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Description of key information

Low absorption potential of the parent substance after oral, dermal and inhalation exposure. Hydrolysis products are expected to be readily absorbed from gastrointestinal tract. Parent substance expected to be hydrolysed to ethylenediamine and palmitic and/or stearic acid. Ethylenediamine probably conjugated in phase II metabolism and excreted via the kidneys in the urine. Fatty acids fed into citrate cycle and degraded to CO2 by beta-oxidation. Not absorbed parent substance to be excreted in the faeces.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential

Additional information

There are no studies available in which the toxicokinetic behaviour of ‘amides, C16-C18 (even), N,N'-ethylenebis’ (EC No. 931-299-4) was investigated. In accordance with Annex VIII, Column 1, Item 8.8.1, of Regulation (EC) No. 1907/2006 and with ‘Guidance on information requirements and chemical safety assessment Chapter R.7c: Endpoint specific guidance’ (ECHA, 2017), an assessment of the toxicokinetic behaviour of the substance was 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 Chapter R.7c Guidance document (ECHA, 2017).

Physico-chemical properties

Amides, C16-C18 (even), N,N'-ethylenebis is an UVCB substance, with three major constituents. The constituents differ in the combination of palmitic acid (C16) and/or stearic acid (C18) attached to the central ethylenediamine moiety; thus, there is a C18-C18 constituent, a C18-C16 constituent and a C16-C16 constituent.

The substance has a molecular weight range from 593.94 g/mol for the C18-C18 constituent to 536.93 g/mol for the C16-C16 constituent. It is a solid at 20 °C and is insoluble in water. The water solubility of the three major constituents have been calculated to be in the range of 5.4xE-07 - 5.9xE-07 mg/L at 25 °C, using the WATERNT QSAR model (US EPA, 2014). The log Pow values of the constituents were estimated to be 12 - 14, indicating an extreme lipophilic character of amides, C16-C18 (even), N,N'-ethylenebis. The vapour pressure at 20 °C was extrapolated to be 2.3E-07 hPa, based on the measured vapour pressure of 1.5 x 10E-05 hPa at 63 °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 can be taken up by micellar solubilisation by bile salts, and this mechanism may be of particular importance for highly lipophilic compounds (log Pow > 4), especially for those that are poorly soluble in water (≤ 1 mg/L) as these substances would otherwise be poorly absorbed (ECHA, 2017).

The molecular weight, log Pow and water solubility of amides, C16-C18 (even), N,N'-ethylenebis all suggest the substance will be poorly absorbed from the GI tract as  relevant parameters fall outside the favourable ranges. However, micellar solubilisation might play a certain role due to the extreme lipophilicity of the substance. A limited absorption after oral exposure to amides, C16-C18 (even), N,N'-ethylenebis is further supported by the lack of systemic toxicity observed in three acute oral toxicity studies as well as in a subchronic (90-day) repeated dose toxicity study. No toxicologically relevant effects have been noted in any of these studies.

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 ‘chemical’ hydrolysis at the extreme acidic pH of the stomach. 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 or apply to a lesser extent (ECHA, 2017).

Amides, C16-C18 (even), N,N'-ethylenebis is the diamide of ethylenediamine with palmitic and/or stearic acid. The amide groups (-C(=O)-NH-) may be hydrolysed in the GI tract - either enzymatically or acid catalysed - to form ethylenediamine and the corresponding acid moieties (palmitic and/or stearic acid). The rate of hydrolysis is not known. The smaller molecules of ethylenediamine and the acid moieties may be absorbed faster than the parent molecule.

In conclusion, based on the available information, amides, C16-C18 (even), N,N'-ethylenebis is predicted to undergo enzymatic hydrolysis in the GI tract and absorption of the hydrolysis products rather than (or in addition to) the parent substance is likely. The absorption rate of the hydrolysis products is expected to be high. Due to the limited information on the hydrolysis products, as a worst-case approach the oral absorption potential is assumed to be high.

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. 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 the 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).

Amides, C16-C18 (even), N,N'-ethylenebis is almost insoluble in water, with the highest water solubility value of its major constituents estimated to be 5.4xE-07 mg/L. This indicates a low dermal absorption. Furthermore, the log Pow values of the constituents indicate a reduced uptake into the stratum corneum. The limited dermal absorption is further supported by the molecular weights of > 500 g/mol.

The dermal permeability coefficient (Kp) can be calculated from log Pow and molecular weight (MW) applying the following equation described in US EPA (2014):

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

Depending on the constituent of amides, C16-C18 (even), N,N'-ethylenebis, the following permeability coefficients and fluxes through the skin barrier have been calculated. For the latter, the respective water solubilities were taken into account.

 

C18-C18 constituent

C18-C16 constituent

C16-C16 constituent

Kp [cm/h]

1280

413

134

Flux [µg/cm2/h]

0.00075

0.00024

0.00007

All values determined indicate a very low dermal absorption potential.

If a substance shows skin irritating or corrosive properties, damage to the skin surface may enhance penetration. The in-vivo skin irritation tests according or similar to OECD guideline 404 performed with the substance indicated only minimal irritating properties; only slight edema (grade 1 according to Draize) was observed on intact skin. Therefore, no enhanced penetration of amides, C16-C18 (even), N,N'-ethylenebis due to skin damage is expected. Moreover, the Local Lymph Node Assay (LLNA) performed with the substance yielded a negative result. The lack of a sensitising potential may indicate that the substance is not systemically available. This further supports the assumption of a very low dermal absorption.

Taking all the available information into account, the dermal absorption potential of amides, C16-C18 (even), N,N'-ethylenebis is considered to be very low.

Inhalation

Amides, C16-C18 (even), N,N'-ethylenebis is a solid and its vapour pressure is very low (2.3E-07 hPa at 20 °C). The substance, therefore, has very low volatility. Consequently, under normal use and handling conditions that avoid the formation of dust, inhalation exposure and availability for respiratory absorption of the substance in the form of vapour, gases or mists is not significant (ECHA, 2017). However, the substance may be available for respiratory absorption in the lung after inhalation of dust particles, if the substance is handled under open process conditions. 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). The granulometric analysis of the substance showed D10, D50, and D90 values of 2.1, 8.76, and 22.5 µm, respectively, which clearly indicate the particles may be inhaled. A large fraction of the particles will reach the alveolar region.

The log Pow and water solubility indicate that the target substance has only a low potential to be absorbed across the respiratory tract epithelium. In addition, the high molecular weight may have a limiting effect on the absorption rate. In an acute inhalation toxicity study performed according to OECD guideline 403, laboured breathing and/or rales, dark material around nose or mouth, decreased activity, urine stain, and thrashing in the cage were observed. These clinical signs can be attributed to the concentration, size and shape of the particles rather than to a systemic effect caused by the chemical structure of the substance after absorption in the respiratory system. Moreover, the clinical signs are considered to be due to local effects of the exposure rather than systemic effects.

In conclusion, the systemic bioavailability of amides, C16-C18 (even), N,N'-ethylenebis in humans is considered to be very limited, e.g. after inhalation of aerosols with an aerodynamic diameter below 15 µm. However, due to the solid particles that might be deposited in the deepest regions of the respiratory system, effects originating from the particulate nature of the substance (local effects as a consequence of the particle deposition, effects driven by the concentration, size and shape of the particles) cannot be excluded. In applying a worst-case scenario, the absorption potential via the inhalation route of exposure is assumed to be the same as via the oral route of exposure.

Distribution and accumulation

Distribution of a substance 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).

No toxicologically relevant systemic effects were observed in any of the studies with amides, C16-C18 (even), N,N'-ethylenebis. The macroscopic examination during necropsy did not show any target organ for acute toxicity with the exception of pale lungs and multifocal dark red foci on the lungs in the acute inhalation study which are attributed to the concentration and size of the particles of the test substance. Furthermore, no lasting effects were seen for any parameters in the subchronic (90-day) study. Therefore, it is not possible to determine from the experimental data available if the substance will be distributed to tissues and organs in the body.

As discussed under oral absorption, amides, C16-C18 (even), N,N'-ethylenebis is expected to undergo enzymatic and/or acid-catalysed hydrolysis in the GI tract to some extent prior to absorption. After being absorbed, the hydrolysis products ethylenediamine, palmitic and stearic acid are expected to be widely distributed, due to the size of the respective molecules and the functional groups that significantly increase their water solubility. The substances absorbed from the GI tract will be transported via the portal vein to the liver, where further metabolism can take place. Substances that are absorbed through the pulmonary alveolar membrane or through the skin (which is considered unlikely for amides, C16-C18 (even), N,N'-ethylenebis) enter the systemic circulation directly before they are transported to the liver where metabolism will take place. The substances are not expected to accumulate in adipose tissue due to the lack of lipophilic groups.

Metabolism

Amides, C16-C18 (even), N,N'-ethylenebis has functional groups that are suitable  for phase I metabolism reactions. The amide groups may be enzymatically hydrolysed by amidases, which are expressed in most tissues and play an important role in most organisms (Gossauer, 2006). The hydrolysis product ethylenediamine may be conjugated (e.g. by glucuronidation) to form a polar molecule suitable for excretion. Palmitic and stearic acid are expected to be further metabolised by the process of β-oxidation for energy generation in the citrate cycle or are esterified with glycerol to form triacylglycerides that can be stored in adipose tissue (Lehninger, 1993).

The potential metabolites following enzymatic metabolism of the three major constituents of the test substance were predicted using the OECD QSAR Toolbox v4.3.1 (OECD, 2019). This QSAR tool predicts which metabolites of the test substance may result from enzymatic activity in the liver and in the skin, and by intestinal bacteria in the GI tract. Essentially, the hepatic metabolic conversions consist of either hydrolysis of one or both amide group(s) or addition of a hydroxyl group to a fatty acid moiety. Hydroxyl groups tend to be introduced toward the end of the lipophilic carbon-chain and are subsequently subject to oxidation reactions, resulting in the formation of aldehydes or ketones. Aldehydes might be further oxidised to carboxylic acids. Depending on the combination of the fatty acid moieties in the parent compound, up to 77 hepatic metabolites are predicted. In contrast to the hepatic metabolism, no hydrolysis reactions are predicted to occur in the skin. Only the introduction of hydroxyl groups is simulated in the skin metabolism prediction. In general, the hydroxyl groups make the substances more water-soluble and susceptible to metabolism by phase II-enzymes. The smaller molecules resulting from hydrolysis of the parent compounds are also expected to have higher water solubility. The metabolites formed in the skin are relatively few, compared with the liver, due to the lower level of enzymes in the skin. The skin metabolites and any absorbed parent substance will enter the blood circulation and have the same fate as the hepatic metabolites. A multitude of up to 114 metabolites were predicted to result from microbiological metabolism. The high number includes many minor variations in the carbon-chain length and number of carbonyl- and hydroxyl groups; reflecting the many microbial enzymes identified. Not all of these reactions are expected to take place in the human GI tract. The results of the OECD Toolbox prediction substantiate the information on metabolism known from the general literature (Lehninger, 1993).

There is no indication that amides, C16-C18 (even), N,N'-ethylenebis is metabolised to mutagenic intermediates under the relevant test conditions. All three experimental studies performed on genotoxicity (Ames test, chromosome aberration test and mouse lymphoma assay) were negative, with and without metabolic activation. In addition, the substance did not show skin sensitising properties in the Local Lymph Node Assay (LLNA), indicating that it is either not taken up through the skin or not metabolised to reactive compounds that would lead to skin sensitisation.

Excretion

Ethylenediamine formed as an hydrolysis product of amides, C16-C18 (even), N,N'-ethylenebis will most likely be conjugated with e.g. glutathione to form more water-soluble compounds and may then be excreted via the urine. The fatty acids palmitic and stearic acid can be utilised for energy generation by entering the citrate cycle. Thus, they are expected to be ultimately degraded to CO2, which will be exhaled.

The fraction of the parent substance that is not absorbed in the GI tract will be excreted via the faeces. If microbial metabolism occurs, the smaller metabolites may be absorbed, entering the systemic circulation. The metabolites are expected to be conjugated as described above and excreted via the urine.

References

ECHA (2017). Guidance on information requirements and chemical safety assessment, Chapter R.7c: Endpoint specific guidance. Version 3.0.

Gossauer, Albert (2006). Struktur und Reaktivität der Biomoleküle, Verlag Helvetica Chimica Acta, Zürich, 2006

Lehninger, A.L., Nelson, D.L. and Cox, M.M. (1993). Principles of Biochemistry. Second Edition. Worth Publishers, Inc., New York, USA. ISBN 0-87901-500-4.

OECD (2019). (Q)SAR Toolbox v4.3.1, https://qsartoolbox.org/, simulation performed 19 November 2019

US EPA (2014).Estimation Programs Interface Suite™ for Microsoft® Windows, v 4.11. United States Environmental Protection Agency, Washington, DC, USA.Downloaded from: http://www.epa.gov/oppt/exposure/pubs/episuite.htm

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