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Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
October 1993 - February 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study
Objective of study:
metabolism
Qualifier:
no guideline followed
Principles of method if other than guideline:
The rate of hydrolysis of 2-ethylhexyl lactate and di-(2-ethylhexyl)-phthalate by homogenates of rat liver, small intestinal mucosa, blood, skin and caecum content was investigated. The esters were incubated with the homogenates at pH 7.0 and 37 °C. The amount of liberated 2-ethylhexanol was quantified at a series of time points, from which the initial rate of hydrolysis was determined. Using a concentration range of 3-50 µM, the enzyme kinetic parameters Km and Vmax were calculated.
GLP compliance:
yes
Radiolabelling:
no
Species:
other: homogenates of rat blood, skin, liver, small intestinal mucosa and caecum content
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
Homogenates were prepared from two control F1-generation male Wistar rats, about 15 weeks old. The rats were exsanguinated whilst under diethylether anaesthesia. Blood was collected in heparinised tubes, and cells were lysed by addition of 3 parts of water. Dorsal skin was shaved, removed and homogenised with three parts of homogenisation buffer (0.01 M Tris.HCl/0.14 M KCl, pH 7.4) by means of a dismembrator. Subsequently, the skin homogenate was diluted 1:1 with homogenisation buffer and centrifuged at 10,000xg. The liver was removed, sliced and homogenised with three parts of buffer. The small intestine was removed, rinsed thoroughly with ice-cold buffer and cut open. The mucosa (s.i. mucosa) was scraped off, collected in ice-cold buffer, and homogenised. The content of caecum was weighed and diluted to 10% (w/v) with ice-cold buffer. The tissue suspensions and caecum contents were homogenised using a Potter Elvejhem tissue homogeniser at 0-4°C. From this step on, the homogenate of the caecum content was flushed with nitrogen during ten minutes to prevent aerobic microbiotic growth. Homogenates were stored at -80 °C in cryo tubes. Of liver, skin and small intestinal mucosa homogenates, as well as whole blood, the protein concentration was determined using the method of Bradford. Esterase activity of the homogenates was measured with p-nitrophenylbutyrate. Briefly, 100 µM p-nitrophenylbutyrate 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. Enzymatic activity was expressed in µmol per min per mg protein using a molar extinction coefficient of 17,700 M-1.cm-1.
Route of administration:
other: incubation with homogenate of rat blood, skin, liver, small intestinal mucosa and caecum content.
Vehicle:
acetone
Details on exposure:
Incubations:
The incubation mixture consisted of the following components:

500 µl 0.1 M potassium-phosphate buffer pH 7.0
100 µl bovine serum albumin serum albumin solution (10 mg/ml)
280 µl water
100 µl (diluted) homogenate
20 µl substrate in acetone

The incubations were started by addition of substrate, and were performed at 37 °C. 2-Ethylhexyl lactate was incubated with homogenates for 3, 6, 9, 12 and 15 minutes, and final concentrations of 3.22, 6.44, 12.9, 25.8 and 51.5 µM. Di-(2-ethylhexyl)-phthalate was incubated with homogenates for 12, 24, 36, 48 and 60 minutes, and final concentrations of 3.13, 6.25 and 12.5 µM. Di-(2-ethylhexyl)-phthalate was incubated at only 3 concentrations because of a maximal solubility in the incubation mixture of approximately 13 µM (determined by the Sponsor). Concentrations lower than approximately 3 µM di-(2-ethylhexyl)-phthalate were not used because of the detection limit of the gaschromatographic method. Incubations with di-(2-ethylhexyl)-phthalate and homogenates of blood, skin and caecum content, were performed with incubation volumes of 3, 3 and 2 ml, respectively.
The reaction was terminated by addition of 100 µl 350 µM 1-dodecanol (internal standard), 2-ethylhexanol was extracted into the hexane layer by vortexing for 1 minute. After centrifugation for 10 minutes at 2,500xg, the hexane layer was transferred into another tube and concentrated to approximately 100 µl with nitrogen, and the concentrated extract was transferred into a glass vial for gaschromatographic analysis. The extracts were stored at 0-10 °C until analysis.
Chemical hydrolysis was measured in incubations without homogenate at 51.5 µM (2-ethylhexyl lactate) and 12.5 µM (di-(2-ethylhexyl)-phthalate), after 15 and 60 minutes, respectively.
Because mono-(2-ethylhexyl)-phthalate is not further hydrolysed to phthalic acid and 2-ethylhexanol, it was not necessary to measure other metabolites than 2-ethylhexanol.
Duration and frequency of treatment / exposure:
Single application: 2-ethylhexyl lactate was incubated with homogenates for 3, 6, 9, 12 and 15 minutes and di-(2-ethylhexyl)-phthalate was incubated with homogenates for 12, 24, 36, 48 and 60 minutes.
Remarks:
Doses / Concentrations:
Final concentrations were 3.22, 6.44, 12.9, 25.8 and 51.5 µM for 2-ethylhexyl lactate and 3.13, 6.25 and 12.5 µM for di-(2-ethylhexyl)-phthalate.
No. of animals per sex per dose:
A single incubation per dose was performed. Different amounts of tissue protein or caecum content were used in the incubation experiments. Incubations with 2-ethylhexyl lactate: 1.23 µg/ml of liver protein, 0.29 µg/ml of small intestinal mucosal protein, 10.78 µg/ml of blood protein and 8.77 µg/ml of skin protein, and 10 µg/ml of caecum content. Incubations with di-(2-ethylhexyl)-phthalate: 73.6 µg/ml of liver protein, 33.4 µg/ml of small intestinal mucosal protein, 5390 µg/ml of blood protein and 263 µg/ml of skin protein, and 10 µg/ml of caecum content.
Control animals:
other: Chemical hydrolysis was determined in incubation experiments without homogenates. Blanks were incubations without test substances.
Positive control:
The esterase acitivities of the various homogenates towards the model substrate p-nitrophenylbutyrate were determined.
Details on study design:
- Dose selection rationale: Di-(2-ethylhexyl)-phthalate was incubated at only three concentrations because of a maximal solubility in the incubation mixture of approximately 13 µM.
Details on dosing and sampling:
Gas chromatocgraphic determination of 2-ethyl-1-hexanol:
After termination of the reactions, the samples were extracted, concentrated, transferred to GC-injection vials, stored at 0-10 °C, and transported to the Sponsor for the determination of 2-ethyl-1-hexanol by gas chromatographic analysis.
Statistics:
Calculations:
The amount of 2-ethylhexanol formed during the incubations was calculated by comparing the integrated peak area with the peak area of 2-ethylhexanol standards. Corrections were made by means of the internal standard (IS) 1-dodecanol, which was added to all incubations after termination of the reaction, but before the extraction procedure.
The concentration of 2-ethylhexanol in the incubation mixture was calculated as follows:
(peak area 2-ethylhexanol (sample) x peak area IS (standard)) / (regression coefficient x peak area IS (sample) x 10 x incub. volume)
The initial enzymatic rates of the slope at t=0. When maximal velocity was observed at another time point than the zero-time point, the slope at this time point was used. This was the case for incubations with 2-ethylhexyl lactate and homogenates of s.i. mucosa, blood, skin and caecum content at 3.22 µM, and with 2-ethylhexyl lactate and blood homogenate at 6.44 µM. The incubation time points with di-(2-ethylhexyl)-phthalate and blood homogenate at 3.13 µM were fitted with linear regression analysis.
The initial rates of hydrolysis were expressed as nmol/min/mg protein (liver, small interstinal mucosa, blood and skin) or nmol/min/g (caecum content).
Kinetic parameters (Km and Vmax) were calculated using the Michaelis Menten equation. The curve-fiitng program for the analysis of enzym kinetic dat "EZ-FIT" was used for this purpose. The weight factor used was 1/y².
Preliminary studies:
Validation of the incubation conditions:
The protein contents of the homogenates and the esterase activity towards the standard substrate p-nitro-phenylbutyrate are presented in Table 1.
To investigate possible metabolism of 2-ethylhexanol by the various homogenates, 2-ethylhexanol (20.7 µM) was incubated for 30 minutes. No difference was observed between incubations with and without homogenate (blank). When compared with non-incubated samples, the amount of 2-ethylhexanol recovered after 30 minutes was somewhat lower. However, this was also observed with the blank (incubation without homogenate). It was concluded that 2-ethylhexanol is not further metabolised at the conditions used. Therefore, measurement of 2-ethylhexanol could be used for determination of enzymatic parameters.
The final dilutions of the homogenates used in the main study, as given in section 'details on exposure', were based on preliminary studies establishing the proper amount of homogenate and incubation conditions used. Incubations with di-(2-ethylhexyl)-phthalate and homogenates of blood, skin and caecum content, were performed with incubation volumes of 3, 3 and 2 ml, respectively, to increase the sensitivity of 2-ethylhexanol detection.
The homogenate dilutions used in this study were found to have no effect on the gaschromatographic analysis of 2-ethylhexanol. The recovery of 2-ethylhexanol (20.7 µM), as added to the various homogenates, was 99-112 %. No chemical hydrolysis (incubations without homogenate) of 2-ethylhexyl lactate and di-(2-ethylhexyl)-phthalate was observed.
Type:
metabolism
Details on absorption:
Not applicable
Details on distribution in tissues:
Not applicable
Details on excretion:
Not applicable
Metabolites identified:
yes
Details on metabolites:
The initial rates of hydrolysis of 2-ethylhexyl lactate and di-(2-ethylhexyl)-phthalate at the various concentrations are presented in Table 2.
The kinetic parameters of the enzymatic hydrolysis of 2-ethylhexyl lactate and di-(2-ethylhexyl)-phthalate are presented in Table 3. No enzymatic parameters could be determined for di-(2-ethylhexyl)-phthalate and skin, because the skin homogenate showed no detectable formation of 2-ethylhexanol.

Table 1. Protein contents and esterase activity towards p-nitro-phenylbutyrate of the homogenates.

Homogenate

Protein content (n=2)

Esterase activity (n=3)

 

(mg/ml)

(nmol/min/mg protein)

Liver

36.8  ± 0.6

 590    ±  25

Mucosa s.i.

11.7  ± 0.3

4550    ± 135

Blood

53.9  ± 0.4

      6.2 ±     0.2

Skin

  2.6 ± 0.01

 110    ±     7

Caecum content

          -

 4001   

1caecum: nmol/min/g

Table 2. Rate of hydrolysis of 2-ethylhexyl lactate and di-(2-ethylhexyl)-phthalate to 2-ethylhexanol by homogenates of liver, small intestinal mucosa, blood, skin and caecum content at various substrate concentrations.

 

rate of formation of 2-ethylhexanol (nmol/min/mg protein)

substrate: 2-ethylhexyl lactate

2-EHL conc. (µM)

liver

mucosa

blood

skin

caecum1

3.22

 83

 281

   8.4

 10.1

 10.8

6.44

211

 769

 16.7

 21.8

 19.4

12.9

249

1092

 24.2

 39.4

 29.7

25.8

389

1215

 47.4

 61.3

 67.1

51.5

654

2114

108

103

128

 

 

rate of formation of 2-ethylhexanol (nmol/min/mg protein)

substrate: di-(2-ethylhexyl)-phthalate

DEPH conc. (µM)

liver

mucosa

blood

skin

caecum1

3.13

1.01

2.10

0.0139

<0.012

3.90

6.25

1.63

2.27

0.0267

<0.015

5.20

12.5

1.23

2.85

0.0503

<0.014

5.61

 

1caecum: nmol/min/g

Table 3. Kinetic parameters of the enzymatic hydrolysis of 2-ethylhexyl lactate and di-(2-ethylhexyl)-phthalate to 2-ethylhexanol by homogenates of liver, small intestinal mucosa, blood, skin and caecum content at various substrate concentrations.

2-ethylhexyl lactate

Km

(µM)

Vmax

(nmol/min/mg protein)

liver

 40

1100

mucosa

 25

2900

blood

175

  450

skin

 65

  225

caecum

175

  5501

 

di-(2-ethylhexyl)-phthalate

Km

(µM)

Vmax

(nmol/min/mg protein)

liver

 2

2

mucosa

 2

3

blood

90

     0.04 

skin

 -

-

caecum

 2

7

1caecum: nmol/min/g

Conclusions:
2-Ethylhexyl-S-lactate is hydrolysed effectively before, during or rapidly after absorption, even at high concentrations, in rat liver, small intestinal mucous, blood and skin tissue, as well as caecum content, under formation of L-lactic acid.
Executive summary:

The rate of hydrolysis of 2-ethylhexyl-S-lactate by homogenates of rat liver, small intestinal mucosa, blood, skin and caecum content was investigated. The esters were incubated with the homogenates at pH 7.0 and 37 °C. The amount of liberated 2-ethylhexanol was quantified at a series of time points, from which the initial rate of hydrolysis was determined. Using a concentration range of 3–50 µM, the enzyme kinetic parameters Km and Vmax were calculated.

Taking into consideration the enzymatic parameters of the various homogenates, it can be concluded that 2 -ethylhexyl-S-lactate will be effectively hydrolysed before, during or rapidly after absorption, even at high concentrations.

Endpoint:
basic toxicokinetics in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
For details and justification of read-across please refer to the attached report in section 13 of IUCLID.
Reason / purpose:
read-across source
Details on absorption:
36.6% of the administered dose was not absorbed and was excreted unchanged in the feces.
Details on distribution in tissues:
1.4 ± 0.6% of dose administered was found in the carcasses of the rats. With the notable exception of abdominal fat and liver, all of the tissues (brain, heart, large and small intestines, lungs, kidneys, spleen and testes) showed levels of radioactivity less than or equal to radioactivity found in the carcasses of the animals. This suggests that the radioactivity in the liver may be associated primarily with water-soluble metabolites whereas that found in the abdominal fat may be less-water-soluble metabolites or parent compound.
Details on excretion:
The mean total recovery of 14C from the 10 experimental animals given a single dose of 100mg/kg DEHT was 93.0 ± 2.2%. Most of the radioactivity was eliminated in the faeces (56.5 ± 12.1%); either unabsorbed as DEHT (36.6 ± 13.4%) or as metabolites (e.g. mono-(2-ethylhexyl)terephthalate (MEHT, 2.5%)) and polar metabolites. In the urine (31.9 ± 10.9%) as metabolic products of MEHT and 2-EH or in expired air (small amount as 14CO2, 3.6 ± 0.9%).
Excretion was fast and by 24 and 48 hours more than 95 and 99% of the total urinary and faecal radioactivity were excreted, respectively. Urinary and faecal excretion reached a peak at about 8 to 10 hours after administration. No further details on expiration of radioactive material was provided.

Metabolites identified:
yes
Details on metabolites:
Major metabolites in urine: terephthalic acid (TPA) and 2-Ethylhexanol (2-EH) and theirs respective metabolites and conjugates (glucuronide and sulphate).
Conclusions:
In an in vivo study, male rats (n=10) were administered a single dose of 100 mg/kg bw 14C di (2-ethylhexyl) terephthalate (DEHT) to investigate absorption and metabolism of DEHT. Urine, faeces and expired air were collected for up to 144 hours and analysed for the presence of radioactivity, and faeces and urine were analysed for unlabelled metabolites. The results of the study indicate that radioactivity was eliminated in faeces (56.5% of dose) primarily as unchanged DEHT, small amounts of mono(2-ethylhexyl)phthalate (MEHT) and polar metabolites; excreted in urine (31.9% of dose) principally as MEHT and metabolic products of 2-ethylhexanol (2-EH); and expired as 14CO2 (3.6% of dose). Less than 2%, of the administered radioactivity was found in the carcass. Small amounts of 14C were found in the tissues with the highest amounts found in liver and abdominal fat. In addition, the major metabolite of DEHT identified in urine included was terephthalic acid (TPA), 2-EH, MEHT and glucuronic and sulphuric acid conjugates.
Executive summary:

In an in vivo study, male rats (n= 10) were administered a single dose of 100 mg/kg bw 14C di (2-ethylhexyl)terephthalate (DEHT) to investigate absorption and metabolism of DEHT. Urine, faeces and expired air were collected for up to 144 hours and analysed for the presence of radioactivity, and faeces and urine were analysed for unlabelled metabolites.

The results of the study indicate that radioactivity was eliminated in faeces (56.5 % of dose) primarily as unchanged DEHT, small amounts of mono(2-ethylhrxyl) phthalate (MEHT) and polar metabolites; excreted in urine (31.9 % of dose) principally as MEHT and metabolic products of 2-ethylhexanol (2-EH); and expired as 14CO2 (3.6% of dose). Less than 2%, of the administered radioactivity was found in the carcass. Small amounts of 14C were found in the tissues with the highest amounts found in liver and abdominal fat. Excretion was fast and by 24 and 48 hours more than 95 and 99% of the total urinary and faecal radioactivity were excreted, respectively. Urinary and faecal excretion reached a peak at about 8 to 10 hours after administration. No further details on expiration of radioactive material was provided. In addition, the major metabolite of DEHT identified in urine included was terephthalic acid (TPA), 2-EH, MEHT and glucuronic and sulphuric acid conjugates.

This information is used in a read-across approach in the assessment of the target substance.

For justification of read-across please refer to the attached read-across report (see IUCLID section 13).

Description of key information

2-ethylhexyl-S-lactate, as all lactate esters, is rapidly hydrolyzed in-vivo into lactic acid and 2-ethylhexanol (see IUCLID section 7.9.4).

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.

The biochemistry of lactic acid has been reviewed and summarized in Sterenborg, 2007 (see the lactic acid registration dossier).

2-ethylhexanol is an alcohol that is metabolized by the normal alcohol metabolism pathways.

Regarding the read-across substance di (2-ethylhexyl)terephthalate (DEHT), Barber et al., 1994 published data from an in vitro study to determine the rate of hydrolysis of di (2-ethylhexyl)terephthalate (DEHT) by enzymes in the gut. The test substance was incubated in vitro with rat intestinal homogenates. The test substance was dissolved in methanol and was then incubated at 37 °C for up to 30 minutes with these gut homogenates. Periodically samples were drawn, and the intestinal enzymes were inactivated. Under the study conditions, it was determined that DEHT was metabolized by enzymes present in the gut to 2-EH and that the hydrolysis followed a first-order kinetics with a disappearance half-life of 53.3 minutes. The calculated stoichiometry at 30 min showed that 1.97 mol ethylhexanol is formed per mol DEHT.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential

Additional information

2-ethylhexyl-S-lactate, as all lactate esters, is rapidly hydrolyzed in-vivo into lactic acid and 2-ethylhexanol (see IUCLID section 7.9.4).

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.

The biochemistry of lactic acid has been reviewed and summarized in Sterenborg, 2007 (see the lactic acid registration dossier).

2-ethylhexanol is an alcohol that is metabolized by the normal alcohol metabolism pathways.

Regarding the read-across substance di (2-ethylhexyl)terephthalate (DEHT), Barber et al., 1994 published data from an in vitro study to determine the rate of hydrolysis of di (2-ethylhexyl)terephthalate (DEHT) by enzymes in the gut. The test substance was incubated in vitro with rat intestinal homogenates. The test substance was dissolved in methanol and was then incubated at 37 °C for up to 30 minutes with these gut homogenates. Periodically samples were drawn, and the intestinal enzymes were inactivated. Under the study conditions, it was determined that DEHT was metabolized by enzymes present in the gut to 2-EH and that the hydrolysis followed a first-order kinetics with a disappearance half-life of 53.3 minutes.The calculated stoichiometry at 30 min showed that 1.97 mol ethylhexanol is formed per mol DEHT.