Benzoic acid

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics

Type of information:

other: assessment based on available data

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP assessment report

Principles of method if other than guideline:

Method not determined: reference not located

For the test substance, information on absorption, distribution, metabolism and excretion is available on experimental animals and on human. An overview of the information is given below.

After oral ingestion of the test substance, there is a rapid absorption from the GItract in experimental animals and humans. In humans peak plasma concentration is reached within 1-2 hr. Chemical carboxylic acid compounds can be metabolized via various pathways, with glycineconjugation being a very common route. For the test substance, glycine conjugation is the mainroute of metabolism in many animals species including human. As a result of this route of metabolism, hippuric acid (benzoyl-glycine) is formed and excreted in urine.

The urinary excretion of orally administered 14C-test substance in man and 20 species of animals was examined by Bridges and coworkers, with hippuric acid (benzoyl-glycine) generally being the major urinary metabolite in the species studied. At an oral dose of 50 mg/kg bw, the test substance was excreted by rodents almost entirely as hippuric acid (95-100% of 24hr excretion)b. After ip administration of 14C-test substance (100 μmol/100 g bw or 200 mg/kg bw), 75- 90% of the 14C excreted in 24 hr, of which 99% hippuric acid and trace of benzoyl glucuronide. An oral dose of 2 mg/kg bw 14C-test substance resulted in only 0.04% of the applied dose in urinary benzoylcarnitine. In man at a dose of 1 mg/kg the test substance was excreted entirely as hippuric acid.

The test substance metabolism, as a function of liver mitochondria, depends on the concentrations of ATP, CoA and glycine in the mitochondrial matrix. Although drug metabolism is primarily a liver function, also kidneys have shown to contain appreciable amounts of metabolizing enzymes. The metabolism of the test substance to hippuric acid and glucuronide has been studied in viable hepatocytes and renal tubule fragments from rat, hamster, ferret and dog. Hippuric acid formation was observed in hepatocytes and renal tubules from rat and hamster; a small amount of glucuronidation occurred in hepatocytes of all species tested. However, the liver is quantitatively the most important organ because the renal medulla has only low synthetic activity and the mass of the liver exceeds that of the kidneys.

Based on the observations on excretion of hippuric acid after oral the test substance administration, it can be concluded that the test substance will be absorbed almost completely in the gastro-intestinal tract. For risk assessment purposes the oral absorption of the test substance is set at 100% absorption. Experiments on the distribution and elimination in the rat have shown no accumulation of the test substance in the body.

There is no information available on absorption via inhalation. The relatively low vapourpressure/high boiling point indicate that the test substance will not be available for inhalation as vapour. The particle size distribution of the test substance (1.5% will be present as <425 μm) indicates that there are no inhalable/respirable particles present. Based on these properties, no repiratory exposure and hence no inhalation absorption is to be expected. If however during use of the substance particles between 100 and 10 μm are generated, these will deposit in the nasopharyngeal region and subsequently be coughed or sneezed out of the body or swallowed. Particles below 10 μm might reach the tracheobronchial or pulmonary regions. Based on its water solubility (2.93 g/L) the test substance will dissolve in the mucus lining of the respiratory tract, and can be absorbed throuigh aqueous pores (MW< 200). Following its modest lipophilic character (log Pow =1.88) the test substance has the potential to be absorbed directly across the respiratory tract epithelium. From a worst case scenario basis it is therefore concluded that when the test substance is inhaled (particles< 10 μm only), absorption of the test substance is to be expected and the inhalation absorption of the test substance for risk assessment purposes is therefore set at 100%.

In vitro skin absorption study with hairless guinea pig showed that the metabolism of the test substance during skin absorption is similar qualitatively to that reported for systemic administration. However, only a small percentage of the test substance was converted to hippuric acid in the skin, in contrast to almost complete conjugation after a systemic dose to humans and many animal species. This was also reported by Naseri-Sina and co-workers, who studied in vitro cutaneous metabolism of [carboxyl-14C] the test substance studies in rat and human, and compared this with metabolism in rat hepatocytes. Tissue-specific differences in metabolism was observed, with conjugation in hepatocytes significantly greater (>98% of radioactivity recovered as glycine conjugate) than in keratinocytes (10.9% and 2.1% glycine conjugate in rat and human skin cells, respectively, during 8 hr incubation). These results indicate that rat and human skin possesses low glycine conjugating activity. Human skin is generally recognized as less permeable to chemicals than is animal skin; human skin may also be less capable of biotransforming certain topically applied chemicals.

In vivo data also showed that the test substance is not completely absorbed by the dermal route. In human subjects, 36% dermal absorption in low dose (4 μg/cm2) was observed within 12 hours with a total uptake of 43% within 5 days. Dermal exposure to a high dose (2000 μg/cm2) resulted in 14% dermal absorption within 24h. In vivo dermal studies with experimental animals confirm the results with human. Absorption ranged from 25% in pigs to 89% in rhesus monkeys. For risk assessment purposes, the dermal absorption of the test substance is set at 43%.

Conclusions:

Interpretation of results (migrated information): no bioaccumulation potential based on study resultsOverall there are signs of systemic absorption via oral and dermal exposures, no evidence of target organs or of excretion. After oral ingestion and dermal absorption, the test substance will be metabolised to hippuric acid. Experiments on the distribution and elimination in the rat have shown no accumulation of the test substance in the body.

Executive summary:

With reviewing the existing data, for the test substance, information on absorption, distribution, metabolism and excretion is available on experimental animals and on human. A toxicokinetic assessment has been performed based on physical chemical properties of the substance and available open literature.

Overall there are signs of systemic absorption via oral and dermal exposures, no evidence of target organs or of excretion. After oral ingestion and dermal absorption, the test substance will be metabolised to hippuric acid. Experiments on the distribution and elimination in the rat have shown no accumulation of the test substance in the body.

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: limited report, test in 3 animals only, non GLP

Objective of study:

toxicokinetics

Qualifier:

no guideline followed

Principles of method if other than guideline:

The experiments can be divided into two groups: the formation and conjugation of the test substance formed from administered toluene, benzyl alcohol, benzaldehyde and benzamide, and the conjugation of the test substance administered as such. Only the second experiment is taken into account in this summary

GLP compliance:

no

Species:

rabbit

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Weight at study initiation: not more than 2 kg- Individual metabolism cages: yes- Diet (e.g. ad libitum): maintained on the constant diet of rabbit pellets in the laboratory- Water (e.g. ad libitum): maintained on the constant water customary in the laboratory No additional data available

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Doses / Concentrations:1) 0.84-1.25 g2) 2.5-3.0 g in presence of additional glycine dose

No. of animals per sex per dose / concentration:

1) 3 animals2) 2 animals

Control animals:

other: pre-treatment values were used as control values

Positive control reference chemical:

None stated

Details on dosing and sampling:

Collection of urine samples: 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. The rabbit was housed in a metabolism cage fitted into a tinned-copper collecting funnel of the usual type, the spout of which was connected to a glass tube delivering into the receivers beneath.Estimation of metabolites: 10 samples were collected over a 7 hour periodEther-soluble acid was determined, fractionation being achieved by extraction of ether-soluble material with light petroleum using three successive volumes of 25 mL. The method was adapted for use on blood samples (2 mL). These were collected from a marginal ear vein directly into H2SO4 (15 mL) and subsequently made up to 25 mL with acid of the same strength. A sample of this (24 mL) was transferred to an extractor and the protein precipitated by means of sodium tungstate (3 mL, 10% w/v). The resulting mixture was allowed to stand for 5 min, further acidified by the addition of 2N-H2SO4 (5 mL) and continuously extracted with ether for 6 hr. The ethersoluble acid was titrated with NaOH. Fractionation was carried out as for urinary acid. Control experiments showed that the test substance and hippuric acid added to normal rabbit blood (100-500 mg/100 mL blood) could be recovered by this method to extents of 101 and 105%, respectively. Ester glucuronide in urine was estimated by the reducing method.

Metabolites identified:

yes

Details on metabolites:

benzoic acid (parent), hippuric acid and glucuronide conjugate

Straight-line excretion curves were sometimes obtained for hippuric acid after the administration of precursors as well as for the test substance itself. This happens when the initial rate of conversion of the precursor is greater than the maximum rate of glycine conjugation. Where these rates are approximately equal, small variations in the rate contant for formation of hippuric acid (kh) may be critical and either exponential curves or initially straight lines may be obtained. When the initial rate of conversion of the precursor exceeds the maximum rate of glycine conjugation the test substance would be expected to accumulate in the blood. In six experiments where sodium benzoate was administered it was found that in fourteen determinations an average of 12% (range 6-24%) of the test substance (the acid) in the body was present in the blood.

Hippuric acid formation takes place at a constant rate and the acid is excreted immediately up to a limiting rate which appears to vary considerably between different rabbits. In two experiments, in which hippuric acid was found in the blood after feeding the test substance and glycine, values of kh of 270 and 480 mg/h were found, assuming that hippuric acid was present only in blood and urine. For the three rabbits used in experiments, in which the test substance alone was given, the average rate of formation and excretion of hippuric acid was 147 mg/h. This suggest that the availability of glycine controls the rate of hippuric acid formation. The conjugation of the test substance with glucuronic acid follows the kinetics of a first-order reaction with a velocity constant of 0.08 hr-1.

Conclusions:

Interpretation of results (migrated information): no bioaccumulation potential based on study results rapid metabolism to hippuric acid and glucuronic acidThis study showed that the test substance is metabolised to hippuric acid with glycine as rate limiting factor. The authors conclude that the percentage excreted as glucuronic acid conjugate decreases when glycine is administered with the dose.

Executive summary:

Rabbits received the test substance as sodiumbenzoate (0.84 -3.0 g by gavage) and blood and urine samples were collected upto 7 hours after administration. Hippuric acid was the primary metabolite with glycine availability being the rate limiting factor. After depletion of glycine conjugation with glucuronic acid was reported.

Reference 3

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non GLP

Objective of study:

metabolism

Qualifier:

no guideline followed

Principles of method if other than guideline:

This study has investigated the metabolism of [carboxy-14C]the test substance in the Sprague-Dawley rat at concentrations ranging from 10 μg/kg to 1 g/kg.

GLP compliance:

not specified

Radiolabelling:

yes

Remarks:

14C

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS- 2-3 rats per sex per dose - Housing: housed for 24 h in all-glass metabolic cages- Diet (e.g. ad libitum): free access to pellet food- Water (e.g. ad libitum): free access to waterHousing together thus pooled urinary samples were investigated.No additional data

Route of administration:

oral: unspecified

Vehicle:

water

Duration and frequency of treatment / exposure:

24 h

Remarks:

Doses / Concentrations:10 and 100 μg/kg, 1, 10 and 100 mg/kg and 1 g/kg

Control animals:

not specified

Positive control reference chemical:

None stated

Details on study design:

None stated

Details on dosing and sampling:

The urine collected in a glass vessel cooled in ice.Samples of each urine collection (approx. 100μL) were subjected to t.l.c. in systems 2, 3, 4 and 5 followed by radiochromatogram scanning.

Statistics:

None stated

Details on metabolites:

The test substance is metabolized to hippuric acid. There was no difference in the extent of metabolism of the test substance over this range of concentrations; within 24 h of dosing, the rat excreted 80-100% of the administered dose in the urine solely as hippuric acid. At a concentration of 10 μg/kg, approx. 3% of the dose was expired as 14CO2 within 24 h.

Conclusions:

Interpretation of results (migrated information): no bioaccumulation potential based on study resultsThere was no difference in the extent of metabolism of the test substance over this range of concentrations; within 24 h of dosing, the rat excreted 80-100% of the administered dose in the urine solely as hippuric acid.

Executive summary:

This study has investigated the metabolism of [carboxy-14C]the test substance in the Sprague-Dawley rat at concentrations ranging from 10 μg/kg to 1 g/kg. There was no difference in the extent of metabolism of the test substance over this range of concentrations; within 24 h of dosing, the rat excreted 80- 100% of the administered dose in the urine solely as hippuric acid.

Reference 4

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: study intended to assess the influence of low protein diet on metabolism of the test substance, non-GLP

Objective of study:

metabolism

Qualifier:

no guideline followed

Principles of method if other than guideline:

Male Wistar rats were fed on either a normal diet (protein content 21%) or marasmickwashiorkor diet (protein content 3.5% )for 5 weeks before receiving a single dose of the 14C-test substance administered i.p. at 200 mg/kg. Urinary excretion patterns were measured over a 24 hour period post-dosing.

GLP compliance:

not specified

Radiolabelling:

yes

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: the University of Ibadan animal house- Age at study initiation: ca. 3 weeks old- Weight at study initiation: between 29 g and 34 g- Diet (e.g. ad libitum): Group 1 (controls) was fed ad lib, on a stock diet purchased from Livestock Feed Ltd.. The diet contained 21.0% crude protein, 71.5% carbohydrate, 4.0% fibre and 3.5% oil. The rats in Group 2 were fed ad lib on a kwashiorkoregenic diet: 3.45% protein (casein), 81.53% carbohydrate (Garri), 8.0% corn oil, 3.0% all-vitamin supplement and 4.0% salt mixture.No additional data

Route of administration:

intraperitoneal

Vehicle:

not specified

Details on exposure:

None stated

Duration and frequency of treatment / exposure:

After 5 weeks the rats were injected with radioactive test substance. The rats on low protein diet were transferred to standard diet 2 days after the injection. During the recovery phase these rats were injected again with the test substance at weekly intervals.

Remarks:

Doses / Concentrations:200 mg/kg body weight

No. of animals per sex per dose / concentration:

8 male rats

Control animals:

yes, plain diet

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 (delete / add / specify): urine- Time and frequency of sampling: the 24-h urine collected into beakers- Method type(s) for identification (e.g. GC-FID, GC-MS, HPLC-DAD, HPLC-MS-MS, HPLC-UV, Liquid scintillation counting, NMR, TLC) Liquid scintillation counting; chromatographed as a band on Whatman No. 1 paper strips (5 cm wide)No additional data

Statistics:

None stated

Preliminary studies:

No information provided

Type:

excretion

Results:

Normal rats: excreted the test substance mainly as hippuric acid (99% of 24 h excretion); Marasmic-kwashiorkor rats: excreted 62-85% as hippuric acid and 14-37% as the glucuronide conjugate.

Metabolites of [14C] the test substance in urine of normal and marasmic-kwashiorkor rats

Type	Dose		14C excreted in 24 h urine (% of dose)	% of 24 h excretion found
	The test substance (mg/kg)	14C (μCi/animal)		The test substance	Hippuric acid	Benzoyl glucuronide
Normal fed rats (Wistar albino)	200	4	82 (75-90)	0	99	trace
Marasmic-kwashiorkor fed rats (Wistar albino)	200	4	81 (70-93)	0	74 (62-85)	25 (14-37)

Within 2 weeks of feeding Group 2 rats on the stock diet during the recovery period, the metabolism of the test substance had returned to normal and most of the benzoate was excreted as hippuric acid. The refeeding experiment showed that the increase in the glucuronide conjugate formation in protein-deficient rats was a transient phenomenon. These results strongly suggested that the alteration in the conjugation pattern of the test substance in marasmic-kwashiorkor fed rats, was mainly due to an effect of protein deficiency on reactions involved in the conjugation systems.

The reduced synthesis of hippuric acid in the marasmic-kwashiorkor rats might have been due to a variety of factors including a reduction in the amount of glycine available for conjugation. The latter might have been directly related to the reduced protein intake or to increased utilization of this amino acid for gluconeogenesis.

A reduction in the formation of the glycine conjugate might be compensated for by an increase in the glucuronide conjugate formation.

Conclusions:

Interpretation of results (migrated information): no bioaccumulation potential based on study resultsRats fed a normal diet with the test substance administered i.p. at 200 mg/kg, excreted the the test substance mainly as hippuric acid (99% of 24 h excretion), while marasmic-kwashiorkor fed rats excreted 62-85% as hippuric acid and 14-37% as the glucuronide conjugate. In the recovery period (after 2 weeks back on normal diet) most of the benzoate was excreted as hippuric acid.Protein depletion and concomitant glycine shortage influences the metabolism of the test substance.

Executive summary:

Male Wistar rats were fed on either a normal diet (protein content 21%) or marasmickwashiorkor diet (protein content 3.5% )for 5 weeks before receiving a single dose of the 14C-test substance administered i.p. at 200 mg/kg. Urinary excretion patterns were measured over a 24 hour period post-dosing.

Rats fed a normal diet excreted the the test substance mainly as hippuric acid (99% of 24 h excretion), while marasmic-kwashiorkor fed rats excreted 62-85% as hippuric acid and 14-37% as the glucuronide conjugate. In the recovery period (after 2 weeks back on normal diet) most of the benzoate was excreted as hippuric acid.

Reference 5

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: non-GLP

Objective of study:

excretion

Qualifier:

no guideline followed

Principles of method if other than guideline:

A healthy male subject received a single oral dose of [13C] the test substance (10 mg) with 200 mL of water after an overnight fast. Urine samples were collected immediately before administration, and then 0-0.5, 0.5-1, 1-2, 2-4, 4-6 h postdose. The stable isotope tracer technique using NMR spectroscopy and the selective 13C labeling of protonated carbons was used in this study.

GLP compliance:

not specified

Radiolabelling:

yes

Species:

human

Strain:

not specified

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Weight at study initiation: 64 kg weight- Fasting period before study: an overnightNo additional data

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

VEHICLE- Concentration in vehicle: 50 mg/LNo additional data

Duration and frequency of treatment / exposure:

6h; A healthy male subject received a single oral doseof [13C] the test substance with 200mL of water after an overnight fast.

Remarks:

Doses / Concentrations:10 mg

No. of animals per sex per dose / concentration:

1 male subject

Control animals:

other: not applicable

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: Urine samples were collected immediately before administration, and then 0-0.5, 0.5-1, 1-2, 2-4, 4-6 h postdose. - Other: The volume of urine collected for each period was in the range of 100-200 mL. The urine samples were stored at -20 ℃ until analyzed.No additional data

Statistics:

None stated

Preliminary studies:

No information provided

Type:

excretion

Results:

The urinary excreted [1,3,5-13C] hippuric acid ([13C] HA) was successfully quantitated without any separation procedure by proton-decoupled 13C-NMR spectroscopy of 10-fold concentrated urine in a 10 min accumulation time.

Details on absorption:

No information provided

Details on distribution in tissues:

No information provided

Details on excretion:

[13C] the test substance was used as a precursor in the present research. [13C] the test substance (10 mg) was administered to a human subject and excreted urine was analyzed by 13C{1H}-NMR after concentration by a factor of 10. There was no appreciable resonance due to endogenous the test substance or HA in the spectrum of control urine under the NMR conditions used, although endogenous resonances probably due to urea, creatinine, and citric acid were observed. In the 13C{1H}-NMR spectra of urine following ingestion of the test substance, additional resonances at δ13C 131.5 and 136.1 were clearly observed, and they were due to C3,5 and Cl of [13C] HA formed from administered [13C] the test substance. The C3,5 resonances of the test substance and HA in the concentrated urine were confirmed to be well separated with the line broadening of 2 Hz. The resonance intensities rapidly decreased with time after dosing. In spite of the low dosage, the spectra showed favorable signal-to-noise (S/N) ratios using only a 10-min accumulation time, which was a significant contrast to the resonances of the quaternary carbon (C1) in the spectra.The urinary excretion of [13C] HA over 4 h amounted to virtually 100% of the injected dose. It was found that the [13C] HA excretion could be almost completely followed under the present experimental conditions. The time course was similar to that of the previous experiment using 100 mg of [7-13C] the test substance as a precursor. These experiments have demonstrated the feasibility of detecting the biotransformation of the test substance to HA in human subject by the selective 13C-labeling and 13C-NMR approach even in such a small dose.

Metabolites identified:

not specified

Details on metabolites:

No information provided

Amount of [13C] HA in 10 mL Urine (A) and Total Volume of Urine (B) Excreted in Each Period

Time (h)	[13C] HA amounts (mg)
	A*	B
0-0.5	0.327	2.776
0.5-1	0.436	8.068
1-2	0.229	3.158
2-4	0.117	1.432#

* 10 mL of urine collected for each period was free-dried and reconstituted in 1 mL of H2O. Approximately 0.4 mL of the supernatant was transferred to the NMR tube followed by 13C-NMR analysis

# The values were not corrected for the contribution by endogenous HA.

Conclusions:

Interpretation of results (migrated information): no bioaccumulation potential based on study resultsThe urinary excreted [1,3,5-13C] hippuric acid ([13C] HA) formed from orally administered [1,3,5-13C] the test substance as a model substrate was successfully quantitated without any separation procedure by proton-decoupled 13C-NMR spectroscopy of 10-fold concentrated urine in a 10 min accumulation time. In spite of the low dosage (10 mg the test substance), the C3,5 resonances of [13C] HA were detected with favorable single-to-noise ratios to quantitated [13C] HA concentration. The administration [13C] the test substance was found to be quantitatively biotransformed to HA and excreted in urine within 4 h. the lower limit of detection was estimated to be 50 nmol in an NMR tube, which was improved about one order of magnitude over that of the test substance labed in the quaternary carbon (C7). The potential of an inverse detection experiment using heteronuclear multiple quantum coherence was also investigated in order to detect [13C] HA in urine with a higher sensitivity. The inverse experiment improved the sensitivity by a factor of 2-3 over 13C{1H}-NMR, although the specificity of detection was relatively poor.

Executive summary:

A healthy male subject received a single oral dose of [13C] the test substance (10 mg) with 200 mL of water after an overnight fast. Urine samples were collected immediately before administration, and then 0-0.5, 0.5-1, 1-2, 2-4, 4-6 h postdose. The stable isotope tracer technique using NMR spectroscopy and the selective 13C labeling of protonated carbons was used in this study.

 

The urinary excreted [1,3,5-13C] hippuric acid ([13C] HA) formed from orally administered [1,3,5-13C] the test substance as a model substrate was successfully quantitated without any separation procedure by proton-decoupled 13C-NMR spectroscopy of 10-fold concentrated urine in a 10 min accumulation time. In spite of the low dosage (10 mg the test substance), the C3,5 resonances of [13C] HA were detected with favorable single-to-noise ratios to quantitated [13C] HA concentration. The administration [13C] the test substance was found to be quantitatively biotransformed to HA and excreted in urine within 4 h. the lower limit of detection was estimated to be 50 nmol in an NMR tube, which was improved about one order of magnitude over that of the test substance labed in the quaternary carbon (C7). The potential of an inverse detection experiment using heteronuclear multiple quantum coherence was also investigated in order to detect [13C] HA in urine with a higher sensitivity. The inverse experiment improved the sensitivity by a factor of 2-3 over 13C{1H}-NMR, although the specificity of detection was relatively poor.

Reference 6

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: non GLP

Objective of study:

other: pharmacokinetic

Qualifier:

no guideline followed

Principles of method if other than guideline:

The test substance was administrated into rats, and the excreted urine was analyzed with NMR spectroscopy in order to determine the accumulation time required for the experiments. The use of 13C-labeling and nuclear magnetic resonance spectroscopy to trace the biotransformation of the test substance to hippuric acid in the rat.

GLP compliance:

not specified

Radiolabelling:

yes

Remarks:

13C

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Weight at study initiation: 410-460 gNo additional data available

Route of administration:

intravenous

Vehicle:

physiological saline

Details on exposure:

The right jugular vein was cannulated with 3 Fr. polythylene catheter. Both ureters were cannulated with polyethylene tubing for urine collection. The test substance was dissolved in 0.5 mL saline containing sodium hydroxide equimolar with the test substance and injected into the jugular vein through the catheter at doses ranging from 2.0 to 2.2 mg/kg.

Remarks:

Doses / Concentrations:2.0 to 2.2 mg/kg

Control animals:

yes

Positive control reference chemical:

None stated

Details on dosing and sampling:

After injection, urine excreted through ureters was collected in 5-min periods for 1h and then in the following 1h while infusing 5% mannitol at a rate of 3 mL/h in order to stimulate urine excretion. The volume of urine sampled for each 5-min period was approximately 100 μL. Urine was stored at -20℃ until analyzed. Whole urine collected in each 5-min period and aliquots of 1-2 h postdose urine were subjected to NMR analysis.

Statistics:

None stated

Only resonances at 129.9, 134.9 and 173.2 ppm were observed in the aromatic region of the spectra, and the three resonances were due to 13C-hippuric acid formed from administered 13C-the test substance. The resonance intensities rapidly decreased with the time after dosing as shown later. It was found that the excretion of hippuric acid could be almost completely followed in only 10 min. No significant signals of endogenous compounds including the test substance and hippuric acid appeared on the spectra of control urine in 10 min .

Conclusions:

Interpretation of results (migrated information): no bioaccumulation potential based on study resultsThe metabolic rate of the test substance to Hippuric acid in the rat is 85 to 99%.

Executive summary:

The test substance was administrated into rats, and the excreted urine was analyzed with NMR spectroscopy in order to determine the accumulation time required for the experiments. The use of 13C-labeling and nuclear magnetic resonance spectroscopy to trace the biotransformation of the test substance to hippuric acid in the rat.

The metabolic rate of the test substance to Hippuric acid in the rat is 85 to 99%.

Description of key information

Metabolism
              
Next to a full toxicokinetic assesment (Pelgrom 2010), several studies investigating metabolism and excretion of the
test substances are available. The test substance is metabolised to hippuric acid with glycine depletion being the rate limiting factor (Michaelis-Menten saturation kinetcs). When this primary pathway is staturated, formation of glucuronide conjugates is obeserved. Total excretion is reached within ca 24-48 hours after administration. The same metabolic pathway is described in rabbits (Bray 1950, rats (Kazuki 1992, Jones 1992) and man (Shigeo 1994) when the testsubstance was administerd via the oral, iv or ip route. Glycine pre-treatment (Bray 1950) showed increases of the amounts of hippuric acid excreted in rabbits, while proteine (thus glycine) low diets in rats gave a decreased amount of hippuric acid in urine (Thabrew 1980). 
Oral absorption: From the outcome of the studies it can be concluded that the test substance is absorbed rapidly via the oral route. The oral absorption is set at 100%
Inhalation absorption: Pelgrom (2010) also indicates that rapid absorption via the inhalation route should be possible, but this is unlikely in view of the particle size of the test substance that does not allow the test substance to penetrate into the deeper layers of the respiratory tract and the lungs. 
Dermal absorption: Based on the available in vivo results the dermal absorption of the test substance is set at 50% in a worst case approach. It has to be noted that a clear species difference becomes apparent. The mass balance was not properly maintained in most of the available studies, which renders the results less reliable. In vitro data support this percentage, but it should be noted that many of the in vivo studies had a relatively long exposure period (24 h standard according to the guideline for reasons of loss of viability) and thus may over estimate absorption rates.
In a weight of eveidence approach the 50% seems to be justified.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - dermal (%):

50

Additional information

Next to a full toxicokinetic assesment (Pelgrom 2010), several studies investigating metabolism and excretion of the test substances are available. The test substance is metabolised to hippuric acid with glycine depletion being the rate limiting factor (Michaelis-Menten saturation kinetics). When this primary pathway is staturated, formation of glucuronide conjugates is obeserved. Total excretion is reached within ca 24-48 hours after administration. The same metabolic pathway is described in rabbits (Bray 1950, rats (Kazuki 1992, Jones 1982) and man (Shigeo 1994) when the testsubstance was administerd via the oral, iv or ip route. Glycine pre-treatment (Bray 1950) showed increases of the amounts of hippuric acid excreted in rabbits, while proteine (thus glycine) low diets in rats gave a decreased amount of hippuric acid in urine (Thabrew 1980).

Based on the available in vivo results the dermal absorption of the test substance is set at 50% in a worst case approach. In vitro data support this percentage, but it should be noted that many of the in vitro studies had a relatively long exposure period (24 h standard according to the guideline for reasons of loss of viability) and thus may over estimate absorption rates.

From the outcome of the studies it can be concluded that the test substance is absorbed rapidly via the oral route. Pelgrom (2010) also indicates that rapid absorption via the inhalation route should be possible, but is unlikely in view of the particle size of the test substance.

In vitro and ex-vivo experiments (Mose 2007, Utoguchi 1999, Poulsen 2009) have shown that the test substance is easily transfered across the placenta or taken up by specific (BoWe) cell-lines. The suitability of the models used to assess placental transfer in vivo needs to be further investigated.