Registration Dossier

Toxicological information

Basic toxicokinetics

Currently viewing:

Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000

Materials and methods

Objective of study:
metabolism
Principles of method if other than guideline:
The study was conducted according to a protocol approved by the sponsor, entitled: Protocol for a study on the in vitro hydrolysis of L-lactate esters by rat nasal olfactory epithelium homogenate (protocol P 352143). The esters were incubated with rat nasal olfactory epithelium homogenate at pH 7.0 and 37°C. The amount of liberated L-lactic acid (buffered) was quantified at a series of time points, from which the initial rate of hydrolysis was estimated. Using a concentration range of 0.05-3.2 mM, the enzyme kinetic parameters Km and Vmax were calculated.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethyl (S)-2-hydroxypropionate
EC Number:
211-694-1
EC Name:
Ethyl (S)-2-hydroxypropionate
Cas Number:
687-47-8
Molecular formula:
C5H10O3
IUPAC Name:
ethyl (2S)-2-hydroxypropanoate
Details on test material:
Batch no.: EL 452 D
Radiolabelling:
no

Test animals

Species:
other: rat nasal olfactory epithelium homogenate
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animals and maintenance:
Male Wistar rats (Crl:(WI)WU.BR), about 10 weeks old on arrival, were obtained from Charles River Wiga GmbH, Sulzfeld, Germany. Upon arrival the rats were checked for overt signs of ill health and anomalies. After an acclimatization period of at least 6 days, ten healthy rats were used for the preparation of the homogenates. From their arrival until the day of sacrifice, the rats were housed in suspended, stainless steel cages, fitted with wire mesh floor and front. The rats were kept in a single room thermostatically maintained at a temperature of 22 ± 3°C and a relative humidity of at least 30%, and exposed to 12 hours fluorescent lighting and 12 hours dark. From their arrival until the day before sacrifice, the rats were fed the Institute's cereal based, powdered stock diet for rats, mice and hamsters. Tap-water was supplied in glass bottles and was available at all times.

Preparation of homogenate:
The rats were exsanguinated whilst under nembutal anaestesia. The head of each rat was removed from the carcass and deskinned, and the skull was split in half sagitally, exposing the nasal cavity, using a surgical scalpel. Both halfs of the skull were placed on ice. Subsequently, the olfactory and respiratory portions of the nasal septum were collected seperately from both skull halfs and kept on ice until further processing. The olfactory epithelium of all rats was pooled. Immediately after removal, tissues were frozen in liquid nitrogen and stored at -80°C. Homogenates of olfactory epithelium were prepared by homogenisation with 3 volumes of 0.01 M Tris.HCl/0.14 M KCl pH 7.0 with a Potter-Elvehjem tissue homogenizer. After centrifugation at 10,000xg, the supernatant was stored at -80°C. The protein concentration of the homogenate was measured using the Bradford method. Esterase acitivity of the homogenate was measured with p-nitro-phenylbutyrate according to the method described by Bogdanffy et al.: 100 µM p-nitro-phenylbutyrate and a suitable amount of nasal epithelium were incubated in 0.1 M phosphate buffer pH 7.8 at 25°C in a total volume of 1 ml. The rate of hydrolysis was measured spectrophotometrically at 400 nm. Enzymatic activity was expressed in µmol per min per mg protein using a molar extinction coefficient of 17,700 /per M per cm.

Administration / exposure

Route of administration:
other: incubation with rat nasal olfactory epithelium homogenate
Vehicle:
acetone
Details on exposure:
Ethyl-(S)-lactate was incubated with 51 µg of nasal epithelium protein and 0.05 M phosphate buffer pH 7.0 in a total volume of 1 ml at 37°C for 5, 10, 20, 40, 60 and 120 minutes. Ethyl-(S)-lactate was added to the incubation mixture in 20 µl acetone to give final concentrations of 50, 100, 200, 400 and 800 µM. Because esterase activity of the nasal epithelium homogenate towards ethyl lactate was rather low, ethyl-(S)-lactate was incubated with additional concentrations of 1.6 and 3.2 mM.
Duration and frequency of treatment / exposure:
Single application. Incubation for 5, 10, 20, 40, 60 and 120 minutes
Doses / concentrations
Remarks:
Doses / Concentrations:
50, 100, 200, 400, 800 , 1600 and 3200 µM
No. of animals per sex per dose / concentration:
51 µg of nasal epithelium protein per dose.
Control animals:
other: Chemical hydrolysis was measured in incubations without epithelium homogenate at all concentrations after 120 minutes. The amounts of L-lactic acid at the start of the incubation studies (t=0) (blanks) were determined by addition of 20 µl of acetone to t
Positive control reference chemical:
L-lactate
Details on dosing and sampling:
Incubations:
Ethyl-(S)-lactate was incubated with 51 µg of nasal epithelium protein and 0.05 M phosphate buffer pH 7.0 in a total volume of 1 ml at 37°C for 5, 10, 20, 40, 60 and 120 minutes. Ethyl-(S)-lactate was added to the incubation mixture in 20 µl acetone to give final concentrations of 50, 100, 200, 400 and 800 µM. Because esterase activity of the nasal epithelium homogenate towards ethyl lactate was rather low, ethyl-(S)-lactate was incubated with additional concentrations of 1.6 and 3.2 mM. The reaction was terminated by addition of 3 ml ethanol and cooling of the incubation mixture to -25°C for a minimum of 20 minutes. Subsequently, the tubes were centrifuged for 7 minutes at 2,500xg. After decantation into new tubes, the supernatant was evaporated to dryness with nitrogen and stored at -25°C until analysis.
Chemical hydrolysis was measured in incubations without epithelium homogenate at all concentrations after 120 minutes.
The amounts of L-lactic acid at the start of the incubation studies (t=0) (blanks) were determined by addition of 20 µl of acetone to the incubaton mixtures.
The metabolism of L-lactic acid (buffered) by nasal epithelium homogenate was investigated by incubating 217.6 nmol of L-lactate with 382.5 µg of nasal epithelium protein for 5 minutes at 37°C.

L-lactic acid assay:
The total amount of L-lactic acid formed was quantified with the "Boeringher test for the enzymatic determination of L-lactic acid in foodstuffs and other materials". The test principle is as follows:

LDH
L-lactate + NAD+ ↔ Pyruvate + NADH + H+

GPT
Pyruvate + L-Glutamate ↔ L-Alanin + α-Ketoglutarate

The amount of NADH produced was determined spectrophotometrically at 340 nm. The NADH production of samples was compared with NADH production by various amounts of the standard (L-lactate). Because of the high rate of chemical hydrolysis of the lactate esters at pH 10, a 0.224 M phosphate buffer pH 7.4 containing 0.02% sodium azide as preservative was used instead of teh glycylglycine buffer. The absorbance of the incubation mixture after addition of the L-lactate dehydrogenase solution was measured after 1 hour. The absorbances were measured using demiwater as reference.
Statistics:
Calculations:
The amount of L-lactic acid formed during the incubations was calulated from a standard curve obtained from the absorbance values of various amounts of L-lactate standard. The standard curve was fitted with the model:
absorbance = a + b.(amount L-lactic acid) + c.(amount L-lactic acid)2
The detection limit of the L-lactic acid assay was determined to be 4 nmol (absorbance 0.020-0.025).
The initial enzymatic rates of hydrolysis at the various concentrations of L-lactate esters were caluclated by fitting the amount of L-lactic acid formed at the various time points with the model:
liberated L-lactic acid = a + b.(time) + c.(time)2
and subsequent calculation of the slope of the fitted curve at the timepoint with the first detectable amount of L-lactic acid. When L-lactic acid was only detectable after 20 minutes or more, the curve was fitted included with the zero timepoint and the slope of the curve was calculated at t=0. The initial hydrolysis rates were corrected for chemical hydrolysis and expressed as nmol per min per mg protein. Chemical hydrolysis was assumed to be a linear chemical process.
Kinetic parameters (Km and Vmax) were calculated using the Michaelis Menten equation. The calculation program "EZ-FIT" was used for this purpose.

Results and discussion

Preliminary studies:
The protein content of the nasal epithelium homogenate was determined to be 15.3 mg per ml.

The increase of absorbance towards p-nitro-phenylbutyrate of the nasal epithelium homogenate using 15.3 µg of protein was 0.191 ± 0.006 (n=2), corresponding to an esterase acitivity of 0.71 ± 0.02 µmol per min per mg of epithelial protein.

The rate of chemical hydrolysis was investigated with ethyl-S-lactate in 0.05 M phosphate buffer pH 7.0 at 37°C. Ethyl-S-lactate was incubated in a concentration of 500 µM for 5 and 20 minutes, and 1, 3 and 19.5 hours. The rate of chemical hydrolysis was very low. Liberated L-lactic acid could not be detected. The additonal 2-hours incubations with concentrations of ethyl-S-lactate of 0.8, 1.6 and 3.2 mM showed detectable chemical hydrolysis at the highest concentration tested only. The rate of chemical hydrolysis, measured as the rate of lactic acid formation, was 0.04 nmol/min at a concentration of 3.2 mM ethyl-S-lactate.

Metabolism of L-lactic acid (buffered) was not observed when L-lactic acid was incubated with nasal epithelium homogenate. The use of inhibitors to prevent enzymatic oxidation to pyruvate was therefore not necessary.

The recovery of L-lactic acid, as added to the incubation mixture in the absence of esters, was 91-93%.

Using a set of enzymatic incubations of ethyl-S-lactate without addition of buffer, the effect on the pH was measured as a funtion of incubation time. At the last timepoint (150 min), the amount of protons detected was approximately 20-fold less than the amount of lactic acid produced. An initial lag phase was observed in the detection of protons: an increase in proton concentration was only measured after 60 minutes of incubation. This lag phase cannot be completely explained by the difference between rate of appearance of lactic acid and protons (as a consequence of the buffer present in the nasal epithelium homogenate), since the slope of the two curves after this lag phase still differed considerably.

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
L-Lactic acid is formed by enzymatic hydrolysis of ethyl-(S)-lactate by carboxylesterase present in rat nasal olfactory tissue.
Kinetic parameters of the enzymatic hydrolysis of ethyl-S-lactate by rat olfactory epithelium homogenate were: Km = 1.1 mM and Vmax = 170 nmol/min/mg protein.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): other: Ethyl-S-lactate is hydrolysed by carboxylesterase present in rat nasal olfactory tissue under formation of L-lactic acid.
Lactic acid is formed by enzymatic hydrolysis of ethyl-(S)-lactate by carboxylesterase present in rat nasal olfactory tissue.
Kinetic parameters of the enzymatic hydrolysis of ethyl-S-lactate by rat olfactory epithelium homogenate were: Km = 1.1 mM and Vmax = 170 nmol/min/mg protein. In general, the olfactory epithelium carboxylesterase showed increasing capacity (increasing Vmax) and afinity (decreasing Km) towards L-lactate esters with increasing molecular weight of the alkyl group. From a large discrepancy between the amount of lactic acid formed and the increase in proton concentration even in very poorly buffered systems it is suggested that a certain defence against acidification exisits.
Executive summary:

The hydrolysis of ethyl-S-lactate by rat nasal olfactory epithelium homogenate was investigated. The ester was incubated with rat (male, Wistar) olfactory epithelium at pH 7.0 and 37°C. The amount of liberated L-lactic acid (buffered) was quantified at a series of time points, from which the initial rat of hydrolysis was estimated. Using a concentration range of 0.05 - 3.2 mM, the enzyme kinetic parametes were calculated to be Km = 1.1 mM and Vmax = 170 nmol/min/mg protein.

Seven other L-lactate esters were also tested. In general, the olfactory epithelium carboxylesterase showed increasing capacity (increasing Vmax) and afinity (decreasing Km) towards L-lactate esters with increasing molecular weight of the alkyl group.

Since the pKa value of lactic acid is 3.80, the formation of lactic acid will (in non-buffered systems) directly result in acidification of the solution. However, even in poorly buffered systems (non-buffered incubation mix) a large discrepancy between the amount of lactic acid formed an the increase in proton concentration is observed. This suggests that a certain defence against acidification exists, and that in vivo, only high doses and/or prolonged exposure will result in acidification of tissues.