DATES: From: To: Not documented
Route of administration:
dermal
Vehicle:
other: Applied neat or as a 50% (v/v) solution in ethanol.
Details on exposure:
- Area of exposure: The animals backs were shaved. The area of application varied. It was 6.25, 12 and 18cm2
- % coverage: Not documented
- Type of wrap if used: The dose was applied to a layer of Kleenex tissue (single or double ply) placed on a piece of aluminium foil of an appropriate area. The dressing was positioned on the shaven area with the tissue next to the skin and was held in place with polyethylene tape which was wrapped around the body and further occluded the application site.
- Time intervals for shavings or clipplings: At the beginning of each experiment animals were shaved.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The shaven areas were washed twice with lint lint swabs wetted with ethanol and the animals were immediately returned to the metabolism cages.
- Time after start of exposure: 6 hours following exposure

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 100, 250 and 500 mg/kg body weight.
- concentration (if solution): 100% or 50% (v/v) solution in ethanol.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Ethanol if applied as a dilution
- Amount(s) applied (volume or weight with unit): Not documented
- Concentration (if solution): Not documented
- Lot/batch no. (if required): Not documented
- Purity: Not documented

USE OF RESTRAINERS FOR PREVENTING INGESTION: no
Duration and frequency of treatment / exposure:
The animals were exposed for 6 hours.
Dose / conc.:
100 mg/kg bw (total dose)
Dose / conc.:
250 mg/kg bw (total dose)
Dose / conc.:
500 mg/kg bw (total dose)
No. of animals per sex per dose:
Not documented
Control animals:
not specified
Positive control:
Not required
Details on study design:
- Dose selection rationale: Not documented
- Rationale for animal assignment (if not random): Not documented
Details on dosing and sampling:
PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled (delete / add / specify): urine, faeces, blood
- Time and frequency of sampling:
- Other: For the tissue distribution study, the liver, heart, lung, brain, kidney, gut wall and gut contents were placed in water and homogenized and levels of 14C were determined following combustion. The skin and subcutaneous fat form the application site and surrounding area were also removed and assayed for radioactivity using alkaline digestion method used to determine levels of residual 14C in carcasses.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled (delete / add / specify): urine
- Time and frequency of sampling: Not documented
- From how many animals: (samples pooled or not) Not documented
- Method type(s) for identification: TLC and HPLC.
- Limits of detection and quantification: Not documented
- Other: The metabolites were identified by comparison of their chromatographic mobilities and colour reactions with those of authentic samples of the metabolites.

TREATMENT FOR CLEAVAGE OF CONJUGATES (if applicable): No information provided
Statistics:
The interdependence of sets of data was assessed by means of the Spearman rank correlation and statistical comparison of data was performed with Student's unpaired t-test.
Type:
absorption
Results:
The absorption of the compound was unaffected by dose size, area of application or the use of ethanol as a vehicle. In all cases, a significant proportion of the dose (28-48%) was recovered from the application site after 6 hr.
Type:
absorption
Results:
A twofold increase in the concentration on the skin resulted in an approximately twofold increase in the amount absorbed per unit area of skin.
Type:
metabolism
Results:
hippuric acid was the major metabolite of the topically applied compound, accounting for c. 95% of urinary 14C.
Type:
metabolism
Results:
Also present were much smaller amounts of benzoyl glucuronide, benzoic acid and benzylmercapturic acid, each accounting for c. 1-2% of urinary radioactivity.
Type:
metabolism
Results:
the proportions of the administered dose excreted as hippuric, benzoic and benzylmercapturic acids were not significantly influenced by dose size.
Type:
distribution
Results:
approximately 79% of the administered dose was recovered
Type:
distribution
Results:
levels of radioactivity in all the organs examined were lower in the animals killed 24 hr after administration of the test compound.
Type:
distribution
Results:
Recovery of 14C in the skin and subcutaneous fat of the application site and surrounding area accounted for 3.7% of the dose immediately after the removal of the dressings and this had declined to 1.2% 24hr after dosing.
Type:
distribution
Results:
Radioactivity remaining in the carcasses of the rats accounted for < 4% of the administered dose.
Details on absorption:
The extent of absorption of the test compound was assessed at three dose levels, two of which were applied to different areas of skin. The absorption of the compound was unaffected by dose size, area of application or the use of ethanol as a vehicle. In all cases, a significant proportion of the dose (28-48%) was recovered from the application site after 6 hr. Most of this (c. 96%) was found on the dressings with the remainder in the skin washings. Most of the absorbed 14C was excreted in the urine within 24 hr, only small amounts being found in urine excreted between 24 and 48 hr (< 2%) and between 48 and 72 hr (< 1%). About 1% of the dose was excreted in the faeces. Overall, the recovery of 14C was 77-88%. Less than 2% of the dose was found in the carcasses of rats 72 hours after dosing.
Details on distribution in tissues:
Levels of radioactivity in all the organs examined were lower in the animals killed 24 hr after administration of the test compound. Recovery of 14C in the skin and subcutaneous fat of the application site and surrounding area accounted for 3.7% of the dose immediately after the removal of the dressings and this had declined to 1.2% 24hr after dosing. Radioactivity remaining in the carcasses of the rats accounted for < 4% of the administered dose.
Details on excretion:
Most of the absorbed 14C was excreted in the urine within 24 hr, only small amounts being found in urine excreted between 24 and 48 hr (< 2%) and between 48 and 72 hr (< 1%). About 1% of the dose was excreted in the faeces.
Metabolites identified:
yes
Details on metabolites:
Hippuric acid was the major metabolite of the topically applied compound, accounting for c. 95% of urinary 14C. Also present were much smaller amounts of benzoyl glucuronide, benzoic acid and benzylmercapturic acid, each accounting for c. 1-2% of urinary radioactivity.
The proportions of the administered dose excreted as hippuric, benzoic and benzylmercapturic acids were not significantly influenced by dose size.

No additional information

Conclusions:
The data presented provides information on the effects of various factors on the absorption and disposition of topically applied benzyl acetate in the rat.
Executive summary:

Methylene-14CBenzyl acetate was applied over an area of 6.25, 12 or 18 cm 2 to the shaved backs of male Fischer 344 rats under an occlusive dressing at dose levels of 100, 250 and 500 mg/kg. The compound was administered either as the neat substance or as a 50% (v/v) solution in ethanol. After 6 hr the dressing was removed, the shaven area was washed with ethanol and the dressing and washings were counted for 14C. Urine and faeces were collected for 72 hr from the start of treatment and urinary metabolites were assayed by radio-TLC and HPLC. Following administration of the neat compound, a significant proportion of the dose was recovered from the application site (28-48%) and a similar proportion (28-46%) was absorbed and excreted in the 0-24-hr urine. Excretion of 14C in the urine over 0-24 hr accounted for c. 95% of absorbed 14C in all cases, and total recovery of radioactivity was 79-84% with <2% of the dose present in the carcass at the end of the experiments. The extent of absorption of benzyl acetate per unit area of skin, as assessed by the recovery of its metabolites in urine, rose with increasing concentration (mg/cm 2) of the test compound on the skin. The absorption of topically applied benzyl acetate was essentially the same when the dose was administered in a 50% ethanolic solution. In all cases, the major urinary metabolite was hippuric acid (c. 95% of urinary ~4C), together with much smaller amounts of benzoyl glucuronide, benzoic acid and benzylmercapturic acid. The distribution of 14C in the tissues was examined 6 and 24 hr after the topical application of 5 mg [methylene-14C]benzyl acetate/kg as a 1% (v/v) solution in ethanol to rats. Radioactivity in all carcasses was <4% of the administered dose and levels in all the organs examined were lower at 24 than at 6 hr.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
toxicokinetics
Qualifier:
no guideline followed
Principles of method if other than guideline:
The study follows standard methodology for evaluation of toxicokinetics following gavage or dietary administration
GLP compliance:
not specified
Specific details on test material used for the study:
- Name of test material (as cited in study report): Benzyl Acetate
- Physical state: Colourless liquid
Radiolabelling:
no
Species:
other: rat and mouse
Strain:
other: F344/N and B6C3F1
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories (Raleigh, NC, USA).
- Age at study initiation: Both rats and mice were 9 weeks old.
- Weight at study initiation: Not documented
- Fasting period before study: Not documented
- Housing: Animals in the oral feed treated group were housed like the following: Rats - 3 or 4 per cage
Mice - 5 per cage
- Individual metabolism cages: No
- Diet (e.g. ad libitum): Not documented
- Water (e.g. ad libitum): Not documented
- Acclimation period: 1 week before the start of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not documented
- Humidity (%): Not documented
- Air changes (per hr): Not documented
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycles with light cycles starting at 7am.

IN-LIFE DATES: From: To: Not documented
Route of administration:
other: oral gavage and oral feed
Vehicle:
other: corn oil for oral gavage treated animals only.
Details on exposure:
Oral Gavage treated animals:
Groups of rats and mice were administered benzyl acetate in corn oil at 500 mg/kg (rat) and 1000mg/kg (mouse). Gavage formulations were prepared by dissolving benzyl acetate in corn oil. The gavage volume was 5 ml/kg for rats and 10 ml/kg for mice.

Oral feed treated animals:
Groups of rats and mice were were dosed with benzyl acetate in feed (10800 ppm for rats and 2700ppm for mice), ad libitum over the course of the entire study. Feed formulations were prepared by blending benzyl acetate directly with NIH-07. Dosed feed was changed daily to minimize loss of benzyl acetate due to evaporation.
Duration and frequency of treatment / exposure:
No information provided
Dose / conc.:
500 mg/kg bw (total dose)
Remarks:
Rat, oral gavage
Dose / conc.:
1 000 mg/kg bw (total dose)
Remarks:
Mouse, oral gavage
Dose / conc.:
10 800 ppm
Remarks:
Rat, dietary
Dose / conc.:
2 700 ppm
Remarks:
Mouse, dietary
No. of animals per sex per dose:
Oral Gavage treated group:
6 rats and 12 mice

Oral feed treated group:
10 rats and mice.
Control animals:
not specified
Positive control:
No information provided
Details on study design:
No information provided
Details on dosing and sampling:
PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled : blood, plasma
- Time and frequency of sampling: Oral Gavage treated group: 5, 10, 20 and 40 minutes and 1, 1.5, 2, 3, 3.7, 6, 9 and 24 hours after administration with one animal per time point.
Oral feed treated group: Blood samples were taken on day 7 at 18:00, 23:00, 05:30 (next day), 07:30 (next day) and 09:00 (next day). 2 rats and 2 mice were bled at each of the five time points.
- Other: Blood samples were collected from the orbital sinus of rats and mice. Approximately 800ul were collected. Plasma concentrations of benzoic acid, benzyl alcohol and hippuric acid were determined. Briefly, an aliquot of plasma (100ul) was transferred to an extraction tube and mixed with 50ul internal standard solution (100ug/ml acetanilide in methanol). Plasma proteins were precipitated by the addition of a saturated aqueous barium hydroxide solution.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled (delete / add / specify): No information provided
- Time and frequency of sampling: No information provided
- From how many animals: (samples pooled or not) No information provided
- Method type(s) for identification (e.g. GC-FID, GC-MS, HPLC-DAD, HPLC-MS-MS, HPLC-UV, Liquid scintillation counting, NMR, TLC) No information provided
- Limits of detection and quantification: No information provided
- Other: No information provided

TREATMENT FOR CLEAVAGE OF CONJUGATES (if applicable): No information provided
Statistics:
No information provided
Preliminary studies:
No information provided
Type:
absorption
Results:
readily absorbed following oral administration
Details on absorption:
Oral gavage treated group:
The results of this study indicate that benzyl acetate was readily absorbed after a corn oil gavage dose of 500 mg/kg for rats and 1000mg/kg for mice.

Oral feed treated group:
No information provided.
Details on distribution in tissues:
There is no information provided on the distribution of benzyl acetate in tissues.
Details on excretion:
No information provided on excretion of benzyl acetate.
Metabolites identified:
not specified
Details on metabolites:
No information provided

The toxicokinetics of benzyl acetate after gavage differed greatly from those after dosed feed administration. The differences may be related to the concentration of intermediate (benzaldehyde) generated, which is postulated to be higher in the gavage study owing to the higher input of benzyl acetate.

Conclusions:
The toxicokinetics of benzyl acetate after gavage differed greatly from those after dosed feed administration. The differences may be related to the concentration of intermediate (benzaldehyde) generated, which is postulated to be higher in the gavage study owing to the higher input of benzyl acetate.
Executive summary:

In a study conducted by Yuan et al, (1995), the effects of gavage versus dosed feed administration on the toxicokinetics of benzyl acetate were studied in male F344 rats and B6C3F1 mice. Benzyl acetate was rapidly hydrolyzed to benzyl alcohol and then oxidized to benzoc acid. After gavage adminstration of benzyl acetate in corn oil at 500 mg/kg (rats) and 1000 mg/kg (mice), high benzoic acid plasma concentrations were observed. In contrast, much lower benzoic acid plasma concentrations were found after dosed feed administration at about 615 mg/kg /day for rats and about 850 mg/kg/day for mice. Results show that although the daily doses of benzyl acetate are comparable bolus gavage administration effectively saturated the benzoic acid elimination pathway whereas dosed feed administration did not. In contrast, hippuric acid plasma concentrations were similar after both gavage and dosed feed administration due to the depletion of the glycine supply pool. The toxicokinetics of benzyl acetate after gavage differed greatly from those after dosed feed administration. The differences may be related to the concentration of intermediate (benzaldehyde) generated, which is postulated to be higher in the gavage study owing to the higher input of benzyl acetate.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1985-1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
other: various studies summarised in NTP review
Qualifier:
no guideline followed
Principles of method if other than guideline:
Information relating to ADME investigations, included in the NTP technical report have been reviewed and summarised for use as read -across supporting evidence for phenyl ethyl acetate and benzyl acetate
GLP compliance:
not specified
Specific details on test material used for the study:
Benzyl acetate, CAS number 140-11-4
vapour pressure 1.99 mm HG at 60°C
density 1.05
Synonyms: alpha-acetoxytoluene; benzyl ethanoate; acetic acid, benzyl ester

Benzyl acetate (ring-UL-14C) was purchased from Midwest Research Institute; unlabeled benzyl acetate was purchased from Aldrich Chemical Co.
Both labeled and unlabeled Benzyl Acetate were determined to be greater than 99% chemically and radiochemically
pure by HPLC analysis.
BA was administered by gavage in corn oil;
a preparation of Emulphor EL-620 : ethanol : water (1:1:4) was used for iv dosing.
Radiolabelling:
other: radiolabelling used in some investigations summarised in NTP technical report
Species:
other: rat and mouse
Strain:
other: F344 rats and B6C3F1 mice
Details on species / strain selection:
Standard strains used in laboratory studies
Sex:
male
Details on test animals and environmental conditions:
Adult (8-10 wk old, 180-220g) male Fischer 344 rats and adult (6-8 wk old, 19-26g) male B6C3Fl mice were purchased from Charles River Breeders (Kingston, N.Y.).
Each animal received pelleted NIH 31 rat chow and water ad libitum throughout the study.
Animals dosed with 14C-labelled Benzyl acetate preparations, were transferred to individual metabolism cages to provide separate collection of urine, faeces, exhaled 14C02, and volatilized benzyl acetate or metabolites.
Route of administration:
other: various routes - refer to details on exposure
Vehicle:
other: various vehicles used
Details on exposure:
For mice, the dosing preparation for intravenous dosing was a solution of Emulphor:ethanol:
water (I: 1 :4) containing 29 µCi benzyl acetate-ring I4C and sufficient unlabelled benzyl acetate (BA) to total 10 mg BA per ml. This preparation was injected into a tail vein at 1 µl/g body wt to deliver a dose of 10 mg/ kg.
For rats, the dosing preparation for intravenous administration was a solution of Emulphor:ethanol: water (1: 1 :8) containing 10 µCi benzyl acetate-ring I4C and sufficient unlabelled BA to total 5 mg BA per ml and was injected into a tail vein to deliver a dose of 5 mg/ kg.
Oral doses for rats and mice were administered in corn oil.
For mice, the oral doses contained approximately 12 µCi benzyl acetate-ring I4C per ml and sufficient unlabelled BA to administer doses at 10, 100 or 1,000 mg/ kg in 10 ml corn oil/ kg body weight. Mice were treated with an 18 gauge feeding needle.
The oral dose for rats contained approximately 2µCi benzyl acetate-ring I4C per ml and sufficient unlabelled BA to administer doses of 5, 50 or 500 mg/ kg in 5 ml corn oil/ kg body weight. Rats were treated with an 18 gauge feeding needle.

For repeated-dose studies rats and mice received unlabelled BA by gavage in corn oil as described above at 500 or I ,000 mg/ kg, respectively. Doses were administered 5 days per week for 14 days. Treatment was staggered to allow sacrifice of mice on Thursday and rats on Friday. Twenty-four hours prior to sacrifice each animal received an identical dose of BA in corn oil, except that it contained benzyl acetate-ring I4C as described above, and the animals were placed immediately in individual metabolism cages. For studies of dermal absorption rats were anesthetized with pentobarbital, hair was clipped from the intrascapular area of the back with surgical clippers and an area of 2 cm2 was marked off with a fine tip marker. BA doses of 0.1, 1.0 or 10.0 mg containing 3.5 µCi benzyl acetate-ring I4C were prepared in ethanol and applied to 2 cm2 ofskin in a total volume of 50 µl. The area ofapplication was immediately covered with a perforated stainless steel shield secured with cyanoacrylate glue and each rat was placed in an individual metabolism cage
Duration and frequency of treatment / exposure:
various studies summarised - the single dose study in rats and mice involved dosing rats at 5, 50 or 500 mg//kg bw and mice at 10, 100 or 1000 mg/kg bw as a radiolabelled bolus administration
Dose / conc.:
10 mg/kg bw (total dose)
Remarks:
For mice, single i.v. dose of 29 µCi benzyl acetate-ring I4C in a dose of 10 mg/ kg.
Dose / conc.:
5 mg/kg bw (total dose)
Remarks:
For rats, single i.v. dose 10 µCi benzyl acetate-ring I4C injected into a tail vein to deliver a dose of 5 mg/ kg.
Dose / conc.:
10 mg/kg bw/day
Remarks:
Oral dose, mice, approximately 12 µCi benzyl acetate-ring I4C per ml
Dose / conc.:
100 mg/kg bw (total dose)
Remarks:
Oral dose, mice, approximately 12 µCi benzyl acetate-ring I4C per ml
Dose / conc.:
1 000 mg/kg bw (total dose)
Remarks:
Oral dose, mice, approximately 12 µCi benzyl acetate-ring I4C per ml
Dose / conc.:
5 mg/kg bw (total dose)
Remarks:
Oral dose, rats, approximately 2 µCi benzyl acetate-ring I4C per ml
Dose / conc.:
50 mg/kg bw (total dose)
Remarks:
Oral dose, rats, approximately 2 µCi benzyl acetate-ring I4C per ml
Dose / conc.:
500 mg/kg bw (total dose)
Remarks:
Oral dose, rats, approximately 2 µCi benzyl acetate-ring I4C per ml
Dose / conc.:
500 mg/kg bw/day
Remarks:
Rat, gavage, unlabelled
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
Mouse, gavage, unlabelled
Dose / conc.:
0.1 other: mg/ 2cm² skin
Remarks:
Dermal absorption; 3.5 µCi benzyl acetate-ring I4C prepared in ethanol and applied to 2 cm2 of skin in a total volume of 50 µl.
Dose / conc.:
1 other: mg/ 2cm² skin
Remarks:
Dermal absorption; 3.5 µCi benzyl acetate-ring I4C prepared in ethanol and applied to 2 cm2 of skin in a total volume of 50 µl.
Dose / conc.:
10 other: mg/ 2cm² skin
Remarks:
Dermal absorption; 3.5 µCi benzyl acetate-ring I4C prepared in ethanol and applied to 2 cm2 of skin in a total volume of 50 µl.
No. of animals per sex per dose:
no information
Control animals:
not specified
Positive control:
Not required
Details on study design:
Male F344/N rats and B6C3F1 mice were given single oral doses at concentrations detailed above. Radiolabelled benzyl acetate wasadministered in corn oil. Radioactivity recoveries were measured and tissue distribution assessed. Metabolites were identified primarily in urine samples.

In a second study (Chidgeyet al 1987) topical application of radiolabelled benzyl acetate was followed by determination of dermal penetration. absorption, carcass residues and urinary elimination.
Details on dosing and sampling:
Urine and faeces were collected separately over a period of 24 hr. C02 and volatiles were collected by pulling air through the metabolism cages at 300-400 ml/ min and then through a series of traps. Volatiles were collected in a series of two traps containing 200 ml cold ethanol. C02 was collected in a series of two traps containing 200 ml ethanolamine: methyl cellosolve (7:3). Traps were changed at 2, 4, 5 and 24 hr. Each animal was sacrificed 24 hr after treatment. At sacrifice, blood, liver, muscle, adipose, skin, lung, kidney, stomach and the site of application (tail for iv, skin for dermal) were collected from each animal. BA-derived 14C in urine and the volatile and C02 trapping solutions were determined by counting triplicate samples in a Beckman Model LS-9800 Liquid Scintillation Counter (Beckman Instrument Co., Fullerton, CA). BA-derived I4C in faeces and tissues was determined by combustion of triplicate 100 mg samples to 14C02 in a Packard Tri-Carb Sample Oxidizer (Packard Instrument Co., Downers Grove, IL) and quantitation of the I4C by liquid scintillation counting. No corrections were made in any data to account for recovery of radioactivity administered.
Statistics:
No information
Preliminary studies:
No information
Details on absorption:
In a metabolism study, benzyl acetate was absorbed from the gastrointestinal tract of rats and mice. Approximately 90% of the administered dose was recovered as various metabolites in the urine within 24 hr. The primary metabolite was hippuric acid, with minor amounts of mercapturic acid, and one or more unidentified metabolites. The capacity for absorption, metabolism, and disposition was unaffected by the amount or number of doses administered.

In a chemical disposition study conducted by the National Toxicology Program, male Fischer 344 rats and male B6C3F1 mice were shown to efficiently absorb and rapidly metabolize and excrete orally administered benzyl acetate. The doses used in this study were 5, 50, or 500 mg/ kg for rats and 10, 100, or I ,000 mgj kg for mice in single-dose corn oil gavage administrations and 500 mg/ kg for rats and 1,000 mg/ kg for mice daily five times per week for two weeks, also administered by gavage in corn oil. Most (90%) of the benzyl acetate-derived radioactivity was recovered in the urine and none was detected in the liver, blood, muscle, adipose tissue, skin, lung, kidney, or stomach of treated rats or mice. The major metabolite isolated in the urine was hippuric acid (94.6%-99.3% of the dose). Other metabolites found were mercapturic acid and benzyl alcohol. Benzyl acetate was not detected in the urine of treated animals. Neither the size of the dose nor the frequency of dosing had any effect on the absorption, metabolism, or excretion of this compound. There was no evidence to indicate any saturation of this metabolizing capacity in either species over the range of doses studied.
Details on distribution in tissues:
No benzyl acetate was detected in adipose tissue, blood, kidney. liver, lung, muscle, skin or stomach tissue. Circa 95-99% of administered radioactivity was recovered as the hippuric acid metabolite, from urine.
In the second study, following topical application residual carcass levels were less than 4% of administered radioactivity.
Details on excretion:
Circa 95-99% of administered radioactivity was recovered as the hippuric acid metabolite, from urine.
Following oral gavage admijistration there was some evidence that the elimination pathway for the major metabolite, hippuric acid, could be saturated. this effect was not evident following dietary administration.
Metabolites identified:
yes
Details on metabolites:
The major metabolite isolated from urine samples was hippuric acid and other urinary metabolites included mercapturic acid and benzyl alcohol.

The clearance of benzyl acetate (BA) 24 hr following iv or oral dosing is presented in Table 1. Elimination of BA as C02 or volatiles was minimal following iv administration and was not determined following oral dosing. The lack of an effect of dose or route of administration on elimination in urine and faeces indicates that absorption following oral administration was nearly complete and was not affected by dose level. Total recovery exceeded 90% of the dose administered in all but one case. The one exception is believed to be an artifact, or possibly an error in dose preparation, recovery or urine analysis, and was not due to slower elimination of BA when administered by gavage at 5 mg/ kg.

The data in Table 1 also indicate that clearance of BA in urine and faeces did not change with repeated dosing at 500 mg/ kg for I4 days. A series of tissues was analyzed for BA-derived radioactivity. The analysis of blood, liver, muscle, adipose, skin, lung, kidney and stomachs of animals that received either oral or iv doses did not indicate the presence of any BA-derived radioactivity. The detection limit for the 5 mg/kg dose was approximately 1 ppm. Such complete clearance of a xenobiotic from all major tissues within 24 hr is quite unusual. Based on these results, it is believed that failure to find less than 100% of the total dose in excreta collected from rats treated with BA is more likely to be due to minor errors in dosing or collection of radioactivity than to retention of this compound or its metabolites in the tissues. Clearance of BA by mice is described in Table 2. As was seen for the rat, absorption of BA from the gastrointestinal tract by mice was almost complete and elimination of BA-derived radioactivity was rapid. Recovery of the 10 mg/kg iv and oral doses was poor, but an analysis of tissues from these animals did not reveal any trace of BA-derived radioactivity. The detection limit for BA-derived radioactivity in mouse tissue was approximately 0.1 ppm. It is believed that the poor recovery of BA in these studies was due to incomplete recovery of urine. Recovery of the 100 and 1000 mg/kg and repeat oral doses was good and no apparent effect of the size or number of doses was observed.

Dermal absorption of BA was studied by applying a range of doses to a fixed area of skin on rats. Following application the rats were placed immediately in individual glass metabolism cages, which permitted the capture of all volatiles as well as urine and faeces. At the end of 24 hr, treated rats were removed from the cages and sacrificed; the major tissues plus skin from the site of BA application were taken for analysis. The results of these studies are presented in Table 3, and data obtained from the study of BA clearance following iv dosing is presented for comparison. The major clearance of BA applied to skin was by volatilization. Loss to volatilization following iv dosing was minimal. It also appears that loss to volatilization was inversely correlated with dose. That is, as the dose increased loss to volatilization decreased and excretion in urine increased. This may have been due at least in part to the fact that the larger dose took longer to evaporate and therefore had more time to be absorbed through the skin. The decreased rate of evaporation was visibly apparent with the 10 mg dose and the spreading of this dose prevented the fixing of the stainless steel shield which was used with the two lower doses. The absence of the shield appeared to decrease retention at the application site, but increased excretion in faeces and urine. These observations may also indicate that a portion of the dose was removed by grooming and subsequent ingestion.

Elimination in C02 appears to be greater following dermal application, but this is likely to result from a small spillover into the C02 trap from the volatile trap. Due to the very large dilution of BA or C02 in these traps, it was not practical to determine the nature of the volatile radioactivity.

It is apparent from the data in Table 3 that dermal absorption of BA was incomplete. However, dermal absorption may be estimated by the following formula:

% dermal absorption = % total dose in urine following dermal application x 100 / % total dose in urine following IV injection

Calculations using this formula indicate dermal absorption of the 0.1, 1.0 and 10.0 mg over 2 cm2 doses was approximately 14%, 19%, and 33% of the respective doses. However, as pointed out earlier, a portion of the 10 mg dose may have been ingested. In any case, results presented in Tables I and 3 indicate that it is not likely that a dose can be absorbed through the skin at levels similar to that achieved by oral administration.

Since urine was the major route for clearance of absorbed BA, the urine from each animal used in this study was analyzed by HPLC for BA and its metabolites. Results obtained on HPLC analysis of urine from rats treated with BA are presented in Table 4. In these studies, benzyl acetate was detected in trace quantities in the urine of only one rat. That rat had received a dermal dose and this trace of BA may represent contamination directly from the skin, but that cannot be confirmed. BA was not detected in the urine of mice (Table 5). The major metabolite of BA detected in all animals was hippuric acid (Tables 4 and 5). The identity of hippuric acid was confirmed by co-chromatography with standard hippuric acid purchased commercially. This metabolite accounted for greater than 90% of the total radioactivity excreted in urine of all dose groups and its formation was apparently unaffected by dose, route or number of exposures. Hippuric acid is the anticipated metabolite formed by hydrolysis of the BA ester to benzyl alcohol and acetic acid, followed by oxidation of benzyl alcohol to benzoic acid and conjugation of benzoic acid with glycine. Hippuric acid is a natural constituent of human urine, but is more concentrated in the urine of herbivores. The formation of hippuric acid from benzoic acid was one of the first biotransformations of xenobiotic chemicals to be described.

Traces of benzyl alcohol were detected in the urine of some rats and mice (Tables 4 and 5,). Its identity was confirmed by co-chromatography with a synthetic standard. This metabolite is believed to represent a small portion of the total benzyl alcohol formed on hydrolysis of BA, which was excreted prior to oxidation to benzoic acid and conjugation with glycine to form hippuric acid. The possibility was considered that the presence of benzyl alcohol in urine might represent excretion of the parent compound in urine and subsequent breakdown prior to analysis. However, collection of urine over dry ice at 2-hr intervals and immediate HPLC analysis failed to show the presence of benzyl acetate, but did indicate the presence of benzyl alcohol. The identity of the third metabolite of BA detected in urine of some rats and mice, benzyl mercapturic acid (Tables 4 and 5) was confirmed by synthesis of the authentic compound and cochromatography. This metabolite is the anticipated product of reaction of BA with glutathione catalyzed by glutathione-S-transferase and subsequent degradation of the glutathione conjugate to this mercapturic acid.

The "Other" metabolite(s) shown in Tables 4 and 5 represent one or occasionally two minor peaks observed in the HPLC analysis of rat and mouse urine. These peaks were not identified further than to confirm that they were quite polar relative to BA or the metabolites which were identified. This metabolite(s) was infrequently present in the urine of all animals of a given group and never accounted for a major portion of the dose.

Conclusions:
No bioaccumulation potential based on study results.
Both rats and mice apparently have a considerable capacity to absorb benzyl acetate from the gastrointestinal tract and metabolize it to hippuric acid and several minor metabolites, which are excreted in the urine. The relative amounts of the dose absorbed, metabolized, and excreted as the various metabolites were apparently unaffected by the size or number of doses administered. There was no evidence to indicate any saturation of this capacity in either species in the dose ranges studied, 5-500 mg/ kg for rats, 10-1,000 mg/ kg for mice. Therefore, it must be assumed that the innate capacity of these strains of rats and mice to metabolize and excrete benzyl acetate is sufficient to handle the doses administered in the National Toxicology Program 2-year studies of toxicity and carcinogenicity. Further, the data indicate that this innate capacity to absorb, metabolize, and excrete benzyl acetate is not measurably altered by repeat administration of the doses used in the two-year assay for chronic toxicity and carcinogenicity.

Studies of dermal absorption, metabolism, and clearance indicate that benzyl acetate is incompletely absorbed from the skin. Most of the dermal dose was lost to volatilization and a very minor portion of the dose remained at the site of application after 24 hr. That portion of the dose which was absorbed was apparently rapidly metabolized and excreted in the same manner as an oral dose. It is unlikely that it will be possible to saturate metabolism and clearance of benzyl acetate by dermal application to rats and mice. These data indicate that the only effects of benzyl acetate which could be demonstrated by skin painting studies which could not be achieved by oral administration would be those produced by direct contact with this chemical.
Executive summary:

Benzyl acetate ADME was summarised in the NTPTechnical Report series No 431 - an amalgamation of data sources including those previously reported in NTP TR250. The findings from five studies are presented in NTP TR431 (NTP, 1986; Abdo, 1985; Chidgey, 1987; Yuan, 1993 and Chidgey & Caldwell, 1986).

Benzyl acetate may be absorbed from the gastrointestinal tract, lung and skin in various test species. It is excreted primarily as urinary metabolites following oral or dermal administration. Benzyl acetate is hydrolysed to benzyl alcohol which is oxidised to benzoic acid and excreted as hippuric acid (major metabolite) or mercapturic acid (minor metabolite). In the Abdo study, the hippuric acid metabolite was present at 95 to 99% of administered radioactivity following single oral doses to rats and mice. The study confirmed no radioactivity was detected in adipose tissue, blood, kidney, liver, lung, muscle, skin or stomach tissues. No evidence of saturation of the absorption, distribution, metabolism or excretion pathways was apparent in the single oral dose study.

Percutaneous absorption was studied in rats following topical application of neat benzyl acetate or a 50% solution in ethanol. Following 24 hours exposure to rat dermis 28 to 48% of the dose was recovered from the exposed skin and a similar proportion was recovered as urinary metabolites and 4% was recovered from the residual carcass. Dermal absorption of neat benzyl acetate was not dissimilar to the absorption of the ethanolic solution.

Chidgey and Caldwell investigated the effect of vehicle on plasma pharmacokinetics in rats. The dose was administered in corn oil or propylene glycol. Peak plasma concentrations appeared earlier in animals given neat benzyl acetate and absorption was slightly delayed and slightly lower following administration in propylene glycol.

The metabolite profile identified in the Yuan study was consistent with other investigations with benzoic acid plasma concentrations higher following oral administration than after dietary administration. Bolus gavage administration saturated the benzoic acid elimination pathway but dietary administration did not.

The influence of age on benzyl acetate distribution in male rats (aged 3-4, 9 or 25 months old) or male mice (2, 13 or 25 months). Hippuric acid formation was not affected by age or dose level in rats or mice. However, the minor routes of excretion (biliary and faecal ) were influenced by age. Faecal excretion of benzyl acetate declined with age and the excretion of benzylmercapturic acid in urine increased with age.

Description of key information

Bolus gavage administration effectively saturated the benzoic acid elimination pathway whereas dietary administration did not. In contrast, hippuric acid plasma concentrations were similar after both gavage and dietary administration due to the depletion of the glycine supply pool. The toxicokinetics of benzyl acetate after gavage differed greatly from those after treated diet administration.

Topical application of neat material or a 50% ethanolic dilution, resulted in recovery of radiolabelled material, from skin from the application site, from urine samples and a small quantity from the residual carcass. Circa 50 % dermal absorption was observed. Metabolites were identified, hippuric acid was identified as the major metabolite.  

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential
Absorption rate - oral (%):
50
Absorption rate - dermal (%):
50
Absorption rate - inhalation (%):
100

Additional information

In a study conducted by Yuan et al, (1995), the effects of gavage versus dosed feed administration on the toxicokinetics of benzyl acetate were studied in male F344 rats and B6C3F1 mice. Benzyl acetate was rapidly hydrolyzed to benzyl alcohol and then oxidized to benzoic acid. After gavage adminstration of benzyl acetate in corn oil at 500 mg/kg (rats) and 1000 mg/kg (mice), high benzoic acid plasma concentrations were observed. In contrast, much lower benzoic acid plasma concentrations were found after treated dietary administration at about 615 mg/kg /day for rats and about 850 mg/kg/day for mice. Results show that although the daily doses of benzyl acetate are comparable, bolus gavage administration effectively saturated the benzoic acid elimination pathway whereas dietary administration did not. In contrast, hippuric acid plasma concentrations were similar after both gavage and dietary administration due to the depletion of the glycine supply pool. The toxicokinetics of benzyl acetate after gavage differed greatly from those after dosed feed administration. The differences may be related to the concentration of intermediate (benzaldehyde) generated, which is postulated to be higher in the gavage study owing to the higher input of benzyl acetate.

In a study conducted by Chidgey (1987), Methylene-14C Benzyl acetate was applied over an area of 6.25, 12 or 18 cm2to the shaved backs of male Fischer 344 rats under an occlusive dressing at dose levels of 100, 250 and 500 mg/kg. The compound was administered either as the neat substance or as a 50% (v/v) solution in ethanol. After 6 hr the dressing was removed, the shaven area was washed with ethanol and the dressing and washings were counted for14C. Urine and faeces were collected for 72 hr from the start of treatment and urinary metabolites were assayed by radio-TLC and HPLC. Following administration of the neat compound, a significant proportion of the dose was recovered from the application site (28-48%) and a similar proportion (28-46%) was absorbed and excreted in the 0-24-hr urine. Excretion of14C in the urine over 0-24 hr accounted for c. 95% of absorbed14C in all cases, and total recovery of radioactivity was 79-84% with <2% of the dose present in the carcass at the end of the experiments. The extent of absorption of benzyl acetate per unit area of skin, as assessed by the recovery of its metabolites in urine, rose with increasing concentration (mg/cm 2) of the test compound on the skin. The absorption of topically applied benzyl acetate was essentially the same when the dose was administered in a 50% ethanolic solution. In all cases, the major urinary metabolite was hippuric acid (c. 95% of urinary 14C), together with much smaller amounts of benzoyl glucuronide, benzoic acid and benzylmercapturic acid. The distribution of 14C in the tissues was examined 6 and 24 hr after the topical application of 5 mg [methylene-14C]benzyl acetate/kg as a 1% (v/v) solution in ethanol to rats. Radioactivity in all carcasses was <4% of the administered dose and levels in all the organs examined were lower at 24 than at 6 hr.