Docosanamide

Endpoint summary

Additional information

Justification for grouping of substances and read-across

The present analogue approach contemplates docosanamide (CAS No. 3061-75-4) as target substance for read-across from the source substance (Z)-docos-13-enamide (erucamide, CAS 112-84-5). Both substances are primary fatty acid amides formed from long-chain saturated (C22, docosanoic (behenic) acid) and mono-unsaturated fatty acids (C22:1ω9, (Z)-docos-13-enoic (erucic) acid) with ammonia. Structural similarities of the target and source substance 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 C22 fatty acids and ammonia. Fatty acids, 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 physico-chemical properties, 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. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across.

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: produced from the reaction between an appropriate fatty acid and ammonia, commonly using a coupling agent.

(ii) Similar structural features: The target substance and the source substance are primary amides of saturated or unsaturated fatty acids, containing only one or no double bonds. Their structures contain a linear carbon chain (i.e. not branched) and the number of carbon atoms generally fall within the range of > C14 and < C24.

(iii) Similar physico-chemical properties: Each of the substances are solid in form and have in common a low water solubility (<0.1 mg/L), high log Pow (> 5) and low vapour pressure (< 0.1 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 due to the ready biodegradability. 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 and the characteristics that the substances were readily biodegradable. 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.

(v) Similar metabolic pathways: The target and source substance are anticipated to be hydrolysed in the gastrointestinal tract and/or liver, resulting in the generation of free ammonia as well as the structurally closely related long-chain fatty acids behenic acid (C22) and erucic acid (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 are finally metabolised for energy generation after transport to the mitochondria. Unsaturated fatty acids like erucic acid 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. Ammonia, a further potential metabolite resulting from the hydrolysis of both the source and target substance, 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. Furthermore, the target and source substances are not mutagenic or clastogenic.

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

Due to the fact that target substance docosanamide (CAS 3061-75-4) and the source substance (Z)-docos-13-enamide (CAS 112-84-5) only differ in a double bond at the omega-9 position of the C22 carbon chain of the fatty acid, the molecular weight of both substances is nearly identical (approximately 340 g/mol). Since the molecular weights are < 500 g/mol, both substances are anticipated to be 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 substance 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 (Z)-docos-13-enamide (CAS 112-84-5) showed an overall LD50 value > 2500 mg/kg bw based on the lack of mortality and systemic effects in rats of different strains (Sanders, 2000; Reijnders, 1988; Whittaker, 1986). Moreover, data on the oral (28-day) repeated dose toxicity in rat is available for the source substance, indicating that dietary application of (Z)-docos-13-enamide (CAS 112-84-5) did not result in any adverse effects at a dose level of 10000 mg/kg bw/day, which is well above the currently applied limit dose of 1000 mg/kg bw/day in repeated dose toxicity testing (WARF, 1960).

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 and source substance resulted in the formation of two metabolites, identified to be the corresponding saturated or mono-unsaturated long-chain fatty acids and ammonia.

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 digestive enzymes (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 in the body. There is evidence provided from the physiologically occurring, bioactive substance oleamide to show that primary amides with 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.

Relative rates for FAAH-catalysed hydrolysis have been reported for a variety of long-chain fatty acid amides (Boger et al., 2000). Among those, (Z)-docos-13-enamide (erucamide), palmitamide and stearamide showed relative hydrolysis rates of 0.83, 0.72 and 0.69, respectively, compared to that of oleamide (representing a hydrolysis rate of 1.0). These results well illustrate that monounsaturated fatty acid amides like (Z)-docos-13-enamide and oleamide are more rapidly hydrolysed than long-chain saturated fatty acid amides (Boger et al., 2000). Indeed, a structure-activity relationship has been identified for FAAH, demonstrating that primary fatty amides, which are in general the preferred class of substrates for this enzyme, are the more effectively hydrolysed the more cis (Z)-double bonds they contain in their fatty acid moiety. In contrast, long-chain saturated fatty amides have been demonstrated to be hydrolysed slower by FAAH with increasing chain length (Boger et al., 2000; Bisogno et al., 2002). Therefore, a lower rate of hydrolysis of the target substance docosanamide (CAS 3061-75-4) with saturated fatty acid moiety compared to the source substance (Z)-docos-13-enamide (CAS 112-84-5) exhibiting a cis-(Z) monounsaturated fatty acid is anticipated.

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, the target substance docosanamide (CAS 3061-75-4) is anticipated to be enzymatically hydrolysed to the long-chain saturated C22 fatty acid docosanoic acid (behenic acid) as well as ammonia. Read-across from the source substance (Z)-docos-13-enamide (CAS 112-84-5), which has been demonstrated to be hydrolysed by FAAH, may rather reflect an overestimation of possible health hazards caused by docosanamide (CAS 3061-75-4), especially with respect to the possible adverse effects reported for its hydrolysis product (Z)-docos-13-enoic acid (erucic acid) (Food Standards Australia New Zealand, 2003).

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 available data on docosanoic acid (behenic acid) indicate a rather poor absorption from dietary sources, resulting in an approximate absorption rate of 25% in piglets (Mattson and Streck, 1974) and 30% in humans (Peters et al., 1991, as cited in Cater and Denke, 2001). In contrast, (Z)-docos-13-enoic acid (erucic acid) containing rapeseed oil has been demonstrated to have a high (99%) digestibility in humans (Deuel et al., 1949).

The second hydrolysis product, ammonia, 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 docosanamide (CAS 3061-75-4) and the source substance (Z)-docos-13-enamide (CAS 112-84-5) 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 docosanamide (CAS 3061-75-4) and the source substance (Z)-docos-13-enamide (CAS 112-84-5) are solids with very low water solubility, thus indicating a poor dermal absorption potential (ECHA, 2012). The high log Pow of both substances suggests that the rate of skin penetration by the substance 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 substance.

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 substance (Z)-docos-13-enamide (CAS 112-84-5) show that no significant skin irritation occurred, which excludes enhanced penetration of the substance due to local skin damage (Daamen, 1988; Warren, 2010). Furthermore, no skin reactions and systemic effects were observed in the skin sensitisation study with the source substance (Vogel, 2010).

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)-docos-13-enamide (CAS 112-84-5), resulting in a dermal LD50 > 2000 mg/kg bw in rat (Braun, 2010). 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 absorbed substance may be derived from these observations.

Overall, based on the available information on physicochemical properties, the dermal absorption potential of docosanamide (CAS 3061-75-4) is predicted to be low.

Inhalation

As the vapour pressure of target substance docosanamide (CAS 3061-75-4) and the source substance (Z)-docos-13-enamide (CAS 112-84-5) is very low (2.97E-02 Pa at 25 °C and 2.30E-05 Pa at 20 °C, respectively), the volatility is also low. Therefore, the potential for exposure 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 0.14% 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 substance, ammonia and the corresponding long-chain fatty acids (C22 and C22:1ω9), available for inhalative absorption.

As for acute toxicity via the oral and dermal route, the occurrence of treatment-related clinical signs of toxicity after acute inhalation exposure may indicate whether a substance has been absorbed (ECHA, 2012). The available study on acute inhalation toxicity of the source substance (Z)-docos-13-enamide (CAS 112-84-5) demonstrates no adverse systemic effects up to the maximum technically feasible concentration of 2.8 mg/L air (Pothman, 2010).

Based on these results, a low potential for acute systemic toxicity has been demonstrated, although no quantitative measure can be derived from these studies.

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, ammonia and the long-chain fatty acids (C22) 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 the source substance (Z)-docos-13-enamide (CAS 112-84-5), 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).

For docosanoic acid (behenic acid), which is one of the anticipated hydrolysis products of the target substance docosanamide (CAS 3061-75-4), no accumulation in the heart was observed in rats fed diets containing hydrogenated rapeseed oil, in which the main fatty acid component erucic acid had been converted to behenic acid via saturation of the double bound (Mattson and Streck, 1974). Based on the measurement of behenic acid in the thoracic lymph duct of the animals, an absorbability of only 25% was determined, which in turn may probably account for the low accumulation potential of behenic acid in heart tissue (Mattson and Streck, 1974).

Therefore, (Z)-docos-13-enoic acid (erucic acid) resulting from the hydrolysis of the source substance (Z)-docos-13-enamide (erucamide, CAS 112-84-5) may rather represent an overestimation of potential health hazards with regard to the target substance and its hydrolysis product.

After hydrolysis of the source and target substance in the gastrointestinal tract, the released ammonia is transported through the portal vein in the form of ammonium and distributed to the liver, where it is efficiently and rapidly detoxified via the urea cycle (Kuntz and Kuntz, 2008; Lehninger, 1993).

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

Metabolism:

The potential metabolism of the target and source substance initially occurs via hydrolysis of the amide bond resulting in saturated and mono-unsaturated fatty acids of C22 chain length and ammonia. 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 and 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 hydrolysis of the source and target substance, may be transported to 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. Only 11 hepatic metabolites and 2 dermal metabolites were predicted for the target substance. The amide bond is cleaved in both the liver and skin, and the hydrolysis products (the very long-chain saturated C22-fatty acid and ammonia) may be further metabolised. Besides hydrolysis, liver and skin metabolites of the target substance are mainly the product of 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 121 metabolites were predicted to result from all kinds of microbiological metabolism of the substance.

Furthermore, the available data from the source substance (Z)-docos-13-enamide (CAS 112-84-5) provide evidence that the target 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 source substance consistently showed negative results independent of metabolic activation (Jones, 1990; Hall, 2010; Wollny, 2010).

Excretion:

The saturated and unsaturated very long-chain fatty acids resulting from hydrolysis of the target and source substance 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 CO₂ or 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 acids (Z)-docos-13-enoic acid (erucic acid), which is a potential hydrolysis product of the source substance (Z)-docos-13-enamide (CAS 112-84-5), have 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 rapidly 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 docosanamide (CAS 3061-75-4) and the source substance (Z)-docos-13-enamide (erucamide, CAS 112-84-5) 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 the structurally closely related long-chain fatty acids behenic acid (C22) and erucic acid (C22:1ω9), which only differ in the saturation of the C22 fatty acid. Based on the common metabolic fate, which is irrespective of the degree of fatty acid saturation, 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. Furthermore, they are neither mutagenic nor clastogenic, and indicate no potential for systemic toxicity after repeated oral exposure.

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

Mammalian toxicity

Chemical Name	Docosanamide (a)	(Z)-Docos-13-enamide (b)
CAS No.	3061-75-4	112-84-5
Acute toxicity oral	RA: CAS 112-84-5	Experimental result: LD50 > 2500 mg/kg bw
Acute toxicity dermal	RA: CAS 112-84-5	Experimental result: LD50 > 2000 mg/kg bw
Acute toxicity inhalation	RA: CAS 112-84-5	Experimental result: LC50 > 2.8 mg/L
Skin irritation	WoE RA: CAS 112-84-5	WoE: Experimental result: not irritating
Eye irritation	RA: CAS 112-84-5	Experimental result: not irritating
Skin sensitisation	RA: CAS 112-84-5	Experimental result: not sensitising
Repeated dose toxicity oral	RA: CAS 112-84-5	Experimental result: NOAEL (rat) ≥ 10000 mg/kg bw/day (28-day study) NOAEL (rat) ≥ 1000 mg/kg bw/day (90-day study)
Genetic Toxicity in vitro: gene mutation in bacteria	RA: CAS 112-84-5	Experimental result: not mutagenic
Genetic Toxicity in vitro: cytogenicity in mammalian cells	RA: CAS 112-84-5	Experimental result: not clastogenic
Genetic Toxicity in vitro: gene mutation in mammalian cells	RA: CAS 112-84-5	Experimental result: not mutagenic
Genetic Toxicity in vivo	Waiving	--
Toxicity to reproduction - Fertility	Waiving	--
Toxicity to reproduction – Developmental toxicity	RA: CAS 112-84-5	NOAEL (rat, maternal and developmental)   ≥1000 mg/kg bw/day (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 toxicity: oral, dermal and inhalation

No study investigating the acute toxicity via the oral, dermal and inhalation route of the target substance docosanamide (CAS 3061-75-4) is available. Therefore, read-across based on the analogue approach from the structurally related source substance (Z)-docos-13-enamide (erucamide, CAS 112-84-5) is performed to cover this endpoint.

Three studies are available investigating the acute oral toxicity of (Z)-Docos-13-enamide (erucamide, CAS 112-84-5) in rat, all showing no mortalities and systemic effects. Therefore, an overall LD50 value > 2500 mg/kg bw for acute oral toxicity in rats was derived from the results of these studies. Similarly, no acute toxicity was observed in the inhalation toxicity study with the source substance, resulting in a LC50 value of > 2.8 mg/L in rat, which was the maximum technically feasible aerosol concentration with respirable particles of the source substance. Furthermore, the source substance was not acutely toxic in rat via the dermal route, as shown by a LD50 value > 2000 mg/kg bw.

Based on the available data on acute oral, inhalative and dermal toxicity of the structural analogue, it may be concluded that docosanamide (CAS 3061-75-4) does not exert acute toxicity via the oral, inhalation and dermal route, either.

Skin and Eye irritation / corrosion

There are no data available on skin and eye irritation of the target substance docosanamide (CAS 3061-75-4). Therefore, hazard assessment is conducted by means of read-across from the structurally related source substance (Z)-docos-13-enamide (erucamide, CAS 112-84-5).

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

Based on the available data on skin and eye irritation of the structurally related source substance, it may be concluded that the target substance docosanamide (CAS 3061-75-4) does not have a skin or eye irritating potential, either.

Skin sensitisation

No study investigating the skin sensitisation potential of the target substance docosanamide (CAS 3061-75-4) is available. Therefore, read-across based on the analogue approach from the structurally related source substance (Z)-docos-13-enamide (CAS 112-84-5) is performed to cover this endpoint.

A skin sensitisation study conducted with the source substance did not show any sensitising properties in mice.

Thus, the available study on the skin sensitisation potential of the structural analogue (Z)-docos-13-enamide (CAS 112-84-5) provides strong evidence that docosanamide (CAS 3061-75-4) is not skin sensitising, either.

Repeated dose toxicity oral

There are no data available on repeated dose toxicity of the target substance docosanamide (CAS 3061-75-4). Therefore, hazard assessment is conducted by means of read-across from the structurally related source substance (Z)-docos-13-enamide (CAS 112-84-5).

A subacute (28-day) oral repeated dose toxicity study in male rats was performed with the source substance (Z)-docos-13-enamide (CAS 112-84-5), resulting in no mortalities and no adverse effects up to the end of the study. Based on the results of this study, the NOAEL was considered to be ≥ 10000 mg/kg bw/day for male rats, which is considerably above the conventionally applied limit dose of 1000 mg/kg bw/day in repeated dose toxicity testing.

However, in light of the very high NOAEL of 10000 mg/kg bw/day derived from the 28-day toxicity study with the structurally closely related source substance, no toxicity is expected to occur after repeated exposure to the target substance docosanamide (CAS 3061-75-4), either.

A key subchronic repeated dose toxicity study (90-days) with the structurally-related substance erucamide (CAS 112-84-5) was performed according to OECD TG 408 and in compliance with GLP. In the available study the test substance was investigated for toxic effects after repeated administration via the oral route with a particular focus on fertility parameters and reproductive organs. Groups of 10 Wistar rats (Crl:WI(Han)) per sex per dose were administered doses of 100, 300 and 1000 mg/kg bw/day (nominal dose) via oral gavage. No treatment-related effects were observed in regard to body weight, food consumption, haematology, clinical chemistry, organ weights, gross and histopathology. No test item related effect on epididymal sperm motility or testicular sperm count was noted at the end of the treatment of this study. The statistical analysis showed no statistically significant changes between the control group and any of the dose groups in the testicular number of sperms/g testis. Evaluation of sperm morphology did not reveal any indicator for toxicity induced by the test item, and percentages of normal and abnormal sperms in treatment groups were comparable with the controls. The test material had no biologically significant effect on the estrous cycle analyzed 4, 8 and 12 weeks after the first administration. In summary, the 90-Day Repeated Dose Oral Toxicity study with CAS 112-84-5 in male and female Wistar rats revealed no major toxicity or mortality. No effects of the test material were found at the dose level of 1000 mg/kg bw/day. Therefore, the NOAEL is considered to be ≥1000 mg/kg bw/day.

Thus, the available data on repeated dose toxicity of the structural analogue (Z)-docos-13-enamide (CAS 112-84-5) provides strong evidence that docosanamide (CAS 3061-75-4) does not cause adverse effects after repeated exposure, either.

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

Genetic toxicity in vitro

No study investigating the genetic toxicity of the target substance docosanamide (CAS 3061-75-4) is available. Therefore, read-across based on the analogue approach from the structurally related source substance (Z)-docos-13-enamide (CAS 112-84-5) is performed to cover this endpoint.

All available in vitro studies with the source substance on the induction of gene mutations in bacteria and mammalian cells as well as on the induction of chromosome aberrations yielded negative results.

Based on the negative results of the available studies on the structurally related source substance, it may be concluded that the target substance docosanamide (CAS 3061-75-4) does not have the potential to induce genetic toxicity, either.

Toxicity to reproduction

There are no data available on the toxicity to reproduction (fertility) of target substance docosanamide (CAS 3061-75-4).

Therefore, hazard assessment was conducted by means of read-across from the structurally related source substance (Z)-docos-13-enamide (CAS 112-84-5), for which a 90-day subchronic oral toxicity study according to OECD 408 with an extended standard protocol was performed. This study included the investigation of fertility parameters as well as detailed gross and histopathological examination of reproduction organs to assess possible effects on fertility. No treatment-related effects were found at the highest dose level of 1000 mg/kg bw/day. Therefore, the NOAEL is considered to be≥1000 mg/kg bw/day (for more information please refer to Repeated dose toxicity).

Thus, the available data on repeated dose toxicity of the structural analogue (Z)-docos-13-enamide (CAS 112-84-5) provides strong evidence that docosanamide (CAS 3061-75-4) does not cause adverse effects on reproduction organs and tissues after repeated exposure, either.

Developmental toxicity

There are no data on developmental toxicity available for the target substance docosanamide (CAS 3061-75-4). Therefore, read-across based on the analogue approach from the structurally related source substance (Z)-docos-13 -enamide (CAS 112 -84 -5) is performed to cover this endpoint.

In the available key study the structurally-related test substance (Z)-docos-13 -enamide (erucamide, CAS 112-84-5) was investigated for prenatal developmental toxicity after repeated oral administration conducted according to OECD TG 414, and in compliance with GLP. Groups of 21 (low dose), 23 (medium dose) and 20 (high dose) pregnant female Crl:WI(Han) rats per dose were administered doses of 100, 300 and 1000 mg/kg bw/day via oral gavage. In summary, administration of (Z)-docos-13 -enamide (CAS 112-84-5) to pregnant female Wistar rats from gestation days 5 to 19 resulted in significant lower female litter weight as well as lower discolouration of the dexter lobe of the liver and significant increase in the litter incidence of incomplete ossification of the interparietal bone. These findings were considered to be not test item related and of no toxicological relevance due to lack of dose dependency and consistency. Therefore, both the maternal and embryo-fetal NOAELs were considered to be ≥ 1000 mg/kg bw/day.

Thus, the available data on developmental toxicity of the structural analogue (Z)-docos-13-enamide (CAS 112-84-5) provides strong evidence that docosanamide (CAS 3061-75-4) does not cause adverse effects on the foetal development, either.

Classification

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