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Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
Not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
A valid study is available for the analogue substance benzyl acetate. It is conducted in line with good scientific principles. The read-across is considered to be suitable based on the structural and “mechanistic action” similarities between the target substance (1-phenylethyl acetate) and source substance (benzyl acetate) and their similar physico-chemical properties.

Data source

Reference
Reference Type:
publication
Title:
Studies on benzyl acetate. I. effect of dose size and vehicle on the plasma pharmacokinetics and metabolism of [methylene-14C]benzyl actetate in the rat
Author:
Chigdey, M.A.J. and Caldwell, J.
Year:
1986
Bibliographic source:
Fd Chem. Toxic. Vol 24, No. 12, pp. 1257-1265

Materials and methods

Objective of study:
excretion
metabolism
Principles of method if other than guideline:
Male Fischer 344 rats were dosed with radiolabelled benzyl acetate by oral gavage either neat, in corn oil or in propylene glycol at doses of 5, 250 or 500 mg/kg. Animals housed in metabolism cages were dosed either with the neat substance or in corn oil. Urine and faeces were collected during the study for assessment. The animals were sacrificed and exsanguinated at various time points in order to assess individual parameters and the effect of the size of the dose and the choice of vehicle on these parameters. Urinary and plasma metabolites were identified by thin layer chromatography and high pressure liquid chromatography.
GLP compliance:
not specified

Test material

Constituent 1
Chemical structure
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 : benzyl acetate- Radiochemical purity (if radiolabelling): 96 %- Smiles notation : c1(COC(C)=O)ccccc1- Structural formula attached as image file: see Fig.1- Molecular formula : C9H10O2- Molecular weight: 150.2 g/mol
Radiolabelling:
yes
Remarks:
[methylene-14C] benzyl acetate (specific activity: 53 mCi/mmol)

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS- Source: Oxford Laboratory Animal Co. (Oxford) or Bantin & Kingman Ltd (Hull)- Weight at study initiation: 200 g- Housing: the first group, used to examine the plasma and carcass examinations, animals were kept in plastic cages, the second group (dosed as the first group) were housed in metabolism cages- Individual metabolism cages: yes- Diet: ad libitum- Water: ad libitum

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: either neat, in corn oil or in propylene glycol
Duration and frequency of treatment / exposure:
One single dose
Doses / concentrations
Remarks:
Doses / Concentrations:5, 250 or 500 mg/kg.
No. of animals per sex per dose / concentration:
At least 3
Control animals:
no
Details on dosing and sampling:
PHARMACOKINETIC STUDY (Absorption, distribution, excretion)- Tissues and body fluids sampled: urine, faeces, cage washes, plasma and carcass- Time and frequency of sampling: Urine and faeces from the metabolism cages were collected daily over a three day period following dosing. Plasma samples were collected at 0.5, 1, 2, 4, 6 and 8 hours post dosing, animals were sacrificed and exsanguinated. Blood (approximately 4 mL) was collected from the blood vessels in the neck. Residual 14C in the carcass of the rats was assayed.- Analytical techniques: Fluids and excreta were assayed by liquid scintillation spectrometry. Faecal samples were assayed following homogenisation and bleaching. Residual 14C in the carcass was assayed following digestion in KOH.METABOLITE CHARACTERISATION STUDIES- Tissues and body fluids sampled : urine and plasma. Plasma was prepared to give only plasma samples containing > 5000 dpm/mL (representing 250 dpm per injection onto the HPLC column). Urine was assessed by TLC and HPLC without further treatment. Further urine samples were prepared after the following treatments:Alkaline hydrolysis (adjusted to pH 9, incubated overnight at rtp and examined by HPLC and TLC), Incubation with β-glucuronidase ((Urine was incubated with β-Glucuronidase at pH 5 for 20 hours at 37 °C, the reaction was stopped by the addition of acetone then analysed by HPLC and TLC) with control experiments carried out in the presence of the specific inhibitor D-saccharic acid acid 1,4-lactone and Ammonolysis of ester glucuronides (urine was streaked onto TLC plates a standard solution of glucuronic acid was spotted separately, the plates incubated overnight in a tank containing ammonia solution. Excess ammonia was removed from the plates, then the plates developed with propanl-ol-water (7: 3, v/v). Sugars were located using the naphthoresorcinol spray).- Method type(s) for identification: Thin layer chromatography and high performance liquid chromatography.Thin-layer chromatographySamples were spotted onto pre-coated silica gel F25 4 plates, 0.2mm thick on aluminium support, the plates were developed for 15 cm by the ascending technique using one of the following solvent systems:(A) benzene-acetone glacial acetic acid (6:2 : 1, by vol.)(B) benzene-l,4-dioxan-glacial acetic acid (90:25: 8, by vol.) (C) butan-l-ol-glacial acetic acid-water (3:2:2, by vol.).Compounds were detected on the plates by inspection under UV light (254 nm), aromatic compounds showing as dark quenching spots against the fluorescent background. The following additional spray reagents were used:Naphthoresorcinol glucuronic acid derivatives appear as a blue spot on a pink background when heated for 10 mins at 100 °C.p-Dimethylaminobenzaldehyde (DMAB): sodium acetate trihydrate: glycine conjugates appear as orange/red spots when the plates are heated at 100°C for 5 min.KeCreO7/AgN03: Compounds containing divalent sulphur appear as orange/yellow spots on a red/brown background.High-pressure liquid chromatographyHPLC system: Waters Associates U6K loop injector and two pumps (Waters Models 6000A and M45), linked to a Cecil 2012 UV detector at 254 nm and a Waters Model 730 Data Module. Column: stainless-steel column (100 x 5 mm) packed with 5 µ ODS-Hypersil (C18 reversed phase).Solvents: A Waters Associates Model 720 System Controller was used to control a linear gradient running from 100% solvent D (acetonitrile-water-glacial acetic acid, 10:88:2, by vol.) to 100% solvent E (acetonitrile-water-glacial acetic acid, 70:28: 2, by vol.) over a period of 20 min and at a flow rate of 1 mL/min.

Results and discussion

Main ADME resultsopen allclose all
Type:
distribution
Results:
The levels of 14C in plasma post dosing were dependent upon the dose size, the vehicle and the time after administration
Type:
metabolism
Results:
In urine, the proportions of administered 14C converted to hippuric, benzoic and benzylmercapturic acids were not significantly influenced by the size of the dose. The dose vehicle had no effect upon the metabolic pattern of benzyl acetate.
Type:
metabolism
Results:
The metabolites detected in plasma following administration were dependent on the dose level, the vehicle and the time after dosing.
Type:
excretion
Results:
Excretion was found essentially to be complete by 72 hours, with the major route of excretion being via the urine

Toxicokinetic / pharmacokinetic studies

Details on distribution in tissues:
The levels of 14C in plasma post dosing were dependent upon the dose size, the vehicle and the time after administration. At 500 mg/kg in corn oil, 14C reached a plateau of around 190 µg benzyl acetate equivalents/mL, which was maintained up to 8 hours. This declined to 3.0 µg equivalents/mL after 24 hours. When the same dose was given in propylene glycol, plasma 14C peaked at 336 µg equivalents/mL at 4 hours and declined monoexponentially to 115 µg equivalents/mL by 8 hours. However, when dosed neat, 14C levels peaked at 1 hour (411 µg/equivalents/mL) and then declined monoexponentially to 36 µg equivalents/mL by 8 hours.After the 250 mg in corn oil, plasma 14C levels were maintained at approximately 85 µg equivalents/mL from 0.5 to 2 hours. Then declined to 6 µg benzyl acetate equivalents/mL monoexponentially by 8 hours. Following administration of the same dose neat, 14C levels peaked at 1 hour (177 µg benzyl acetate equivalents/mL) and then fell monoexponentially to negligible levels by 8 hours.Following administration at 5 mg/kg in corn oil or propylene glycol, by 0.5 hours peak levels of radioactivity were reached (approximately 1.6 and 1.9 µg equivalents/mL, respectively) which then proceeded to fall monoexponentially.
Details on excretion:
No systematic variation in the rate or route of excretion of 14C occurred with dose size or vehicle. In all cases, the major route of excretion was the urine, with 70-89% of the dose being recovered within 24 hours. Recovery of 14C in the 24-48- and 48-72 hours urine was low (around 3 and 1%, respectively). A small proportion of the dose (ca. 4%) was excreted in the faeces, and very little of the dose (< 1%) was found in the carcasses of the rats 72 hours after dosing, indicating that excretion was essentially complete.

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
- Plasma:At least six metabolites were identified in plasma samples, although not all were present in every sample. Based on the known properties of the expected metabolites, the compounds eluting at 5.3, 7.6, 10.8 and 12.5 minutes were determined to be hippuric acid, benzyl alcohol, benzoic acid and benzylmercapturic acid, respectively. Two additional unknown metabolites, one of very high polarity and one of moderate polarity, were also detected. Benzyl acetate was not detected.The metabolites detected were dependent on the dose level, vehicle and time after dosing. In all samples after the 500 mg/kg dose the major metabolite was benzoic acid, at around 150 µg equivalents/mL for up to 8 hours when dosed in corn oil. When administered as the neat substance or in a propylene glycol vehicle, levels of benzoic acid reached a maximum at 1 and 4 hours, respectively, then declined monoexponentially. Hippuric acid levels were similar (around 15 µg equivalents/mL) in all samples. When dosed in corn oil, benzyl alcohol in the plasma increased until a maximum value of 22 µg benzyl acetate equivalents/mL was reached at 2 hours then declined thereafter. Benzyl alcohol was only detected at early time points after administration of benzyl acetate as the neat substance or dissolved in propylene glycol. In addition, small amounts of radioactivity (< 5% of total plasma 14C) were present as unknown metabolites of high and moderate polarity, although neither was detected in all samples.Benzoic acid was also the major plasma metabolite at the 250-mg/kg dose level. When dosed in corn oil, levels were maintained at around 70 µg equivalents/mL for 0.5-2 hours after dosing but then declined. In contrast, a peak level of 159 µg equivalents/mL was reached at 1 hour following neat administration. Hippuric acid levels were maintained around 15 µg equivalents/mL over the time period investigated. Benzyl alcohol was only detected at early times (1-2 hours) after administration of corn oil. The very minor unknown metabolites were again observed after administration in corn oil or neat.At 5-mg/kg in either vehicle, the major metabolite was hippuric acid, which declined monoexponentially with time. Around 20% of the 14C in plasma was present as the polar unknown metabolite, although this became relatively less important with time. Small amounts of benzoic acid were also detected in each sample. With propylene glycol, benzylmercapturic acid was also detected in the plasma. This metabolite was not detected when a corn oil was used and was not found with any other dose.- Urine:HPLC of the 0-24-hours urine revealed four metabolites. From the known properties of the expected metabolites the compounds detected with the following retention times 5.3, 6.2, 10.8 and 12.5min were determined to be hippuric acid, benzoyl glucuronide, benzoic acid and benzylmercapturic acid, respectively. Analysis by TLC using solvent system A revealed only three areas of 14C activity with RF values of 0.01, 0.34 and 0.91 determined to be benzoyl glucuronide, hippuric acid and benzoic acid, respectively. An intense blue band (RF 0.01), indicated a glucuronic acid conjugate, when plates were sprayed with naphthoresorcinol reagent. The band at RF 0.01 disappeared on treatment with β-glucuronidase with a concurrent increase in the band corresponding in R F to benzoic acid. This change was prevented by the addition of the β-glucuronidase inhibitor D-saccharic acid 1,4-1actone to control samples incubated concurrently. The glycoside was examined further by ammonolysis, and the only sugar liberated co-chromatographed with glucuronic acid and gave an identical colour reaction. This metabolite (RF 0.01; solvent system A) was labile in alkaline solution, giving rise to benzoic acid. The metabolite located at RF 0.34 (solvent system A) gave an orange/red colour with the DMAB reagent, and corresponded in RF and colour reaction to hippuric acid. An orange band, indicated the presence of a mercapturic acid at RF 0.39 following treatment of the plates with the K2Cr2OT/AgNO3 spray reagent. The 14C associated with the latter minor metabolite was obscured by the major 14C peak due to hippuric acid at RF 0.34. Chromatograms of blank urine gave no colour reaction when sprayed with the appropriate reagents. These results indicated that the metabolites located at RF 0.34 and RF 0.39 (solvent system A) are the glycine conjugate, hippuric acid, and benzylmercapturic acid, respectively. Results obtained by TLC in the other solvent systems were consistent with those obtained using solvent system A. Four metabolites were identified in the urine of rats following administration of the compound as the neat substance or in corn oil at various doses. The major urinary metabolite in all cases was the glycine conjugate, hippuric acid. Benzoyl glucuronide, benzoic acid and benzylmercapturic acid were also detected in the urine at all dose levels. There occurred a significant decrease in the portion of the dose converted to benzoyl glucuronide as the dose level was lowered (P < 0.001 by the Spearman rank correlation, R(s) = 0.95). However, the proportions of administered 14C converted to hippuric, benzoic and benzylmercapturic acids were not significantly influenced by the size of the dose. The dose vehicle had no effect upon the metabolic pattern of benzyl acetate.

Any other information on results incl. tables

Table 2: Excretion of 14C following gavaged doses of [methylene-14C] benzyl acetate

Dose (mg/kg)

Vehicle

% of dose collected in urine

% of dose collected in faeces 0-72 hours

Total*

0-24 hours

24-48 hours

48-72 hours

500

Corn oil

79.7 (76.1-84.1)

3.2 (2.3-4.4)

1.3 (0.5-2.4)

4.8 (1.9-7.9)

93.4 (87.2-97.7)

None

76.0 (73.5-79.0)

2.7 (2.5-2.9)

0.8 (0.7-1.0)

5.0 (4.3-5.4)

87.7 (86.0-89.5)

250

Corn oil

80.4 (75.7-84.0)

2.9 (2.3-3.9)

1.0 (0.8-1.2)

4.3 (3.6-5.5)

94.7 (93.6-95.5)

None

70.5 (69.7-72.0)

1.6 (0.7-2.2)

0.5 (0.3-0.8)

3.8 (3.0-5.3)

78.5 (77.6-79.2)

5

Corn oil

77.5 (69.5-88.6)

2.5 (1.8-3.4)

0.8 (0.5-1.2)

2.4 (1.7-4.1)

87.0 (79.2-94.6)

* Including cage washed after 72 hours but not that found in the carcass (always <1 % of the dose)

Values are mean of three or four animals with ranges in parentheses.

 

Table 3: Urinary metabolites

Dose (mg/kg)

Vehicle

% of dose in urine collected (0-24 hours) as:

Hippuric acid

Benzoyl glucuronide

Benzoic acid

Benzylmercapturic acid

500

Corn oil

66.2 (63.5-73.0)

10.5 (8.3-12.4)

1.5 (0.8-2.1)

1.5 (1.2-1.9)

None

59.7 (56.5-63.7)

11.7 (8.3-14.4)

3.6 (1.1-6.8)

1.1 (0.9-1.2)

250

Corn oil

71.8 (65.1-75.7)

4.1 (3.2-5.6)

2.7 (0.8-3.9)

1.9 (1.7-2.2)

None

62.3 (62.2-62.5)

5.0 (3.4-7.1)

2.1 (1.7-2.7)

1.1 (1.1-1.1)

5

Corn oil

71.6 (64.6-83.0)

2.5 (2.0-2.8)

2.5 (1.5-4.2)

1.0 (0.8-1.2)

Values are mean of three or four animals with ranges in parantheses.

ANALOGUE APPROACH JUSTIFICATION:

- See attached “Justification for read-across” document for full details.

- In summary, important considerations for the use of read-across for acute toxicity are: i) 1-phenylethyl acetate (the target chemical) has similar physico-chemical properties as benzyl acetate (the source substance), ii) there are structural similarities between the two chemicals, iii) the OECD QSAR Toolbox assigns an identical toxicity profiles to both chemicals, and iv) both chemicals have been tested for acute oral toxicity, which demonstrated that neither substance requires classification for acute toxicity and that the benzyl acetate will represent a worst-case scenario, and are adequate for classification and labelling and risk assessment purposes.

The information reported in this summary is included to demonstrate comparability between the source (benzyl acetate) and target (1-phenyl-ethyl acetate) substance.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): low bioaccumulation potential based on study resultsUnder the conditions of the test, the levels of metabolites in the plasma were found to be affected by dose size, test substance and time after dosing indicating the potential for the metabolic pathway to become saturated with high doses. In urine the proportion of administered 14C converted to hippuric, benzoic and benzylmercapturic acids were not significantly influenced by the size of the dose. The dose vehicle had no effect upon the metabolic pattern of benzyl acetate. Excretion of the test substance was found to be rapid, and was considered complete by 72 hours less than 1 % was found to be retained by the carcass after 72 hours.
Executive summary:

Male Fischer 344 rats were dosed with radiolabelled benzyl acetate by oral gavage either neat, in corn oil or in propylene glycol at doses of 5, 250 or 500 mg/kg. Animals housed in metabolism cages were dosed either with the neat substance or in corn oil. Urine and faeces were collected during the study for assessment. The animals were sacrificed and exsanguinated at various time points in order to assess individual parameters and the effect of the size of the dose and the choice of vehicle on these parameters. Urinary and plasma metabolites were identified by thin layer chromatography and high pressure liquid chromatography. Under the conditions of the test, the levels of metabolites in the plasma were found to be affected by dose size, test substance and time after dosing indicating the potential for the metabolic pathway to become saturated with high doses. In urine the proportion of administered 14C converted to hippuric, benzoic and benzylmercapturic acids were not significantly influenced by the size of the dose. The dose vehicle had no effect upon the metabolic pattern of benzyl acetate. Excretion of the test substance was found to be rapid, and was considered complete by 72 hours less than 1 % was found to be retained by the carcass after 72 hours.