Registration Dossier

Administrative data

Link to relevant study record(s)

Description of key information

Please refer to expert statement regarding toxicokinetic behaviour given under "Toxicokinetics, metabolism and distribution" (see IUCLID sections 7.1 and 13).

Key value for chemical safety assessment

Additional information

Justification for grouping of substances and read-across

The present analogue approach contemplates (Z)-N-octadecyldocos-13-enamide (CAS 10094-45-8) as target substance for read-across from the source substances N-octadecylstearamide (CAS 13276-08-9) and (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6). The target and source substances are secondary fatty acid amides formed from reaction of mono-unsaturated fatty acids (C22:1ω9) with the primary fatty amine stearylamine (target substance) and long-chain saturated (C16 and C18) fatty acids with the primary fatty amines stearylamine and oleylamine (source substances), respectively. Structural similarities of the target and source substances are reflected by similar physico-chemical properties and mode of action. The target and source substance have a common metabolic fate that involves hydrolysis of the amide bond to the corresponding long-chain saturated or monounsaturated fatty acids and ammonia. Fatty acids of different carbon chain lengths, representing the main difference in the structure of the target and source substance, are metabolised via β-and ω-oxidation and fed into further physiological pathways such as the citric acid cycle. Ammonia, the second potential metabolite resulting from the hydrolysis of both the source and target substance, is rapidly absorbed, distributed, metabolised and excreted.

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

The substances within the analogue approach are considered to apply to these general rules and the similarity is justified on basis of scope of variability and overlapping of composition, representative molecular structure, physico-chemical properties, toxicological and ecotoxicological profiles and supported by various QSAR methods. There is convincing evidence that these chemicals of this analogue approach have an overall common profile. The key points that the target and source substance share are:

(i) Common origin: secondary N-alkyl alkyl amides produced from a coupling reaction between an appropriate fatty acid and a fatty amine.

(ii) Similar structural features: The target substance and the source substances are secondary amides which belong to the chemical class of N-Fatty Alkyl Amides of Saturated and Unsaturated Fatty Acids. Their structures consist of two linear carbon chains (i.e. not branched but potentially containing a C=C double bond) of chain length C14 to C24, linked by an amide moiety, -C(=O)NH-, and with the total number of carbon atoms generally falling within the range of > C32 and < C42.

(iii) Similar physico-chemical properties: Each of the substances is solid in form and has in common a low water solubility (< 0.01 mg/L), high log Pow (> 5) and low vapour pressure (< 0 Pa at 25 °C).

(iv) Common properties for environmental fate & eco-toxicological profile: Considering the low water solubility and the potential for adsorption to organic soil and sediment particles, the main compartment for environmental distribution is expected to be soil and sediment. Nevertheless, once this contact takes place, these substances are expected to be removed from the water column to a significant degree as the substance will be physically removed in sewage treatment plants due to the low water solubility and high adsorption potential to sewage sludge. Thus, discharged concentrations of these substance (if at all) into the aqueous/sediment and soil compartment are likely to be low. Evaporation into air and the transport through the atmospheric compartment is not expected since the target substance and the source substances are not volatile based on the low vapour pressure. Moreover, bioaccumulation is assumed to be low based on the insolubility of amides of saturated and unsaturated fatty acids. It is not likely that they can be found in the aquatic environment in high concentrations. Additionally, a bioaccumulation test is technically hardly feasible due to the high insolubility of the substances and is not necessary due to the non-hazardous character of the substances.

Available data for the target and the source substances showed that the substances are of low toxicity to aquatic organisms as no effects were observed in acute studies up to the limit of water solubility (fish, aquatic invertebrates and algae). Target and source substance did not exhibit any effects on aquatic microorganisms. Therefore, effects on the microorganism community and the degradation process in sewage treatment plants are not anticipated.

(v) Similar metabolic pathways: The target and source substances are anticipated to be hydrolysed in the gastrointestinal tract and/or liver, resulting in the generation of primary fatty amines (stearyl amine or oleylamine) as well as the long-chain saturated and mono-unsaturated fatty acids (C16:0, C18:0, or C22:1ω9). Hydrolysis represents the first chemical step in the absorption, distribution, metabolism and excretion pathways assumed to be similar between the target substance and the source substance. Following hydrolysis of fatty acid amides, fatty acids are readily absorbed by the intestinal mucosa and distribute systemically in the organism. The resulting long-chain fatty acids are primarily degraded via peroxisomal β-oxidation and the breakdown products are finally metabolised for energy generation after transport to the mitochondria. Unsaturated fatty acids like erucic acid (C22:1ω9) require additional isomerization prior to entering the β-oxidation cycle. Alternative pathways for (very) long chain fatty acids (≥ C22) may also involve omega-oxidation in the endoplasmic reticulum at high concentrations, resulting in the formation of long-chain dicarboxylic acids that are further degraded to short-chain dicarboxylic acids and finally excreted via urine. The second hydrolysis product, the primary fatty amine (oleylamine or stearylamine) may be oxidatively deaminated by monoaminooxidases to yield the corresponding aldehyde and ammonia. The aldehyde may be further oxidised via the enzymatic action of aldehyde dehydrogenase to the corresponding carboxylic acid, which may be fed into further metabolic pathways such as beta-oxidation. Ammonia resulting from the oxidative deamination of the hydrolysis product stearylamine and oleylamine is likewise readily absorbed and distributed within the body, especially in liver, where it is detoxified via the urea cycle. The resulting urea is transported to the kidneys, where it will either be re-absorbed and fed into physiological pathways, or directly excreted via urine.

(vi) Common levels and mode of human health related effects: The available data indicate that the target and source substances have similar toxicokinetic behaviour (low bioavailability of the parent substance; anticipated hydrolysis of the amide bond followed by absorption, distribution, metabolism and excretion of the breakdown products) and that the constant pattern consists in a lack of potency change of properties. Thus, based on the available data, the target and the source substance of the analogue approach show a low acute oral, dermal and inhalation toxicity and no potential for skin or eye irritation and no skin sensitisation properties.

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

 

Similar metabolic pathways

Toxicokinetic, metabolism and distribution

Absorption:

Absorption is a function of the potential for a substance to diffuse across biological membranes. The most useful parameters to provide information on this potential are the molecular weight, octanol/water coefficient (log Pow) value and water solubility (ECHA, 2012). The log Pow value provides information on the relative solubility of the substance in water and lipids (ECHA, 2012).

Oral

The molecular weight of the target substance (Z)-N-octadecyldocos-13-enamide (CAS 10094-45-8) and the source substances N-octadecylstearamide (CAS 13276-08-9) and (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6) is higher than 500 g/mol, indicating that all substances are poorly available for absorption (ECHA, 2012). 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 hardly soluble in water (≤ 1 mg/L), which would otherwise be poorly absorbed (ECHA, 2012). The high log Pow in combination with the very low water solubility suggests that any absorption of the target and source substances will likely happen via micellar solubilisation by bile salts (ECHA, 2012).

The absorption potential of a substance may also be derived from oral toxicity data, in which e.g. treatment-related systemic toxicity was observed (ECHA, 2012).

The available acute oral toxicity data on the source substance N-octadecylstearamide (CAS 13276-08-9) and (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6) consistently showed an oral LD50 value > 2400 mg/kg bw based on the lack of mortality and systemic effects in rats (Thouin, 1986a; Thouin, 1986b). Moreover, data on the oral repeated dose toxicity in rat is available for the target substance, indicating that continuous dietary application of the substance for 90 days did not result in any treatment-related adverse effects up to and including the highest dose level of 1000 mg/kg bw/day (Brownlie, 1998). Furthermore, no adverse effects were observed in rats after receiving the target substance via oral gavage for 5 consecutive days at the relatively high dose level of 5000 mg/kg bw/day (Levenstein, 1963).

Overall, the available data indicate that both the target and source substance have a low potential for toxicity via the oral route, although no assumptions can be made regarding the actual amount absorbed based on these experimental data.

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 pH-dependent hydrolysis, metabolism by GI flora, or by enzymes released into the GI tract. These changes will alter the physicochemical characteristics of the substance and hence predictions based upon the physico-chemical characteristics of the parent substance may no longer apply (ECHA, 2012).

Possible metabolites following hydrolysis of the target and source substance were predicted using the OECD QSAR Toolbox version 3.0 (OECD, 2012). The simulation of acidic and basic hydrolysis of both the target substance resulted in the formation of two metabolites, identified to be the corresponding mono-unsaturated long-chain fatty acid (Z)-docos-13-enoic acid (erucic acid, C22:1ω9) and the primary amine stearylamine after partial hydrolysis of the parent compound.

However, having regard to the in vivo situation, acidic hydrolysis of the target and source substance in the stomach is not expected to occur, since both the target and source substance show a very low solubility in water. This assumption is further supported by hydrolysis data on the structurally related water-insoluble long-chain fatty acid amide oleamide, showing a negligible rate of hydrolysis after incubation for 4 h at 37 °C in simulated gastric fluid containing the hydrolase pepsin (Cooper et al., 1995). In contrast, simulated intestinal fluid enriched with a mixture of several digestiveenzymes(pancreatin) and bile salts was found to significantly increase the rate of hydrolysis of oleamide to about 95% after incubation for 4 h at 37 °C, suggesting that the environmental conditions in the intestinal fluid in vivo may likewise favour hydrolysis of water-insoluble fatty acid amides. However, only in the presence of bile salts a complete hydrolysis of the fatty acid amide oleamide in intestinal fluid was achieved, indicating that spontaneous micelle formation by the involvement of bile salts seems to be an important prerequisite for the hydrolysis of long-chain fatty acid amides.

In contrast to the predicted acid- or base-catalysed chemical hydrolysis, data from naturally occurring long-chain fatty acid amides suggest that the target and source substance may rather be cleaved via enzymatic action of intestinal hydrolases after uptake into the body.

There is evidence provided from the physiologically occurring, bioactive substances oleamide and anandamide (arachidonylethanolamide) to show that primary and secondary amides derived from long-chain fatty acids are substrates for fatty acid amide hydrolase (FAAH), a serine hydrolase enzyme widely distributed in the body, including small intestine, liver, kidney and brain (Bisogno et al., 2002; Boger et al., 2000; Wei, et al. 2006). In humans, the liver is one of the organs with the highest FAAH expression and, in contrast to rat and mice, both forms of fatty acid amide hydrolase (FAAH-1 and FAAH-2) are expressed here (Wei et al., 2006). Therefore, the final and complete hydrolysis of those minor amounts of fatty acid amides, which might have escaped hydrolysis in the lumen and the cells of the small intestine so far, may be hydrolysed by FAAH enzymes located in the liver.

Data on the in vitro digestion of the target substance (Z)-N-octadecyldocos-13-enamide (CAS 10094-45-8) in freshly prepared rat liver homogenate, which is known to contain typical mammalian amidases such as FAAH, is available (FDRL, 1963). In this study, increasing quantities of the substance (3, 10 and 100 mg) were incubated for up to 6 h with 2 g of rat liver homogenate additionally enriched with bile salts and simulated intestinal fluid excluding pancreatin. Liberated fatty acids were neutralised with 0.1 N base and excess base was analysed after back titration with 0.1 N acid. The degree of enzymatic hydrolysis of the parent compound was determined relative to total available acidity as measured by acid hydrolysis of the sample in 6.0 N hydrochloric acid for 3 hours, followed by acidification, extraction and titration. The results showed that 100 mg sample was digested up to ca. 40% within 6 h in liver homogenate, whereas for progressively lower levels of substance, the relative degree of hydrolysis proportionately increased up to 73% and 105% for the 10 mg and 3 mg sample, respectively. Although complete hydrolysis of the target substance was demonstrated at lower levels, the amount of the enzyme in the liver was the limiting factor for digestion of higher amounts of the substance.

In summary, for the in vivo situation, it cannot be directly ruled out if the parent substance or a fraction of it may be absorbed unchanged by micellar solubilisation and be hydrolysed within the body. Therefore, in a worst case approach, target substance (Z)-N-octadecyldocos-13-enamide (CAS 10094-45-8)is anticipated to be enzymatically hydrolysed tomono-unsaturated long-chain omega-9 fatty acid (Z)-docos-13-enoic acid (erucic acid, C22:1ω9)as well as the primary amine oleyl amine stearylamine.

In general, free fatty acids are readily absorbed by the intestinal mucosa after hydrolysis from triglycerides. Within the epithelial cells, fatty acids are (re-)esterified with glycerol to triglycerides. In general, short-chain or unsaturated fatty acids are more readily absorbed than long-chain, saturated fatty acids (Greenberger et al., 1966; IOM, 2005; Mattson and Volpenhein, 1962, 1964).

The second hydrolysis product, stearylamine, is anticipated to be oxidatively deaminated by monoaminooxidases to yield the corresponding aldehyde and ammonia (Hayes, 2001).

Ammonia, which is liberated from the hydrolysis product oleylamine, is an endogenously occurring molecule resulting from various metabolic processes, including the catabolism of amino acids, amines, nucleic acids, glutamine and glutamate in peripheral tissues (especially in muscle, liver and kidney). Most of the naturally occurring ammonia (ca. 0.23 mol/day) is formed in the gastrointestinal tract, especially in the colon, by hydrolysis of dietary proteins. In the intestine, ammonia is also produced from glutamine or by the rehydrolysation from urea to ammonium (Kuntz and Kuntz, 2008). Ammonia is freely diffusible and toxic to the mammalian organism. However, under physiological conditions, more than 90% of ammonia resulting from metabolic degradation is available as non-diffusible ammonium, resulting in cellular accumulation (Kuntz and Kuntz, 2008; Lehninger, 1993). In the gastrointestinal system, ammonia is readily absorbed in the portal circulation and to a great part detoxified in liver via the urea cycle (Kuntz and Kuntz, 2008; Lehninger, 1993).

In conclusion, based on the available information, the physicochemical properties and molecular weight of the target substance (Z)-N-octadecyldocos-13-enamide (CAS 10094-45-8) and the source substances N-octadecylstearamide (CAS 13276-08-9) and (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6) suggest poor oral absorption. However, due to the strong structural similarity, both substances are anticipated to undergo enzymatic hydrolysis in the gastrointestinal tract and absorption of the hydrolysis products may also be relevant.

Dermal

In general, the physical state may already be taken into consideration for a crude estimation of the absorption potential of a substance, which means that 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. Furthermore, the dermal uptake of substances with a high water solubility of > 10 g/L (and log Pow < 0) will be low, as the substance may be too hydrophilic to cross the stratum corneum. Log Pow values between 1 and 4 favour dermal absorption (values between 2 and 3 are optimal), in particular if water solubility is high. In contrast, log Pow values < –1 suggest that a substance is not likely to be sufficiently lipophilic to cross the stratum corneum, therefore dermal absorption is likely to be low (ECHA, 2012).

The target substance (Z)-N-octadecyldocos-13-enamide (CAS 10094-45-8) and the source substances N-octadecylstearamide (CAS 13276-08-9) and (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6) are solids with very low water solubility, thus indicating a poor dermal absorption potential (ECHA, 2012). The high log Pow of these substances suggests that their rate of skin penetration may be limited by the rate of transfer between the stratum corneum and the epidermis (ECHA, 2012).

Apart from the physico-chemical properties, further criteria may apply to assume the dermal absorption potential of the target and source substances.

In general, substances that show skin irritating or corrosive properties may enhance penetration by causing damage to the surface of the skin. Furthermore, if a 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, 2012).

The experimental in vitro and animal data on the source substances N-octadecylstearamide (CAS 13276-08-9) and (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6) show that no significant skin irritation and no signs of systemic intoxication occurred, which excludes enhanced penetration of the substance due to local skin damage (Thouin, 1986c; Thouin, 1986d). Furthermore, no skin reactions attributable to a sensitisation reaction and no systemic effects were observed in the skin sensitisation study with both source substances (Weterings, 1986a; Weterings, 1986b).

Furthermore, data on dermal toxicity may indicate whether a substance may be absorbed, if signs of systemic toxicity were clearly attributable to treatment (ECHA, 2012).

Consistent with the data on skin irritation and sensitisation, there is no indication for clinical signs of toxicity and any other treatment-related adverse effects from the acute dermal toxicity study with the source substance (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6), resulting in an dermal LD50 > 2000 mg/kg bw in rat (Bradshaw, 2012). Thus, consistent with the data from acute oral toxicity, a low potential for acute dermal toxicity has been demonstrated, although no information on the actual amount of dermally absorbed substance may be derived from these observations.

Overall, based on the available information on physicochemical properties, the dermal absorption potential of target substance (Z)-N-octadecyldocos-13-enamide (CAS 10094-45-8) is predicted to be low.

Inhalation

As the vapour pressure of target substance (Z)-N-octadecyldocos-13-enamide (CAS 10094-45-8) and the source substances N-octadecylstearamide (CAS 13276-08-9) and (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6) is very low (4.9E-05 Pa at 20°C, 8.3E-05 Pa at 20 °C and 0 Pa at 25°C, respectively), the volatility is also low. Therefore, the potential for exposure to vapours and subsequent absorption via inhalation during normal use and handling is considered to be negligible.

In general, particles with an aerodynamic diameter < 100 μm have the potential to be inhaled, whereas only particles with an aerodynamic diameter < 50 μm can reach the thoracic region and those < 15 μm may enter the alveolar region of the respiratory tract (ECHA, 2012). Data on the particle size distribution of the target substance demonstrate that the inhalable fraction of the target substance is considerably low, as it contains only 0.07% of particles with an aerodynamic diameter < 500 µm (Croda Europe Limited, 2011). Therefore, under normal conditions of handling, human exposure to the target substance via the inhalation route is negligible. Moreover, if any inhalation exposure may occur, the molecular weight, log Pow and water solubility of the target substance are suggestive of very low absorption across the respiratory tract epithelium, preferably by micellar solubilisation.

Hydrolases present in the lung lining fluid may also hydrolyse the substance, hence making the hydrolysis products of the target and source substances, the primary fatty amines stearylamine and oleylamine as well as the corresponding long-chain fatty acids (C16, C18 and C22:1ω9), available for inhalative absorption.

However, due to the information available (low volatility and no inhalable particle size fraction), absorption via inhalation route is assumed to be unlikely, but in case exposure via inhalation should actually occur, absorption is expected to be identical compared to the oral route which is considered to be sufficiently conservative for hazard assessment.

Distribution and Accumulation:

Distribution of a compound within the body depends on the physicochemical 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 extracellular concentration, particularly in fatty tissues (ECHA, 2012).

Considering the worst case situation, both the target and source substance will mainly be absorbed in the form of the hydrolysis products. Therefore, the primary fatty amines (oleylamine and stearylamine) and the long-chain fatty acids (C16, C18 and C22:1ω9) are the most relevant components to assess for the target and source substance.

After being absorbed, fatty acids are (re-)esterified along with other fatty acids into triglycerides and released in chylomicrons, which 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 tissue and oxidised in order to generate energy, or they are released into the systemic circulation and transported in chylomicrons or lipoproteins and returned to the liver (IOM, 2005; Johnson, 1990; Lehninger, 1993; Stryer, 1996).

Very long-chain monounsaturated fatty acids (≥ C22) from dietary triglycerides, such as (Z)-docos-13-enoic acid (erucic acid), which is one of the proposed hydrolysis products of target substance (Z)-N-octadecyldocos-13-enamide (CAS 10094-45-8), have been shown to be related to fatty accumulation in the heart, presumably due to the slow oxidative breakdown of long-chain fatty acids in the mitochondria (Bremer and Norum, 1982).

In detail, an association between dietary (Z)-docos-13-enoic acid (erucic acid) and myocardial lipidosis has been shown in rats and nursling pigs, and an association between dietary (Z)-docos-13-enoic acid (erucic acid) and heart lesions has been demonstrated in rats, although at very high daily exposure levels. However, there is no evidence that dietary (Z)-docos-13-enoic acid (erucic acid) can be correlated to either of these effects in humans; nevertheless, concerning what is known about (Z)-docos-13-enoic acid (erucic acid) metabolism a possible susceptibility of humans to myocardial lipidosis following high levels of (Z)-docos-13-enoic acid (erucic acid) cannot be completely excluded (Food Standards Australia New Zealand, 2003).

There is strong evidence that primary amines like stearyl amine and oleyl amine will be readily distributed within the organism, as experimental animal data on several alkyl amines, e.g. octanamine, demonstrated that alkyl amines are rapidly distributed to the lung, brain, heart, spleen, kidneys and liver (Committee for Risk Assessment, 2011).

Taken together, the potential hydrolysis products of the target and the source substances are anticipated to distribute systemically in the organism.

Metabolism:

The potential metabolism of the target and source substances initially occurs via hydrolysis of the amide bond resulting in saturated and mono-unsaturated long-chain fatty acids (C16, C18, or C22:1ω9) and primary fatty amines (stearylamine and oleylamine, respectively). Besides chemical hydrolysis, fatty acid amides may be cleaved via enzymatic action of hydrolases, e.g. FAAH, present in the GI tract and other compartments of the body, e.g. the liver. In contrast, substances which are absorbed through the pulmonary alveolar membrane or through the skin may enter the systemic circulation directly before entering the liver where hydrolysis is likely to take place (ECHA, 2012).

A major metabolic pathway for linear fatty acids is the beta-oxidation which is one of the main mechanisms required 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. The complete oxidation of mono-unsaturated fatty acids such as oleic or erucic acid requires an additional isomerisation step. Further oxidation via the citric acid cycle leads to the formation of H2O and CO2(Lehninger, 1993). In addition to mitochondria, beta-oxidation may also take place in the cellular peroxisomes, which is especially important for the oxidation of naturally occurring, very long chain fatty acids comprising chain lengths of > C22. The oxidation in peroxisomes takes place via a similar enzymatic pathway, but the enzymes mediating the first oxidation step of beta-oxidation reduce oxygen, resulting in the formation of hydrogen peroxide that needs to be detoxified by the action of catalase. Since peroxisomes do not contain enzymes of the citric cycle, acetyl-CoA molecules resulting from beta-oxidation cannot be further metabolised at this place, and thus have to be transported to the mitochondria for energy generation (Lehninger, 1993). However, very long-chain fatty acids, especially those with saturated carbon back bone, decrease the activity of beta-oxidising enzymes in the peroxisomes, thereby decelerating oxidation of those fatty acids (Bremer and Norum, 1982).

Alternative pathways for (very) long-chain fatty acids include the omega-oxidation at high concentrations (WHO, 1999). The first step in the fatty oxidation via this pathway involves hydroxylation of the terminal (omega) carbon atom of the fatty acid by enzymes of the cytochrome P450 family and further oxidation to omega-carboxylic acids via alcohol and aldehyde dehydrogenase in the endoplasmic reticulum (Sanders, 2006). The resulting dicarboxylic acid may then be further oxidised in peroxisomes and/or mitochondria to short-chain dicarboxylic acids, which are finally excreted via urine (Ferdinandusse et al., 2004; Sanders et al., 2006).

Ammonia, resulting from the deamination of the hydrolysis products stearylamine and oleylamine , may be transported to the liver, where it will be converted to urea via the urea cycle. About two-thirds of the ammonia transported to the liver is detoxified via the urea cycle in the periportal hepatocytes, whereas the remaining part is trapped by periportal hepatocytes involved in the glutamine cycle (Kuntz and Kuntz, 2008; Lehninger, 1993). The urea formed in periportal hepatocytes diffuses into the blood and is then transported to the kidneys for re-absorption or final excretion (Lehninger, 1993). However, urea transported in the blood stream may also be taken up into the lumen of the gastro-intestinal tract, in a process termed ‘urea nitrogen salvaging’, where bacterial ureases can cleave urea to provide nitrogen for the synthesis of amino acids and peptides, which may also be reabsorbed by the host mammalian circulation (Stewart and Smith, 2005). Glutamine, the non-toxic transport form of ammonia, is generated in periportal hepatocytes via the action of the enzyme glutamine synthetase (Kuntz and Kuntz, 2008). Hepatic glutamine may be released into blood, distributed to other tissues and fed into the synthesis of amino acids (Lehninger, 1993).

The potential metabolites following enzymatic metabolism of the target substance were predicted using the OECD QSAR Toolbox version 3.0 (OECD, 2012). This QSAR tool predicts which metabolites may result from enzymatic activity in the liver and in the skin, and by intestinal bacteria in the gastrointestinal tract. Thirty-nine hepatic metabolites and 8 dermal metabolites were predicted for the target substance. The amide bond is cleaved in both the liver and skin, and the hydrolysis products (the long-chain unsaturated fatty acids (C22:1ω9) as well as the primary fatty amine stearylamine) may be further metabolised. Besides hydrolysis, liver and skin metabolites of the target substance are either the product of beta-oxidation of the C18 fatty acid resulting from the oxidative deamination of stearylamine and subsequent aldehyde dehydrogenase-dependent oxidation of the corresponding aldehyde or omega-oxidation of the very long-chain unsaturated C22 fatty acid followed by beta-oxidation to short-chain dicarboxylic acids. The metabolites formed in the skin are expected to enter the blood circulation and have the same fate as the hepatic metabolites. Up to 170 metabolites were predicted to result from all kinds of microbiological metabolism after hydrolysis of the substance.

Furthermore, the available data on the target substance (Z)-N-octadecyldocos-13-enamide (CAS 10094-45-8) provide evidence that the substance is not activated to reactive metabolites in the presence of an artificial metabolic system in vitro, since studies performed on genotoxicity (Ames test, gene mutation in mammalian cells in vitro, chromosome aberration assay in mammalian cells in vitro) with the target substance consistently showed negative results independent of metabolic activation (Jones, 1990,; Collins, 1997; Kelly, 1997).

Excretion:

The saturated and unsaturated long-chain fatty acids resulting from hydrolysis of the target and source substances will be further metabolised in order to generate energy or stored as lipids in adipose tissue or used for further physiological functions, e.g. incorporation into cell membranes (Lehninger, 1993). Therefore, the fatty acid metabolites are not expected to be excreted to a significant degree via the urine or faeces, but they are expected to be excreted via exhaled air as CO2or stored as described above.

In the case of omega-oxidation of the very long-chain fatty acids, the resulting very long-chain dicarboxylic acids will be further beta-oxidised to short-chain dicarboxylic acids, which are finally excreted via urine (Ferdinandusse, 2004; Sanders, 2006).

However, the very long-chain fatty acid (Z)-docos-13-enoic acid (erucic acid), which is a potential hydrolysis product of the target substance (Z)-N-octadecyldocos-13-enamide (CAS 10094-45-8), has also been reported to be directly excreted in faeces without any metabolisation (Food Standards Australia New Zealand, 2003).

Most of the urea resulting from the detoxification of ammonia in the liver will be transported to the kidneys, where it will either be re-absorbed or directly passed into the urine (Lehninger, 1993).

Taken together, the available data support the assumption that the major portion of the target and source substance may be cleaved after absorption, and the resulting hydrolysis products may either be utilised in physiological pathways or may be excreted from the organism.

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

 

Similar mammalian toxicity profiles

The toxicological properties show that the target substance (Z)-N-octadecyldocos-13-enamide (CAS 10094-45-8) and the source substances N-octadecylstearamide (CAS 13276-08-9) and (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6) have similar toxicokinetic behaviour, including low bioavailability of the parent substance, but anticipated hydrolysis of the amide bond followed by absorption, distribution, metabolism and excretion of the breakdown products ammonia as well as free long-chain, saturated or mono-unsaturated fatty acids (C16:0, C18:0, C18:1 or C22:1ω9). Based on the common metabolic fate, which is irrespective of the degree of fatty acid saturation and carbon chain length, the target and source substance show no acute oral, dermal or inhalative toxicity, no potential for skin and eye irritation and no skin sensitisation properties.

An overview on the target and source substance and their mammalian toxicity profiles is given below:


Mammalian toxicity

 

Target

Source 1

Source 2

Chemical Name

(Z)-N-octadecyldocos-13-enamide (a)

(Z)-N-octadec-9-enylhexadecan-1-amide (b)

N-octadecylstearamide

CAS No.

10094-45-8

16260-09-6

13276-08-9

Acute toxicity oral

RA: CAS 13276-08-9
RA: CAS 16260-09-6

Experimental result:
LD50 > 2400 mg/kg bw

Experimental result:
LD50 > 2400 mg/kg bw

Acute toxicity dermal

RA: CAS 16260 -09 -6

Experimental result:

LD50 > 2000 mg/kg bw

--

Acute toxicity inhalation

Waiving

Waiving

--

Skin irritation

RA: CAS 13276-08-9
RA: CAS 16260-09-6

Experimental result:
not irritating

Experimental result:
not irritating

Eye irritation

RA: CAS 13276-08-9
RA: CAS 16260-09-6

Experimental result:
not irritating

Experimental result:
not irritating

Skin sensitisation

RA: CAS 13276-08-9
RA: CAS 16260-09-6

Experimental result:
not sensitising

Experimental result:
not sensitising

Repeated dose toxicity oral

Experimental result:
NOAEL (rat) ≥ 1000 mg/kg bw/day
(90-day study)

Experimental result:
NOAEL (rat) ≥ 1000 mg/kg bw/day
(90-day study)

--

Genetic Toxicity in vitro: gene mutation in bacteria

Experimental result:
not mutagenic

Experimental result:
not mutagenic

--

Genetic Toxicity in vitro: cytogenicity in mammalian cells

Experimental result:
not clastogenic

Experimental result:
not clastogenic

--

Genetic Toxicity in vitro: gene mutation in mammalian cells

Experimental result:
not mutagenic

Experimental result:
not mutagenic

--

Genetic Toxicity in vivo

Waiving

Waiving

--

Toxicity to reproduction - Fertility

Waiving

Waiving

--

Toxicity to reproduction – Developmental toxicity

Waiving

Testing proposal: OECD 414

--

(a) The substance subject to registration is indicated in bold font.

(b) Reference (read-across) substance is indicated in normal font. Lack of data for a given endpoint is indicated by “--“.

 

Acute toxicityoral, dermal and inhalation

No study investigating the acute toxicity via the oral and dermal route of the target substance (Z)-N-octadecyldocos-13-enamide (CAS 10094-45-8) is available. Therefore, read-across based on the analogue approach from the structurally related source substances N-octadecylstearamide (CAS 13276-08-9) and (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6) is performed to cover this endpoint.

For both source substances, studies on the acute oral toxicity in rat are available, all showing no mortalities or systemic effects. Therefore, an overall LD50 value > 2400 mg/kg bw for acute oral toxicity in rats was derived from the results of these studies. Furthermore, in an acute dermal toxicity study with the source substance (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6) no mortalities and systemic effects were observed, thus resulting in a dermal LD50 value > 2000 mg/kg bw.

No data are available on the acute toxicity via the inhalation route, since the target substance is a solid with very low vapour pressure (4.9E-05 Pa at 20 °C) and does not contain respirable particles in a significant amount. Thus, exposure of humans via inhalation is unlikely and testing for acute toxicity by the inhalation route is not appropriate.

Based on the available data on acute oral and dermal toxicity of the structural analogues, it may be concluded that (Z)-N-octadecyldocos-13-enamide (CAS 10094-45-8) does not exert acute toxicity via the oral and dermal route, either.

Skin and Eye irritation / corrosion

There are no data available on skin and eye irritation of the target substance (Z)-N-octadecyldocos-13-enamide (CAS 10094-45-8). Therefore, hazard assessment is conducted by means of read-across from the structurally related source substances N-octadecylstearamide (CAS 13276-08-9) and (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6).

Several studies on skin and eye irritation in rabbit demonstrate that the source substances are neither eye nor skin irritating.

Based on the available data on skin and eye irritation of the structurally related source substances, it may be concluded that the target substance (Z)-N-octadecyldocos-13-enamide (CAS 10094-45-8) does not have a skin or eye irritating potential, either.

Skin sensitisation

No study investigating the skin sensitisation potential of the target substance (Z)-N-octadecyldocos-13-enamide (CAS 10094-45-8) is available. Therefore, read-across based on the analogue approach from the structurally related source substances N-octadecylstearamide (CAS 13276-08-9) and (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6) is performed to cover this endpoint.

The skin sensitisation studies conducted with the source substances did not show any sensitising properties in guinea pigs.

Thus, the available studies on the skin sensitisation potential of the structural analogues provide strong evidence that the target substance (Z)-N-octadecyldocos-13-enamide (CAS 10094-45-8) is not skin sensitising, either.

Repeated dose toxicity oral

A subchronic (90-day) oral repeated dose toxicity study in male and female rats was performed with the target substance (Z)-N-octadecyldocos-13-enamide (CAS 10094-45-8), demonstrating no adverse effects up to and including the highest dose tested. Based on the results of this study, the NOAEL was considered to be ≥ 1000 mg/kg bw/day for males and females, respectively.

Furthermore, data on the subacute oral toxicity study with the target substance in female rats are available, resulting in no mortalities and no adverse effects after repeated oral gavage administration of the substance for 5 consecutive days. Based on the results of this study, the NOAEL was considered to be ≥ 5000 mg/kg bw/day for female rats.

Since adequate and reliable data are available for the target substance, no read-across is necessary to cover this endpoint.

There are no data available on repeated dose toxicity by the inhalation and dermal routes.

Genetic toxicity in vitro

All available in vitro studies with the target substance (Z)-N-octadecyldocos-13-enamide (CAS 10094-45-8) on the induction of gene mutations in bacteria and mammalian cells as well as on the induction of chromosome aberrations yielded negative results.

Since adequate and reliable data are available for the target substance, no read-across is necessary to cover this endpoint.

Toxicity to reproduction

There are no data available on the toxicity to reproduction (fertility) of target substance (Z)-N-octadecyldocos-13-enamide (CAS 10094-45-8). However, the available subchronic oral (90-day) toxicity study in male and female rats conducted with the target substance does not indicate any adverse effects on reproductive organs or tissues as assessed by gross and histopathological examination up to the conventional limit dose value of 1000 mg/kg bw/day. Therefore, on behalf of animal welfare, the conduct of animal studies on the reproduction toxicity with the target substance by any route of exposure is considered scientifically unjustified.

Developmental toxicity

There are no data on developmental toxicity available for the target substance (Z)-N-octadecyldocos-13-enamide (CAS 10094-45-8). Therefore, hazard assessment will be conducted by means of read-across from the structurally related source substance (Z)-N-octadec-9-enylhexadecan-1-amide (CAS 16260-09-6), for which a pre-natal developmental toxicity study via the oral route according to OECD Guideline 414 is proposed.

As soon as available, the results of the pre-natal developmental toxicity study with the source substance will be evaluated with regard to hazard assessment of the target substance.

Classification

According to the classification criteria of Regulation (EC) No. 1272/2008 (CLP) and Directive 67/548/EEC (DSD), the target and source substances are not classified for physical hazards, environmental hazards, or human health hazards.