Ethyl (S)-2-hydroxypropionate

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

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

Objective of study:

metabolism

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

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

Species:

other: homogenates of rat blood, skin, liver, nasal olfactory epithelium, small intestinal mucosa and caecum content

Strain:

Wistar

Sex:

male

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.

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).

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.

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.

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. V_maxis 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

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.

Reference 2

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

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

Objective of study:

metabolism

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)

Specific details on test material used for the study:

- Name of the test material: ethyl-(S)-lactate
- Batch No.: EL 452 D
- Appearance: clear, colourless liquid

Radiolabelling:

no

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.

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

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. 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 study design:

n.a.

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 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 "Boehringer 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-Alanine + 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 the 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 calculated 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 calculated 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 time point 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 time point 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.

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.

Type:

metabolism

Results:

The following kinetic parameters of the enzymatic hydrolysis ofethyl (S)-lactate by rat olfactory epithelium homogenate: KM: 1.1 mM; Vmax: 170 nmol/min/mg protein

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.

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. The calculated half-life of the enzymatic hydrolysis of n-butyl lactate is 0.045 min or 0.27 sec.

Conclusions:

In a in vitro study to assess the hydrolysis of L-lactate esters by rat nasal olfactory epitheliun homogenate the following observations were made:
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. The calculated half-life of the enzymatic hydrolysis of n-butyl lactate is 0.045 min or 0.27 sec.

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.

Reference 3

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

absorption

metabolism

Qualifier:

no guideline followed

Principles of method if other than guideline:

Absorption and hydrolysis of ethyl-L-lactate were measured by estimating the concentration of ethyl-L-lactate, L-lactate and ethanol in samples of blood obtained from the portal vein of rats at several times after intragastric administration of ethyl-L-lactate, water (negative control) or a mixture of ethanol and L-lactate (positive control).

GLP compliance:

no

Radiolabelling:

no

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

Male Wistar rats (Cpb: WU, Wistar random) were obtained from the Central Institute for the Breeding of Laboratory Animals TNO, Zeist, The Netherlands. At the time of the experiment their body weights were 300-400 g.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Rats, which had been fasted for 16-24 hrs, were anesthetized by intraperitoneal injection of urethane (1.3-1.5 g/kg). Body temperature was continuously monitored and kept between 35.5 and 37.5 °C by means of a heating lamp. Undisturbed respiration was ensured by insertion of trachea cannula.

The abdomen was opened by a tranverse flank incision of 3.5-4.5 cm. A polypropylene cannula (PP100) was inserted via the oesophagus into the stomach and fixed in this position by a ligature around the abdominal oesaphagus to prevent reflux of administered fluids. Subsequently a side branch of the portal vein was cannulated with a polyethylene cannula (PE10). The tip of the cannula was placed just inside the lumen of the portal vein, with minimal disturbance of the portal blood flow, and fixed in this position by two ligatures around the side branch. The cannula was filled with a heparin solution in saline (approximately 10 IU/ml). Blood samples of 0.4 ml were drawn through the cannula with a syringe connected to a three-way valve. Samples were drawn before and at 5, 10, 20 and 40 minutes after administration of test-fluids. After sampling, the cannula was flushed with approximately 0.4 ml of the heparin solution. During the experiment the abdominal incision was kept covered with gauze wetted in warm saline. Presence of gut peristaltic activity was checked several times during the experiment as an indication of continuing intestinal function.

The rats received approximately 1 mL either of a 10% (0.84 mL/L) solution of ethyl-L-lactate in water (rats 1-4), or water (rats 5-8) or a solution of 0.84 mol/L L-lactate and 0.84 mol/L ethanol in water (rats 9-12).

Duration and frequency of treatment / exposure:

Single application

Dose / conc.:

0 other: mol/L

Remarks:

Control (1 mL of water)

Dose / conc.:

0.84 other: mol/L

Remarks:

Ethyl-L.lactate, 1 mL of a 10% solution (0.84 mol/L)

Dose / conc.:

0.84 other: mol/L

Remarks:

Positive control (1 mL of a solution of 0.84 mol/L L-lactate and 0.84 mol/L ethanol in water)

No. of animals per sex per dose / concentration:

4 male rats per dose

Control animals:

yes, concurrent vehicle

Positive control reference chemical:

Yes, 1 ml of a solution of 0.84 mol/L Li-L-lactate and 0.84 mol/L ethanol in water

Details on dosing and sampling:

Blood sample preparation:
Blood samples obtained from the portal vein were immediately mixed with ca 25 µL fluoride-EDTA solution (Boehringer Mannheim) in pre-cooled tubes. The samples were stored on ice for maximal 40 minutes. Plasma was prepared by centrifugation in a refrigerated centrifuge at 3000 g for 10 minutes at a temperature of 0-2 °C. Plasma was collected, frozen immediately in liquid nitrogen and stored at -20 °C till analysis.

Determination of ethyl-L-lactate and ethanol:
Ethyl-L-lactate and ethanol concentrations were determined by gas chromatography on a glass capillary column (length 50 m) coated with carbowax 400. Plasma samples of 0.5 µL were directly injected. The gas chromatograph (Intersmat IGC-16 equipped with a flame ionization detector) was isothermically operated at 80 °C. The temperature of the injector was 200°C, the detector temperature 250 °C. Helium was used as a carrier gas.
Peak identification was based on retention times compared to standard solutions of ethanol and ethyl-L-lactate.
Concentrations were calculated by measuring peak heights and comparing them to known standards.

Determination of L-lactate:
L-lactate concentrations were determined enzymatically using the Boehringer L-lactate monotest (Boehringer Mannheim GmbH Nr 149993).

Details on absorption:

The data (see Table 1-3 in attached background material) show that ethyl-L-lactate appears in the portal bloodstream when given intragastrically. There is a large variation in the pattern of ethyl-L-lactate absorption among the individual animals. In animal 1 ethyl-L-lactate concentrations showed a maximum after 5 min., in animal 2 after about 20 min., in animal 3 at approximately 40 min., while in animal 4 the concentration remained very low, with a possible minor peak at 5 min. No ethyl-L-lactate was found in either the negative or the positive controls.

Metabolites identified:

yes

Details on metabolites:

Ethanol concentrations after administration of ethyl-L-lactate to rats were always higher than the concentrations of ethyl-L-lactate. Like the ethyl-L-lactate concentrations, the pattern of the ethanol concentration showed some inter-animal variation but this pattern was more consistent. In animal 1 the ethanol concentration showed a peak at 5 minutes. In the other animals the ethanol concentration increased gradually during the 40 min. observation period. Small amounts of ethanol were nearly always present at t=0 min. and in the negative controls. In the positive control group ethanol was found in the portal blood but in smaller amounts than after the gift of an equivalent dose of ethyl-L-lactate.

Relatively large concentrations of L-lactate were present in the portal blood of all animals. With a few exceptions the L-lactate concentrations increased continuously during the course of the experiment. Only in animal 1 there was a definite peak in the L-lactate concentrations which correlated with the ethyl-L-lactate and ethanol peaks.

When ethyl-L-lactate was mixed with rat plasma to a final concentration of 100 ppm (0.85 mmol/L) and left at room temperature for 60 minutes, 80% of the ethyl-L-lactate had disappeared.

Conclusions:

From the present results it is concluded that ethyl-L-lactate is at least partially absorbed unhydrolyzed from the gastro-intestinal tract of rats. A quantitative estimation of the fraction of ethyl-L-lactate which is absorbed prior to hydrolysis is not possible. Absorbed ethyl-L-lactate is hydrolysed in rat blood and possibly in other organs.

Executive summary:

Solutions of ethyl-L-lactate in water, solutions of ethanol and L-lactate in water or water were administered intragastrically to fasting male Wistar rats. Before and at 5, 10, 20 and 40 minutes after administration blood samples were obtained from the portal vein by cannulation of a side branch of this vein in urethane anesthetized rats. Ethyl-L-lactate and ethanol concentrations in the portal plasma were determined by gas-chromatography. L-lactate concentrations were determined enzymatically.

Ethyl-L-lactate and ethanol were detected in portal blood after intragastric administration of ethyl-L-lactate. The uptake pattern in time differed between animals. Ethanol concentrations in blood from animals administered ethanol and L-lactate were lower compared to ethyl-L-lactate administered animals. This implies that part of the ethyl-L-lactate taken up from the gastrointestinal tract is hydrolyzed before arrival in the blood at the portal vein. This is supported by the in vitro study in which 80% of 100 ppm ethyl-L-lactate supplied to rat plasma hydrolyzed in 60 minutes at room temperature. From the results it can be concluded that ethyl-L-lactate administered at high doses to fastening rats is at least partially absorbed without prior hydrolysis, but the data also shows that absorbed ethyl-l-lactate is hydrolyzed in rat blood.

Reference 4

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

August 1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

metabolism

Qualifier:

no guideline followed

Principles of method if other than guideline:

The enzymatic hydrolysis in vitro of ethyl-L-lactate by pancreatin and by an enzyme preparation isolated from porcine small intestinal mucosa was studied. The degree of hydrolysis was measured either by enzymatically determining the lactic acid formed in the reaction or by GC measurement in the incubation mixture the decrease in ethyl lactate concentration and the production of ethanol.

GLP compliance:

no

Radiolabelling:

no

Species:

pig

Strain:

not specified

Sex:

not specified

Details on test animals or test system and environmental conditions:

Pancreatin solution: 80 mg pancreatin (Merck, Art. 7132) were mixed with 40 mL 0.1 M buffer solution. The suspension was kept in a refrigerator, occasionally shaken and after 20 h filtered through Schleicher & Schüll Nr. 589² paper.
Intestinal mucosa: 80 mg of freeze dried preparation of porcine small intestinal mucosa were mixed with 40 mL 0.1 M buffer solution. The suspension was kept in a refrigerator, occasionally shaken and after 20 h filtered through Schleicher & Schüll Nr. 589² paper.

Route of administration:

application in vitro

Vehicle:

water

Details on exposure:

Incubation procedure:
Equal volumes (2 mL) of ethyl lactate solution (containing 2 mg ester per mL demineralized water, prepared shortly before the start of the experiment) and either the 0.1 M buffer, pancreatin or intestinal mucosa solution were mixed and incubated at 37 °C in all-glass, stoppered test tubes.

Duration and frequency of treatment / exposure:

single application for 1 and 2 hours.

Dose / conc.:

2 other: mg ester/mL water

No. of animals per sex per dose / concentration:

not applicable

Control animals:

other: Ethyl lactate in buffer solution without pancreatine or intestinal mucosa

Details on study design:

see box "Details on dosing and sampling".

Details on dosing and sampling:

Reagents:
Ethyl-L-lactate was supplied by the principal.
0.1 M Buffer solution: 50 mg of sodium azide were dissolved in 100 mL 0.1 M potassium phosphate buffer of pH 7.5.

Analytical methods:
Determination of L-lactic acid: The L-lactic acid content of the various incubation mixtures was determined enzymatically with L-lactate dehydrogenase (UV-test, Boehringer-Mannheim GmbH) after diluting 0.5 mL sample with 4.5 mL demineralized water.
Gas-chromatographic analysis: The ethyl lactate and the ethanol concentrations in the digests were determined on a 150 m x 0.75 mm capillary column coated with Carbowax 400. The gas-chromatograph (Intersmat IGC 120, equipped with a flame ionization detector) was isothermally operated at 90 °C. Helium was used as a carrier gas at a flow of 15 mL/min. The injection temperature was 250 °C, the detector temperature 225 °C.
The ethyl lactate concentrations were determined by measuring peak heights; n-butanol was used as an internal standard.
To determine the ethanol concentrations a calibration standard containing 0.063 mg ethanol and 0.2 µL n-butanol per mL was used.
Samples of 0.5 µL of the various mixtures were directly injected into the gas-chromatograph.


Experiments:
Two experiments were conducted. In the first experiment (Experiment A) "blank" samples (containing only ester in buffer) were taken at zero time and after 1 h and "enzyme" samples (containing ester and either pancreatin or the intestinal mucosa preparation) only after 1 h to be analyzed for lactic acid content as described above.
In the second experiment (Experiment B) samples of both blank and enzyme samples were taken at zero time and then after 1 and 2 h. In this experiment the ester solution contained also 0.2 µL n-butanol per mLas an internal standard.

Metabolites identified:

yes

Details on metabolites:

The results of Experiment A are summarized in Table 1 and those of experiment B in Table 2.
The data show that under the conditions applied in Experiment A the lactic acid concentration increased in all samples, indicating hydrolysis of ethyl-L-lactate. From the data of the buffer ("blank") solution it appears that the chemical hydrolysis proceeded very slowly (<1 per cent in 1 h). With pancreatin only a small amount of ester (< 10 per cent) was hydrolyzed, but with the intestinal mucosa preparation a substantial amount of ester (> 50 per cent) was split.
Comparable results were obtained in Experiment B. The decrease in ethyl lactate concentration was accompanied by an increase in the ethanol content of the incubation mixtures. In this experiment, too, only the intestinal mucosa preparation induced a substantial hydrolysis of the ester.
It should be mentioned that in the enzymatic lactic acid assay the samples were incubated at 25°C and pH 9 for 1 h. It is likely that under these conditions some chemical hydrolysis will occur. Hence, the lactic acid concentrations reported are probably somewhat higher than those acutally present in the digests at the end of the experiment. By contrast, in the GC analysis no extra hydrolysis of ethyl lactate is likely to occur after the samples have been injected.

Table 1. Chemical and enzymatic hydrolysis of ethyl-L-lactate.

Experiment A: Enzymatic determination of lactic acid formed in the reaction

	lactic acid (µg/mL)		% hydrolysis^*)
	0 h	1 h	After 1 h
ethyl-L-lactate (1 mg/mL)
+ buffer pH = 7.5	26	32	0.8
+ pancreatin	n.d.^**)	82	7
+ intestinal mucosa	n.d.	428	52

*)          corrected for "blank" value (= hydrolysis measured in the buffer sample at zero time or after 1 h)

**)        n.d. = not determined

 

Table 2. Chemical and enzymatic hydrolysis of ethyl-L-lactate.

Experiment B: Gaschromatographic determination of the decrease in ester concentration and of the ethanol formed in the reaction

	ethyl-L-lactate				ethanol
	mg/ml		% hydrolysis		mg/ml		% hydrolysis
	1 h	2 h	1 h	2 h	1 h	2 h	1 h	2 h
ethyl-L-lactate (1 mg/mL)
+ buffer pH = 7.5	1.01	0.98	-1	2	4	8	1	2
+ pancreatin	0.99	0.93	1	7	20	35	5	9
+ intestinal mucosa	0.52	0.27	48	73	218	335	56	86

 

Conclusions:

Under physiological conditions (pH 7.5; t = 37 °C) ethyl-L-lactate is relatively stable in aqueous solutions. Ethyl-L-lactate is hydrolyzed by enzymes in the intestinal tract, particularly by the enzymes of the small intestinal mucosa. Orally administered ethyl-L-lactate in concentrations below 1 mg/mL will probably be completely hydrolyzed in the intestinal tract. This implies that for systemic toxicity ethyl-L-lactate may be evaluated as a mixture of ethanol and L-lactic acid.

Executive summary:

The data of the present study show that under physiological conditions (pH 7.5; t = 37 °C) ethyl-L-lactate is relatively stable in aqueous solutions, but that the compound is hydrolyzed by the enzymes of the intestinal tract, particularly by the enzymes of the small intestinal mucosa. In view of the enzyme concentrations used in the study and the relative large amounts of mucosa present in the small intestines, one might expect that orally administered ethyl-L-lactate in concentrations below l mg/mL will be completely hydrolyzed in the "milieu exterieur" , viz. the intestinal tract. The toxicological implication of this feature, as far as the "milieu interieur" is concerned, is that ethyl-L-lactate may be evaluated as a mixture of ethanol and L-lactic acid.

Reference 5

Endpoint:

basic toxicokinetics

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

For details and justification of read-across please refer to the read-across report attached to IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

The role of lactic acid in metabolism has kept researchers occupied for a long time. For many years, lactic acid was considered a dead-end waste product of the glycolysis, the conversion of glucose into pyruvate (producing a relatively small amount of ATP), in the absence of oxygen. Recently, the role of lactic acid in metabolism was reconsidered, and L-lactate is considered as a functional metabolite and mammalian fuel. It was observed that lactate can be transferred from its site of production (cytosol) to neighbouring cells and other organs, as well as intracellularly, where its oxidation or continued metabolism can occur. This "lactate shuttle" results in the distribution of lactic acid to other cells, where it is directly oxidised, re-converted back to pyruvate or glucose, allowing the process of glycolysis to restart and ATP provision maintained.

Conclusions:

In the evaluation of the use of lactic acid as the active substance in biocidal products, the natural occurrence of lactic acid in human food and the human body, as well as the role of the compound in human metabolism and physiology should be taken into account. This means that, when the risk for its use in biocidal products is assessed, the natural exposure to lactic acid in food and via endogenous sources, as well as exposure via the use of lactic acid as a food additive should be considered.
In the present report it is concluded that lactic acid can no longer be considered as a “dead-end” waste product of human metabolism, but should instead be seen to play an important role in cellular, regional, and whole body metabolism. Lactic acid has been detected in blood, several other body fluids and tissues. Concentrations of lactic acid increase significantly during intense exercise. At rest, blood concentrations have been reported of 1-1.5 mMol/L (90.1-135.12 mg/L), which can increase up to 10 mMol/L (900.8 mg/L) during exercise.
External human exposure to lactic acid can occur via its natural presence in food, for example in fruit, vegetables, sour milk products, and fermented products such as sauerkraut, yogurt and beer. Based on the available information on concentrations of lactic acid in some of these products, an estimate of the daily consumption of lactic acid due to its natural presence in food was made using the ‘FAO/WHO standard European diet’. A (minimum) daily intake of 1.175 g/person/day was calculated using the available information.
Another source of external exposure is its use as food additive; as such it is authorized in Europe (E270) and the United States (generally recognized as safe = GRAS). A daily intake of 1.65-2.76 g/person/day was estimated using the “Per Capita times 10” method, based on the amount of lactic acid put onto the market (EU and USA) as a food additive by Purac.
Based on the high levels of lactic acid in the human body and in human food, and its use as food additive, the evaluation of the human health effects of lactic acid should first and for all be based on a comparison of this background exposure and the potential contribution of lactic acid in biocidal products to these levels. Therefore, a risk assessment should not be based on the comparison with effects of exposure, but on the comparison with the total daily intake of lactic acid via food, both naturally and as food additive, which was estimated to be 2.8 g/person/day. When the application of Purac’s products will not result in a systemic exposure that contributes substantially to the total systemic exposure, many of the standard human toxicological studies dealing with systemic effects are deemed superfluous.

Executive summary:

The natural occurrence of lactic acid in human food and the human body, as well as the role of the compound in human metabolism and physiology is of primary importance in the understanding of the metabolism and toxicology of lactic acid. This means that, in risk assessment, the natural exposure to lactic acid in food and via endogenous sources, as well as exposure via the use of lactic acid as a food additive should be considered.

In the present report it is concluded that lactic acid, in contrast to previously held belief, can no longer be considered as a “dead-end” waste product of human metabolism, but should instead be seen to play an important role in cellular, regional, and whole body metabolism. Lactic acid has been detected in blood, several other body fluids and tissues. Concentrations of lactic acid increase significantly during intense exercise. At rest, blood concentrations have been reported of 1-1.5 mMol/L (90.1-135.12 mg/L), which can increase up to 10 mMol/L (900.8 mg/L) during exercise.

External human exposure to lactic acid can occur via its natural presence in food, for example in fruit, vegetables, sour milk products, and fermented products such as sauerkraut, yogurt and beer. Based on the available information on concentrations of lactic acid in some of these products, an estimate of the daily consumption of lactic acid due to its natural presence in food was made using the ‘FAO/WHO standard European diet’. A (minimum) daily intake of 1.175 g/person/day was calculated using the available information.

Another source of external exposure is its use as food additive; as such it is authorized in Europe (E270) and the United States (generally recognized as safe = GRAS). A daily intake of 1.65-2.76 g/person/day was estimated using the “Per Capita times 10” method, based on the amount of lactic acid put onto the market (EU and USA) as a food additive by Purac.

Based on the high levels of lactic acid in the human body and in human food, and its use as food additive, the evaluation of the human health effects of lactic acid should first and for all be based on a comparison of this background exposure and the potential contribution of lactic acid in biocidal products to these levels. Therefore, a risk assessment should not be based on the comparison with effects of exposure, but on the comparison with the total daily intake of lactic acid via food, both naturally and as food additive, which was estimated to be 2.8 g/person/day. When the application of Purac’s products will not result in a systemic exposure that contributes substantially to the total systemic exposure, many of the standard human toxicological studies dealing with systemic effects are deemed superfluous.

This information is used in a read-across approach in the assessment of the target substance. For details and justification of read-across please refer to the read-across report attached to IUCLID section 13.

Description of key information

Ethyl (S)-lactate, as all lactate esters, is rapidly hydrolysed in vivo into lactic acid and ethanol.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Additional information

Ethyl (S)-lactate, as all lactate esters, is rapidly hydrolysed in vivo into lactic acid and ethanol. A summary of the data collected is presented below:

In a hydrolysis study of ethyl (S)-lactate by rat nasal olfactory epithelium homogenate, lactic acid was formed by enzymic reaction of carboxylesterase; the kinetic parameters were: Km = 1.1 mM and Vmax = 170 nmol/min/mg protein. 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.

In another study, solutions of ethyl (S)-lactate in water, solutions of ethanol and L-lactate in water or water were administered intragastrically to rats. The results revealed that ethyl (S)-lactate is at least partially absorbed unhydrolyzed from the gastro-intestinal tract of rats, but it mainly enzymatically hydrolysed rapidly after uptake.

Aqueous solutions of ethyl (S)-lactate, containing 1 mg of ester/mL, were incubated in a 0.05 M phosphate buffer pH 7.5 with or without pancreatin or a porcine intestinal mucosa preparation at 37 °C. The degree of hydrolysis was determined after 1–2 h. The results of the study suggested that orally administered ethyl (S)-lactate in concentrations below 1 mg/mL will be completely hydrolysed in the intestinal tract.

In another study the rates of hydrolysis of ethyl (S)-lactate to ethanol and L-lactic acid by homogenates of male rat liver, blood, skin, small intestinal mucosa and nasal olfactory epithelium and caecum content homogenates, was examined. All homogenates showed esterase activity to ethyl (S)-lactate. Nasal olfactory epithelium, liver and skin were, in this order, the most efficient tissues with respect to the hydrolysis of ethyl (S)-lactate. Enzymatic hydrolysis of ethyl (S)-lactate in vivo would be much faster than chemical hydrolysis.

Lactic acid is a ubiquitous and essential biological molecule, in humans and other mammals, but also in most if not all vertebrate and invertebrate animals, as well as in many micro-organisms. As such the biokinetics, metabolism and distribution of lactic acid have to be considered in the context of its normal biochemistry; exogenous lactic acid will be indistinguishable from endogenous lactic acid and will follow the same biochemical pathways as endogenous lactic acid, at least up to a certain systemic level. Ethanol is an alcohol that is metabolised by the normal alcohol metabolism pathways.