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EC number: 211-694-1 | CAS number: 687-47-8
- 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
Basic toxicokinetics
Administrative data
- Endpoint:
- basic toxicokinetics in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 1997-06-06 to 1997-07-30
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- test procedure in accordance with national standard methods
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 1 997
- Report date:
- 1997
Materials and methods
- Objective of study:
- metabolism
Test guideline
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- In the present study, the hydrolysis of ethyl-(L)-lactoyl lactate and ethyl-(L)-lactoyl lactoyl lactate to ethanol and L-lactic acid, was compared to that of ethyl-(L)-lactate. Incubation experiments with various rat tissue homogenates were performed for this purpose. The lactate ester concentrations used were 100, 250, 500, 1250 and 3000 µM.
- GLP compliance:
- yes
Test material
- 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 2-hydroxypropanoate
Constituent 1
- Specific details on test material used for the study:
- - Name of test material used: Ethyl-(L)-lactate (EL)
- Batch number: HK 157EN
- Purity: 99%
- Storage conditions: ambient temperature - Radiolabelling:
- no
Test animals
- Species:
- other: homogenates of rat blood, skin, liver, nasal olfactory epithelium, small intestinal mucosa and caecum content
- Strain:
- Wistar
- Sex:
- male
Administration / exposure
- Route of administration:
- other: incubation with homogenate of rat blood, skin, liver, small intestinal mucosa, nasal olfactory epithelium and caecum content.
- Vehicle:
- unchanged (no vehicle)
- Details on exposure:
- General incubation conditions:
The ethyl lactate esters were incubated at 37 °C in 1 mL incubation mixtures containing 0.10 M potassium phosphate buffer pH 7.4. The chemical hydrolysis was determined in incubation experiments without homogenates. Blanks were homogenates without test substances. The amounts of tissue protein or caecum content used in the incubation experiments, are:
Ethanol experiments: 2.86 µg of nasal olfactory epithelium protein, 96.2 µg of small intestinal mucosal protein, 6.37 µg of liver protein, 32.8 µg of skin protein and 1.46 mg of blood protein, and 10.2 mg of caecum content.
L-lactic acid experiments: 4.76 µg of nasal olfactory epithelium protein, 192.4 µg of small intestinal mucosal protein, 14.9 µg of liver protein, 54.6 µg of skin protein and 1.46 mg of blood protein, and 10.2 mg of caecum content.
a) Determination of ethanol:
Incubations were performed in 2 mL HPLC vials, which were capped immediately after addition of the test substances. The reaction was terminated by heating the samples up to 90-95 °C for 1.5 min. Subsequently, the sample vials were placed on ice. After centrifugation for 10 min. at 4 °C and 4,300 x g, the supernatant was used for immediate determination of liberated ethanol. No loss of the ethanol was observed, as determined, with ethanol-standard solution, when heating the samples in capped vials.
b) Determination of L-lactic acid:
Incubations were performed in open tubes. The reaction was terminated by addition of 3 mL of ice-cold ethanol. After placing the samples in the freezer for at least 20 minutes, the tubes were centrifuged for 10 min. at 4,300 x g (room temperature) and decanted into new tubes. The samples were evaporated to dryness with nitrogen and stored at ≤ -15 °C until analysis.
Times and concentrations:
The ethyl lactate esters were incubated with the various homogenates for 5, 10, 20, 40 and 120 minutes. Chemical hydrolysis was measured by incubating the substrates without homogenates during 60 min. The ethyl lactate ester concentrations used were 100, 250, 500, 1250 and 3000 µM. - Duration and frequency of treatment / exposure:
- Single application for 5, 10, 20, 40 and 120 minutes.
Doses / concentrationsopen allclose all
- Dose / conc.:
- 3 000 other: µM
- Dose / conc.:
- 1 250 other: µM
- Dose / conc.:
- 500 other: µM
- Dose / conc.:
- 250 other: µM
- Dose / conc.:
- 100 other: µM
- Positive control reference chemical:
- The esterase acitivities of the various homogenates towards the model substrate p-nitrophenylbutyrate were determined.
- Details on dosing and sampling:
- Analysis:
a) Determination of ethanol
The liberated amount of ethanol was determined by using the Boehringer test for the determination of ethanol in foodstuffs and other materials. Instead of the potassium phosphate buffer (pH 9.3), included in the kit, a 1:1 mixture of this buffer with 0.1 M potassium phosphate buffer pH 7.4 (final pH 8.7) (assay buffer) was used in order to minimize hydrolysis of the lactate esters during the measurement of the liberated amount of ethanol. The detection limit of the method was arbitrarily decided to be 5 nmol (absorption ~0.02).
b) Determination of L-lactic acid
The liberated amount of L-lactic acid was determined by using the Boehringer test for the determination of L-lactic acid in foodstuffs and other materials. Instead of the glycylglycine buffer (pH 10), included in the kit, a 0.1 M potassium phosphate buffer pH 7.4 (assay buffer) was used, in order to minimize hydrolysis of the lactate esters during the measurement of the liberated amount of L-lactic acid. The detection limit of the method was arbitrarily decided to be 10 nmol (absorption ~ 0.02). - Statistics:
- Calculations:
The amounts of ethanol and L-lactic acid formed during the incubations were calculated from the respective standard curves.
The rates of hydrolysis were corrected for the chemical hydrolysis, which was assumed to be a linear chemical process.
The initial rates of hydrolysis were calculated from the amounts of liberated ethanol/L-lactic acid (corrected for chemical hydrolysis) with the regression model:
liberated ethanol/L-lactic acid (nmol) = a . (time) + b . (time)²,
or with the regression model
liberated ethanol/L-lactic acid (nmol) = a.(time),
with a = regression coefficient of the linear component and b = regression coefficient of the quadratic component.
The regression coefficient 'a' represents the initial rate of hydrolysis expressed as nmol/min.
After calculating the initial rates of hydrolysis expressed as nmol/min/mg S9 protein or nmol/min/g caecum content, the enzyme kinetic parameters Km and Vmax were determined by the curve-fitting program for the analysis of enzyme kinetic data "EZ-FIT" (Perella, 1988).
Results and discussion
- Preliminary studies:
- Protein concentrations:
The protein concentrations, and the esterase activities towards the model substrate p-nitrophenylbutyrate of the various tissue homogenates are presented in Table 1 in box "Any other information on results incl. tables". These results show that esterase activity was present in the various homogenates and thus could be used for the measurement of esterase activity towards the ethyl lactate esters.
Chemical hydrolysis:
The amounts of ethanol and L-lactic acid formed by chemical hydrolysis after 1 hour incubations are presented in Table 2 in box ""Any other information on results incl. tables". From the results it can be concluded, that the rate of chemical hydrolysis of ethyl-L-lactate at pH 7.4 and 37 °C is very low.
Metabolite characterisation studies
- Metabolites identified:
- yes
- Details on metabolites:
- The initial rates of hydrolysis obtained for the various incubation time periods are presented in Table 3 in box "Any other information on results incl. tables". Theoretically, the initial rates of hydrolysis of ethyl-L-lactate to ethanol and L-lactic acid have to be similar, which is reflected by the data. Expressed as nmol/min/mg protein, ethyl-L-lactate was hydrolyzed most efficiently in nasal olfactory epithelium and liver homogenates. The lowest activities were found for blood and small intestinal mucosa.
The enzyme kinetic parameters Km and Vmax, presented in Table 4, were calculated from the initial rates of hydrolysis of ethyl-L-lactate. The hydrolysis of ethyl-L-lactate by the homogenates of nasal epithelium, liver and skin showed Km values in the same order of magnitude (range 163-362 µM). Caecum content showed an intermediate Km value, while blood and small intestinal mucosa showed a high Km value, or first order kinetics in the tested concentration range. With respect to the obtained Vmax values it is observed that ethyl-L-lactate was most efficiently hydrolyzed by nasal olfactory epithelium and liver homogenate.
Compared to a previous study with ethyl-L-lactate and nasal epithelium (TNO report V92.339), a 4-fold lower Km value and a 10-fold higher Vmax value were obtained. However, the activities towards teh model substrate p-nitrophenylbutyrate from both studies are well in agreement with each other. Therefore, differences in kinetic parameters are probably due to different pH values of the incubation experiments. In the first study, incubations were performed at pH 7.0, while in the present study the more physiological pH 7.4 was used. This higher pH value may be optimal for the esterase activity.
In order to extrapolate the obtained kinetic parameters in terms of disappearance rates of ethyl-L-lactate in the organs/tissues, the obtained kinetic parameters were scaled up to hydrolysis rates expressed per weight of tissue, by using the total amount of protein/gram of tissue. Subsequently, the disappearance in time of the compound in the organs/tissues was calculated by the Michaelis-Menten or first order equation and the data presented in Table 4. A starting concentration of 500 µM was used. However, it had to be assumed that the equilibrium of the reactions are completely on the side of the hydrolyzed compounds. The calculated disappearance rates would be higher in vivo. The times were calculated in which at least 99% of the ester would be hydrolyzed (Table 5). Table 5 shows that nasal olfactory epithelium and liver are the most efficient tissues with respect to the hydrolysis of the ethyl-L-lactate, while caecum content, blood and small intestinal mucosa were much less efficient. Table 5 also shows that chemical hydrolysis, compared to the enzymatic hydrolysis by nasal olfactory epithelium and liver, is negligible.
Any other information on results incl. tables
Table 1. Mean protein concentrations, and esterase activities towards p-nitrophenylbutyrate (mean ± sd) of the various rat tissue homogenates.
Homogenate |
Protein concentration homogenate (mg/ml) |
Esterase activity towards p-nitrophenylbutyrate |
|
|
|
Amount of protein used in assay (µg) |
Activity (µmol/min/mg protein) |
Nasal olfactory |
7.14 |
11.9 |
1.044 ± 0.030 |
Small intestinal mucosa |
9.62 |
2.41 |
7.298 ± 0.006 |
Liver |
22.3 |
11.2 |
0.910 ± 0.018 |
Skin |
2.52 |
63.0 |
0.120 ± 0.001 |
Blood |
41.6 |
104 |
0.0102 ± 0.0002 |
Caecum content |
- |
508¹ |
2.63 ± 0.09² |
¹ µg of caecum content; ² µg/min/g caecum content
Table 2. Chemical hydrolysis of ethyl-L-lactate to ethanol and L-lactic acid.
Product |
nmol of ethanol/L-lactic acid formed at the various concentrations |
||||
100 µM |
250 µM |
500 µM |
1250 µM |
3000 µM |
|
Ethanol |
< 5 |
< 5 |
< 5 |
10.9 |
19.5 |
L-lactic acid |
< 10 |
< 10 |
< 10 |
< 10 |
31.7 |
Table 3. The hydrolysis of ethyl-L-lactate by various rat tissue homogenates to ethanol or L-lactic acid. Enzyme activities are expressed as nmol/min/mg protein or nmol/min/g caecum content.
|
Concentration (µM) |
Initial rates of hydrolysis |
|||||
Nasal olfactory epithelium |
Small intestinal mucosa |
Liver |
Skin |
Blood |
Caecum content |
||
Ethanol |
100 |
834 |
1.1 |
340 |
64.8 |
0.5 |
66.0 |
250 |
1395 |
2.5 |
595 |
121.8 |
1.1 |
111.2 |
|
500 |
1746 |
6.2 |
765 |
151.9 |
2.2 |
182.8 |
|
1250 |
1740 |
18.4 |
817 |
172.0 |
4.7 |
254.6 |
|
3000 |
2774 |
58.4 |
1320 |
255.1 |
11.4 |
371.5 |
|
L-lactic acid |
100 |
470 |
2.6 |
356 |
54.3 |
* |
71.5 |
250 |
986 |
5.2 |
638 |
102.8 |
1.9 |
105.2 |
|
500 |
1360 |
8.6 |
744 |
149.5 |
2.2 |
138.5 |
|
1250 |
1677 |
19.5 |
855 |
190.4 |
6.7 |
213.4 |
|
3000 |
1834 |
43.1 |
961 |
185.6 |
10.1 |
201.1 |
* initial rate of hydrolysis could not be determined accurately
Table 4. Enzyme kinetic parameters (mean ± sd) of the hydrolysis of ethyl-L-lactate. Vmaxis expressed as nmol/min/mg protein or nmol/min/g caecum content.
Homogenate |
Product |
Km (µM) |
Vmax |
Nasal olfactory epithelium |
Ethanol |
256 ± 128 |
2640 ± 368 |
L-lactic acid |
275 ± 29 |
2030 ± 61 |
|
Small intestinal mucosa |
Ethanol |
first order¹ v = 0.01856xS |
|
L-lactic acid |
first order¹ v = 0.01466xS |
||
Liver |
Ethanol |
362 ± 171 |
1320 ± 193 |
L-lactic acid |
163 ± 19 |
996 ± 29 |
|
Skin |
Ethanol |
338 ± 126 |
259 ± 29 |
L-lactic acid |
250 ± 53 |
214 ± 13 |
|
Blood |
Ethanol |
first order¹ v = 0.003813xS |
|
L-lactic acid |
2740 ± 1300 |
19.6 ± 5.3 |
|
Caecum content |
Ethanol |
795 ± 165 |
455 ± 37 |
* v = rate expressed as nmol/min/mg protein; S = ester concentration
Table 5. Calculated times (seconds) in which at least 99% of the ethyl-L-lactate would be hydrolyzed. The starting concentration was 500 µM. The reactions were assumed to be completely oriented towards the hydrolyzed compounds.
Homogenate |
Product |
Time (s) |
Nasal olfactory epithelium |
Ethanol |
0.6 |
L-lactic acid |
0.9 |
|
Small intestinal mucosa |
Ethanol |
330 |
L-lactic acid |
425 |
|
Liver |
Ethanol |
1.1 |
L-lactic acid |
0.8 |
|
Skin |
Ethanol |
22 |
L-lactic acid |
20 |
|
Blood |
Ethanol |
425 |
L-lactic acid |
230 |
|
Caecum content |
Ethanol |
530 |
Chemical hydrolysis |
Ethanol |
> 3240 |
L-lactic acid |
> 1590 |
Applicant's summary and conclusion
- Conclusions:
- In the present study, the hydrolysis of ethyl-(L)-lactate was studied by conducting incubation
experiments with various rat tissue homogenates. It was found that the lactate ester is hydrolyzed to ethanol and L-lactic acid. - Executive summary:
The rates of hydrolysis of ethyl-L-lactate to ethanol and L-lactic acid by homogenates of liver, blood, skin, small intestinal mucosa and nasal olfactory epithelium and caecum content homogenates, prepared from healthy mal Wistar rats, was studied. Enzym kinetic parameters Km and Vmax were established, where possible.
All homogenates showed esterase activity to ethyl-L-lactate. Nasal olfactory epithelium, liver and skin were, in this order, the most efficient tissues with repect to the hydrolysis of ethyl-L-lactate. Enzymatic hydrolysis of ethyl-L-lactate in vivo would be much faster than chemical hydrolysis.
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