Fatty acids, C8-10, oxybis(2-hydroxy-3,1-propanediyl) esters

Endpoint summary

Link to relevant study record(s)

Reference

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1 - 11 Mar 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Objective of study:

other: hydrolysis in intestinal fluid simulant

Qualifier:

equivalent or similar to guideline

Guideline:

other: EFSA Note for Guidance for Food Contact Materials Annex 1 to Chapter III MEASUREMENT OF HYDROLYSIS OF PLASTICS MONOMERS AND ADDITIVES IN DIGESTIVE FLUID SIMULANTS (30/07/2008)

Deviations:

yes

Remarks:

no hydrolysis test in saliva and gastric juice simulants

GLP compliance:

not specified

Radiolabelling:

no

Species:

other: pancreatin from porcine pancreas

Strain:

not specified

Sex:

not specified

Details on test animals or test system and environmental conditions:

INTESTINAL FLUID SIMULANT
- Description: the intestinal fluid simulant contains pancreatin from porcine pancreas as hydrolytic catalyst.
- Preparation: reported to have been done according to the guideline.
- Source of Pancreatin: SIGMA P7545 Lot SLBD06040V; 8 x USP; CAS: 8049-47-6

Route of administration:

other: mixing

Vehicle:

other: tetrahydrofuran (THF) (presolution)

Details on exposure:

PREPARATION OF INTERNAL STANDARD SOLUTION:
An internal standard solution with a concentration of approx. 0.6 mg/g n-heptadecane (C17 n-alkane; Aldrich Chemistry) in heptane:pyridine (vol. 2:1) was prepared.

PREPARATION OF BLANK SAMPLE:
A 100 mL sample of intestinal fluid simulant was extracted (see “Details on dosing and sampling”).

PREPARATION OF TMS REAGENT FOR SILYLATION DERIVATISATION:
N, O bis-trimethylsilyl trifluoro-acetamide (BSTFA) was mixed 100:1 (v/v) with trimethyl chlorosilane (TMCS) for preparation of the TMS reagent.

PREPARATION OF CALIBRATION SAMPLE:
50 mg of the calibration sample (trade name) was weighted into a glass container with screw cap, 12 mL of internal standard solution was added and the container was tightly closed and reweighed. After mixing, 500 µL of the sample solution was transferred to a crimp vial and 100 µL TMS reagent was added. Then, the crimp vial was closed using a TEFLON® coated seal and mixed well. The vial was placed in a thermostatic heating block for 15 min at 60 °C. Approx. 1 µL of the sample solution was injected into the GC system

HYDROLYSIS WITH INTESTINAL-FLUID SIMULANT:
For the hydrolysis investigation, 50 mg of test item was dissolved in 2 mL tetrahydrofuran (THF). THF was gently removed in a stream of compressed air and a thin film of the product was formed on the bottom of the flask. 50 mL of simulant without enzyme was added and the test substance was dispersed in the aqueous phase while stirring for 2 min by magnetic stirring. Samples were taken after 0, 1, 2 and 4 h.

Duration and frequency of treatment / exposure:

0, 1, 2 and 4 h

Dose / conc.:

50 other: mg

No. of animals per sex per dose / concentration:

not applicable, the test was performed in triplicates

Control animals:

other: for GC: blank samples from test digestive simulants and samples of reference materials (parent substance and hydrolysis products)

Details on dosing and sampling:

EXTRACTION OF HYDROLYSATES
1. 1 mL of formic acid was added to the 250 mL Bluecap flask containing the hydrolysate while stirring.
2. 50 mL of methyl tert-butyl ether (MTBE) was added and the stirring continued for 2 min.
3. 25 mL of P-ether (40-60) was added and the stirring continued for 15 sec.
4. While stirring 10-20 mL of 96% ethanol was added and the stirring was stopped.
5. The phase separation was improved by very gently adding up to 10 mL ethanol to the organic upper phase.
6. The clear upper phase was transferred to a 250 mL round bottomed flask.
7. Steps 2-6 were repeated twice.
8. The solvent was removed by means of a rotatory evaporator (approx. 70 °C, 15 mmHg)
9. 12 mL of internal standard (Heptadecane) solution was weighed into the round-bottomed flask containing the dried hydrolysate.
10. A 500 mL aliquot of the solution was transferred to a 2 mL crimp vial and 100 µL TMS reafgen was added.
11. The crimp vial was sealed and heated to 60 °C for 15 min.
12. A 1 µL aliquot was injected into the GC system by means of an auto sampler.

DETERMINATION OF HYDROLYSIS PRODUCTS (GC ANALYSIS)
- Principle:
The hydrolysis products of the test substance were extracted by means of tetrahydrofuran (THF), methyl tert-butyl ether (MTBE), petroleum ether (bp. 40-60 °C) and ethanol (96%). Formic acid was added prior to extraction to convert fatty acid salt (soaps) into free fatty acids. Free hydroxyl groups and free acids in the extract were protected by means of trimethyl silyl groups (silylation) and non-hydrolysed diglycerol monooleate was quantified by means of GC.
- Calibration standard:
A sample of pure test material was analysed.
- Internal standard:
The calibration was carried out by an internal standard calibration procedure. The internal standard was n-Heptadecane (C17 n-alkane).
- Apparatus:
GC instrument: Perkin Elmer Clarus 600 equipped with an autosampler, FID detector and a programmable split/splitless injector operated in the cold split mode.
GC Integration system: Perkin Elmer Turbochrom Workstation ver. 6.3.2.0646
- Blank sample preparation:
To identify any blank peaks in the GC chromatograms100 ml samples of intestinal fluid simulant was extracted and analysed by means of GC.
- Optimisation of instrumentation:
The GC system was optimised for analysis of silylated glycerides. This was carried out by optimising the instrumental conditions in order to comply with the repeatability values given for analysis of glycerides in mono- and diglyceride concentrates given in American Oil Chemists’ Society, Champaign Illinois; Official Methods and Recommended Practices of the AOCS 5 th. ed.; Official Method Cd 11b-91: “Determination of Mono- and Diglycerides by Capillary Gas Chromatography”.
- Calculation of components.
Calculation of the content of hydrolysis components in the sample was carried out using the formula:
w/w% component = [Response factor (component) x Area (component) x mg Internal standard x 100%] / [Area (Internal standard) x mg Sample prior to hydrolysis]

- Determination of response factors:
The Response factor for the main component of the hydrolysis products (= parent substance), inclusing all diglcerol monooleate isomers, was determined experimentally relative to n-heptadecane. The Response factor (Rf)for a retention time range of main components (X) in the chromatogram was calculated using the following formula:
Rf (X) = [Area (Internal standard) x mg Calibration Sample] / [Area (x) x mg Internal Standard], where mg Internal Standard is calculated from: mg Internal standards (IS) = IS solution (g) x concentration of IS solution (F) [mg/g C17 alkane]

CONFIRMATION ASSAYS
The identification of all components was carried out by comparing retention times of peaks in chromatograms.

RECOVERY
The overall recovery of the method was determined by a simple recovery experiment. The test substance was analysed in triplicate according to the analytical method using a sample of intestinal fluid without enzyme added and with 0 h of hydrolysis. A mean recovery of 95.8% was found (see table 2 under “Any other information on results incl. tables”).

Statistics:

Mean values of triplicates were calculated.

Type:

other: ester hydrolysis in intestinal fluid simulant

Results:

84, 90, and 94.3 % after 1, 2 and 4 h, respectively

Details on absorption:

not applicable

Details on distribution in tissues:

not applicable

Details on excretion:

not applicable

Metabolites identified:

no

Table.1 Hydrolysis of the test item with intestinal-fluid simulant

Hydrolysis study
Concentration of internal standard (I.S.) solution (F): 0.6422 mg/g C17 alkane
Experiment No.	1535	1536	1537	1539	1540	1541	1545	1546	1547	1548	1549	1550
Time of hydrolysis	0 hours			1 hours			2 hours			4 hours
Data:
mg sample	51.80	49.31	47.10	51.34	48.26	48.16	53.46	49.25	50.50	51.77	52.73	49.98
I.S. solution (g)	9.5952	9.7220	9.5071	9.5686	9.4879	9.4755	9.3912	9.5178	9.5252	9.5616	9.5043	9.4770
mg I.S (calculated)	6.162	6.243	6.106	6.145	6.093	6.083	6.031	6.112	6.117	6.1414	6.104	6.086
Area (I.S.)	336862	352376	335097	338291	340040	340192	337381	342221	340950	344348	358211	338235
Area (X)	1892921	1891083	1863661	368325	369106	339607	247633	229627	225207	132125	149457	132971
Rf	1.226	1.226	1.226	1.218	1.218	1.214	1.214	1.214	1.214	1.216	1.216	1.216
Results:
% test item	81.95	83.31	88.39	15.87	16.69	15.36	10.05	10.11	9.71	5.53	5.87	5.82
Mean	84.6			16.0			10.0			5.7

 

Table 2. Recovery experiment (0 hours without enzyme added)

Recovery study
Concentration of internal standard solution (F): 0.6422 mg/g C17 alkane
Experiment No.	1542	1543	1544
Time of hydrolysis	0 hours
Data:
mg sample	47.27	50.36	50.48
Internal standard solution (g)	9.4822	9.4909	9.4631
mg internal standard solution (calculated)	6.090	6.095	6.077
Area (internal standard)	330728	340028	334857
Area (X)	2036744	2227788	2201882
Rf	1.209	1.209	1.209
Results:
% PGE O80	95.91	95.87	95.71
Mean	95.8

Conclusions:

Interpretation of results: no bioaccumulation potential based on study results

Description of key information

Oral absorption

Based on available data, absorption after oral ingestion is predicted to be limited as rapid hydrolysis in the gastrointestinal tract is expected to occur. Resulting hydrolysis products are expected to be readily absorbed.

Dermal absorption

The high 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.

Inhalative absorption

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.

Distribution and accumulation

The available information indicates that the intact parent compound is not assumed to distribute throughout the body due to fast hydrolysis. In contrast, wide distribution within the body is expected for the hydrolysis products diglycerol 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 that the hydrolysis rate is fast. If hydrolysis occurs, a major metabolic pathway for linear fatty acids is the β-oxidation for energy generation. In contrast, diglycerol is absorbed rapidly and mainly excreted unchanged without metabolic transformation.

Excretion

A fast rate of hydrolysis is expected via the gastrointestinal tract. Thus, the substance is considered to be excreted only to a minor extent. 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. Diglycerol is not metabolised 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 the target substance Fatty acids, C8-10, oxybis(2-hydroxy-3,1-propanediyl) esters (CAS No. 92044-91-2) 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), an assessment of the toxicokinetic behavior 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 source substances from which data was used for read-across to cover data gaps.

Fatty acids, C8-10, oxybis(2-hydroxy-3,1-propanediyl) esters (CAS No. 92044-91-2) is an UVCB substance composed of a variety of different esterification products of C8 fatty acid, C10 fatty acid and diglycerol. Its composition is characterised by highly variable concentrations of the different constituents. The fatty acids feedstock used to produce the substance contains C8 fatty acid in an amount of 50 – 60% while the content of C10 fatty acid is in the range of 40 – 50%. Additional variation is originating from different esterification degrees. The content of diglycerol monoesters, diesters and triesters is 30 – 50%, 20 – 40% and 5 – 15%, respectively. The target substance is a liquid with a water solubility of 1.265 g/L (Emery, 2017a). The molecular weight of its various constituents ranges between 292.37 – 628.90 g/mol, the log Pow was estimated to be > 1 (QSAR models: VEGA / ALogP version 1.0.0 and EPI Suite / KOWWIN version 1.68) and the calculated vapour pressure is < 0.0001 Pa at 20 °C (QSAR, ARChem SPARC. version 4.6).

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 g/mol are favorable for oral absorption (ECHA, 2017). As the molecular weight of the target substance ranges between 292.37 and 628.90 g/mol, absorption in the gastrointestinal tract is expected only for the low molecular weight constituents, i.e. those originating from C8 (caprylic acid) fatty acid and/or the monoesters. Absorption after oral administration is also expected when the “Lipinski Rule of Five” (Lipinski et al., 2001; Ghose et al., 1999) is applied as all rules are fulfilled except for the log Pow, which is likely above the given range of ‑0.4 to 5.6. The log Pow of > 1 suggests that the high molecular weight constituents might also be absorbed by micellar solubilisation, as this mechanism is of importance for highly lipophilic substances (log Pow > 4), who are poorly soluble in water (1 mg/L or less). Although the water solubility of the complete target substance has been measured to be 1.265 g/L, it is expected that the high molecular weight constituents are much less water soluble and hence exhibit an even higher log Pow.

The potential of a substance to be absorbed from the gastrointestinal tract (GIT) may be influenced by chemical changes taking place in gastrointestinal fluids, for instance due to metabolism by gastrointestinal flora or by enzymes released into the gastrointestinal tract or by hydrolysis. This is especially relevant for substances with a high solubility in water, as is the case for the target substance. These changes will alter the physico-chemical characteristics of the substance and hence predictions based upon the physico-chemical characteristics of the parent substance may in some cases no longer apply (ECHA, 2017). After oral ingestion, fatty acid esters with glycerol (glycerides) are rapidly hydrolised by ubiquitously expressed esterases and almost completely absorbed (Mattsson and Volpenhein, 1972a; Michael and Coots, 1971). On the contrary, a lower rate of enzymatic hydrolysis in the GIT was demonstrated for compounds with more than 3 ester groups (Mattson and Volpenhein, 1972a,b).

As a consequence of the hydrolysis of fatty acid esters, the respective alcohol as well as the fatty acids are formed. In the case of the target substance fatty acids, C8-10, oxybis(2-hydroxy-3,1-propanediyl) esters (CAS No. 92044-91-2), the predicted metabolites are the free C8 fatty acid (caprylic acid) and C10 fatty acid (capric acid) and the polyglycerol diglycerol. This assumption was confirmed by an in vitro hydrolysis test performed with the source substance 1 ‘oleic acid, monoester with oxybis(propanediol)’ (CAS No. 49553-76-6) in intestinal fluid simulant, which was conducted according to EFSA Note for Guidance for Food Contact Materials Annex 1 to Chapter III “Measurement of hydrolysis of plastics monomers and additives in digestive fluid simulants (30/07/2008)” (DuPont, 2013). After incubation with the intestinal fluid, the remaining content of oleic acid, monoester with oxybis(propanediol) was extracted and measured by gas chromatography. The in vitro experiment demonstrated that approximately 94% of the total oleic acid, monoester with oxybis(propanediol) was hydrolysed by intestinal fluid simulant within 4 hours at 37°C (DuPont, 2013). Both hydrolysis products (fatty acids and diglycerol) are anticipated to be rapidly absorbed in the gastro-intestinal tract. The highly lipophilic fatty acids are absorbed by micellar solubilisation (Ramirez et al., 2001), whereas the alcohol is readily dissolved into the gastrointestinal fluids and absorbed from the gastrointestinal tract. Moreover, data from metabolism studies with fatty acid-labelled polyglycerol esters have shown that more than 90% of triglycerol moieties from respective esters were absorbed. Furthermore, it was shown that hydrolysis of the polyglycerol esters occurred to a large extent prior to absorption (Michael and Coots, 1971).

Therefore, hydrolysis of the target substance, i.e. the parent compound, is expected to be high resulting in low systemic exposure to the parent compound itself, but absorption of metabolites, caprylic acid, capric acid and the polyglycerol diglycerol, from the gastrointestinal tract is expected to be high.

The available data on acute and repeated dose oral toxicity are also considered for assessment of oral absorption. Acute oral toxicity investigations have been performed with the source substances Hexanedioic acid, mixed esters with decanoic acid, 12-hydroxyoctadecanoic acid, isostearic acid, octanoic acid, 3,3'-oxybis[1,2--ropanediol] and stearic acid (CAS No. 130905-60-1) and 1,2,3-Propanetriol, homopolymer, diisooctadecanoate (CAS No. 63705-03-3). A single administration of 2000 mg/kg bw (Sasol, 1990a) and 5000 mg/kg bw (BASF, 1988a) test material to male and female rats did not induce any mortality or any signs of systemic toxicity. Moreover, in a subacute repeated dose toxicity study performed with the source substance Hexanedioic acid, mixed esters with decanoic acid, 12-hydroxyoctadecanoic acid, isostearic acid, octanoic acid, 3,3'-oxybis[1,2-propanediol] and stearic acid (CAS No. 130905-60-1) and a combined repeated dose and reproduction / developmental screening with source substance 1,2,3-Propanetriol, homopolymer, diisooctadecanoate (CAS No. 63705-03-3) oral exposure of male and female rats over a period of 4 weeks did not yield any toxicologically relevant adverse effects and hence resulted in NOAEL values of ≥ 1000 mg/kg bw/day, corresponding to the highest doses tested (Sasol, 1990b; BASF, 2013). However, it must be noted that the lack of systemic toxicity observed in the studies can also be attributed to a low degree of absorption. The lack of systemic toxicity is therefore only an indicator rather than a proof of no or a low toxicity after oral exposure. Based on these results, no final conclusions on the oral absorption potential is possible.

In summary, the physico-chemical properties of the target substance discussed above and experimental data on in vitro hydrolysis and oral toxicity obtained with adequate source substances and further supported by literature data do indicate hydrolysis to the respective fatty acids (caprylic acid and capric acid) and the alcohol component diglycerol before absorption in the GIT can take place. On the basis of the above mentioned data, oral absorption of the test material is not expected to occur in a significant amount. The hydrolysis products, however, are predicted be readily absorbed.

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). As the molecular weight of the target substance ranges between 292.37 and 628.90 g/mol, a dermal absorption cannot be excluded.

If the substance is a skin irritant or corrosive, damage to the skin surface may enhance penetration (ECHA, 2017). To this regard primary skin irritation studies conducted with the structurally related source substances Hexanedioic acid, mixed esters with decanoic acid, 12-hydroxyoctadecanoic acid, isostearic acid, octanoic acid, 3,3'-oxybis[1,2-propanediol] and stearic acid (CAS No. 130905-60-1) and 1,2,3-Propanetriol, homopolymer, diisooctadecanoate (CAS No. 63705-03-3) showed no sign of skin irritation (Sasol, 1990c) or only minor erythema (BASF, 1988b). Therefore, also the target substance 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.

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). As the water solubility of the target substance is 1.265 g/L and the log Pow is estimated to be > 1, dermal uptake is considered to be low. However, it cannot be excluded and finally assessed based on these physico-chemical parameters alone. The low potential for dermal absorption is also supported by QSAR calculations of the dermal absorption rate. Calculations with the Episuite 4.1, DERMWIN 2.02 tool yielded dermal permeability constants Kp of 1.93E-04 cm/h (medium low) and 2.49E-06 cm/h (very low) for the low molecular weight and high molecular weight constituents, respectively. Based on these values, the substance has a low potential for dermal absorption.

Overall, the calculated low dermal absorption potential, the high molecular weight (> 100 g/mol), the high log Pow value of > 1 and the fact that the substance is not irritating to skin all lead to the conclusion that dermal uptake of Fatty acids, C8-10, oxybis(2-hydroxy-3,1-propanediyl) esters in humans is limited.

Inhalation absorption

Fatty acids, C8-10, oxybis(2-hydroxy-3,1-propanediyl) esters (CAS No. 92044-91-2)  has a low vapour pressure of less than 0.0001 Pa at 20 °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. Particles deposited in the nasopharyngeal/thoracic region will mainly be cleared from the airways by the mucocilliary mechanism and swallowed (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. As discussed above, due to the high molecular weight of the parent substance, absorption is mainly driven by enzymatic hydrolysis of the parent compound. The respective metabolites (caprylic acid, capric acid and diglycerol) are then subsequently readily absorbed. Overall, a systemic bioavailability of the target substance  in humans is considered likely after inhalation of aerosols with aerodynamic diameters below 15 μm.

Accumulation and distribution

Distribution of a compound within the body through the circulatory system depends on the physico-chemical properties of the substance; especially the molecular weight, the lipophilic character and the water solubility. In general, the smaller the molecule, the wider is the distribution. 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. Furthermore, the concentration of a substance in blood or plasma and subsequently its distribution depends on the rates of absorption. 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 > 1 of Fatty acids, C8-10, oxybis(2-hydroxy-3,1-propanediyl) esters is therefore indicative of the potential to accumulate in adipose tissue (ECHA, 2017). However as the absorption of the parent substance is considered to be very low as a consequence of rapid hydrolysis in the gastric juice, the potential of its bioaccumulation is very low.

As discussed in length under oral absorption, esters of polyglycerols, e.g. diglycerol, and fatty acids will undergo fast esterase-catalysed hydrolysis, leading to the hydrolysis products polyglycerol, e.g. diglycerol, and fatty acids. Therefore, an assessment of distribution and accumulation of the hydrolysis products is considered more relevant. Diglycerol is a rather small substance (MW = 166.18 g/mol) of high water solubility and log Pow < 0 (Danish QSAR database, 2013). It will be distributed in aqueous compartments of the organism by diffusion through aqueous channels and pores and may also be taken up by different tissues (Michael and Coots, 1971). There is no protein binding assumed and it is distributed poorly in fatty tissues. Consequently, there is no potential to accumulate in adipose tissue. After being absorbed, fatty acids are (re-)esterified along with other fatty acids into triglycerides and released in chylomicrons into the lymphatic system. Fatty acids of carbon chain lengths ≤ 12 may be transported directly to the liver via the portal vein as the free acid bound to albumin, instead of being re-esterified. Chylomicrons are transported in the lymph to the thoracic duct and subsequently to the venous system. On contact with the capillaries, enzymatic hydrolysis of chylomicron triacylglycerol fatty acids by lipoprotein lipase takes place. Most of the resulting fatty acids are taken up by adipose tissue and re-esterified into triglycerides for storage. Triacylglycerol fatty acids are likewise taken up by muscle and oxidized to derive energy or they are released into the systemic circulation and returned to the liver, where they are metabolised, stored or re-enter the circulation (IOM, 2005; Johnson, 1990; Johnson, 2001; Lehninger et al., 1998; NTP, 1994; Stryer, 1996; WHO, 2001). There is a continuous turnover of stored fatty acids, as they are constantly metabolised to generate energy and then excreted as CO₂. Accumulation of fatty acids takes place only if their intake exceeds the caloric requirements of the organism.

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

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 hydrolysed 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. Thus, Fatty acids, C8-10, oxybis(2-hydroxy-3,1-propanediyl) esters is expected to be hydrolysed to diglycerol, caprylic acid and capric acid. The hydrolysis of fatty acid esters containing more than 3 ester groups is assumed to be slow as already discussed above. In vivo studies in rats demonstrated a decrease in absorption with increasing esterification grade. For example, for Pentaerythritol tetraoleate 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).

In an in vitro enzymatic digestion method using fresh pancreatic juice plus bile described by King et al. (1971), fatty acid labelled polyglycerol esters were studied. Thin layer chromatography (TLC) and radio-assay procedures were used to determine the distribution of 14C among the products of digestion. After enzymatic digestion of an oleate-labelled polyglycerol ester, 89 - 92% of the recovered 14C was present as free oleic acid, whereas the remaining 8 and 11% was unhydrolysed or partially hydrolysed starting material. Hydrolysis of the eicosanoate-labelled polyglycerol ester was much slower than the oleate ester and only 21% of the 14C was recovered as free eicosanoic acid (Michael and Coots, 1971). This finding is further supported by the in vitro experiment conducted with the source substance Oleic acid, monoester with oxybis(propanediol) (CAS No. 49553-76-6). The study shows that approximately 94% of the substance is hydrolysed by an intestinal fluid simulant within 4 h at 37 °C. The main isomer of Oleic acid, monoester with oxybis(propanediol) is fully hydrolysed within 1 h of hydrolysis, whereas other positional isomer of the test item have a lower rate of hydrolysis (DuPont, 2013).

After hydrolysis of the target substance Fatty acids, C8-10, oxybis(2-hydroxy-3,1-propanediyl) esters, the first hydrolysis products, caprylic acid (C8 fatty acid) and capric acid (C10 fatty acid), are degraded by mitochondrial β-oxidation which takes place in most animal tissues and uses an enzyme complex for a series of oxidation and hydration reactions, resulting in the cleavage of acetate groups in the form of acetyl-CoA. The alkyl chain length is reduced by 2 carbon atoms during each β-oxidation cycle. Alternative pathways for oxidation can be found in the liver (ω-oxidation) and the brain (α-oxidation). Iso-fatty acids such as isooctadecanoic acid have been found to be activated by acyl coenzyme A synthetase of rat liver homogenates and to be metabolised to a large extent by ω-oxidation. Each C2-unit resulting from β-oxidation enters the citric acid cycle as acetyl-CoA, through which they are completely oxidized to CO₂ (CIR, 1983, 1987; IOM, 2005; Lehninger, 1998; Stryer, 1996; Matulka, 2009). The second hydrolysis product, the polyol diglycerol, is assumed to be rapidly excreted and metabolism via cleavage of the ether bond to glycerol will not occur as for the related triglycerol (Michael and Coots, 1971). In this study the authors concluded that polyglycerols like triglycerol were not catabolized and that ether linkages within the molecule are inert of normal enzymatic hydrolysis.

The potential metabolites following enzymatic degradation of the target substance were also predicted using the QSAR OECD Toolbox (OECD, 2017). This QSAR tool predicts the primary and secondary metabolites of the parent compound that may result from enzymatic activity in the liver, in the skin and by the micro-flora in the GIT. Between 30 and 47 hepatic metabolites, depending on the exact structure taken for the prediction, and 6 dermal metabolites were predicted for three representative constituents of Fatty acids, C8-10, oxybis(2-hydroxy-3,1-propanediyl) esters. As can be expected, the typical hepatic metabolic transformation of the parent compound is the cleavage of the various ester bonds yielding the fatty acids and diglycerol ester with reduced esterification degree. Moreover, introduction of a hydroxyl function in at various positions of the aliphatic chains of the fatty acids is also predicted, accounting for additional oxidation processes. Finally, oxidation of diglycerol to structures containing an additional carboxyl group is predicted. Metabolites formed in the skin are hydrolysis products, i.e. the fatty acids and the diglycerol. No addition of a hydroxyl groups to any alkyl chain is calculated. Following the first reaction step, hydrolysis products may be metabolised further. No metabolite resulting from cleavage of the ether bond in diglycerol is anticipated. The resulting liver and skin metabolites are the products of α-, β- or ω-oxidation (i.e. addition of a hydroxyl group). In the case of ω-oxidation, it is followed by further oxidation to the aldehyde, which is then oxidised to the corresponding carboxylic acid. The ester bond may also remain intact, in which case a hydroxyl group is added to an alkyl chain. In general, the hydroxyl groups make the substances more water-soluble and susceptible to metabolism by phase II enzymes. The metabolites formed in the skin are expected to enter the blood circulation and have the same fate as the hepatic metabolites. Between 40 and 91 metabolites were predicted to result from all kinds of microbiological metabolism for the three representative constituents considered. This rather high number includes many minor variations in the C-chain length and number of carbonyl and hydroxyl groups, reflecting the diversity of many microbial enzymes identified. Not all of these reactions are expected to take place in the human GIT. In conclusion, the results of the OECD Toolbox simulation support the information on metabolism routes retrieved in the literature.

Excretion

As a consequence of the rapid enzymatic hydrolysis anticipated for Fatty acids, C8-10, oxybis(2-hydroxy-3,1-propanediyl) esters, it is considered to be excreted only to a very minor extent. Diglycerol is assumed to be metabolised only to a certain degree and therefore to be excreted almost quantitatively in the urine (Michael and Coots, 1971). 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, 1996), as discussed in detail above. 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. Thus, fatty acids are not expected to be excreted to any significant amount via the urine or faeces.

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A detailed reference list is provided in the technical dossier (see IUCLID, section 13) and in the CSR.