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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:
other: stud in 5 animals, limited information on experimental period, additional glycine provided in 4/5 animals, non GLP.
Objective of study:
metabolism
Qualifier:
no guideline followed
Principles of method if other than guideline:
The rate and amount of the formation and conjugation of benzoic acid formed from administered test substance in rabbit was assessed. The concentration of test substance was 0.5, 1.0, 1.5 g/kg (as equivalents benzoic acid).The urine and blood samples were collected and reaction kinetics was measured.
GLP compliance:
no
Species:
rabbit
Strain:
not specified
Sex:
not specified
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Weight at study initiation: more than 2 kg
- Fasting period before study: The experiments were carried out on fasting animals.
- Housing: Housed in a metabolism cage.
- Diet (e.g. ad libitum): on the constant diet of rabbit pellets, fasted durin the experiment
- Water (e.g. ad libitum): on the constant and water customary, water was supplied during the test period

No additional data
Route of administration:
oral: gavage
Vehicle:
not specified
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Fasted during the test period. A food ration was given at approx. 7 p.m. when most of the administered compound had been excreted.

No additional data
Duration and frequency of treatment / exposure:
None stated, but from the result section it can be derived that the observation period has been between 7 and 24 hours.
Remarks:
Doses / Concentrations:
0.5, 1.0, 1.5 g/kg (as equivalents benzoic acid)
No. of animals per sex per dose / concentration:
0.5 g/kg: No.299
1.0 g/kg No.299, 301, 309 (with glycine)
1.5 g/kg No.262 (with glycine)
Control animals:
not specified
Positive control reference chemical:
None stated
Details on study design:
None stated.
Details on dosing and sampling:
METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, faeces
- Time and frequency of sampling: For this purpose a fraction collector was constructed. It consisted of a brass turntable which carried round its periphery forty-eight receiving tubes each of capacity 140 mL. Its movement was weight-activated and controlled by an escapement operated by an electromagnet activated by impulses from a timing clock. In this way the receiver could be changed automatically at predetermined time intervals ranging from 1 min. to 1 hr.
- From how many animals: 5 (not pooled)

No additional data
Statistics:
None stated.
Preliminary studies:
No information provided.
Type:
metabolism
Results:
The processes of conversion of test substance to benzoic acid also follow first-order reaction kinetics with velocity constants 0.33 /hr.
Details on excretion:
0-5% excreted in urine as the glucuronide and 95-100% as hippuric acid
Metabolites identified:
yes
Details on metabolites:
The formation of benzoylglucuronide follows the kinetics of a first-order reaction with velocity constant 0.08/hr. The processes of conversion of test substance to benzoic acid also follow first-order reaction kinetics with velocity constants 0.33 /hr.
Mathematical treatment of the observed kinetics gives expressions from which theoretical values can be derived which are in agreement with experimental observations.

The species differences in the degree of conjugation of benzoic acid with glucuronic acid is explained by the availability of glycine.

Species

Dose

level (equv benzoic acid)

 

Percentage of dose

excreted as glucuronide

Rate of glycine

mobilization

(mg./kg./hr.)

 

 

Calc. Found

 

 

Dog

0.45

68     66*

3.5*

Man

0.043

9       5t

9.0*

Pig

0.50

41     31**

15.0*

Rabbit

0.50

24     19§

50.0§

*Quick (1931)    t Unpublishedresults  **Csonka(1924)§ Bray (1951)

 

Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results

This study showed that in rabbit, benzaldehyde is rapidly oxidised to benzoic acid, which is then excreted as hippuric acid. The processes of conversion follows first-order reaction kinetics with velocity constants 0.33 hr-1. Hippuric acid appears to be the only major metabolite present in urine (95-100%) and there was no signficant excretion of benzoyl glucoronide observed (0-5%). An approximate rate constant of 0,33 h-1 was determined.
Executive summary:

The test of the formation and conjugation of benzoic acid formed from administered test substance in rabbit was made (period 7 -24 hours). The concentration of test substance was 0.5, 1.0, 1.5 g/kg (as equivalents benzoic acid). Urine and blood samples were collected and reaction kinetics was measured.

The processes of conversion of test substance to benzoic acid follow first-order reaction kinetics with velocity constants 0.33 hr-1. The recovery of the test substance in urine was 0-5% as the glucuronide and 95-100% as hippuric acid. It has to be noted that a clear species difference is expected regarding the ratio glucuronide/hippuric acid.

This study showed that in rabbit, benzaldehyde is rapidly oxidised to benzoic acid, which is then excreted as hippuric acid. Hippuric acid appears to be the only major metabolite present in urine and there was no signficant excretion of benzoyl glucoronide observed.

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:
other: mass balance not reported, non-GLP
Objective of study:
toxicokinetics
Qualifier:
no guideline followed
Principles of method if other than guideline:
Rats exposed to 11C-labelled test substance for 2 min via inhalation, the kinetics and biodistribution were studied based on the decay characteristics of short-lived gamma-emitting radioisotopes.
GLP compliance:
not specified
Radiolabelling:
yes
Remarks:
11C-labelled the test substance
Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 8-10 weeks old
- Weight at study initiation: a mean weight of 212 g
- Fasting period before study: 24 hour
- Individual metabolism cages: yes
- Diet (e.g. ad libitum): commercial chow
- Water (e.g. ad libitum): water ad libitum

No additional data
Route of administration:
inhalation: vapour
Vehicle:
not specified
Details on exposure:
The animal to be exposed was lightly anaesthetized with Ethrane and placed into a glass restrainer. However, by the time the animal was exposed to the test substance it had always fully regained consciousness. A latex mask, with a small hole to permit exposure of the nostrils, was placed over the animal's protruding nose and fastened to the restrainer with an O-ring which provided an air-tight seal for the nose cone. A respirator pump was used to provide an airflow of 120 mL/min through the nose cone. The exposure to the test substance was begun by redirecting the air flow from the by-pass tubing through the delivery line. For all experiments described, exposures were for 2 min, during which time the gas chromatograph trap was heated with a hot air gun to aid in the rapid volatilization of the test substance. After the exposure period, the amount of the test substance accumulated by the animal was immediately determined by removing the restrainer containing the rat from the dosing apparatus and measuring the radioactivity in an ionization chamber. The animal was then removed and the restrainer checked for radioactivity to verify that the nose mask had not leaked, which would have resulted in contamination of the glass restrainer as well as the animal's fur. After removal from the restrainer, in which the animals spent approx. 5 min, the dosed animal was placed in a separate cage until killed.
Duration and frequency of treatment / exposure:
2 min
Remarks:
Doses / Concentrations:
2.5 ug test substance during 2 min
No. of animals per sex per dose / concentration:
five animals in all
Control animals:
no
Positive control reference chemical:
None stated
Details on study design:
None stated
Details on dosing and sampling:
For collection of exhaled 11CO2 and trapping of expired organic metabolites, some of the dosed rats were placed into a chamber through which ambient air was pulled, at the rate of 1 litre per minute, with a vacuum pump. In line, between the animal chamber and the pump were: a 2 litre 2 M NaOH trap, a cold (liquid nitrogen) activated charcoal trap, an additional CO2/moisture trap (barium hydroxide lime), and a flow meter. After a collection period of 35 min the traps were examined for radioactivity.
Urine collected from some of the exposed animals was chromatographed on silica gel G t.l.c. plates using the organic phase of a dichloromethane-water-acetic acid mixture (2 : 2 : 1) as a solvent. The RF values of the radiolabel were compared with the test substance, benzoic acid and hippuric acid standards.
At 1.5, 5, 12, 20 and 40 min after exposure, dosed animals were killed by cervical dislocation and the retained dose of the test substance rapidly determined by placement into the ionization chamber. The animal was then bled by decapitation and the major organs removed or sampled to determine the distribution of radiolabel. The total lungs, bronchi, trachea, heart, spleen, kidney (left) and brain were removed. Samples of the following organs were taken: blood, liver, small intestine (anterior-most portion, mean wt. 0.4 g), muscle (right femoral), skin (tip of ear) and perirenal fat. Urine was collected when available and the contents of the anterior-most portion of the small intestine were also collected for counting. The tissues were blotted free of blood and placed into preweighted counting vials and the vials sealed. The samples were counted in a Packard sodium iodide well counter.
Statistics:
None stated
Type:
absorption
Results:
Inhaled the test substance was rapidly absorbed and at 1.5 min after exposure only 0.8%, of the administered dose was resident in the lungs.
Type:
distribution
Results:
The test substance was quickly distributed with the peak radioactivity in the organs occurring at 1.5 min after exposure.
Type:
excretion
Results:
The test substance was rapidly excreted via the renal system with the kidneys containing 17% of the total administered activity at 5 min.
Details on absorption:
Using the dynamic nose exposure apparatus the amount of the test substance taken up by the animal varied as a linear function of the amount of compound in the gas chromatograph trap( figure 1). The total amount accumulated by the exposed rats during the 2 min exposures ranged from 0.26 to 5.79 μg. The test substance inhaled by the rat was rapidly absorbed from the lungs. At 1.5 min after exposure, only 1.2% of the administered dose was resident in the respiratory tract, with 0.8% in the lungs.
Details on distribution in tissues:
The ratios of the test substance in the sampled tissues to that of the blood, on a per weight basis, are presented in table 1. The absorption of the test substance from the respiratory tract was so rapid that the blood contained greater radioactivity than the lungs as early as 1.5 min after exposure (figure 2). The distribution coefficient of the lungs, as well as that of most other well perfused organs, remained essentially unchanged from the 1.5 min values during the 40 minutes after exposure. The poorly perfused tissues such as the skin, muscle and adipose tissue, continued to accumulate radiolabel for longer periods, e.g. up to 12 min in the skin (figure 2).
Details on excretion:
To investigate routes of the test substance elimination, the expired breath, the urine and the contents of the anterior portion of the small intestine (to indirectly collect the bile) were sampled. Neither 11CO2 nor radiolabelled organic metabolites could be detected in the expired breath of the exposed animals. The low radioactivity of the intestinal contents (table 2) indicated that the bile was not a major route of the test substance excretion. Elimination of the test substance from the rat appeared to be totally via the renal system with detectable radioactivity in the urine as early as 1.5 minutes after the exposure (table 2). Because inhaled the test substance was rapidly excreted into the urine, the percentage of the administered dose retained by the animals (figure 3) until the time of sacrifice was largely dependent upon when the rat last urinated and the amount of urine present in the bladder. Samples of the urine voided during 20 to 40 minutes after exposures were subjected to t.1.c. on silica gel to identify the excreted compound. Over 90% of the radiolabel on the plate cochromatographed with the hippuric acid standard. The biological half-life of the test substance in the blood of exposed rats was 8.1 minutes, and elimination of radiolabel from most organs paralleled the clearance of the test substance from the blood. Organs with a limited blood flow eliminated the test substance more slowly with a net loss of radiolabel recorded only after 5 to 12 minutes following exposure (figure 2).
Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results

The biological half-life of benzaldehyde in the blood was 8.1 minutes and elimination via the renal system from most organs was rapid and linear over time. There were no 11CO2 metabolites measured in the expired air and there were very low levels of radiolabel measured in the intestinal content. It was observed that 11C-benzaldehyde was almost exclusively eliminated by the kidneys in the form of hippuric acid (>90%). It can be concluded that benzaldehye is almost completely absorbed following inhalation exposure, without indications of a potential for accumulation in the body.
Executive summary:

This study was an investigation into the biodistribution and excretion of 11C-benzaldehyde in the rat after two-minute inhalation exposures. The kinetics and biodistribution were studied based on the decay characteristics of short-lived gamma-emitting radioisotopes.

Rats exposed to 11C-labelled benzaldehyde accumulated an average of 2.5 μg of the test substance. The inhaled test substance was rapidly absorbed and at 1.5 min after exposure, only 0.8% of the administered dose was resident in the lungs. The test substance was quickly distributed with the peak radioactivity in the organs occurring at 1.5 min after exposure. Subsequent loss of radiolabel from tissues was rapid and paralleled the removal from the blood. The adipose tissue cleared most slowly. The test substance was rapidly excreted via the renal system with the kidneys containing 17% of the total administered activity at 5 min. The excreted radiolabelled compound co-chromatographed with hippuric acid on t.l.c.

The biological half-life of benzaldehyde in the blood was 8.1 minutes and elimination via the renal system from most organs was rapid and linear over time. There were no 11CO2 metabolites measured in the expired air and there were very low levels of radiolabel measured in the intestinal content. It was observed that 11C-benzaldehyde was almost exclusively eliminated by the kidneys in the form of hippuric acid (>90%). It can be concluded that benzaldehye is almost completely absorbed following inhalation exposure, without indications of a potential for accumulation in the body.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
metabolism
Principles of method if other than guideline:
To investigate the metabolism of benzaldehyde in Sprague-Dawley rats following repeated oral administration.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Test material: Benzaldehyde
- Source: Aldrich Chemical Company (98 + % pure)
- Purity: Purified by distillation under reduced pressure (62°C at 10 mm Hg) and the purity was checked by gas chromatography

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Kept under nitrogen in small dark glass bottles stored in well--ventilated fume hoods

Radiolabelling:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Quebec, Canada)
- Age at study initiation: not specified
- Weight at study initiation: males= 230-245g and females= 170-185g
- Housing: not specified
- Diet (e.g. ad libitum): not specified
- Water (e.g. ad libitum): not specified
- Acclimation period: the animals were kept for 10 days
Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on exposure:
ADMINISTRATION OF TEST MATERIAL
- Several groups of randomised female and male Sprague-Dawley rats were divided into three groups with 5 rats/sex/group:
1. Low level (400 mg/kg)
2. Medium level (750 mg/kg)
3. High level (1000 mg/kg)

Duration and frequency of treatment / exposure:
The rats were administered undiluted benzaldehyde by oral gavage once a day for 13 consecutive days.
Dose / conc.:
400 mg/kg diet
Remarks:
Males/Females
Dose / conc.:
750 mg/kg diet
Remarks:
Males/Females
Dose / conc.:
1 000 mg/kg diet
Remarks:
Males/Females
No. of animals per sex per dose / concentration:
5 rats/sex/group
Control animals:
yes, concurrent no treatment
Positive control reference chemical:
Not applicable
Details on study design:
TEST ANIMALS
- Randomised groups of male and female Sprague-Dawley rats were obtained.

COLLECTION OF URINE
- Immediately after dosing, the animals were placed in stainless steel metabolism cages where they had access to food and water ad libitum.
- The cages were equipped with special deflectors in order to prevent the fecal contamination of urine by deviating boluses outside the collection vessel.
- Urine was collected for 24 hours after the test material was administered on the Day 2, 8 and 13, and placed in a beaker over a Dewar flask containing liquid nitrogen.
- Urine samples were thawed in the dark at room temperature and were immediately analysed to prevent the biological oxidation of the urinary metabolites.

IDENTIFICATION AND QUANTITATIVE DETERMINATION OF BENZYLMERCAPTURIC ACID
> Identification
- 1 mL aliquots of pooled urine from test rats were acidifed (pH:2.0) with 6 N H2SO4.
- After the addition of ammonium sulphate (1g), the urine sample was extracted with ethyl acetate (4 mL) and the extract was dried over anhydrous sodium sulphate.
- The extract was evapourated and the residue diluted with an ether-methanol mixture (9/1; v/v) and methylated with diazomethane.
- The resulting residue was diluted in methanol (1 mL) and the methalonic extract (1 µL) was then introduced into a Finnigan 4000 GC/MS (EI-mode).
- Conditions: initial oven temperature = 50°C (0.1 min) and then programmed to 100°C (15°C/min) and then to 270°C (5°C/min); average linear velocity = 22 cm/sec; helium as a carrier gas; injector temperature = 200°C; separator temperature = 240°C; ion source temperature = 250°C.

> Quantitative determination
- Experimental conditions can be found in Table 1.
- A calibration curve was prepared using freshly voided urines of control Sprague-Dawley rats spiked with various amounts of benzylmercapturic acid (0.01 -1.00 mg/mL)
- The methyl ester of benzylmercapturic acid showed a retention time of 24.54 minutes.
- The equation of the curve was: y = 0.4355 x 10^ -6 with a correlation coefficient of 0.9992.
Details on dosing and sampling:
TOXICOKINETIC / PHARMACOKINETIC STUDY (excretion)
- Tissues and body fluids sampled: urine
- Time and frequency of sampling: Day 2, 8 and 13 after dosing

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine
- Time and frequency of sampling: Day 2, 8 and 13 after dosing
- From how many animals: urine samples pooled from test rats
- Method type(s) for identification: GC-MS
- Limits of detection and quantification: l imit of detection was 0.5 µg/mL and the recovery was 94% for the benzylmercapturic acid metabolite (Range: 92 -96%)
Type:
metabolism
Results:
Benzylmercapturic acid: retention time = 24.54 minutes
Metabolites identified:
yes
Details on metabolites:
- Benzylmercapturic acid was extracted from pooled urines of test rats dosed with benzaldehyde and shown to be methylated.
- GC/MS results showed that the metabolite was absent from the urine of control rats.
- Benzylmercapturic acid was observed to have a retention time of 24.54 minutes, characterised by the following peaks m/z (%): 91(100), 176 (27), 208 (22), 43 (19), 88 (13), 117 (10), 134 (19) and M+ 267 (2).

 

 Table 1: Gas chromatography conditions used for microdetermination of benzylmercapturic acid (BENZM) in rat urine.

 Instrument:

 Perkin-Elmer gas chromatograph, model Sigma 3, equipped with a flame ionisation detector (FID)

 Column

 Capillary column (J&W) D8 -5, 30 m x 0.32 mm I.D.

 Temperature  275°C for injector and detector. Oven was set at 60°C isothermal for 3 minutes, programmed at 10°C/minute to 230°C, then held 15 minutes at 230°C
 Carrier Gas  Hellium at 20.8 cm/second
 Injection  Splitless 0.6 µL
 Retention time  24.54 minutes for benzylmercapturic acid methyl ester

   

 Table 2: Urinary excretion of benzylmercapturic acid in the Sprague-Dawley rat orally administered benzaldehyde over a 13 -day period
 Sex  Level (mg/kg)  2 doses  8 doses  13 doses
 F  400  0.13  0.13  0.23
 F  750  0.27  0.21  0.46
 F  1000  0.85  0.77  1.64
         
 M  400  0.30  0.29  0.35
 M  750  0.75  0.82  0.92
 M  1000  1.39  1.93  2.05
Conclusions:
The mass spectrometry results indicate the metabolite present in the urine of male and female Sprague-Dawley rats were characteristic of benzylmercapturic acid. The metabolite was found in the urine samples of all male and female rats treated with benzaldehyde. Monitoring of urines from both female and male rats revealed a dose-related increase of benzylmercapturic acid, whihc was found to be a reliable indicator of exposure to benzaldehyde.

Benzaldehyde is reduced to a minor extent to form benzyl alcohol, that may then react with glutathione in the presence of glutathione S-transferase, for which sulphate esters are substrates to finally form benzylmercapturic acid. The second metabolic pathway seems to occur only after the primary elimination pathway has been saturated.
Executive summary:

This study was an investigation into the biological conversion of benzaldehyde into benzylmercapturic acid in the rat.

Male and female Sprague-Dawley rats were used in this study, which were randomised into groups. 5 animals/sex/group were split into low level (400 mg/kg), medium level (750 mg/kg) and high level (1000 mg/kg) groups. The animals were then exposed to a repeated oral administration of undiluted test material by gavage once a day for 13 consecutive days. Control groups (5 rats/sex/group) received tap water (1000 mg/kg) over the same period. Urine was collected and analysed for the presence of metabolites. The acifidication of the pooled urine samples (pH 2:0) with 6 N H2SO4 was followed by ethyl acetate extraction, evapouration of the extract and methylation with diazomethane. The metabolites were identified via comparison with a synthetic sample of benzylmercapturic acid and conducted via gas chromatography.

Mass spectrometry of the metabolite revealed that the peaks were characteristic of benzylmercapturic acid. The metabolite was found in the urine samples of all male and female rats treated with benzaldehyde. Monitoring of urines from both female and male rats revealed a dose-related increase of benzylmercapturic acid, whihc was found to be a reliable indicator of exposure to benzaldehyde.

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:
other: urinary metabolites investigated, limited report, non-GLP
Objective of study:
metabolism
Qualifier:
no guideline followed
Principles of method if other than guideline:
The test substance administered two groups of male New Zealand White rabbits by gavage, whereas water was given orally to the third group. Urine of all groups was collected daily for 15 consecutive days. The quantitative metabolism of the test substance was measurement.
GLP compliance:
not specified
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
Source: Aldrich Chemical Company (98+%)
Purity: Purified by distillation under reduced pressure (62°C at 10 mm Hg). Purity was checked by gas chromatography, ultraviolet, infrared and mass spectrometry. There was no evidence of organic peroxide or any other chemical impurity structurally related to benzaldehdyde such as benzyl alcohol, benzoic acid, benzyl chloride or benzyl acetate.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Under nitrogen in small dark glass bottles stored in a well-ventilated fume hood..
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:
Species:
rabbit
Strain:
New Zealand White
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Obtained from Maple Lane Farm, Clifford, Ontario
- Weight at study initiation: 3100 - 3250 g
- Acclimation period: 14 days

No additional data
Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on exposure:
None stated
Duration and frequency of treatment / exposure:
a single dose of the test substance
Dose / conc.:
0.35 other: g/kg/rabbit
Remarks:
Low-dose
Dose / conc.:
0.75 other: g/kg/rabbit
Remarks:
High-dose
No. of animals per sex per dose / concentration:
3 male rabbits/group
Control animals:
yes
Details on study design:
COLLECTION OF URINE
- Immediately after dosing, the animals were placed in stainless steel rabbit metabolism cages, where they had access to food and water ad libitum.
- Urine was collected daily for 15 consecutive days in a beaker placed over a Dewar flask containing liquid nitrogen.
- Urine samples were thawed in the dark at room temperature and were immediately analysed to prevent photochemical oxidation of the urinary metabolites.

IDENTIFICATION AND QUANTITATIVE DETERMINATION OF BENZALDEHYDE METABOLITES IN RABBIT URINE
a) Acidic metabolites
- 1mL aliquot volume of pooled urine from the test rabbits was acidified (pH 1.5) with 6 N H2SO4.
- 1g ammonium sulphate was added and then the urine sample was extracted with ethyl acetate (4 mL) and then treated with 2 mL of an aqueous NaHCO3 solution (5% w/v).
- The aqueous layer was acidified (pH 1.5) with 6 N H2SO4, extracted with ethyl acetate and the extract was dried over anhydrous Na2SO4.
- It was then evapourated and the residue was diluted with an ether-methanol mixture (9/1; v/v) and methylated with diazomethane.
- The residue was then diluted with 1 mL methanol and the methanolic extract (1µL) was then introduced in a Finnigan 4000 GC/MS (EI-mode).
- Conditions: initial oven temperature 50°C (0.1 min) then programmed to 100°C (15°C/min) and then to 270°C (5°C/min); average linear velocity 22 cm/sec using helium as carrier gas; injector temperature 200°C; separator temperature 240°C; ion source temperature 250°C
- Quantitaive determination of free benzoic acid (FBA), hippuric acid (HA) and benzylmercapturic acid (BMA) was developed by gas chromatography.

b) Glucuronides
- Benzoylglucuronic acid and benzyl glucuronide were determined by a previously described method [1].

c) Benzyl sulphate ester
- This compound was determined by the method described by Bray and Thorpe [2].

d) Free benzyl alcohol
- 10 mL extraction of urine previously acidified with 6 N H2SO4 (pH 1.5) was carried out with ethyl acetate (40 mL) and the extract was washed with a 5% (w/v) aqueous solution of NaHCO3 (20 mL).
- After drying (anhydrous Na2SO4) and concentration of the solvent phase, 1 µL was then introduced in a Finnigan 4000 GC-MS (EI-mode) equipped for capillary column analysis.
Details on dosing and sampling:
METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine
- Time and frequency of sampling: Urine was collected daily for 15 consecutive days
- From how many animals: 9 rabbits (three groups)
- Method type(s) for identification: An analytical method for the quantitative determination of free benzoic acid (FBA), hippuric acid (HA) and benzylmercapturic acid (BMA) was developed by gas chromatography. Benzoylglucuronic acid and benzyl glucuronide were determined by a previously described method. Benzyl sulfate ester was determined by the method described by Bray and Thorpe. Free benzyl alcohol was determined by GC-MS.
- Other: The total amount of individual metabolites excreted in urine percentage of the dose administered orally to the rabbits

No additional data
Statistics:
None stated
Type:
metabolism
Results:
The test substance was metabolism, excreted in urine, as hippuric acid (HA), free (FBA) and conjugated benzoic acid (benzoylglucuronic acid: BGA), benzyl glucuronide (BG) and benzyl mercapturic acid (BMA)
Metabolites identified:
yes
Details on metabolites:
Urinary Excretion of the test substance metabolites:
a. Acidic metabolites
For comparison purposes, pooled urines of control rabbits were collected every day and similarly treated. The GC results showed the presence of free benzoic acid (FBA), hippuric acid (HA) and benzylmercapturic acid (BMA) in the urines of rabbits administered the test substance. The small amounts of FBA and HA found in control rabbits did not exceed the baseline values previously established. The retention times were quite specific for the following acidic metabolites: 10.75 minutes for benzoic acid, 19.03 minutes for hippuric acid and 24.54 minutes for benzylmercapturic acid.
Benzylmercapturic acid was present in trace amounts (<0.01%) in the urine of treated rabbits. It was detected during the first three days in the urine from the high-dose group and only in the first-day urine from the low-dose group. It was not detected in control rabbits.
Taking into account the baseline values, the average FBA excreted expressed as a percentage of the oral dose received was 1.6% in the low-dose group vs. 1.4% in the high-dose group. Similarly, the average HA excreted was higher in the low-dose group (69.9%) than in the high-dose group (66.7%).
b. Glucuronides
The average urinary excretion of BGA in the high-dose group (11.2%) was significantly higher than in the low-dose group (8.8%). In addition, the average BG excreted in the high-dose group (3.0%) was higher than in the low-dose group (2.9%). None of these metabolites was found in control rabbits.
c. Benzyl sulfate ester
This metabolite was not detected in treated or control rabbits.
d. Free benzyl alcohol
No free benzyl alcohol could be found in the urine of treated or control rabbits.

[1] Bray, H.G., Thorpe, W.T. and White K., Kinetic studies of the metabolism of foreign organic compounds. I. The formation of benzoic acid from benzamide, toluene, benzyl alcohol and benzaldehyde and its conjugation with glycine and glucuronic acid in the rabbit. Biochem J., 48, 88 -96, 1951

[2] Bray, H.G. and Thorpe, W.T., Analysis of phenolic ompounds of interest in metabolism. Methods of Biochemical Analysis, 1. 27 -52, 1958.

Conclusions:
Interpretation of results (migrated information): bioaccumulation potential cannot be judged based on study results

This study investigated the principal metabolic pathway of benzaldehyde and found a single oral dose of the test substance when administered to rabbits, is oxidised mainly to benzoic acid and excreted predominantly as hippuric acid (~68%). Other metabolites which were detected included benzoyl glucuronic acid (10%), benzyl glucuronide (3%), free benzoic acid (1.5%) and trace amounts of benzylmercapturic acid (<0.01%).

Daily monitoring indicated the urinary excretion of the following metabolites: hippuric acid (HA), free (FBA) and conjugated benzoic acid (benzoylglucuronic acid: BGA), benzyl glucuronide (BG) and benzyl mercapturic acid (BMA). Although the total amount of urinary metabolites excreted by the low-dose group (83.2% of the total dose) was not significantly different from that excreted by the high-dose group (82.3% of the total dose), some metabolites showed differences in their urinary output: HA, 69.9% in the low-dose group vs. 66.7% in the high-dose group; BGA, 8.8% in the low-dose group vs. 11.2% in the high-dose group; BG, 2.9% in the low-dose group vs. 3.0% in the high-dose group; FBA, 1.6% in the low-dose group vs. 1.4% in the high-dose group. In both treated groups, BMA was present in trace amounts (<0.01% of the total dose).
Executive summary:

This study investigated the principal metabolic pathway of benzaldehyde.The test substance administered two groups of male New Zealand White rabbits by gavage, whereas water was given orally to the third group. Urine of all groups was collected daily for 15 consecutive days. The quantitative metabolism of the test substance was measurement.

Daily monitoring indicated the urinary excretion of the following metabolites: hippuric acid (HA), free (FBA) and conjugated benzoic acid (benzoylglucuronic acid: BGA), benzyl glucuronide (BG) and benzyl mercapturic acid (BMA). Although the total amount of urinary metabolites excreted by the low-dose group (83.2% of the total dose) was not significantly different from that excreted by the high-dose group (82.3% of the total dose), some metabolites showed differences in their urinary output: HA, 69.9% in the low-dose group vs. 66.7% in the high-dose group; BGA, 8.8% in the low-dose group vs. 11.2% in the high-dose group; BG, 2.9% in the low-dose group vs. 3.0% in the high-dose group; FBA, 1.6% in the low-dose group vs. 1.4% in the high-dose group. In both treated groups, BMA was present in trace amounts (<0.01% of the total dose).

It can be concluded that when a single oral dose of benzaldehyde is administered to rabbits under the test conditions of the study, the test substance is oxidised mainly to benzoic acid and excreted predominantly as hippuric acid (~68%). Other metabolites which were detected included benzoyl glucuronic acid (10%), benzyl glucuronide (3%), free benzoic acid (1.5%) and trace amounts of benzylmercapturic acid (<0.01%).

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1970
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: limited report, non-GLP
Objective of study:
excretion
Qualifier:
no guideline followed
Principles of method if other than guideline:
The test substance dissolved in physiological saline were administered by intraperitoneal route to female albino rats. The 24 h-urine following injection was collected, and the amount of hippuric acid present in the rat urines was determined.
GLP compliance:
no
Species:
rat
Strain:
other: albino rats
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Diet (e.g. ad libitum): food pellets
- Water (e.g. ad libitum): tap water

No additional data

Route of administration:
intraperitoneal
Vehicle:
physiological saline
Details on exposure:
The test substance was administered in suspension by addition of physiological salinc, with a few drops of Tween 80 added.
Duration and frequency of treatment / exposure:
24 hour
Dose / conc.:
0.41 other: mmole
No. of animals per sex per dose / concentration:
4
Control animals:
yes
Positive control reference chemical:
None stated
Details on study design:
None stated
Details on dosing and sampling:
PHARMACOKINETIC STUDY (excretion)
- Tissues and body fluids sampled (delete / add / specify): urine
- Time and frequency of sampling: The 24 h-urine following injection was collected over 2 mL 2 N hydrochloric acid, in order to prevent oxidation of sensitive substances.
- Other: At the end of the experiment, the cage bottoms and collecting funnels were washed with a few ml of distilled water. The washings were combined with the urine.
Statistics:
None stated
Type:
excretion
Results:
The conversion of the test substance into hippuric acid takes place in the rat.
Details on excretion:
It appears from our experiments that the test substance is converted to about 29.3% of the dose to hippuric acid

Hippuric acid excretion of rats, after administration of possible aromatic and hydroaromatic precursors.

Administered

compound

Dose in

mmole

Number

Of experiments

µg hipp. ac.

per 10 µL

concentrated

extr. (mean

of 6 analyses)

S. D.

Urine

vol.

in mL

Total hipp. ac. Cont. of urine sample in mg

Surplus hipp. ac. excrn. In mg

% of inj. Compound excreted as hipp. acid

Mean

The test substance

0.41

4

70.9

1.2

9.2

32.6

27.3

37.1

29.3

37.7

1.0

13.0

24.5

19.2

26.1

95.4

1.2

6.2

29.6

24.3

32.9

76.0

1.9

5.5

20.9

15.6

21.2

Conclusions:
Interpretation of results (migrated information): bioaccumulation potential cannot be judged based on study results

It can be concluded that the test substance is converted to about 29.3% of the dose to hippuric acid in rats.
Executive summary:

This study was a first-time investigation into the quantitative aspect of the possible hippuric acid precursor capacity in the laboratory rat. The test substance dissolved in physiological saline were administered by intraperitoneal route to female albino rats. 24 -hours following injection, urine was collected, and the amount of hippuric acid present in the rat urines was determined.

It can be concluded that the test substance is converted to about 29.3% of the dose to hippuric acid in rats. The addition of glycine to benzoic effect did not affect the hippuric acid output in the rat under the experimental conditions, which indicates that glycine was not rate-limiting during the hippuric acid formation.

Description of key information

A study conducted by Bray H.G.et al., 1951, showed that in the rabbit, benzaldehyde is rapidly oxidised to benzoic acid, which is excreted as hippuric acid and appears to be the only major metabolite present in urine. There was no significant excretion of benzoyl glucoronide observed. However, there is a lack of numerical or graphical data to fully demonstrate rates of biotransformation.

Teuchy, H.et al., 1971 further reported that 29.3% of the benzaldehyde administered by intraperitoneal injection to rats was excreted in urine as to hippuric acid.

The Kutzman, R.S. et al., 1980 study was an investigation into the biodistribution and excretion of 11C-benzaldehyde in the rat after two-minute inhalation exposures. The kinetics and biodistribution were studied based on the decay characteristics of short-lived gamma-emitting radioisotopes. The biological half-life of benzaldehyde in the blood was 8.1 minutes and eIimination via the renal system from most organs was rapid and linear over time. There were no 11CO2 metabolites measured in the expired air and there were very low levels of radiolabel measured in the intestinal content. It was observed that 11C-benzaldegyde was almost exclusively eliminated by the kidneys in the form of hippuric acid (>90%). It can be concluded that benzaldehyde is almost completely absorbed following inhalation expoSure, without indications of a potential for accumulation in the body.

The Laham S., et al. 1987 study, investigated the biological conversion of benzaldehyde to benzylmercapturic acid in the Sprague-Dawley rat. Benzaldehyde is reduced to a minor extent to form benzyl alcohol, that may then react with glutathione in the presence of glutathione S-transferase, for which sulphate esters are substrates to finally form benzylmercapturic acid. The second metabolic pathway seems to occur only after the primary elimination pathway has been saturated.

The Laham S., et al., 1988, was a study looking into the metabolism of benzaldehyde in New Zealand white rabbits. This study investigated the prinicipal metabolic pathway of benzaldehyde and found a single oral dose of the test substance when administered to rabbits, is oxidised mainly to benzoic acid and excreted predominantly as hippuric acid (~68%). Other metabolites which were detected included benzoyl glucuronic acid (10%), benzyl glucuronide (3%), free benzoic acid (1.5%) and trace amounts of benzylmercapturic acid (<0.01%). 

 

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential

Additional information

Altogether, available data suggests that the principal pathway of metabolism of benzaldehyde includes oxidation to yield benzoic acid, which is subsequently conjugated with glycine and excreted as hippuric acid.In parallel, benzaldehyde is reduced, to a minor extent, to benzyl alcohol, which as the sulfate conjugate may react with glutathione to form benzylmercaturic acid.

After re-evaluation of the available data for the ADME of benzaldehyde, the Consortium agrees with ECHA’s comment (CCH-D-2114378524 -42 -01 -F) that the published data are somewhat limited. Although all of these published studies are considered as good quality Klimisch category 2 peer reviewed reports, none of them are GLP or conducted according to OECD test guideline 417. For some of them, we recognize that there is a lack of numerical or graphical data to fully demonstrate rates of biotransformation (Bray et al., 1951). In some of the studies, no control group was included (Kutzman et al., 1980).

However, the Consortium considers that these data provide useful supportive evidence to indicate that the half-life of benzaldehyde in the body is short, and that the principal pathway of metabolism of benzaldehyde includes oxidation to yield benzoic acid. Since the Consortium cannot provide a Klimisch category 1 robust study summary, we propose a tiered-approach to ECHA to first clarify the ADME of benzaldehyde by conducting a GLP toxicokinetic study in accordance with OECD guideline 417.

The Consortium considers that the results of this definitive GLP ADME study with benzaldehyde will provide robust support for the previously-published ADME studies and will therefore demonstrate that the read across hypothesis is valid.