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EC number: 611-025-7 | CAS number: 53651-69-7
- 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:
- key study
- Study period:
- 1998-11-16 to 1999-01-26
- 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:
- 1 999
- Report date:
- 1999
Materials and methods
- Objective of study:
- metabolism
Test guideline
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- S9 homogenates of liver, blood, skin, small intestinal mucosa and nasal olfactory epithelium, prepared from healthy male Wistar rats, were used. The rates of hydrolysis of the lactate esters by these homogenates were determined, and the enzyme kinetic parameters Km and Vmax were established, where possible. The S9 homogenates used were similar to those used in a previous in vitro hydrolysis study with L-lactic acid esters (TNO-report V97.552). The esterase acitivities of the various homogenates towards the model substrate p-nitrophenyl-butyrate, were determined according to the method described by Bogdanffy et al. (1987): 100 µM p-nitrophenyl-butyrate and a suitable amount of homogenate 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.
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- propyl (2S)-2-hydroxypropanoate
- EC Number:
- 611-025-7
- Cas Number:
- 53651-69-7
- Molecular formula:
- C6H12O3
- IUPAC Name:
- propyl (2S)-2-hydroxypropanoate
Constituent 1
- Specific details on test material used for the study:
- - Name of test material used in the report: n-Propyl-(L)-lactate
- Batch no.: MJE-8
- Purity: >99%
- Storage: ambient temperature - Radiolabelling:
- no
Test animals
- Species:
- other: incubation with homogenate of rat blood, skin, liver, small intestinal mucosa and nasal olfactory epithelium.
- Strain:
- Wistar
- Sex:
- male
Administration / exposure
- Route of administration:
- other: incubation with homogenate of rat blood, skin, liver, small intestinal mucosa and nasal olfactory epithelium
- Vehicle:
- unchanged (no vehicle)
- Details on exposure:
- General incubation conditions:
The 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. 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 4300 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 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 120 min. The lactate ester concentrations used were 50, 100, 250, 500 and 1250 µM.
Analysis:
The liberated amount of L-lactic acid was determined by using the Boehringer test for the determination of L-lactic acid in foodstuffs. The liberated amount of D-lactic acid was determined by using the Boehringer test for the determination of D- and 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 amounts of L- and D-lactic acid. - Duration and frequency of treatment / exposure:
- Single application for 5, 10, 20, 40 and 120 minutes.
Doses / concentrationsopen allclose all
- Dose / conc.:
- 1 250 other: µM
- Dose / conc.:
- 500 other: µM
- Dose / conc.:
- 250 other: µM
- Dose / conc.:
- 100 other: µM
- Dose / conc.:
- 50 other: µM
- No. of animals per sex per dose / concentration:
- The amounts of tissue protein used in the incubation experiments, are: 2.38 µg of nasal olfactory epithelium protein, 192.4 µg of small intestinal mucosal protein, 7.43 µg of liver protein, 50.4 µg of skin protein and 832 µg of blood protein.
- Control animals:
- other: Chemical hydrolysis was determined in incubation experiments without homogenates. Blanks were homogenates without test substances.
- 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:
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. The liberated amount of D-lactic acid was determined by using the Boehringher test for the determination of D- and 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- and D-lactic acid.
The detection limit of the method was arbitrarily fixed at 10 nmol (absorption ~ 0.02). - Statistics:
- Calculations:
The amounts L-lactic acid or D-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 proces.
The initital rates of hydrolysis were calculated from the amounts of liberated L-lactic acid/D-lactic acid (corrected for chemical hydrolysis) with the regression model:
liberated lactic acid (nmol) = a . (time) + b . (time)²,
or with the regression model
liberated 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' respresents the initital rate of hydrolysis expressed as nmol/min.
After calculating the initial rates of hydrolysis expressed as nmol/min/mg S9 protein, the enzyme kinetic parameters Km and Vmax were determined by the curve-fitting program "EZ-FIT" (version 4.13 for MS Windows).
Results and discussion
Metabolite characterisation studies
- Metabolites identified:
- yes
Any other information on results incl. tables
The protein concentrations, and the esterase activities towards the model substrate p-nitrophenylbutyrate of the various tissue homogenates are presented in Table 1. 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 various lactate esters.
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 |
1.013 ± 0.028 |
Small intestinal mucosa |
9.62 |
2.41 |
7.836 ± 0.064 |
Liver |
22.3 |
11.2 |
0.852 ± 0.010 |
Skin |
2.52 |
63.0 |
0.113 ± 0.003 |
Blood |
41.6 |
104 |
0.0134 ± 0.0001 |
The amounts of lactic acid formed by chemical hydrolysis after 2 hour incubations are presented in Table 2. From the results it can be concluded that the rate of chemical hydrolysis of n-propyl-L-lactate at pH 7.4 and 37°C is very low.
Table 2: Chemical hydrolysis of n-propyl-L-lactate to L-lactic acid.
Lactate ester |
nmol of L-lactic acid formed at the various concentrations |
||||
50 µM |
100 µM |
250 µM |
500 µM |
1250 µM |
|
n-propyl-(L)-lactate |
< 10 |
< 10 |
< 10 |
< 10 |
15.3 |
The initial rates of hydrolysis obtained for the various incubation time periods are presented in Table 3. The hydrolyses of n-propyl-L-lactate was most efficiently catalyzed by nasal olfactory epithelium and liver homogenates. The lowest activities were found for blood.
Table 3. Initial rates of hydrolysis of n-propyl-L-lactate by various rat tissue homogenates to L-lactic acid. Enzyme activities are expressed as nmol/min/mg protein.
|
Concentration (µM) |
Initial rates of hydrolysis |
|||||
Nasal olfactory epithelium |
Small intestinal mucosa |
Liver |
Skin |
Blood |
|
||
n-propyl-(L)-lactate |
50 |
260 |
3.7 |
165 |
23.6 |
0.7 |
|
100 |
559 |
7.0 |
283 |
40.0 |
1.3 |
|
|
250 |
982 |
13.0 |
516 |
75.0 |
2.8 |
|
|
500 |
1128 |
21.4 |
674 |
104.3 |
4.9 |
|
|
1250 |
1216 |
56.9 |
795 |
127.8 |
8.9 |
|
The enzyme kinetic parameters Km and Vmax, presented in Table 4, were calculated from the initial rates of hydrolysis of n-propyl-L-lactate. The hydrolysis of n-propyl-L-lactate by the homogenates of nasal epithelium, liver and skin showed Km values in the same order of magnitude (range 150-278 µM), 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 n-propyl-L-lactate was most efficiently hydrolyzed by nasal olfactory epithelium and liver homogenate.
Table 4. Enzyme kinetic parameters (mean ± sd) of the hydrolysis of n-propyl-L-lactate. Vmax is expressed as nmol/min/mg protein.
Homogenate |
Km (µM) |
Vmax |
Nasal olfactory epithelium |
150 ± 37 |
1420 ± 109 |
Small intestinal mucosa |
first order¹ v = 0.0455xS |
|
Liver |
220 ± 17 |
949 ± 25 |
Skin |
278 ± 18 |
158 ± 4 |
Blood |
1450 ± 62 |
19.2 ± 0.5 |
* v = rate expressed as nmol/min/mg protein; S = ester concentration
In order to extrapolate the obtained kinetic parameters in terms of disappearance rates of n-propyl-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 n-propyl-L-lactate were hydrolysed very rapidly by nasal olfactory epithelium and liver homogenate, while blood and small intestinal mucosa were much less efficient. Table 5 also shows that chemical hydrolysis, compared to the enzymatical hydrolysis by nasal olfactory epithelium and liver, is not detectable.
Table 5. Calculated times (seconds) in which at least 99% of n-propyl-L-lactate would be hydrolyzed. The starting concentration was 500 µM. The reactions were assumed to be completely oriented towards the hydrolyzed compounds.
Homogenate |
Time (s) |
Nasal olfactory epithelium |
0.9 |
Small intestinal mucosa |
136 |
Liver |
1.0 |
Skin |
31 |
Blood |
132 |
Chemical hydrolysis |
- |
- = no detectable rate of hydrolysis
Applicant's summary and conclusion
- Conclusions:
- In the present study, the hydrolysis of propyl (S)-lactate was studied by conducting incubation experiments with various rat tissue homogenates. It was found that the propyl (S)-lactate is hydrolysed very rapidely by the different tissue homgenates.
- Executive summary:
The rates of hydrolysis of propyl (S)-lactate to L-lactic acid by homogenates of liver, blood, skin, small intestinal mucosa and nasal olfactory epithelium, prepared from healthy mal Wistar rats, was studied. Enzym kinetic parameters Km and Vmax were established, where possible.
All homogenates showed esterase activity to propyl (S)-lactate. Nasal olfactory epithelium, liver and skin were, in this order, the most efficient tissues with repect to the hydrolysis of propyl (S)-lactate. Enzymatic hydrolysis of n-propyl-L-lactate in vivo would be much faster than chemical hydrolysis.
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