Monopentaerythritol tetraesters and dipentaerythritol hexaesters of 2-ethylhexanoic and n-valeric acids

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Reason / purpose for cross-reference:

reference to same study

Lipids are not only structural building blocks of cells and tissues but at the same time suppliers of C atoms for a number of biosynthetic pathways as well as carriers of essential fatty acids and fat-soluble vitamins. In addition, fatty acids are precursors of prostaglandins and other eicosanoids and therefore have important metabolic functions.

Fatty acids can be divided into three groups, saturated, monounsaturated, and polyunsaturated fatty acids.

Each class of fatty acids has a preferential specific role.

- Saturated fatty acids (medium or long-chain) are more devoted to energy supply, but one should not forget their specific structural role.

- The polyunsaturated fatty acids of the n–3 and n–6 families have very important structural and functional roles and ideally should not be utilized for energy purposes.

 

Table 1:

Role of different classes of fatty acids

Fatty acids	Energy	Structure	Function
Medium-chain saturated fatty acids	+++	0	0
Long-chain fatty acids
Saturated	++	++	(+)
Monounsaturated	++	++	(+)
Polyunsaturated
Linoleic or n-6 family	0	+++	+++
Linolenic or n-3 family	0	+++	+++

 0, +, ++, +++ : Emphasis of contribution, increasing in rank order

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

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:

absorption

Principles of method if other than guideline:

The mechanism of the intestinal fat absorption has been studied with 14C labeled fat in rats with the intestinal lymph duct cannulated.

GLP compliance:

no

Radiolabelling:

yes

Remarks:

14C labeled fat

Species:

rat

Strain:

not specified

Sex:

not specified

Route of administration:

oral: gavage

Duration and frequency of treatment / exposure:

single oral exposure
(at least 18 hours after surgery)

Remarks:

Doses / Concentrations:
A) 0.5 mL corn oil + 2.5 mg active palmitic acid-1-14C
B) 0.5 mL corn oil transesterified with 2.5 mg active palmitic acid-1-14C
C) 0.5 mL hydrolysed corn oil + 2.5 mg active palmitic acid-1-14C

No. of animals per sex per dose / concentration:

5-6

Control animals:

no

Details on absorption:

24 hours after administration of the different fats the mean recovered activities in lymph were as following:
A) 0.5 mL corn oil + 2.5 mg active palmitic acid-1-14C: 57.0 %
B) 0.5 mL corn oil transesterified with 2.5 mg active palmitic acid-1-14C: 61.7 %
C) 0.5 mL hydrolysed corn oil + 2.5 mg active palmitic acid-1-14C: 62.3 %

In all three groups of experiments maximum recoveries were found after 24 hours, i.e. 80.9, 85.0 and 87.5 % of the activity given.
Free fatty acids administered alone or together with glycerides appear in the lymph in glycerides and phospholipids.
No free fatty acids or soaps appear in the lymph.
The intestinal wall supplies a quantitatively important part of phospholipids to the blood during fat absorption.
The recoveries in the lymph of the fat fed varied widely. Diarrhea occured in some animals especially after feeding hydrolysed corn oil.

Details on distribution in tissues:

Absorbed fat is mainly transported via lymphatic channels to the systemic circulation whether fed as glycerides or as fatty acids.

Details on metabolites:

A complete hydrolysis of the fat in the intestinal lumen might occur in the rat.

The proportions of neutral fat and phospholipids in the lymph were in all three cases about the same. 90% of the fatty acids were present in the neutral fat and the remaining 10 % in phospholipids. The neutral fat consisted chiefly of triglycerides; cholesterol and cholesterol esters representing only a minor part of this fraction. No free fatty acids or soaps appeared in the lymph.

The results indicated that glycerides might be completely hydrolysed in the intestinal lumen of the rat and then resynthesized in the intestinal wall.

Conclusions:

Mean absorption rate of corn oil combined with palmitic acid was between 57 - 62 %.

Reference 3

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

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

Principles of method if other than guideline:

The lipolytic activity of human gastric and duodenal juice against medium chain and long chain triglycerides was compared.

GLP compliance:

no

Radiolabelling:

yes

Remarks:

Glyceryl trioctanoate-1-14C

Species:

human

Route of administration:

other: in vitro testing

Enzymatic Lipolysis by Gastric and Duodenal Juice:

All samples of gastric juice showed lipolytic activity against trioctanoin and triolein. Hydrolysis of emulsified trioctanoin was greater than of emulsified triolein. Hydrolysis of unemulsified trioctanoin was less and more variable.

Duodenal juice was more active, even against unemulsified trioctanoin and triolein. Duodenal juice was more active against unemulsified substrate than gastric juice against emulsified substrate.

Table 1: Hydrolysis of trioctanoin and triolein*

	Substrate and form (μmoles)	Hydrolysis (%)
		Trioctanoin	Triolein
Gastric juice	30, unemulsified	21	1
	60, emulsified	33	16
Duodenal juice	30, unemulsified	40	34
	45, emulsified	42	35
	105, emulsified	45	36

*Gastric or duodenal juice (1 mL) was incubated (1 hour, continuous shaking, 37ºC) with 1 mL of buffer and unemulsified substrate or 1 mL of substrate emulsified in 10 mM sodium taurodeoxycholate, pH6.

pH Optimum

In the presence of bile acids, gastric lipolytic activity against trioctanoin had a broad pH optimum, between 4 and 7. The lipolytic activity of duodenal juice had a sharper pH optimum, between 6 and 8. The pH optimum was lower for short chain triglycerides, indicating that pH optimum values for lipases must be defined for a particular substrate.

Chain Length Specificity

Lipolysis rates increased with decreasing chain lengths for pure triglycerides.

Tributyrin was cleaved more rapidly than trihexanoin which in turn was cleaved more rapidly than trioctanoin (ratio of rates, 100:69:53). Because the pH optimum of gastric lipase is lower for short chain triglycerides than for MCT, trihexanoin and tributyrin were cleaved much more rapidly than, for example, trioctanoin at pH5.

Esterification and Fatty Acid Acceptors by Gastric and Duodenal Lipases

Gastric and duodenal lipases did not induce esterification of the fatty acid acceptor, glyceryl 2 -monooleyl ester, by octanoic acid over the pH range of 2 to 6. However, it was esterified by oleic acid in the presence of gastric juice, duodenal juice, or pancreatic fistula juice when bile acids were added. Esterification, calculated by disappearance of titratable fatty acid, was confirmed by TLC which showed the formation of compounds having the mobilities of a monoether monoester and a monoether diester. Control incubations without enzyme showed no loss of oleic acid or appearance of new lipids by TLC. To determine the amount of disubstituted and trisubstituted glyceryl derivatives which were formed, 14C-labeled glyceryl 2 -monooleyl ether was used and the products of the reaction were examined by zonal scanning. The glyceryl 2 -monooleyl ether was not cleaved during the incubation procedure. The amounts of ester bonds formed estimated by titration and by zonal scanning were in good agreement.

Products of Lipolysis and Positional Specificity

The specificity of pancreatic lipase for the 1 -ester bond in LCT has been demonstrated previously by establishing the formation of 2 -monoglycerides and fatty acid as end products of lipolysis. This procedure cannot be used for MCT because medium chain 2 -monoglycerides are either cleaved by pancreatic lipase or rapidly isomerized to the 1 -isomer which is rapidly hydrolyzed or both. Indeed, chromatographic examination of the products of hydrolysis of trioctanoin-14C showed only a small fraction of monoglyceride present.

Table 2: Products of hydrolysis of trioctanoin by gastric juice*

	Radioactivity distribution** (%)				Lipolysis (%)
	Monoglyceride	Diglyceride	Fatty acid	Triglyceride
Buffer (control)	0	0	0	100	0
Gastric juice 1 mL	3	26	26	44	34
	3	28	24	43	33
	4	28	25	43	36
	4	28	25	43	36
Duodenal juice
0.4 mL	4	9	15	72	26
0.5 mL	4	14	20	62	40

*Glyceryl trioctanoate-1-¹⁴C was added to 1 mL of emulsified trioctanoin (60 μmoles) and incubated for 1 hour at 37ºC with buffer (blank) or gastric or duodenal juice. The reaction mixture was extracted and a 50 μL aliquot was analyzed by TLC and zonal scanning. A 3 mL aliquot was titrated to quantify fatty acids liberated.

Discussion:

The work confirmed extensive literature showing that gastric juice contains lipolytic activity, that ingested triglyceride is hydrolyzed in the stomach, even after pancreatic diversion, that lipase may be demonstrated histochemically in gastric mucosa, and that gastric mucosal homogenates have lipolytic activity. Pancreatic lipase has some activity at the pH of gastric content, which is between pH6 and pH3 in normal subjects.

Reference 4

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

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:

absorption

Principles of method if other than guideline:

The absorbability of the fatty acid moiety of the complete, oleate esters of alcohols containing from one to six hydroxyl groups was determined by the fat balance technique in adult rats. Similarly, the absorbability of sucrose octaoleate and sucrose monooleate was determined.

GLP compliance:

no

Radiolabelling:

no

Species:

rat

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS

- Source: no data
- Age at study initiation: young adult
- Weight at study initiation: approx. 200 g
- Housing: Individually in cages with raised screen bottoms
- Diet (e.g. ad libitum): ad libitum

Route of administration:

oral: feed

Duration and frequency of treatment / exposure:

10 Days, diet ad libitum

Remarks:

Doses / Concentrations:
10% and 25 % of dietary fat

Details on absorption:

The fatty acids of the compounds containing less than four ester groups, methyl oleate, ethylene glycol dioleate, glycerol trioleate, and sucrose monooleate, were almost completely absorbed. As the number of ester groups was increased - erythritol and pentaerythritol tetraoleate and xylitol pentaoleate - the absorbability decreased. The fatty acids of sorbitol hexaoleate and sucrose octaoleate were not absorbed. These differences in absorbability are related to the activity and specificity of the lipolytic enzymes in the lumen of the intestinal tract.

Test fat	Percentage of dietary fat	Absorbability [%]
Methyl Oleate	10	100
	25	96
Ethylen Glycol Oleate	10	100
	25	92
Glycerol Trioleate	100	(100)
Erythritol Tetraoleate	10	-
	25	72
Pentaerythritol Tetraoleate	10	90
	25	64
Xylol Pentaoleate	10	50
	25	24
Sorbitol hexaoleate	10	0
	25	0
Sucrose Octaoleate	5	0
	10	0
	15	0
Sucrose Monooleate	5	100
	10	100
	15	100

Conclusions:

Absorption rates were between 0 an 100 %, depending on the amount of ester groups present in the substance fed. Pentaerythritole tetraoleate had an absorption rate of 90% (10% of dietary fat) and 64% (25% of dietary fat), respectively. Erythritole tetraoleate had an absorption rate of 72% (25% of dietary fat).

Reference 5

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Only secondary data Short review on metabolism from previous publications.

Objective of study:

metabolism

The metabolism of Medium chain triglycerides in the canine is a process whereby lipases from the buccal cavity and pancreas release the fatty acids in the gastrointestinal tract where they are absorbed. Unlike long chain triglycerides (LCT), where long chain fatty acids (LCFA) form micelles and are absorbed via the thoracic lymph duct, MCFA are most often transported directly to the liver through the portal vein and do not necessarily form micelles. Also, MCFA do not re-esterify into MCT across the intestinal mucosa. MCFA are transported into the hepatocytes through a carnitine-independent mechanism, and are metabolized into carbon dioxide, acetate, and ketones through b-oxidation and the citric acid cycle.

Description of key information

Oral absorption

Based on available data, absorption after oral ingestion is predicted to be limited. Due to the rather high number of ester bonds, only slow hydrolysis in the GIT is expected to occur, resulting in hydrolysis products that may be readily absorbed. If absorption of the intact parental compound or the respective metabolites occurs, it is predicted to cause a low order of systemic toxicity.

Dermal absorption

The low water solubility, the high molecular weight, the high log Pow value and the lack of potential for skin irritation / corrosion indicate that dermal uptake in humans is likely to be low. Again, a low order of systemic toxicity is expected, if absorption via the dermal route of exposure occurs.

Absorption by inhalation

A systemic bioavailability in humans after inhalation exposure cannot be excluded, e.g. after inhalation of aerosols with aerodynamic diameters below 15 μm. The absorption rate is not expected to be higher than that following oral exposure. Applying a worst-case approach, the absorption potential via the inhalation route of exposure is assumed to be the same as via the oral route of exposure. After absorption of the parent compound or its predicted hydrolysis products, a low order of systemic toxicity is expected.

Distribution and accumulation

The available information indicates that the intact parent compound is not assumed to distribute throughout the body due to limited absorption. In contrast, wide distribution within the body is expected for the hydrolysis products pentaerythritol and the fatty acids. However, no significant bioaccumulation of both the parent substance and its anticipated hydrolysis products in adipose tissue is expected.

Metabolism

Esters of fatty acids are hydrolysed to the corresponding alcohol and fatty acids by ubiquitously expressed esterases. It is assumed, however, that the hydrolysis rate is low as a result of steric hindrance due to the number of ester bonds and the complexity of the parent molecule. If hydrolysis occurs, a major metabolic pathway for linear and simple branched fatty acids is the beta-oxidation for energy generation. In contrast, pentaerythritol (PE) is absorbed rapidly and mainly excreted unchanged without metabolic conversion.

Excretion

A low absorption rate is expected via the gastrointestinal tract. Thus the biggest part of the ingested substance is assumed to be excreted in the faeces. Following the potential hydrolysis of the parent molecule, the fatty acids are not expected to be excreted to a significant degree via the urine or faeces but excreted via exhaled air as CO2. Pentaerythritol is not metabolized but excreted mainly unchanged via urine.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Additional information

Basic toxicokinetics

There are no studies available in which the toxicokinetic behaviour of Monopentaerythritol tetraesters and dipentaerythritol hexaesters of 2-ethylhexanoic and n-valeric acids has been investigated. Therefore, in accordance with Annex VIII, Column 1, Item 8.8 of Regulation (EC) No. 1907/2006 (REACH) and with the Guidance on information requirements and chemical safety assessment Chapter R.7c: Endpoint specific guidance (ECHA, 2017), assessment of the toxicokinetic behaviour was conducted based on relevant available information. This comprises a qualitative assessment of the available substance-specific data on physico-chemical and toxicological properties and taking into account further available information on the polyol esters category from which data was used for read-across to cover data gaps.

Monopentaerythritol tetraesters and dipentaerythritol hexaesters of 2-ethylhexanoic and n-valeric acids is a UVCB substance as it contains esters of pentaerythritol and dipentaerythritol with saturated linear and branched fatty acids. It is a liquid which is poorly water soluble with a water solubility in the range of 1.5 - 5.1 mg/L with a molecular weight ranging from 472.61 to 1011.45 g/mol, a log Pow of 6.74 to > 10 and a vapour pressure of <0.0001 Pa at 25 °C (QSAR).

General considerations on absorption

Absorption is a function of the potential for a substance to diffuse across biological membranes. The most useful parameters providing information on this potential are the molecular weight, the octanol/water partition coefficient (log Pow) value and the water solubility. The log Pow value provides information on the relative solubility of the substance in water and lipids (ECHA, 2017).

Oral absorption

The smaller the molecule, the more easily it will be taken up. In general, molecular weights below 500 are favourable for oral absorption (ECHA, 2017). As the molecular weight of Monopentaerythritol tetraesters and dipentaerythritol hexaesters of 2-ethylhexanoic and n-valeric acids ranges between 473 and 1011 g/mol, absorption of the molecule in the gastrointestinal tract is not likely. Absorption after oral administration is also unexpected when the “Lipinski Rule of Five” (Lipinski et al. (2001), refined by Ghose et al. (1999)) is applied. The log Pow value is well above the given range of ¬0.4 to 5.6 and the molecular weight is for three of the four main constituents well above 500 g/mol.

The log Pow > 6.7 of Monopentaerythritol tetraesters and dipentaerythritol hexaesters of 2-ethylhexanoic and n-valeric acids is favourable for absorption by micellar solubilisation, as this mechanism is of importance for highly lipophilic substances (log Pow > 4), which are poorly soluble in water (1 mg/L or less). However, for large molecules the gastrointestinal absorption is not likely to occur. In the gastrointestinal (GI) tract, metabolism prior to absorption via enzymes of gut microflora or in the GI mucosa may occur. In fact, after oral ingestion, fatty acid esters with glycerol (glycerides) are rapidly hydrolised by ubiquitously expressed esterases and almost completely absorbed (Mattsson and Volpenhein, 1972a). On the contrary, lower rates of enzymatic hydrolysis in the GIT were demonstrated for compounds with more than 3 ester groups (Mattson and Volpenhein, 1972a, b). The in vitro hydrolysis rate of Pentaerythritol esters was about 2000 times slower in comparison to glycerol esters (Mattson and Volpenhein, 1972a, b). Moreover in vivo studies in rats demonstrated the incomplete absorption of the compounds containing more than three ester groups. This decrease became more pronounced as the number of ester groups increased, probably the results of different rates of hydrolysis in the intestinal lumen (Mattson and Volpenhein, 1972c).

The available data on oral toxicity of structurally related analogue substances are also considered for assessment of the oral absorption. Acute oral toxicity studies conducted with a variety of analogue source substances covering C-chain distributions of C5-9, C5-0, C6, and C9, with saturated and linear as well as branched alkyl moieties, did not show any signs of systemic toxicity at concentrations of up to 2000 mg/kg bw in rats. In addition, the structurally related substance Pentaerythritol ester of pentanoic acids , mixed esters with pentaerythritol, isopentanoic and isononanoic acid (CAS 146289-36-3) showed no systemic effects up to the highest dose group in a 90-day repeated dose subchronic toxicity study resulting in a NOAEL ≥ 1000mg/kg bw/day. Therefore, if absorption of the intact parent compound or the respective metabolites occurred, it resulted in a low order of systemic toxicity. These findings are supported by investigations of the short-term repeated dose toxicity (28-day or screening study) performed with Fatty acids, C7, C8, C10 and 2-ethylhexanoic acid, tetraesters with pentaerythritol (CAS 68424-31-7), Dipentaerythritol with fatty acids, C5 and C9 iso (CAS 647028-25-9), and Hexanoic acid, 2-ethyl-, 2,2-bis [ [(2-ethyl-1-oxohexyl)oxy] methyl] -1,3-propanediyl ester (CAS 7299-99-2). All results available suggest that the test substance is of low systemic toxicity, either due to low toxicity potency or by a low absorption in combination with a low systemic toxicity.

In general, after oral ingestion, aliphatic esters of polyhydroxy alcohols (Polyol) and 1 – 6 fatty acids will undergo chemical changes in the gastro-intestinal fluids as a result of enzymatic hydrolysis. Pentaerythritol (PE, parental polyol) as well as the fatty acids (C10-C28) will be formed, even though it was shown in-vitro that the hydrolysis rate of Pentaerythritol tetraoleate when compared with the hydrolysis rate of the triglyceride Glycerol trioleate was very slow (Mattson and Volpenhein, 1972). The physical-chemical characteristics of the hydrolysis products (e.g. physical form, water solubility, molecular weight, log Pow, vapour pressure) will be different from those of the parent substance before absorption into the blood stream takes place, and hence the predictions based upon the physical-chemical characteristics of the parent substance do no longer apply. However, also for both hydrolysis products (fatty acids and pentaerythritol), it is anticipated that they will be readily absorbed in the gastro-intestinal tract.

The highly lipophilic fatty acids will be absorbed by micellar solubilisation (Ramirez et al., 2001). A study by Mattson and Nolen (1972) investigated the potential for absorption of the fatty acid moiety of the complete oleate esters of alcohols containing one to six hydroxyl groups. The fatty acids of the compounds containing less than four ester groups were almost completely absorbed. As the number of ester groups was increased (erythritol and pentaerythritol tetraoleate and xylitol pentaoleate) the potential for absorption of the fatty acids decreased but was still present. Pentaerythritol, having a low molecular weight (136.15 g/mol) and being a highly water-soluble substance (25 g/L, OECD SIDS, 1998), will readily dissolve into the gastrointestinal fluids. After oral administration of 10 mg/kg 14C-labled PE to mice, almost half of the administered dose was absorbed from the gastrointestinal tract within 15 minutes (DiCarlo et al., 1965).

In summary, the physical-chemical properties of Monopentaerythritol tetraesters and dipentaerythritol hexaesters of 2-ethylhexanoic and n-valeric acids and relevant data from available literature on fatty acid esters with more than three ester bonds do not indicate rapid hydrolysis before absorption of the parent substance.

Dermal absorption

Similar to oral absorption, dermal absorption is favoured for small molecules. In general, a molecular weight below 100 g/mol favours dermal absorption, while a molecular weight above 500 g/mol may be considered too large (ECHA, 2017). The molecular weight of Monopentaerythritol tetraesters and dipentaerythritol hexaesters of 2-ethylhexanoic and n-valeric acids ranges between 472.62 and 1011.48 g/mol.

If a substance is a skin irritant or corrosive, damage to the skin surface may enhance penetration through the skin. As Monopentaerythritol tetraesters and dipentaerythritol hexaesters of 2-ethylhexanoic and n-valeric acids was not tested for skin irritation or corrosion, read-across from a variety of structural analogue substances was applied. The structurally related source substances chosen cover C-chain distributions of C5 to C9, with saturated and linear as well as branched alkyl moieties. None of the source substances was identified as skin irritant or corrosive to skin. Therefore, also Monopentaerythritol tetraesters and dipentaerythritol hexaesters of 2-ethylhexanoic and n-valeric acids is not considered to be irritating or corrosive to skin in humans and an enhanced penetration of the substance due to local skin damage can be excluded.

The dermal permeability coefficient (Kp) can be calculated from log Pow and molecular weight (MW) applying the following equation described in US EPA (2012), using the Epi Suite software:

log(Kp) = -2.80 + 0.66 log Pow – 0.0056 MW

Using DermWin v2.02 the Kp and dermal flux were calculated for the four main constituents of the target substance using the SMILES codes and entering a water solubility of 0.0051 mg/cm³ and a log Kow of 6.74 (selected edge values).

Constituent	Molecular weight	Molecular formula	Kp [cm/h]	Dermal flux [mg/cm²h]
C5 PE tetra	472.62	C25 H44 O8	0.105	0.0102
2EHA PE tetra	640.95	C37 H68 O8	0.0119	0.00345
C5 DiPE hexa	759.00	C40 H70 O13	0.0026	0.00161
2EHA DiPE hexa	1011.48	C58 H106 O13	9.95e-005	0.000315

The calculated dermal flux rates indicated very low dermal absorption potential.

For substances with a log Pow above 4, the rate of dermal penetration is limited by the rate of transfer between the stratum corneum and the epidermis, but uptake into the stratum corneum will be high. For substances with a log Pow above 6, the rate of transfer between the stratum corneum and the epidermis will be slow and will limit absorption across the skin, and the uptake into the stratum corneum itself is also slow. The substance must be sufficiently soluble in water to partition from the stratum corneum into the epidermis (ECHA, 2017). The water solubility of Monopentaerythritol tetraesters and dipentaerythritol hexaesters of 2-ethylhexanoic and n-valeric acids was measured to be in the range of 1.5 – 5.1 mg/L at 20 °C, and log the Pow was estimated to be 6.74 to > 10. Based on the available physical data, dermal uptake is expected to be low.

The available data on dermal toxicity of the structurally related substance Dipentaerythritol with fatty acids, C5 and C9 iso (CAS 647028-25-9) is also considered for the assessment of dermal absorption. At a limit dose of 2000 mg/kg bw in rats no signs of systemic toxicity were observed. Moreover, in a subchronic (90-day) dermal repeated dose toxicity study performed with the Fatty acids, C5-9, tetraesters with pentaerythritol (CAS 67762-53-2), no toxicologically relevant effects were noted up to and including the highest dose level of 2000 mg/kg bw/day in male and female rats.

Overall, the calculated low dermal absorption potential, the low-moderate water solubility, the high molecular weight (> 470 g/mol), the high log Pow values and the fact that the substance is not irritating or corrosive to skin indicates that dermal uptake of Monopentaerythritol tetraesters and dipentaerythritol hexaesters of 2-ethylhexanoic and n-valeric acids in humans will be very limited.

Inhalation absorption

Monopentaerythritol tetraesters and dipentaerythritol hexaesters of 2-ethylhexanoic and n-valeric acids has a low vapour pressure of < 0.0001 Pa at 25 °C thus being of low volatility. Therefore, under normal use and handling conditions, inhalation exposure and thus availability for respiratory absorption of the substance in the form of vapours, gases or mists is not expected to be significant. However, the substance may be available for respiratory absorption in the lung after inhalation of aerosols, if the substance is sprayed. In humans, particles with aerodynamic diameters below 100 μm have the potential to be inhaled. Particles with aerodynamic diameters below 50 μm may reach the thoracic region and those below 15 μm the alveolar region of the respiratory tract (ECHA, 2017).

Lipophilic compounds with a log Pow > 4, that are poorly soluble in water (1 mg/L or less) can be taken up by micellar solubilisation. Esterases present in the lung lining fluid may also hydrolyse the substance, hence making the resulting alcohol and fatty acids available for respiratory absorption. Due to the high molecular weight of the parent substance, absorption is driven by enzymatic hydrolysis of the ester to the respective metabolites and subsequent absorption. However, as discussed above, hydrolysis of fatty acid esters with more than 3 ester bounds is considered to be slow (Mattson und Volpenhein, 1972a) and the possibility of the target substance to be hydrolysed enzymatically to the respective metabolites and their respective absorption is considered to be low as well.

The available data on inhalation toxicity of the three structurally related substances Fatty acids, C5-10 (linear and branched), tetraesters with pentaerythritol (CAS 68424-31-7), Fatty acids, C5-9, tetraesters with pentaerythritol (CAS 67762-53-2) and Fatty acids, C5-9, mixed esters with dipentaerythritol and pentaerythritol (CAS 85536-35-2) are also considered for assessment of inhalation absorption. An acute inhalation toxicity studies conducted with these three source substances the acute LC50 was found to be > 5 mg/L air. In one additional study with Fatty acids, C5-9, tetraesters with pentaerythritol (CAS 67762-53-2) the LC50 was found to be > 4.06 mg/L air, which was the highest attainable concentration in this study.

Moreover, in a subchronic (90-day) repeated (inhalation) dose toxicity study performed with the Fatty acids, C5-9, tetraesters with pentaerythritol (CAS 67762-53-2), no toxicologically relevant effects were observed up to and including the highest dose level of 0.5 mg/mL air in male and female rats. Therefore, respiratory absorption of the test substance is considered not to be higher than absorption through the intestinal epithelium.

Overall, a systemic bioavailability of Monopentaerythritol tetraesters and dipentaerythritol hexaesters of 2-ethylhexanoic and n-valeric acids in humans is considered likely after inhalation but not expected to be higher than following oral exposure. It is important to note that due to the low vapour pressure, exposure via the inhalation route is expected only if aerosols/droplets of an inhalable size (i.e. < 15 µm) are generated, e.g. in spray applications.

Accumulation

Highly lipophilic substances in general tend to concentrate in adipose tissue, and - depending on the conditions of exposure - may accumulate. Although there is no direct correlation between the lipophilicity of a substance and its biological half-life, it is generally accepted that substances with high log Pow values have long biological half-lives. The high log Pow of 6.74 to >10 of Monopentaerythritol tetraesters and dipentaerythritol hexaesters of 2-ethylhexanoic and n-valeric acids is therefore indicative of the potential to accumulate in adipose tissue (ECHA, 2017). However as the absorption of the substance is considered to be very low, the potential of its bioaccumulation is very low as well.

As will be discussed in the section metabolism below, esters of pentaerythritol and fatty acids will undergo slow esterase-catalysed hydrolysis, leading to the hydrolysis products pentaerythritol and the fatty acids. The log Pow of the hydrolysis product pentaerythritol is < 0.3 and it is highly soluble in water (25 g/L) (OECD SIDS, 1998). Consequently, there is no potential for pentaerythritol to accumulate in adipose tissue. The second hydrolysis product (fatty acids) can be stored as triglycerides in adipose tissue depots or be incorporated into cell membranes. Fatty acids are also required as a source of energy. Thus, stored fatty acids underlie a continuous turnover as they are permanently metabolised and excreted. Bioaccumulation of fatty acids only takes place, if their intake exceeds the caloric requirements of the organism.

Overall, the available information indicates that no significant bioaccumulation in adipose tissue of the parent substance and its hydrolysis products is anticipated.

Distribution

Distribution within the body through the circulatory system depends on the molecular weight, the lipophilic character and water solubility of a substance. In general, the smaller the molecule, the wider it will be distributed. If the molecule is lipophilic, it is likely to distribute into cells and the intracellular concentration may be higher than its extracellular concentration particularly in fatty tissues (ECHA, 2017). Furthermore, the concentration of a substance in blood or plasma and subsequently its distribution depends on the rates of absorption. As has been discussed above, absorption of Monopentaerythritol tetraesters and dipentaerythritol hexaesters of 2-ethylhexanoic and n-valeric acids is considered low based on its physical-chemical characteristics (low water solubility and high molecular weight).

However, it can be anticipated that a small amount of the substance will be subject to slow enzymatic hydrolysis, leading to the hydrolysis products pentaerythritol and various fatty acids. Pentaerythritol is highly water-soluble (25 mg/L and log Pow < 0.3) with a low molecular weight. It will be distributed in aqueous fluids by diffusion through aqueous channels and pores. There is no protein binding assumed and it is distributed poorly in fatty tissues (OECD SIDS, 1998). The fatty acids are also distributed in the organism and can be taken up by different tissues. They can be stored as triglycerides in adipose tissue depots or they can be incorporated into cell membranes (Masoro, 1977).

Metabolism

Esters of fatty acids are hydrolysed to the corresponding alcohol and fatty acid by esterases (Fukami and Yokoi, 2012). Depending on the route of exposure, esterase-catalysed hydrolysis takes place at different places in the organism. After oral ingestion, esters are hydrolised already in the gastro-intestinal fluids. In contrast, esters which are absorbed through the pulmonary alveolar membrane or through the skin enter the systemic circulation directly before they are transported to the liver where hydrolysis will basically take place.

The hydrolysis of fatty acid esters containing more than 3 ester groups is assumed to be slow as already discussed above. In in- vivo studies in rats, a decrease in absorption was observed with increasing esterification grade. For example, for Pentaerythritol tetraoleate ester an absorption rate of 64% and 90% (25% and 10% of dietary fat) was observed, respectively, while an absorption rate of 100% was observed for Glycerol trioleate when ingested at 100% of dietary fat (Mattson and Nolen, 1972). In addition it has been shown in-vitro that the hydrolysis rate of pentaerythritol tetraoleate was lower when compared with the hydrolysis rate of the triglyceride Glycerol trioleate (Mattson and Volpenhein, 1972a). Nonspecific lipase completely hydrolysed erythritol tetraoleate to free erythritol (Mattson and Volpenhein, 1972b).

Thus, Monopentaerythritol tetraesters and dipentaerythritol hexaesters of 2-ethylhexanoic and n-valeric acids is expected to be hydrolysed to pentaerythritol and the respective fatty acids by esterases, even though the hydrolysis rate is expected to be low.

Pentaerythritol (PE) with a molecular weight of 136 g/mol is absorbed rapidly but excreted unchanged. DiCarlo et al. (1965) reported that 10 mg/kg 14C-labled PE orally administered to mice was absorbed and excreted rapidly from the gastrointestinal tract. Almost half of the administered dose left the gastrointestinal tract within 15 minutes and 68% of the dose appeared as unchanged PE in the urine and faeces after 4 hours already. Fatty acids are stepwise degraded by means of beta -oxidation based on enzymatic removal of C2 units in the matrix of the mitochondria in most vertebrate tissues. The C2 units are cleaved as acyl-CoA, the entry molecule for the citric acid cycle. For the complete catabolism of unsaturated fatty acids such as oleic acid, an additional isomerization reaction step is required.

Using the OECD QSAR Toolbox v4.1 metabolites were predicted for the four main constituents of the target substance:

		# Metabolites	# Metabolites	# Metabolites
Constituent	Molecular weight	Skin metabolism simulator	Rat liver S9 simulator	Microbial metabolism simulator
C5 PE tetra	472.62	2	7	23
2EHA PE tetra	640.95	4	9	41
C5 DiPE hexa	759.00	2	6	42
2EHA DiPE hexa	1011.48	4	9	64

Excretion

On the basis of its low absorption, one route of excretion for Monopentaerythritol tetraesters and dipentaerythritol hexaesters of 2-ethylhexanoic and n-valeric acids is expected to be by biliary excretion via faeces. Hydrolysis of the parent compound yields fatty acids and pentaerythritol. The fatty acids will be metabolised for energy generation or stored as lipids in adipose tissue or used for further physiological processes, e.g. incorporation into cell membranes (Lehninger, 1970; Stryer, 2002). Therefore, the fatty acid components are not expected to be excreted to a significant degree via the urine or faeces but excreted via exhaled air as CO2 or stored as described above. Pentaerythritol is not metabolized but excreted unchanged via urine. 10 mg/kg 14C-labled pentaerythritol orally administered to mice was absorbed from the gastrointestinal tract rapidly and excreted via urine. Almost half of the administered dose left the gastrointestinal tract within 15 minutes and 68% of the dose appeared as unchanged PE in the urine and faeces after 4 hours (DiCarlo et al., 1965). The amount found in faeces was assumed to be contamination from urine due to the setup of the metabolic cages. Additionally, Kutscher (1948) found 85 - 87% of unaltered PE in the urine of humans ingesting pentaerythritol.

References

DiCarlo F.J., Hartigan J.M. Jr., Couthino, C.B. and Phillips, G.E. (1965). Absorption, distribution and excretion of Pentaerythritol and Pentaerythritol Tetranitrate by mice. Proceedings of the Society for Experimental Biology and Medicine. 118: 311-314

ECHA (2017). Guidance on information requirements and chemical safety assessment - Chapter 7c: Endpoint specific guidance; Version 3; June 2017; European Chemicals Agency, Helsinki, Finland

Fukami, T. and Yokoi, T. (2012). The Emerging Role of Human Esterases. Drug Metab Pharmacokinet 27(5): 466-477.

Ghose et al. (1999). A Knowledge-Based Approach in Designing Combinatorial or Medicinal Chemistry Libraries for Drug Discovery. J. Comb. Chem. 1 (1): 55-68.

Kutscher, W. (1948). Über das Verhalten des Pentaerythrits im Stoffwechsel. Hoppe-Seyler´s Zeitschrift für physiologische Chemie , Volume 283 (5-6)

Lehninger, A.L. (1970). Biochemistry. Worth Publishers, Inc.

Lipinski et al. (2001). Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Del. Rev. 46: 3-26.

Masoro (1977). Lipids and lipid metabolism. Ann. Rev. Physiol.39: 301-321.

Mattson F.H. and Nolen G.A. (1972). Absorbability by rats of compounds containing from one to eight ester groups. J Nutrition, 102: 1171-1176.

Mattson F.H. and Volpenhein R.A. (1972a). Hydrolysis of fully esterified alcohols containing from one to eight hydroxyl groups by the lipolytic enzymes of rat pancreatic juice. J Lip Res 13, 325-328

Mattson F.H. and Volpenhein R.A. (1972b). Digestion in vitro of erythritol esters by rat pancreatic juice enzymes. J Lip Res 13, 777-782

Mattson F.H. and Volpenhein R.A. (1972c). Rate and extent of absorption of the fatty acids of fully esterified glycerol, erythritol, xylitol, and sucrose as measured in thoracic duct cannulated rats. J Nutr 102, 1177-1180

OECD SIDS (1998). Pentaerythritol, CAS 115-77-5, SIDS Initial Assessment Report for 8th SIAM, http://www.inchem.org/documents/sids/sids/115775.pdf, (last accessed 2017-09-08)

Ramirez et al. (2001). Absorption and distribution of dietary fatty acids from different sources. Early Human Development 65 Suppl.: S95–S101.

Stryer, L. (2002). Biochemistry. 5th Edition. W.H. Freeman, New York, USA. ISBN 0‐7167‐3051‐0.