Fatty acids, C16-18 and C18-unsatd., Me esters, epoxidized, reaction products with ethylene glycol

Endpoint summary

Link to relevant study record(s)

Reference

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Remarks:

performed with a group of esterified alcohols

Adequacy of study:

key study

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 the esters of methanol, ethylene glycol, glycerol, erythritol, pentaerythritol, adonitol, sorbitol, and sucrose in which all alcohols groups were esterified with oleic acid was studied. Various preparations of rat pancreatic juice, including pure lipase, were used as the sources of enzymes. To distinguish lipase form non-specific lipase activity, incubations containing sodium taurocholate and pancreatic juice treated with proteolytic enzymes were included.

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material (as cited in study report):methanol, ethylene glycol, glycerol, erythritol, pentaerythritol, adonitol, sorbitol, sucrose and oleic acid.
- Analytical purity: Oleic acid - 99% pure by Gas-liquid chromatography.
- Other: The alcohols, except ethylene glycol, were transesterified with an excess of methyl oleate to form the complete esters. Ethylene glycol dioleate was pre- pared by acylation of the alcohol with oleoyl chloride

Details on test animals or test system and environmental conditions:

Preparation of enzymes:
- Combination fluid - The common bile-pancreatic duct was cannulated at a point near its entrance into the duodenum. In the 24 -hr period following the cannulation, the combination of bile and pancreatic fluid was collected at 4°C. The lipolytic enzymes in this preparation retained their activity for at least 48 hr.
- Untreated pancreatic juice solids - Pancreatic juice, free of bile, was obtained by cannulating separately the bile and pancreatic ducts. The pancreatic fluid was collected at 4°C and freezed-dried. On the day of use, the solids were reconstituted, 3 mg/ mL, in 0.01 M histidine, pH 7.0.
- Treated pancreatic juice solids - To inactivate nonspecific lipase in the study, 150 mg of crude pancreatic juice solids and 1 mg of α-chymotrypsin was dissolved in 80 mL of 0.01 M histidine, pH 9.0.
- Purified Lipase - Lipase (EC 3.1.1.3) was isolated from the untreated pancreatic juice solids using the method of Vandermeers, A and Christophe, J (Biochim. Biophy. Acta. 154:110 -129).

Route of administration:

other: In incubation medium

Details on exposure:

- The esters were digested in a cylindrical, flat bottomed glass tube 30 mm (I.D.) x 90 mm. Four 3-mm indentations in the side wall prevented vortexing during stirring.
- Each digest contained 100 mg of substrates, 85 µmoles of CaCl2, 3.5 mg of histidine (final concentration 0.002 M), 152 mg of NaCl (final concentration 0.15 M), and the enzyme in a total volume of 10 mL. For incubations with untreated pancreatic juice, treated pancreatic juice and purified lipase incubations with and without 200 mg of sodium taurocholate (final concentration 37 mM) were included. Prior to the addition of the enzyme, all the components of the digest were stirred for 5 min.
- The digestions were carried out at pH 9.0 and at 27°C. Under these conditions all substrates were liquids.
- The pH was maintained with the aid of a pH stat by the addition of 0.02 N KOH.

Dose / conc.:

100 other: mg

Control animals:

other: sodium taurocholate

Details on dosing and sampling:

The rate of addition of alkali (0.02 N KOH) was linear during the first several minutes of digestion and was reported as the rate of hydrolysis: µmoles of free fatty acid released per minute per milligrams or per millilitre of enzyme preparation.

Metabolites identified:

no

Details on metabolites:

- The combination of bile-pancreatic fluid digested all substrates with the exception of sorbital hexaoleate and sucrose octaoleate. This failure of hydrolysis was obtained in spite of using 400 times as much combination bile-pancreatic fluid as was used when triolein was the substrate.
- When pancreatic juice without bile was used as a source of the enzymes, the esters that were hydrolysed depended on the presence or absence of added sodium taurocholate. In the absence of sodium taurocholate, only those substrates that contained less than four ester groups were hydrolysed. The addition of sodium taurocholate to the digest permitted the hydrolysis also of the substrates containing four and five ester groups. There were marked differences in the rates of hydrolysis of the oleate esters of methanol, ethylene glycol, and glycerol if taurocholate was not present, but these differences disappeared if this bile salt was added to the digest.
- In the absence of sodium taurocholate, the pattern of digestion by treated pancreatic juice was similar to that seen with the untreated pancreateic juice. However, in the presence of added sodium taurocholate, pancreatic juice that had been treated with the proteolytic enzyme could not digest any of the substrates.
- The final set of results was obtained with purified pancreatic lipase. If sodium taurocholate was not present, this enzyme hydrolysed methyl oleate, ethylene glycol dioleate, and triolein, but did not hydrolyse the substrates that contained more than three ester groups. The additions of sodium taurocholate blocked completely the hydrolytic activity of this enzyme.
- The observation that four and five ester groups were hydrolysed by certain preparations of pancreatic juice is attributed to the enzyme nonspecific lipase. This enzyme also hydrolysed esters of primary alcohols.
- Details are provided in 'any other information on results incl. tables'.

The rate of hydrolysis of the esters of the eight different alcohols by the various preparations of rat pancreatic juice is given in the table below. The numbers in parentheses are the volume or weight of enzyme preparation that was used in that particular digest.

Table 1. Relative rates of hydrolysis by rat pancreatic juice enzymes of the complete oleate esters of the listed alcohols.

	Pancreatic-Bile Juice	Untreated Pancreatic Juice		Treated Pancreatic Juice^a		Purified Lipase
	No TC^b	No TC	TC added	No TC	TC Added	No TC	TC added
	µmoles FFA min/ mL	µmoles FFA min/ mg		µmoles FFA min/ mg		µmoles FFA min/ mg
Methanol, 1^c	54    (0.05)^d	2.6 (1.2)	4.0 (1.2)	2.5 (1.2)	0 (1.2)	63 (0.02)	0 (0.3)
Ethylene glycol, 2	160 (0.025)	10  (0.3)	4.3 (0.3)	7.7 (0.3)	0 (0.3)	200 (0.01)	0 (0.1)
Glycerol, 3	2100 (0.005)	73 (0.075)	6.0 (0.15)	70 (0.06)	0 (0.3)	1900 (0.002)	0 (0.02)
Erythritol, 4	1.9   (1)	0   (6)	1.4 (3)	0 (6)	0 (6)	0 (0.1)	0 (0.1)
Pentaerythritol, 4	1.1   (2)	0   (6)	1.1 (3)	0 (6)	0 (6)	0 (0.1)	0 (0.1)
Adonitol, 5	0.53 (2)	0   (6)	0.25 (3)	0 (6)		0 (0.1)	0 (0.1)
Sorbitol, 6	0      (2)	0   (6)	0   (12)	0 (6)	0   (12)	0 (0.1)	0 (0.1)
Sucrose, 8	0      (2)	0   (6)	0   (12)	0 (6)	0   (12)	0 (0.1)	0 (0.1)

^a Nonspecific lipase was inactivated by treatment with α-chymotrypsin.

^b TC, Sodium taurocholate.

^c Number of ester groups.

^d The number in parentheses is the volume or weight of the enzyme preparation that was used.

Description of key information

In an in vitro metabolism study, the hydrolysis of oleic acid esterified methanol,  ethylene glycol, glycerol, erythritol, pentaerythritol, adonitol, sorbitol, and sucrose was studied. The hydrolysis was assessed in incubations with various preparations of rat pancreatic juice, including pure lipase. Incubations with sodium taurocholate were included to distinguish lipase from non-specific lipase activity. Lipase did not catalyse the hydrolysis of substances with more than three ester groups. Compounds with four and five ester groups were hydrolysed by the endogenous enzyme non-specific lipase. Compounds containing six or eight ester groups were not hydrolysed by the pancreatic juice (Mattson and Volpenhain 1972).

Additional information

Toxicokinetic information

The target substance can be divided into a mono-, di- and triadduct. With increasing size of the adduct, the physico-chemical properties vary, and thus the absorption potential decreases. The triadduct is not expected to be absorbed at all. After uptake, the mono- and diadduct are considered to be metabolized by phase I and II enzymes, leading to an excretion of the degradation/conjugation products mainly via bile.

1.    Chemical and physico-chemical description of the substance

The substance to be registered is a reaction product of fatty acids (C16-18 and C18-unsaturated) with methanol and ethylene glycol (EG). It can be described with the CAS # 211450-54-3 (CAS name: Fatty acids, C16-18 (even numbered, C18 unsaturated), Me esters, epoxidized, reaction products with ethylene glycol (1:2)).

The reaction process starts with a fatty acid methyl ester. Due to the high percentage of oleic acid in the fatty acid composition, the main product at this stage is oleic acid methyl ester. Besides, methyl esters of saturated fatty acids are present, but would not further react because they lack a double-bond. In the second and third production step, this unsaturated double-bond is epoxidized and ring-opened with EG. Due to the remaining hydroxy group of the EG component, the monoadduct can further react with a second epoxidized oleic acid methyl ester to form a diadduct. In the following, the two major components of the target substance – mono- and diadduct with oleic acid – are discussed. According to analytical investigations, the monoadduct accounts for appr. 69% of the target substance the diadduct for appr. 25%, and a triadduct detected is present at 6%.

Description of the physico-chemical properties:

- physical state (20°C): liquid

- vapour pressure (20°C):<0.000001 hPa (<0.0001 Pa)

- molecular weight: appr. 375 Da (monoadduct)

- log Kow (25 °C): 5.29 (monoadduct), 11.77 (diadduct) and 18.26 (triadduct)

- water solubility: 53-216 mg/L at 20 °C (experimental data with 1 or 10 g/L weighted sample)

- Boiling point: substance decomposes at about 200°C

 

The substance is characterized by a lipophilic nature, a low volatility and relatively low water solubility.

2.     Toxicokinetic assessment

No experimental data on absorption, metabolism and distribution are available for the substance. Based on the structure and the physico-chemical properties of the substance, the toxicokinetic behaviour can be evaluated.

The toxicokinetic behaviour of the target substance is dominated by the metabolism of the monoadduct, which accounts for 69%. The influence of the triadduct is considered to be negligible due to the low percentage as well as the very low predicted absorption (high log Kow of 18.26 at 25°C, high molecular weight).

 2.1 Absorption:

The amount that is expected to be absorbed decreases in general with an increase in molecular weight / log Kow. Taking this into account, the uptake by eaqch possible route of exposure is considered to be negligible for the triadduct, which has a high molecular weight and a log Kow of 18.26. The potential for absorption is little better for the diadduct: though smaller in the molecular weight, it still has a high log Kow (11.77) and consequently a very low water solubility. The water solubility of the target substance was measured in an experiment, and resulted in a value of 53-216 mg/L (for 1 or 10 g/L added). It was concluded that the mono-adduct is primarily responsible for the water solubility. Altogether, the proportion of mono-, di- and triadduct present in the target substance is expected to shift even more towards the monoadduct in the body, as the absorption potential of the di- and triadduct is very limited.

With regard to absorption after inhalation, the target substance has a low vapour pressure of <0.0000019 Pa (< 0.0001 Pa) and decomposes at about 200°C, indicating that inhalation as a vapour will be negligible. If the substance reaches the respiratory tract, passive diffusion is unlikely due to the high log Kow, the relatively low water solubility and the rather high molecular weight. Theoretically, a systemic uptake of the mono-/diadduct could take place after micellular solubilisation.

In the gastro-intestinal tract, the lipophilic target substance with limited water solubility and a relatively high molecular weight (>= 375 Da) is unlikely to be absorbed by passive diffusion. An uptake due to micellular solubilisation could be expected for the monoadduct and also for the diadduct. Altogether, a worst case oral absorption of 100% is assumed for the target substance.

With a molecular weight of >500 Da and a log Kow of 11.77 or 18.26, the di- and triadduct are unlikely to be absorbed dermally. Even the size and the lipophilicity of the monoadduct (375 Da, log Kow = 5.29) is not favourable for a dermal uptake. It is expected that the monoadduct enters the stratum corneum, but the transfer to the epidermis (and thus the bioavailability after dermal contact) will be limited.

Altogether, a worst case dermal absorption of 10% is assumed for the target substance.

 2.2 Metabolism and Excretion:

Once absorbed, a metabolic reaction could in principle take place: at the epoxide- / the ether site or involving the free hydroxy group of the target substance.

A metabolic reaction at the ether site is unlikely, as ethers are considered to be relatively physiologically stable.

The free hydroxy group at the ethylene glycol site is a good substrate for conjugation by metabolic Phase II enzymes (e.g. glutathione transferase). This reaction improves the water solubility of the molecule and enables the excretion via bile or urine. In consideration of the high molecular weight (>500 Da after conjugation), an excretion via bile is more likely.

The ester function is likely to be metabolized like dietary fats. As shown by Mattson and Volpenhain, esters of fatty acids and different alcohols (methanol, ethylene glycol, glycerol…) are potential substrates for endogenous lipases in the bile-pancreatic fluid (Mattson and Volpenhain 1972). These enzymes catalyse the hydrolysis to the corresponding alcohol and acid. As cleavage products of the target substance, methanol as well fatty acid ether with ethylene glycol is formed.

The mammalian metabolization of methanol is well investigated. It occurs mainly in the liver, where methanol is initially converted to formaldehyde, which is in turn converted to formate. Formate is converted to carbon dioxide and water. In humans and monkeys, the oxidation to formaldehyde is mediated by alcohol dehydrogenases and basically limited to the capacity of those enzymes. In rodents, the oxidation to formaldehyde predominantly employs the catalase-peroxidase pathway which is of less capacity than the ADH-pathway in humans, but on the other hand produces oxygen radicals which may be involved into the developmental effects in rodents which - in contrast to humans - tolerate high methanol levels without signs of CNS or retinal toxicity. The last oxidation step, conversion of formate to carbon dioxide employes formyl-tetrahydrofolate synthetase a co-enzyme, is of comparably low capacity in primates which leads to a low clearance of formate, possibly also from sensitive target tissues (such as CNS and the retina) .

The fatty acid ether with EG formed after ester cleavage could in theory be further metabolized like dietary fatty acids, at least up to the site where EG is bound. In this oxidation process, carbon dioxide and water are formed. However, the fatty acid ether with EG could well be water-soluble enough to be excreted via bile, especially if the free hydroxy groups are further conjugated by metabolic phase II enzymes.

In consideration of the limited uptake and the enzymatic processes in place for metabolic degradation, the target substance is not expected to have a bioaccumulation potential.