L-97-034-PB

Endpoint summary

Additional information

Basic toxicokinetics

There are no studies available in which the toxicokinetic behaviour of Reaction products of pentaerythritol and trimethylol propane with 2-methylbutyric acid, n-pentanoic acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid and n-decanoic acid (EC 430-320-1) has been investigated. Therefore, in accordance with Annex VIII, Column 1, Item 8.8 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), an assessment of the toxicokinetic behaviour was conducted based on relevant available information. This consists of a qualitative assessment of the available substance-specific data on physico-chemical properties, toxicological properties, and predictions made using the OECD QSAR Toolbox and EpiSuite software.

Reaction products of pentaerythritol and trimethylol propane with 2-methylbutyric acid, n-pentanoic acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid and n-decanoic acid is a UVCB substance. It contains esters of pentaerythritol and trimethylol propane with C5-C10 fatty acids, where the fatty acids have linear or iso-branched C5-C10 alkyl chains. The esters are tri- and tetraesters. The substance is a liquid with water solubility < 0.065 mg/L at 20 °C. The vapour pressure is 2.7E-8 Pa at 25 °C and the log Pow > 6.2 (at pH 6.6). The molecular weight is 414-697 g/mol.

Absorption

Absorption is a function of the potential for a substance to diffuse across biological membranes. The most relevant 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 absorption of a substance can occur at different sites and with different mechanisms along the gastrointestinal (GI) tract, and depends primarily on the physico-chemical characteristics of the substance. A molecular weight below 500 g/mol is favourable for absorption while a molecular weight above 1000 g/mol does not favour absorption (ECHA, 2017). The molecular weight of the substance is in the range 414-697 g/mol, indicating a relatively low potential for oral absorption.

Micellar solubilisation is a mechanism of importance for highly lipophilic substances (with a log Pow > 4), that are poorly soluble in water (1 mg/L or less), as the substances are unlikely to dissolve into GI fluids. The low water solubility (< 0.065 mg/L) and the relatively high log Pow (> 6.2) of the registered substance indicate that micellar solubilisation is the most likely mechanism for oral absorption. Absorption following oral administration can also be assessed applying the “Lipinski Rule of Five” in the OECD QSAR Toolbox (OECD, 2019). The registered substance fails to satisfy two rules for good bioavailability, as the molecular weight is > 500 g/mol and the log Pow is > 5. The bioavailability is expected to be limited, according to this set of rules.

The potential of a substance to be absorbed in the GI tract can be affected during the passage through the GI tract. Substances can undergo chemical changes in the 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, which means that predictions made using the physico-chemical characteristics of the parent substance may no longer be accurate (ECHA, 2017).

Following ingestion, the ester group of a fatty acid ester may be hydrolysed by ubiquitously expressed esterases (Lehninger, 1993). The hydrolysis rate for fatty acid esters with glycerol (glycerides) is rapid and extensive, while the hydrolysis rate for substances with more than 3 ester groups is less rapid (Mattson and Volpenhein, 1972a,b). It was demonstrated that the in vitro hydrolysis rate of a pentaerythritol ester was 70-2000 times slower than that of a glycerol ester, depending on the enzyme preparation (Mattson and Volpenhein, 1972a,b). Consequently, for the registered substance, the number of ester groups will affect the hydrolysis rate, and the rate is expected to be moderate. In addition to the hydrolysis rate, the subsequent absorption of the hydrolysis products from the GI tract will determine the potential systemic exposure to the hydrolysis products. In vivo studies performed in the rat showed that a dietary fatty acid from substances with more than 3 ester groups was absorbed more slowly and incompletely, compared with the same fatty acid from triglycerides, indicating a slower and incomplete hydrolysis process (Mattson and Nolen, 1972; Mattson and Volpenhein, 1972a).

Polyols – like the hydrolysis products pentaerythritol (PE) and trimethylol propane (TMP) – have physico-chemical properties (low molecular weight, low log Pow, and high water solubility) that indicate they are easily absorbed from the GI tract. PE, with a water solubility of 27 g/L and log Pow < 0.3, will readily dissolve into the GI fluids (OECD, 1998). Of 10 mg/kg 14C-labelled PE orally administered to mice, almost half of the administered dose left the GI tract within 15 minutes (DiCarlo et al., 1965). TMP is similarly expected to be rapidly absorbed, based on its molecular weight (134 g/mol), water solubility (100 g/L) and log Pow (-0.47) (ECHA, 2019a).

The fatty acids present as hydrolysis products have short C-chains (C5-C10) that are linear or branched. Their physico-chemical properties (low molecular weight, low-moderate log Pow, and moderate-high water solubility) indicate they are easily absorbed from the GI tract. Pentanoic acid has a log Pow of 1.8 and the water solubility is 37.5 g/L, while decanoic acid has a log Pow of 4.1 and the water solubility is 0.062 g/L (ECHA, 2019b,c).

The hydrolysis products of the parent substance Reaction products of pentaerythritol and trimethylol propane with 2-methylbutyric acid, n-pentanoic acid, n-heptanoic acid, 3,5,5 -trimethylhexanoic acid, n-octanoic acid and n-decanoic acid (free alcohols and free fatty acids) are expected to be extensively absorbed following oral exposure and hydrolysis of the parent substance. The hydrolysis rate of the parent substance will therefore primarily determine the overall absorption rate of parent substance and hydrolysis products.

The oral LD50 value was > 5000 mg/kg bw and no systemic effects were observed following the single exposure of the registered substance. In a 28-day repeated dose toxicity study male and female Sprague Dawley rats were administered 150, 500 and 1000 mg/kg bw/day the registered substance. Hepatic and renal effects were observed at the highest dose level, indicating that the registered substance and/or its hydrolysis products are absorbed to a certain extent.

In conclusion, Reaction products of pentaerythritol and trimethylol propane with 2-methylbutyric acid, n-pentanoic acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid and n-decanoic acid and in particular the hydrolysis products are expected to have a moderate-high absorption rate following oral exposure.

Dermal absorption

In general, a molecular weight below 100 g/mol favors dermal absorption, while a molecular weight above 500 g/mol may be considered too large (ECHA, 2017). As the molecular weight of Reaction products of pentaerythritol and trimethylol propane with 2-methylbutyric acid, n-pentanoic acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid and n-decanoic acid is 414-697 g/mol, the dermal absorption, even of the low molecular weight constituents of the substance, is expected to be very limited.

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 also be sufficiently soluble in water to partition from the stratum corneum into the epidermis (ECHA, 2017). The water solubility of the substance is < 0.065 mg/L and the log Pow is > 6.2, indicating that the dermal absorption rate will be very low. The dermal LD50 value was > 2000 mg/kg bw and no systemic effects were observed following the single exposure of the substance. These results suggest that the dermal absorption rate is low and/or that the systemic toxicity of the substance is low.

The dermal permeability coefficient (Kp) can be calculated with the DERMWIN tool which is part of the EpiSuite software from the (estimated) log Pow and molecular weight (MW) applying the following equation described in US EPA (2012):

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

The Kp was calculated for 6 representative constituents of Reaction products of pentaerythritol and trimethylol propane with 2-methylbutyric acid, n-pentanoic acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid and n-decanoic acid (see Table 1). The calculated dermal flux rates indicate a low dermal absorption potential.

Table 1: EpiSuite results for 6 representative constituents

Component	Simplified Molecular Input Line Entry Specification (SMILES) code	Estimated log Pow	Dermal flux (mg/cm^2/h)
PE C7 tetraester	O=C(CCCCCC)OCC(COC(=O)CCCCCC)(COC(=O)CCCCCC)COC(=O)CCCCCC	10.67	0.000656
PE C7,C7,C7,C9i tetraester	CCCCCCC(=O)OCC(COC(=O)CCCCCC)(COC(=O)CCCCCC)COC(=O)CC(C)CC(C)(C)C	11.47	0.00155
PE C7,C7,C10,C9i tetraester	CCCCCCCCCC(=O)OCC(COC(=O)CCCCCC)(COC(=O)CCCCCC)COC(=O)CC(C)CC(C)(C)C	12.94	0.00858
TMP C7 triester	CCCCCCC(=O)OCC(CC)(COC(=O)CCCCCC)COC(=O)CCCCCC	9.17	0.000287
TMP C7,C7,C9i triester	CCCCCCC(=O)OCC(CC)(COC(=O)CCCCCC)COC(=O)CC(C)CC(C)(C)C	9.96	0.000676
TMP C7,C7,C10 triester	CCCCCCCCCC(=O)OCC(CC)(COC(=O)CCCCCC)COC(=O)CCCCCC	10.64	0.00159

* The water solubility for the UVCB (0.065 mg/L) was used to calculate the dermal flux

If a substance is a skin irritant or corrosive, damage to the skin surface caused by the substance may enhance its penetration through the skin. Reaction products of pentaerythritol and trimethylol propane with 2-methylbutyric acid, n-pentanoic acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid and n-decanoic acid did not show skin irritation/corrosion potential in an in vivo study performed in the rabbit according to OECD guideline 404, nor in an in vivo skin sensitisation study performed in guinea pigs according to OECD guideline 406.

A simulation of the potential metabolites of the parent substance was performed using the OECD QSAR Toolbox (version 4.3). The 6 constituents listed in Table 1 were run in the “Skin metabolism simulator”. Between 3 and 6 metabolites were predicted for each of the representative constituents. All the predicted metabolites were a result of the introduction of a hydroxyl group in a fatty acid chain. The resulting substances are expected to have similar physico-chemical properties to the parent substance, although the hydroxyl group will make the molecule slightly more polar, and therefore the dermal absorption rate of the metabolites will not increase significantly compared with the parent substance.

The physico-chemical characteristics, experimental data and QSAR calculations all indicate that the dermal absorption potential of Reaction products of pentaerythritol and trimethylol propane with 2-methylbutyric acid, n-pentanoic acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid and n-decanoic acid is very low.

Inhalation absorption

Reaction products of pentaerythritol and trimethylol propane with 2-methylbutyric acid, n-pentanoic acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid and n-decanoic acid has low volatility, with a low vapour pressure of 2.7E-8 Pa at 25 °C. Therefore, under normal use and handling conditions, inhalation exposure and therefore an availability for respiratory absorption of the substance in the form of vapours, gases, or mists is not expected. However, the substance may be available for respiratory absorption in the lung after inhalation of aerosols, if the substance is sprayed, for example in formulated products. 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), like the substance, may be taken up by micellar solubilisation. The parent substance may be hydrolysed enzymatically to the respective hydrolysis products, for which absorption is expected to be higher than for the parent substance. The rate of enzymatic hydrolysis of the parent substance will limit the amount of hydrolysis products available for respiratory absorption. The systemic bioavailability of the registered substance in humans cannot be excluded, e.g. after inhalation of aerosols with aerodynamic diameters below 15 μm.

In applying a worst-case approach due to the above-mentioned uncertainty, the absorption potential via the inhalation route of exposure is assumed to be the same as via the oral route of exposure for Reaction products of pentaerythritol and trimethylol propane with 2-methylbutyric acid, n-pentanoic acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid and n-decanoic acid.

Distribution

The distribution of a substance via the circulatory system to the organs and tissues depends primarily on the molecular weight, the lipophilic character and the water solubility of the substance. In general, the smaller the molecule, the more widely it will be distributed. If the molecule is lipophilic, it is likely to distribute into the cells and the intracellular concentration may be higher than the extracellular concentration, particularly in adipose tissues (ECHA, 2017).

The absorption rate of Reaction products of pentaerythritol and trimethylol propane with 2-methylbutyric acid, n-pentanoic acid, n-heptanoic acid, 3,5,5 trimethylhexanoic acid, n-octanoic acid and n-decanoic acid is expected to be low and therefore only a small fraction of the dose will be systemically available for distribution and metabolism. The absorbed hydrolysis products are expected to be extensively distributed to the organs and tissues of the body based on their water solubility and log Pow values. Following absorption into the intestinal lumen, fatty acids may be esterified with glycerol to triacylglycerides and included into chylomicrons for transportation via the lymphatic system and the blood stream to the liver, or absorbed from the small intestine directly into the bloodstream (Lehninger, 1993). Hepatic and renal effects were observed in an oral 28-day repeated dose toxicity study performed with male and female Sprague Dawley rats administered Reaction products of pentaerythritol and trimethylol propane with 2-methylbutyric acid, n-pentanoic acid, n-heptanoic acid, 3,5,5 trimethylhexanoic acid, n-octanoic acid and n-decanoic acid, showing that the registered substance and/or its hydrolysis products are distributed to the organs and tissues of the body. In the study performed by DiCarlo et al. (1965), the concentration of 14C-labelled pentaerythritol administered via the oral route to mice was measured in various tissues 15 minutes, and 4 and 24 hours after administration. 15 minutes after administration, 54% PE was measured in the GI tract, 22% PE was present in the carcass, 3.7% was present in adipose tissue and 4.6% PE was retrieved from the liver. 24 hours after administration the amount of PE in the GI tract was 3.1%, in the carcass 3.5%, in adipose tissue 0.7% and from the liver 0.8% PE was retrieved. It is likely that the distribution of TMP will follow a similar pattern, based on the similar molecular structure. The distribution of free fatty acids is predicted to be extensive, based on the moderate water solubility and low-moderate log Pow values.

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 the case that substances with high log Pow values have long biological half-lives. Its high log Pow (> 6.2) implies that Reaction products of pentaerythritol and trimethylol propane with 2-methylbutyric acid, n-pentanoic acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid and n-decanoic acid (parent substance) may have the potential to accumulate in adipose tissue. However, absorption is a prerequisite for accumulation within the body. The absorption rate of the parent substance is predicted to be low and therefore only a small fraction will be absorbed via any of the exposure routes. Depending on the route of exposure, the parent substance may primarily be metabolised in the liver before reaching the adipose tissues (oral exposure) or may be distributed in the body via the circulatory system to the organs and tissues (inhalation and dermal exposure). The hydrolysis products, derived from the hydrolysis of the parent substance prior to absorption or following absorption, are more water soluble and have a lower log Pow compared with the parent substance. Furthermore, the extensive metabolism and/or excretion of the free alcohols and fatty acids indicates that these substances will not be stored to any great extent. Therefore, the hydrolysis products are unlikely to accumulate in adipose tissues.

Metabolism

The parent substance will be metabolised primarily in the liver, due to the high concentration of esterases present in the liver, and may also be metabolised in other tissues with esterases (Lehninger, 1993). In the initial reaction, an ester group will be cleaved to give a fatty acid and a triester (from PE) or a diester (from TMP), after which the remaining ester groups may be hydrolysed to give free PE/TMP and free fatty acids.

The primary metabolic pathway for fatty acids is the beta-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 the 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, 1993).

The linear fatty acids pentanoic acid, heptanoic acid, octanoic acid and decanoic acid are metabolised via the beta-oxidation and citric acid pathway. The linear fatty acids with an odd number of carbon atoms yield acetyl-CoA and propionyl-CoA, where propionyl-CoA is converted to succinyl-CoA before entering the citric acid pathway. The branched-chain fatty acids (2-methylbutyric acid and 3,5,5-trimethylhexanoic acid) will also be metabolised via endogenous metabolic pathways, via several additional steps: The methyl-group may be cleaved in an additional step, before the beta-oxidation cycle can continue. If the methyl group is located at the beta-position, alpha-oxidation will occur to yield acid fragments that allow complete metabolism (WHO, 1999).

Pentaerythritol was reported to be excreted primarily via the urine in the mouse and human (DiCarlo et al, 1965 and references therein), which indicates that the substance is excreted unchanged or metabolised by phase II enzymes (glucuronides, sulphates, etc.) into more polar products. The metabolism and excretion of TMP will most likely follow a similar pattern as PE, based on the similar molecular structure.

The genotoxicity data from Reaction products of pentaerythritol and trimethylol propane with 2-methylbutyric acid, n-pentanoic acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid and n-decanoic acid did not show any genotoxic properties. An Ames test according to OECD guideline 471 and an in-vitro mammalian gene mutation assay according to OECD guideline 476 both showed negative results. Therefore there is no indication of reactivity towards DNA of the substance.

Metabolite simulation using the OECD QSAR Toolbox

A simulation of the potential metabolites of the registered substance was performed using the OECD QSAR Toolbox (version 4.3) to compare the predicted metabolites with the hydrolysis products and secondary metabolites resulting from the metabolic pathway described above. Six constituents were selected to represent the alcohol moieties and fatty acid moiety present in the UVCB substance Reaction products of pentaerythritol and trimethylol propane with 2-methylbutyric acid, n-pentanoic acid, n-heptanoic acid, 3,5,5-trimethylhexanoic acid, n-octanoic acid and n-decanoic acid. The 6 constituents are listed in Table 1 under “Dermal absorption”. The same kind of chemical conversions and, subsequently, the same kind of metabolites can be expected for the constituents that were not included in the metabolite prediction, as these have very similar structures. The variation lies in the specific combination of fatty acid chain length and branching. The metabolism and transformation simulators used to identify potential metabolites are listed below together with the results of the simulation exercise. The predictions for the metabolism simulators are consistent with the available information from public literature, which is presented in the paragraphs above.

Hydrolysis simulator (acidic and basic)

The Hydrolysis simulator (acid and basic) was selected to assess the possibility of the ester groups being hydrolysed in an acidic environment (e.g. the stomach) and in a basic environment. The prediction was the same both for the acidic and basic simulator, showing the relevant mono-, di-, and triesters, the free PE and TMP, and the free fatty acids. This is the result of a “chemical” hydrolysis (as opposed to an enzymatically catalysed reaction) of all the ester groups in turn, indicating that hydrolysis also without enzymes is possible in the GI tract. The hydrolysis rate cannot be predicted in the simulator, but is assumed to be much lower than the hydrolysis rate for enzymatically catalysed reactions.

Rat liver S9 metabolism simulator

Between 8 and 23 metabolites were predicted for each of the representative constituents, depending on the combination of fatty acids. The products resulting from the hydrolysis of the ester groups (mono-, di-, and triesters, and the free fatty acids) were present. In addition, the secondary metabolites expected following the steps of the beta-oxidation pathway were represented. This included fatty acids present in the parent substance from which a C2-unit is removed (e.g. pentanoic acid originating from heptanoic acid following the removal of a C2-unit) and acetic acid. In addition, free fatty acids that had undergone oxidation to introduce a hydroxyl group and subsequent oxidation to an aldehyde were predicted. The same oxidation sequence was predicted for the free OH-group of PE.

Skin metabolism simulator

Between 3 and 6 metabolites were predicted for each of the representative constituents, depending on the combination of fatty acids. All the predicted metabolites were a result of the oxidation of a carbon atom in a fatty acid chain to introduce a hydroxyl group. The resulting substances are expected to have similar physico-chemical properties to the parent substance, although the hydroxyl group will make the molecule slightly more polar.

In vivo rat metabolism simulator

Between 28 and 75 metabolites were predicted for each of the representative constituents, depending on the combination of fatty acids. The products resulting from the hydrolysis of the ester groups (mono-, di-, and triesters, and the free fatty acids) were present. In addition, the secondary metabolites expected following the steps of the beta-oxidation pathway were represented. This included fatty acids present in the parent substance from which a C2-unit is removed (e.g. octanoic acid: decanoic acid following the removal of a C2-unit) and acetic acid. In addition, oxidation of a carbon molecule to introduce a hydroxyl group and subsequent oxidation to an aldehyde were predicted for free fatty acids and fatty acids bound to an alcohol. The same oxidation sequence was predicted for the free OH-group of PE. Considering the relatively rapid metabolism of fatty acids via the endogenous pathway, this is likely to be a less prevalent route of metabolism.

Excretion

The fraction of the parent substance that is not absorbed or hydrolysed in the GI tract will be excreted in the faeces.

The fatty acids that reach the circulation system will be metabolised for energy generation (Lehninger, 1993). Therefore, the fatty acids will be excreted mainly via exhaled air as CO2.

Pentaerythritol was reported to be excreted primarily via the urine in the mouse and human (DiCarlo et al, 1965 and references therein), which indicates that the substance is excreted unchanged or conjugated by phase II enzymes (glucuronides, sulphates, etc.) to derive more polar products. The similarity of the structure between PE and TMP indicates that TMP will follow the same excretion route as described above for PE.