Propane-1,2,3-triyl 3,5,5-trimethylhexanoate

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:

29 Aug - 30 Nov 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to 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

Deviations:

yes

Remarks:

no hydrolysis test in saliva and gastric juice simulants, limited details in reporting

GLP compliance:

not specified

Radiolabelling:

no

Species:

other: not specified; presumably pig in accordance with the test method used

Strain:

not specified

Sex:

not specified

Details on test animals or test system and environmental conditions:

TEST DIGESTIVE SIMULANTS
INTESTINAL FLUID SIMULANT
- Description: intestinal fluid simulant according to “Note of Guidance for Food Contact Materials”, no further details given.

Route of administration:

other: mixing

Vehicle:

other: acetonitrile

Details on exposure:

HYDROLYSIS WITH INTESTINAL-FLUID SIMULANT:
A triplicate was performed. For the hydrolysis investigation the esters were dissolved in acetonitrile. These solutions were added to the intestinal-fluid simulant tempered to 37 °C. The concentration of acetonitrile in the reaction mixture was about 0.1%. Samples were taken after 0, 1, 2 and 4 hours.

Duration and frequency of treatment / exposure:

0, 1, 2 and 4 h

Dose / conc.:

24.34 ppm

No. of animals per sex per dose / concentration:

triplicate determinations

Control animals:

no

Details on dosing and sampling:

DETERMINATION OF HYDROLYSIS PRODUCTS
- Principle: Following incubation, a naphthalene solution in acetone was added as an internal standard to the samples and the enzyme was precipitated by the addition of ice-cold acetone. After filtration the acetone was evaporated. The aqueous solutions were acidified with 0.1 M hydrochloric acid (pH 1.2) and were extracted three times with dichloromethane. After addition of an alkane standard (tridecane) and derivatization with N-Methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) at 60°C for one hour the concentrated dichloromethane solutions were analysed by gas chromatography coupled with a mass spectrometer (GC/MS). Quantification of the esters and the hydrolysis products was performed specifically by external calibration curves.
- Recovery assays: A duplicate of three different concentrations of the acid (hydrolysis product) were performed. For the recovery investigations the acid was dissolved in acetonitrile. These solutions were added to the intestinal-fluid simulant tempered to 37°C. After 4 hours a naphthalene solution in acetone was added as an internal standard to the samples and the enzyme was precipitated by the addition of ice-cold acetone. Work-up and quantification was performed as described above.
Recovery of the ester was determined using a hydrolysis sample analogue to the "0 hour" assay.

Statistics:

Mean values of triplicate determinations were calculated.

Type:

other: ester hydrolysis in intestinal fluid simulant

Results:

19, 23.3 and 29.2% 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:

yes

Details on metabolites:

The following esters of glycerol were quantitatively determined (in ppm) after 0, 1, 2 and 4 h hydrolysis in intestinal fluid simulant, respectively:
Diester with 3,5,5-trimethylhexanoic acid (sum of two stereoisomers): 0.96, 1.07, 1.10 and 1.13 ppm
Triester with 3,5,5-trimethylhexanoic acid (parent substance): 24.34, 19.72, 18.67 and 17.22 ppm
(No monoester was detected)

The following free fatty acid was quantitatively determined (in ppm) after 0, 1, 2 and 4 h hydrolysis in intestinal fluid simulant, respectively:
3,5,5-trimethylhexanoic acid: 0.35, 1.90, 3.07 and 4.56 ppm

Table 1. Hydrolysis of Propane-1,2,3-triyl-3,5,5-trimethylhexanoate with intestinal-fluid simulant

Contact time (h)	Results (1)
	Triester (ppm)	Triester (%)	Diester (ppm) (2)	Acid (ppm)
0	24.34	100.0	0.96	0.35
1	19.72	81.0	1.07	1.90
2	18.67	76.7	1.10	3.07
4	17.22	70.8	1.13	4.56

 

(1) No Monoester was detected.

(2) Sum of two stereo isomers

 

Table 2. Mass balance of the ester hydrolysis

Contact time (h)	Results (1)
	Triester (µmol)	Diester (µmol)	Acid (µmol) (calc.) (1)	Acid (µmol) (exp.)
0	0.233	0.013	0.000	0.011
1	0.192	0.014	0.094	0.060
2	0.182	0.015	0.124	0.097
4	0.168	0.015	0.165	0.144

 

(1) Amount of diester is considered; one ester reacts to three acids.

 

Table 3. Recoveries of ester and acid

Results
ppm (calc.)	ppm (exp.)	Recovery (%)
Ester
23.91	28.32	118.5
Acid
9.86	10.89	110.4
21.92	25.57	116.7
38.36	43.67	113.8

Conclusions:

Interpretation of results: bioaccumulation potential cannot be judged based on study results

Description of key information

Absorption:

Based on the physico-chemical properties low oral absorption is suggested. Substantial enzymatic hydrolysis in the GI tract is expected and absorption of hydrolysis products is likely. The dermal absorption potential is considered to be low to very low. Systemic bioavailability is considered likely after inhalation of aerosols with aerodynamic diameters below 15 µm.

Distribution and Accumulation:

Distribution of parent substance is considered not relevant as enzymatic hydrolysis is expected. Hydrolysis products are anticipated to be distributed widely and are expected to be fed into natural physiological processes. No bioaccumulation potential is predicted for either the parent compound or the hydrolysis products.

Metabolism:

Ultimately, hydrolysis is expected for the parent compound, either in the gastrointestinal tract or the liver. Glycerol is catabolised entirely by oxidative physiologic pathways ultimately leading to the production of carbon dioxide and water. Fatty acids are oxidised by various mechanisms (β-, α- and ω-oxidation) for energy generation.

Excretion:

Not hydrolysed and absorbed parent compound is excreted in the faeces. Glycerol can readily be excreted in the urine. Oxidation of branched fatty acids leads to the formation of various diols, hydroxyl acids, ketoacids or dicarbonic acids. These metabolites may be conjugated to glucuronides or sulphates and can subsequently be excreted via urine or bile or cleaved in the gut with the possibility of reabsorption (entero-hepatic circulation).

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Additional information

Basic toxicokinetics

In accordance with Annex VIII, Column 1, Item 8.8.1, of Regulation (EC) No. 1907/2006 and with the Guidance on information requirements and chemical safety assessment Chapter R.7c: Endpoint specific guidance (ECHA, 2017), assessment of the toxicokinetic behaviour of the target substance propane-1,2,3-triyl 3,5,5-trimethylhexanoate is conducted to the extent that can be derived from the relevant available information. This comprises a qualitative assessment of the available substance specific data on physico-chemical and toxicological properties according to the relevant Guidance (ECHA, 2017) and taking into account further available information from read-across analogue substances. There are no studies available in which the toxicokinetic behaviour of propane-1,2,3-triyl 3,5,5-trimethylhexanoate (CAS 56554-53-1) has been investigated.

The substance propane-1,2,3-triyl 3,5,5-trimethylhexanoate is a triester of glycerol and 3,5,5-trimethylhexanoic (isononanoic) acid. Propane-1,2,3-triyl 3,5,5-trimethylhexanoate has a molecular weight of 512.78 g/mol. The substance is a white liquid at 20 °C with a pour point of < -20 °C at normal pressure, water solubility of < 0.05 mg/L at 20 °C and pH=6.4 - 6.8, calculated log Pow > 10 and calculated vapour pressure < 0.0001 Pa at 20 °C.

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

In general, molecular weights below 500 g/mol and log Pow values between -1 and 4 are favourable for absorption via the gastrointestinal (GI) tract, provided that the substance is sufficiently water soluble (> 1 mg/L). Lipophilic compounds may be taken up by micellar solubilisation by bile salts, but this mechanism may be of particular importance for highly lipophilic compounds (log Pow > 4), in particular for those that are poorly soluble in water (≤ 1 mg/L) which would otherwise be poorly absorbed (Aungst and Chen, 1986; ECHA, 2017).

The physicochemical characteristics (log Pow and water solubility) of the substance and the molecular weight are in a range suggestive of moderate absorption from the GI tract subsequent to oral ingestion.

The potential of a substance to be absorbed in the GI tract may be influenced by chemical changes taking place in GI fluids as a result of metabolism by GI flora, by enzymes released into the GI tract or by hydrolysis. 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 no longer apply (ECHA, 2017).

It is well-accepted knowledge that triglycerides (e.g. from dietary fat) undergo hydrolysis by lipases (a class of ubiquitous carboxylesterases) prior to absorption; and there is sufficient evidence to assume that propane-1,2,3-triyl 3,5,5-trimethylhexanoate will likewise undergo enzymatic hydrolysis in the GI tract as the first step in its absorption, distribution, metabolism and excretion (ADME) pathways as summarised below.

In the GI tract, gastric and intestinal (pancreatic) lipase activities are the most important. Triglycerides are hydrolysed by gastric and pancreatic lipases with high specificity for the sn1- and sn3-positions. For the remaining monoester at the sn2-position (2-monoacylglycerol), there is evidence that it can either be absorbed as such by the intestinal mucosa or isomerise to 1-monoacylglycerol, which can then be hydrolysed. The rate of hydrolysis by gastric and intestinal lipases depends on the carbon chain length of the fatty acid moiety. Thus, triesters of short-chain fatty acids are hydrolysed more rapidly and to a larger extent than triesters of long-chain fatty acids. (Barry et al., 1966; Cohen et al.,1971; Greenberger et al., 1966; IOM, 2005; Mattson and Volpenhein, 1964, 1966, 1968; WHO, 1967, 1975). In a recent study conducted with the substance glycerides, castor-oil-mono, hydrogenated, acetates (CAS 736150-63-3), rapid ester hydrolysis in intestinal fluid simulant was confirmed (Jensen, 2002). In contrast, in vitro studies with triglycerides of 2- and 3-methyl-branched fatty acids have been shown to be subject to significantly lower pancreatic lipase activity due to steric hindrance by the branched chain triglycerides compared to triglycerides with linear fatty acid moieties (Tagiri, 1992).

An in vitro hydrolysis test with propane-1,2,3-triyl 3,5,5-trimethylhexanoate in intestinal fluid simulant demonstrated that about 30% of the triglyceride is hydrolysed completely after a reaction time of 4 h (FABES, 2012). For the in vivo situation, it cannot be ruled out, that the parent substance or a fraction of it may be absorbed unchanged by micellar solubilisation and be hydrolysed within the body, e.g. by esterases in the liver. Therefore, in a worst case approach, 100% hydrolysis and bioavailability of the hydrolysis products glycerol and 3,5,5-trimethylhexanoic acid is assumed for the purpose of the hazard assessment.

Following hydrolysis, the resulting products free glycerol, free fatty acids and (in the case of di- and triglycerides) 2-monoacylglycerols are absorbed by the intestinal mucosa. Within the epithelial cells, triglycerides are reassembled, primarily by re-esterification of absorbed 2-monoacylglycerols. Thus, free glycerol is readily absorbed independently of the fatty acids and little of it is re-esterified. As for hydrolysis, the absorption rate of free fatty acids is chain length-dependent. The absorption of short-chain fatty acids can therefore begin already in the stomach. In general, for intestinal absorption short-chain linear or unsaturated fatty acids are more readily absorbed than long-chain, saturated fatty acids. However, the absorption of saturated long-chain fatty acids is increased if they are esterified at the sn2-position of glycerol (Greenberger et al., 1966; IOM, 2005; Mattson and Volpenhein, 1962, 1964). Furthermore, short-chain (< C8) branched chain aliphatic acids and related esters have also been shown to be rapidly absorbed in the GI tract (WHO, 1999). Recently a study was conducted with 12-[1-14C]acetoxy-octadecanoic acid-2,3-diacetoxy-propyl ester, serving as surrogate for the substance glycerides, castor-oil-mono, hydrogenated, acetates (CAS 736150-63-3) to investigate the pharmacokinetics, tissue distribution, excretion and mass balance of radioactivity in rats after a single oral dose of the test material (St-Pierre, 2004). The results of the study showed that the test material, specifically the fatty acid moiety, was readily absorbed from the GI tract, systemically distributed and metabolised. Based on the reported data on mass balance of radioactivity, absorption was higher than 80%.

In conclusion, based on the available information, the physico-chemical properties and molecular weight of propane-1,2,3-triyl 3,5,5-trimethylhexanoate suggest low oral absorption. However, the substance is anticipated to undergo enzymatic hydrolysis in the GI tract and absorption of the hydrolysis products rather than the parent substance is likely. The absorption rate of the hydrolysis products is considered to be high. It is also likely that the parent substance may be absorbed unchanged by micellar solubilisation.

Dermal

The dermal uptake of liquids and substances in solution is higher than that of dry particulates, since dry particulates need to dissolve into the surface moisture of the skin before uptake can begin. Molecular weights below 100 g/mol favour dermal uptake, while for those above 500 g/mol the molecule may be too large. Dermal uptake is anticipated to be low, if the water solubility is < 1 mg/L; low to moderate if it is between 1-100 mg/L; and moderate to high if it is between 100-10000 mg/L. Dermal uptake of substances with a water solubility > 10000 mg/L (and log Pow < 0) will be low, as the substance may be too hydrophilic to cross the stratum corneum. Log Pow values in the range of 1 to 4 (values between 2 and 3 are optimal) are favourable for dermal absorption, in particular if water solubility is high. For substances with a log Pow above 4, the rate of penetration may be limited by the rate of transfer between the stratum corneum and the epidermis, but uptake into the stratum corneum will be high. Log Pow values above 6 reduce the uptake into the stratum corneum and decrease the rate of transfer from the stratum corneum to the epidermis, thus limiting dermal absorption (ECHA, 2017).

The physico-chemical properties (log Pow and water solubility) of the substance and the molecular weight are in a range suggestive of low absorption through the skin.

The dermal permeability coefficient (Kp) can be calculated from log Pow and molecular weight (MW) applying the following equation described in US EPA (2004):

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

The Kp is thus 8.48 cm/h. Considering the water solubility (0.00005 mg/cm³), the dermal flux is estimated to be ca. 0.0004 mg/cm²/h.

If a substance shows skin irritating or corrosive properties, damage to the skin surface may enhance penetration. If the substance has been identified as a skin sensitiser then some uptake must have occurred although it may only have been a small fraction of the applied dose (ECHA, 2017).

The available data demonstrate that the substance did not cause skin irritation in human-derived epidermal keratinocytes in vitro. Thus, no conditions are favoured that enhance penetration of the substance through the skin. However, there are indications from a LLNA for a moderate skin sensitisation potential of the substance. This suggests that some uptake of the substance must have occurred, although it may only have been a small fraction of the applied dose.

Overall, taking all available information into account, the dermal absorption potential is considered to be low to very low.

Inhalation

Propane-1,2,3-triyl 3,5,5-trimethylhexanoate is a liquid with very low vapour pressure (< 0.0001 Pa at 20 °C), thus being of very 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 significant.

However, the substance may be available for respiratory absorption in the lung after inhalation of aerosols, if the substance is sprayed (e.g. as a formulated product). 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).

As for oral absorption, the molecular weight, log Pow and water solubility are suggestive of absorption across the respiratory tract epithelium by micellar solubilisation.

Overall, systemic bioavailability is considered likely after inhalation of aerosols with aerodynamic diameters below 15 µm.

Distribution and Accumulation

Distribution of a compound within the body 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 (ECHA, 2017).

As discussed under oral absorption, mono-di- and triesters of glycerol undergo enzymatic hydrolysis in the GI tract prior to absorption. Therefore, assessment of distribution and accumulation of the hydrolysis products is considered more relevant.

Absorbed glycerol is readily distributed throughout the organism and can be re-esterified to form endogenous triglycerides, be metabolised and incorporated into physiological pathways or be excreted in the urine. 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 length ≤ 12 may be transported as the free acid bound to albumin directly to the liver via the portal vein, instead of being re-esterified. Chylomicrons are transported in the lymph to the thoracic duct and eventually to the venous system. Upon 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 oxidised for energy or they are released into the systemic circulation and returned to the liver (IOM, 2005; Johnson, 1990; Johnson, 2001; Lehninger, 1998; NTP, 1994; Stryer, 1996; WHO, 2001).

Stored fatty acids underlie a continuous turnover as they are permanently metabolised for energy generation and excreted as CO2. Bioaccumulation of fatty acids takes place, if their intake exceeds the caloric requirements of the organism.

In the study by St-Pierre (2004) with 12-[1-14C]acetoxy-octadecanoic acid-2,3-diacetoxy-propyl ester systemic distribution of the radiolabelled material was confirmed in rats. Radioactivity was detected in all tissues and organs sampled (adipose tissue, GI tract and content, kidneys and adrenals, liver, thymus and the remaining carcass) with highest levels recovered in the GI tract, liver and the remaining carcass. Due to excretion and absorption of the radiolabelled material, the radioactivity content in the GI tract decreased rapidly over the course of the study (168 h). This was similar for the radioactivity recovered in liver, whereas the radioactivity found in the carcasses was nearly constant at the selected time points, indicating that the radiolabelled material may have been distributed to other tissues than the ones selected for analysis. Based on the results of this study, no bioaccumulation potential was observed for 12-acetoxy-octadecanoic acid-2,3-diacetoxy-propyl ester.

Metabolism

Glycerol can be metabolised to dihydroxyacetone phosphate and glyceraldehyde-3-phosphate, which can then be incorporated in the standard metabolic pathways of glycolysis and gluconeogenesis. A major metabolic pathway for linear and branched fatty acids is the β-oxidation for energy generation. In this multi-step process, the fatty acids are at first esterified into acyl-CoA derivatives and subsequently transported into cells and mitochondria by specific transport systems. In the next step, the acyl-CoA derivatives are broken down into acetyl-CoA molecules by sequential removal of 2-carbon units from the aliphatic acyl-CoA molecule. Further oxidation via the citric acid cycle leads to the formation of H2O and CO2 (Lehninger, 1998). Branched-chain acids can be metabolised via the same β-oxidation pathway as linear fatty acids, depending on the steric position of the branching point, but at lower rates (WHO, 1999). The alternative α- and ω-oxidation pathways are a major metabolic pathway for branched-chain fatty acids where a methyl substituent at the β-position blocks certain steps in the β-oxidation (Mukherji, 2003). Generally, a single carbon unit is cleaved off the branched acid in an additional step before the removal of 2-carbon units continues.

Excretion

As far as glycerides are not hydrolysed in the GI tract, they are excreted in the faeces. In general, the hydrolysis product glycerol is catabolised entirely by oxidative physiologic pathways ultimately leading to the production of carbon dioxide and water. Glycerol, being a polar molecule, can also readily be excreted in the urine.

The branched fatty acid resulting from the hydrolysis of propane-1,2,3-triyl 3,5,5-trimethylhexanoate is unlikely to be used for energy generation and storage, since saturated aliphatic, branched-chain acids are described to be subjected to α- and/or ω-oxidation due to steric hindrance by the methyl groups at uneven positions, which results in the formation of various diols, hydroxyl acids, ketoacids or dicarbonic acids. In contrast to the products of β-oxidation, these metabolites may be conjugated to glucuronides or sulphates, which subsequently can be excreted via urine or bile or cleaved in the gut with the possibility of reabsorption (entero-hepatic circulation) (WHO, 1998).

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