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Justification for grouping of substances and read-across

The short chain methyl esters category (SCAE Me) covers fatty acid esters of methanol. The category contains both mono-constituent substances, with fatty acid C-chain lengths ranging from C6 to C18 and UVCB substances, composed of single methyl esters in variable proportions. Fatty acid esters are generally produced by chemical reaction of an alcohol (e.g. methanol) with an organic acid (e.g. lauric acid) in the presence of an acid catalyst (Radzi et al., 2005). The esterification reaction is started by the transfer of a proton from the acid catalyst to the acid to form an alkyloxonium ion. The carboxylic acid is protonated on its carbonyl oxygen followed by a nucleophilic addition of a molecule of the alcohol to the carbonyl carbon of the acid. An intermediate product is formed. This intermediate product loses a water molecule and proton to give an ester (Liu et al., 2006; Lilja et al., 2005; Gubicza et al., 2000; Zhao, 2000). Monoesters are the final products of esterification of fatty acids with methanol.

In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across).

Having regard to the general rules for grouping of substances and read-across approach laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006, whereby substances may be considered as a category provided that their physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity, the substances listed below are allocated to the category of SCAE Me.

Table: The SCAE Me category members include:

CAS

EC name

MW

Fatty acid chain length

Type of alcohol

Molecular formula

106-70-7 (a)

Methyl hexanoate

130.18

C6

Methanol

C7H14O2

111-11-5

Methyl octanoate

158.24

C8

Methanol

C9H18O2

110-42-9

Methyl decanoate

186.29

C10

Methanol

C11H22O2

111-82-0

Methyl laurate

214.35

C12

Methanol

C13H26O2

124-10-7 (b)

Methyl myristate

242.41

C14

Methanol

C15H30O2

112-39-0

Methyl palmitate

270.46

C16

Methanol

C17H34O2

112-62-9

Methyl oleate

296.49

C18:1 (cis)

Methanol

C19H36O2

112-63-0

Methyl linoleate

294.48

C18:2 (cis)

Methanol

C19H34O2

112-61-8

Methyl stearate

298.51

C18

Methanol

C19H38O2

68937-83-7

Fatty acids, C6-10, methyl esters

130.18-186.29

C6-10

Methanol

C7H14O2; C11H22O2

67762-39-4

Fatty acids, C6-12, methyl esters

130.18-214.35

C6-12

Methanol

C7H14O2; C13H26O2

85566-26-3

Fatty acids, C8-10, methyl esters

158.24-186.29

C8-10

Methanol

C9H18O2; C11H22O2

67762-40-7

Fatty acids, C10-16, methyl esters

186.29-270.46

C10-16

Methanol

C11H22O2; C17H34O2

61788-59-8

Fatty acids, coco, methyl esters

214.35-242.40

C12-14

Methanol

C13H26O2; C15H30O2

308065-15-8

Fatty acids, C12-14 (even numbered), methyl esters

214.35-242.40

C12-14

Methanol

C13H26O2: C15H30O2

1234694-02-0

Fatty acids, C12-16 (even numbered) and C18-unsatd., methyl esters

270.46-296.49

C16-18; C18uns.

Methanol

C17H34O2; C19H38O2; C19H36O2

68937-84-8

Fatty acids, C12-18, methyl esters

214.35-296.49

C12-18

Methanol

C13H26O2; C19H38O2

67762-26-9

Fatty acids, C14-18 and C16-18-unsatd., methyl esters

242.40-298.51

C14-18; C16-18uns.

Methanol

C15H30O2; C19H38O2; C17H32O2; C19H36O2

61788-61-2

Fatty acids, tallow, methyl esters

270.46-298.51

C16-18; C18:1

Methanol

C17H34O2; C19H38O2; C19H36O2

67762-38-3

Fatty acids, C16-18 and C18-unsatd., methyl esters

270.46-298.51

C16-18; C18uns.

Methanol

C17H34O2; C19H38O2; C19H36O2

85586-21-6

Fatty acids, C16-18, methyl esters

270.46–298.51

C16-18

Methanol

C17H34O2; C19H38O2

111-62-6 (c)

Ethyl oleate

310.52

C18:1 (cis)

Ethanol

C20H38O2

544-35-4 (c)

Ethyl linoleate

308.51

C18:2 (cis)

Ethanol

C20H36O2

68171-33-5 (c)

Isopropyl isostearate

326.56

C18iso

Iso-propanol

C21H42O2

123-95-5 (c)

Butyl stearate

340.60

C18

Butanol

C22H44O2

22047-49-0 (c)

2-ethylhexyl stearate

396.70

C18

2-ethyl-hexanol

C26H52O2

91031-48-0 (c)

Fatty acids, C16-18, 2-ethylhexyl esters

368.65-396.70

C16-18

2-ethyl-hexanol

C24H48O2; C26H52O2

57-10-3 (c)

Palmitic acid

256.43

C16

--

C16H32O2

 

(a) Category members subject to the REACh Phase-in registration deadline of 31 May 2013 are indicated in bold font. Only for these substances a full set of experimental results and/or read-across is given.

(b) Substances that are either already registered under REACh or not subject to the REACh Phase-in registration deadline of 31 May 2013 are indicated in normal font. Lack of data for a given endpoint is indicated by “--“.

(c)   Surrogate substances are either chemicals forming part of a related category of structurally similar fatty acid esters or precursors/breakdown products of category members (i.e. alcohol and fatty acid moieties). Available data on these substances are used for assessment of (eco )toxicological properties by read-across on the same basis of structural similarity and/or mechanistic reasoning as described below for the present category.

 

Grouping of substances into this category is based on:

(1) common functional groups:

All category members are esters with the ester group being the common functional group of all substances. The substances are monoesters of aliphatic alcohols (methanol) and fatty acids with the chain length C6 to C18 and C18 unsaturated. The fatty acid chains comprise carbon chain lengths ranging from C6 (e.g. methyl hexanoate, CAS 106-70-7) to C18 (e.g. methyl stearate, CAS 112-61-8), mainly saturated but also mono and di unsaturated C18 (e.g. methyl oleate, CAS 112-62-9 and methyl linoleate, CAS 112‑63‑0).

(2) common precursors and the likelihood of common breakdown products via biological processes, which result in structurally similar chemicals:

All members of the category result from esterification of the alcohol with the respective fatty acid(s). Esterification is, in principle, a reversible reaction (hydrolysis). Thus, the alcohol and fatty acid moieties are simultaneously precursors and breakdown products of the category members. For the purpose of grouping of substances, enzymatic hydrolysis in the gastrointestinal tract and/or liver is identified as the biological process, by which the breakdown of the category members result in structurally similar chemicals. Following hydrolysis, fatty acids are enzymatically degraded primarily via beta-oxidation. Alternative oxidation pathways (alpha- and omega-oxidation) are available and are relevant for degradation of branched fatty acids. Unsaturated fatty acids require additional isomerization prior to enter the beta-oxidation cycle. The methanol is slowly oxidized in the liver by the enzyme alcohol dehydrogenase (ADH) to formaldehyde, which itself is oxidized very rapidly by the enzyme aldehyde dehydrogenase (ALDH) to formic acid. Finally, formic acid is slowly metabolised to CO2 and H2O (ICPS, 2002).

(3) constant pattern in the changing of the potency of the properties across the category:

a) Physicochemical properties:

The molecular weight of the category members ranges from 130.19 to 298.5 g/mol. The physical appearance is related to the chain length of the fatty acid moiety and the degree of saturation. Thus, methyl esters up to a fatty acid chain length of C12 are liquid, the C14 methyl ester (methyl myristate, CAS 124-10-7) is a semi solid substance, methyl esters with fatty acid chain lengths of C16 and C18 are solids (methyl palmitate and methyl stearate, CAS 112-39-0 and 112-61-8). Methyl esters with unsaturated fatty acids (C18:1, C18:2) are liquid (methyl oleate and methyl linoleate, CAS 112-62-9 and 112-63-0). For all category members the vapour pressure decreases with the chain length from 496 Pa (C6 methyl ester) to circa 0.002 Pa (C18 methyl ester), for C18 unsaturated esters it is even lower. The octanol/water partition coefficient increases with increasing fatty acid chain length, ranging from log Pow = 2.34 (C6) to log Pow = 8.35 (C18), for C18 unsaturated log Pow = 7.45 (C18:1) and 6.82 (C18:2) respectively. This trend can be observed also of the water solubility where 1330 mg/L for C6 methyl ester to 0.003 mg/L for C18 methyl ester is measured. The solubility of C18 unsaturated esters are slightly higher than for saturated components. It is observed that interactions between molecules of different single substances in water result in a decrease of water solubility comparing with water solubilities of mono-constituents, e.g. for C8-C10 it is 0.3 mg/L, while for C8 (64.4 mg/L) and for C10 (10.62 mg/L) can be found. Also for C10-C16 the value (<0.06 mg/L) is much closer to the lower value for C16 (0.004 mg/L) than to the C10 value of 10.62 mg/L, or C12 value of ca. 7.8 mg/L. It is generally concluded and experimentally confirmed that all UVCBs in the category are characterized with low water solubility (<1 mg/L).

b) Environmental fate and ecotoxicological properties:

The members of the SCAE Me category are readily biodegradable and show low bioaccumulation potential in biota. Hydrolysis is not a relevant degradation pathway for these substances, due to their ready biodegradability and estimated half-lives in water > 1 year at pH 7 and 8 (HYDROWIN v2.00). The majority of the SCAE Me category members have log Koc values > 3, indicating potential for adsorption to solid organic particles. Therefore, the main compartments for environmental distribution of these substances are expected to be soil and sediment, with the exception of methyl hexanoate (CAS 106-70-7) and methyl octanoate (CAS 111-11-5), for which log Koc < 3 are reported. Therefore, these two substances will be most likely available in the water phase. Nevertheless, all substances are readily biodegradable, indicating that persistency in the environment is not expected. The vapour pressure values of methyl hexanoate and methyl octanoate (> 10 Pa) indicate potential for volatilization of these substances into air. But if released into the atmosphere both these substances are susceptible to indirect photodegradation, with half-lives of 81.6 h and 51 h, respectively (AOPWIN v1.92). This degradation pathway is not relevant for all other category members, since their vapour pressure values are low (0.0001 Pa-4.93 Pa) and no significant release into the atmosphere is expected. Regarding the aquatic toxicity profile in the category, the LC50 values reported show that fish species are the least sensitive aquatic organisms when exposed to the SCAE Me category members. On the other hand, aquatic invertebrates and algae showed the highest sensitivity to these substances. A trend in the toxicity of the monoconstituent substances of the SCAE Me category to aquatic invertebrates and algae is observed, related to the toxicity mode of action (narcosis). The toxicity increases at increasing fatty acid C-chain length (starting from C6 (CAS 106-70-7)) up to a toxicity peak at C12 (for which L(E)C50s and NOECs < 1 mg/L have been reported). With decreasing water solubility at longer C-chain lengths (C14), no toxicity up to the highest attainable concentration is observed in Daphnia and algae. Based on the available data, no toxicity to aquatic microorganisms and terrestrial organisms is to be expected for the substances of the SCAE Me category.

 

c) Toxicological properties:

The toxicological properties indicate that all the category members show similar toxicokinetic behaviour. The ester bond will be hydrolysed in the gastrointestinal tract before absorption and the breakdown products will be metabolised. Based on the available and reliable data, none of the category members caused acute oral, dermal or inhalation toxicity, or skin or eye irritation, or skin sensitisation. No treatment-related effects were noted up to and including the limit dose of 1000 mg/kg bw/day after repeated oral exposure, indicating that the category members have a very limited potential for toxicity. The substances did not show a potential for toxicity to reproduction, fertility and development. No mutagenic or clastogenic potential was observed.

 

The available data allows for an accurate hazard and risk assessment of the category and the category concept is applied for the assessment of environmental fate, environmental and human health hazards. Thus where applicable, environmental and human health effects are predicted from adequate and reliable data for source substance(s) within the group by interpolation to the target substances in the group (read-across approach) applying the group concept in accordance with Annex XI, Item 1.5, of Regulation (EC) No 1907/2006. In particular, for each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across.

A detailed justification for the grouping of chemicals and read-across is provided in the technical dossier (see IUCLID Section 13).

Basic toxicokinetics

There are no studies available in which the toxicokinetic behaviour of methyl hexanoate (CAS No. 106-70-7) has been investigated.

Therefore, in accordance with Annex VIII, Column 1, Item 8.8.1 of Regulation (EC) 1907/2006 and with Guidance on information requirements and chemical safety assessment Chapter R.7c: Endpoint specific guidance (ECHA, 2008), assessment of the toxicokinetic behaviour of the substance methyl hexanoate (CAS No. 106-70-7) 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 Guidance on information requirements and chemical safety assessment Chapter R.7c: Endpoint specific guidance (ECHA, 2008) and taking into account further available information on the SCAE Me category.

The substance methyl hexanoate is an ester of methanol and hexanoic acid and meets the definition of a mono-constituent substance based on the analytical characterization.

Methyl hexanoate is liquid at room temperature and has a molecular weight of 130.19 g/mol and a water solubility of 1330 mg/L at 20 °C (Epiwin Database, 2011). The log KOW is estimated to be 2.34 (López Parrón, 2011) and the vapour pressure is calculated to be 496 Pa at 25 °C (Perry and Green, 1984).

 

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 KOW) value and the water solubility. The log KOW value provides information on the relative solubility of the substance in water and lipids (ECHA, 2008).

 

Oral:

The smaller the molecule, the more easily it will be taken up. In general, molecular weights below 500 are favourable for oral absorption (ECHA, 2008). As the molecular weight of methyl hexanoate is 130.19 g/mol, absorption of the molecule in the gastrointestinal tract is expected. Absorption after oral administration is also expected when the “Lipinski Rule of Five” (Lipinski et al. (2001), refined by Ghose et al. (1999)) is applied to the substance methyl hexanoate. The log KOW of 2.34 suggests that methyl hexanoate is favourable for absorption by passive diffusion (ECHA, 2008).

 

After oral ingestion, fatty acid methyl esters will undergo stepwise chemical changes in the gastro-intestinal fluids as a result of enzymatic hydrolysis. Methanol as well as the fatty acid will be formed, even though it was shown in-vitro that the hydrolysis rate of methyl oleate was lower when compared with the hydrolysis rate of the triglyceride Glycerol trioleate (Mattson and Volpenhein, 1972). The physico-chemical characteristics of the cleavage products (e.g. physical form, water solubility, molecular weight, log POW, vapour pressure, etc.) will be different from those of the parent substance before absorption into the blood takes place, and hence the predictions based upon the physico-chemical characteristics of the parent substance do no longer apply (ECHA, 2008). However, also for both hydrolysis products are anticipated to be well absorbed in the gastro-intestinal tract. The lipophilic fatty acid will be absorbed by passive diffusion (Ramirez et a., 2001), whereas the methanol, being a highly water-soluble substance, will readily dissolve into the gastrointestinal fluids and pass through aqueous pores or be carried through the epithelial barrier (ICPS, 2002).

Exemplarily, experimental data of the structurally similar Ethyl Oleate (CAS No. 111-62-6) confirmed this assumption: The absorption, distribution, and excretion of 14C labelled Ethyl Oleate was studied in Sprague Dawley rats after a single, oral dose of 1.7 or 3.4 g/kg bw. It was shown that the test material was well (approximately 70–90%) absorbed (Bookstaff et al., 2003).

Overall, a systemic bioavailability of methyl hexanoate and/or the respective cleavage products in humans is considered likely after oral uptake of the substance.

 

Dermal:

The smaller the molecule, the more easily it may be taken up. In general, a molecular weight below 100 favours dermal absorption, above 500 the molecule may be too large (ECHA, 2008). As the molecular weight of methyl hexanoate is 130.19 g/mol, a dermal absorption of the molecule cannot be excluded.

If the substance is a skin irritant or corrosive, damage to the skin surface may enhance penetration (ECHA, 2008). As methyl hexanoate is not considered as skin irritating in humans, an enhanced penetration of the substance due to local skin damage can be excluded.

Based on QSAR calculations dermal absorption of 0.013 mg/cm2/event (moderate) for methyl hexanoate was calculated (Danish EPA 2010). Based on this value, the substance has a low potential for dermal absorption.

For substances with a log KOW between 1 and 4 favour dermal absorption (values between 2 and 3 are optimal) particularly if water solubility is high (ECHA, 2008). As the log KOW of methyl hexanoate is 2.34 and the water solubility is 1330 mg/L, dermal uptake is likely to be moderate to high.

 

Overall, the calculated low dermal absorption potential, the moderate to high water solubility, the high molecular weight (>100) and the fact that the substance is not irritating to human skin implies that dermal uptake of methyl hexanoate in humans is considered as moderate.

 

Inhalation:

Methyl hexanoate has a low vapour pressure of 496 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 100μm have the potential to be inhaled. Particles with aerodynamic diameters below 50μm may reach the thoracic region and those below 15μm the alveolar region of the respiratory tract (ECHA, 2008).

Moderate log KOW values (between -1 and 4) are favourable for absorption directly across the respiratory tract epithelium by passive diffusion.

Overall, a systemic bioavailability of methyl hexanoate in humans is considered likely after inhalation of aerosols with aerodynamic diameters below 15μm.

 

Accumulation:

Substances with log KOW values of 3 or less would be unlikely to accumulate with the repeated intermittent exposure patterns normally encountered in the workplace but may accumulate if exposures are continuous (ECHA, 2008). The log KOW of 2.34 implies that methyl hexanoate may have the potential to accumulate in adipose tissue (ECHA, 2008).

However, as further described in the section metabolism below, esters of methanol and fatty acids will undergo esterase-catalysed hydrolysis, leading to the hydrolysis products methanol and fatty acid C6.

The log KOW of methanol is < 0, indicating a high solubility in water. Consequently, there is no potential for methanol to accumulate in adipose tissue. Fatty acids can be stored as triglycerides in adipose tissue depots or be incorporated into cell membranes. At the same time, fatty acids are also required as a source of energy. Thus, stored fatty acids underlie a continuous turnover as they are permanently metabolized 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 of the parent substance in adipose tissue is expected.

 

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 is the distribution. If the molecule is lipophilic, it is likely to distribute into cells and the intracellular concentration may be higher than extracellular concentration particularly in fatty tissues (ECHA, 2008).

Esters of methanol and fatty acids will undergo chemical changes as a result of enzymatic hydrolysis, leading to the cleavage products methanol and fatty acid C6.

Methanol, a small, polar water-soluble substance (log KOW < 0), will be distributed in aqueous fluids by diffusion through aqueous channels and pores. There is no protein binding and it is distributed poorly in fatty tissues (ICPS, 2002).

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).

 

Experimental data of the structurally similar Ethyl Oleate (CAS No. 111-62-6, ethyl ester of oleic acid) were regarded exemplarily. The absorption, distribution, and excretion of 14C labelled Ethyl Oleate was studied in Sprague Dawley rats after a single, oral dose of 1.7 or 3.4 g/kg bw. At sacrifice (72 h post-dose), mesenteric fat was the tissue with the highest concentration of radioactivity in both groups. The other organs and tissues had very low concentrations of test material-derived radioactivity (Bookstaff et al., 2003).

Overall, the available information indicates that the cleavage products, methanol and fatty acid, C6, will be distributed in the organism.

 

Metabolism:

Methyl hexanoate is hydrolysed to the corresponding alcohol (methanol) and fatty acid by esterases (Fukami and Yokoi, 2012), even though it was shown in-vitro that the hydrolysis rate of methyl oleate was lower when compared with the hydrolysis rate of the triglyceride Glycerol trioleate (Mattson and Volpenhein, 1972). Depending on the route of exposure, esterase-catalysed hydrolysis takes place at different places in the organism: After oral ingestion, esters of methanol and fatty acids will undergo chemical changes already in the gastro-intestinal fluids as a result of enzymatic hydrolysis. In contrast, substances that are absorbed through the pulmonary alveolar membrane or through the skin enter the systemic circulation directly before entering the liver where hydrolysis will basically take place.

The C6 fatty acid is stepwise degraded by 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 thecitric acid cycle. For the complete catabolism of unsaturated fatty acids such as oleic acid, an additional isomerization reaction step is required (see attached document). The omega- and alpha-oxidation, alternative pathways for oxidation, can be found in the liver and the brain, respectively (CIR, 1987).

Methanol is slowly oxidized in the liver by the enzyme alcohol dehydrogenase (ADH) to formaldehyde, which itself is oxidized very rapidly by the enzyme aldehyde dehydrogenase (ALDH) to formic acid. Finally, formic acid is slowly metabolised to CO2 and H2O (ICPS, 2002).

 

Excretion:

For methyl hexanoate the main route of excretion is expected to be by expired air as CO2 after metabolic degradation. The second route of excretion is expected to be by biliary excretion with the faeces. For methanol it is known that about thirty per cent of ingested substance is excreted unchanged by the respiratory tract; the kidney excretes less than 5% of unchanged methanol (ICPS, 2002).

Experimental data of the structurally similar Ethyl Oleate (CAS No. 111-62-6, ethyl ester of oleic acid) are regarded exemplarily. The absorption, distribution, and excretion of 14C labelled Ethyl Oleate was studied in Sprague Dawley rats after a single, oral dose of 1.7 or 3.4 g/kg bw. At sacrifice (72 h post-dose), mesenteric fat was the tissue with the highest concentration of radioactivity. The other organs and tissues had very low concentrations of test material-derived radioactivity. The main route of excretion of radioactivity in the groups was via expired air as CO2. Excretion of (14C) CO2 was rapid in the groups such that by 12 h after dosing 40-70% of the administered dose was excreted in expired air (consistent with beta-oxidation of fatty acids). The females had a higher percentage of radioactivity expired as CO2 than the corresponding males. A second route of elimination of radioactivity was via the faeces. Faecal elimination of Ethyl Oleate appeared to be dose-dependent. At the dose of 1.7 g/kg bw, 7–8% of the administered dose was eliminated in the faeces. At the dose of 3.4 g/kg bw, approximately 20% of the administered dose was excreted in the faeces.Renal elimination was minimal, with approximately 2% of the radioactivity recovered in urine over 72 h post-dose for the groups (Bookstaff et al., 2003).

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