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EC number: 248-704-9 | CAS number: 27871-49-4
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
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Basic toxicokinetics
Administrative data
- Endpoint:
- basic toxicokinetics in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1992-05-12 to 2000-02-17
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 000
- Report date:
- 2000
Materials and methods
- Objective of study:
- metabolism
Test guideline
- Qualifier:
- no guideline followed
- 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
- Reference substance name:
- Methyl (S)-(-)-lactate
- EC Number:
- 248-704-9
- EC Name:
- Methyl (S)-(-)-lactate
- Cas Number:
- 27871-49-4
- Molecular formula:
- C4H8O3
- IUPAC Name:
- methyl 2-hydroxypropanoate
Constituent 1
- Radiolabelling:
- no
Test animals
- Species:
- other: rat nasal olfactory epithelium homogenate
- Strain:
- Wistar
- Sex:
- male
- 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 acclimatisation 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 anaesthesia. The head of each rat was removed from the carcass and deskinned, and the skull was split in halves sagitally, exposing the nasal cavity, using a surgical scalpel. Both halves of the skull were placed on ice. Subsequently, the olfactory and respiratory portions of the nasal septum were collected separately from both skull halves 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 10000 × g, the supernatant was stored at -80 °C. The protein concentration of the homogenate was measured using the Bradford method. Esterase activity 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 17700 per M per cm.
Administration / exposure
- Route of administration:
- other: incubation with rat nasal olfactory epithelium homogenate
- Vehicle:
- acetone
- Details on exposure:
- Methyl (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. Methyl (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 methyl lactate was rather low, methyl (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 / concentrationsopen allclose all
- Dose / conc.:
- 50 other: µM
- Dose / conc.:
- 100 other: µM
- Dose / conc.:
- 200 other: µM
- Dose / conc.:
- 400 other: µM
- Dose / conc.:
- 800 other: µM
- Dose / conc.:
- 1 600 other: µM
- Dose / conc.:
- 3 200 other: 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.
- Positive control reference chemical:
- L-lactate
- Details on study design:
- n.a.
- Details on dosing and sampling:
- Incubations:
Methyl (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. Methyl (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 methyl lactate was rather low, methyl (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 2500 x g. 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 methyl (S)-lactate in 0.05 M phosphate buffer pH 7.0 at 37 °C. Methyl (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 was detetced only after 19.5 hours (6.1 nmol corresponding to 1.2% of the total amount of ester).
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 methyl (S)-lactate by carboxylesterase present in rat nasal olfactory tissue.
Kinetic parameters of the enzymatic hydrolysis of methyl (S)-lactate by rat olfactory epithelium homogenate were not established as even at the highest concentration of 3.2 mM the rate of formation of L-Lactic acid remained linear. This indicates that the enzyme carboxyylesterase was not yet saturated and enzyme saturation is necessary for estimation of kinetic parameters.
Applicant's summary and conclusion
- Conclusions:
- In an in vitro study to assess the hydrolysis of L-lactate esters by rat nasal olfactory epithelium homogenate the following observations were made:
L-Lactic acid is formed by enzymatic hydrolysis of methyl (S)-lactate by carboxylesterase present in rat nasal olfactory tissue. Kinetic parameters of the enzymatic hydrolysis of methyl (S)-lactate by rat olfactory epithelium homogenate were not established as even at the highest concentration of 3.2 mM the rate of formation of L-Lactic acid remained linear. This indicates that the enzyme carboxylesterase was not yet saturated and enzyme saturation is necessary for estimation of kinetic parameters. In general, the olfactory epithelium carboxylesterase showed increasing capacity (increasing Vmax) and affinity (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 exists. - Executive summary:
The hydrolysis of methyl (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. L-Lactic acid is formed by enzymatic hydrolysis of methyl (S)-lactate by carboxylesterase present in rat nasal olfactory tissue. Kinetic parameters of the enzymatic hydrolysis of methyl (S)-lactate by rat olfactory epithelium homogenate were not established as even at the highest concentration of 3.2 mM the rate of formation of L-Lactic acid remained linear. This indicates that the enzyme carboxylesterase was not yet saturated and enzyme saturation is necessary for the estimation of kinetic parameters. In general, the olfactory epithelium carboxylesterase showed increasing capacity (increasing Vmax) and affinity (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 exists.
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.
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