N,N'-ethane-1,2-diylbisoleamide

Endpoint summary

Description of key information

Low absorption following oral and dermal exposure or after inhalation is predicted for the substance. However, the substance is anticipated to undergo enzymatic hydrolysis in the gastrointestinal tract, and absorption of the hydrolysis products is also relevant. Regarding the available information, the absorption rate of the hydrolysis products is considered to be high.

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.

Additional information

Justification for grouping of substances and read-across

The present analogue approach contemplates Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis (related to CAS 110-31-6) as target substance for read-across from the source substance Amides, C16-C18 (even), N,N'-ethylenebis (related to CAS 110-30-5). Both substances are UVCBs consisting mainly of mixed secondary fatty acid amides formed primarily from long-chain saturated (C16, palmitic acid and C18, stearic acid) and/or mono-unsaturated fatty acids (C18:1, oleic acid) with the primary, bifunctional amine ethylenediamine. 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 fatty acid and ethylenediamine. Fatty acids, representing the main difference in the structure of the target and source substance, are metabolised via β-oxidation and fed into physiological pathways like the citric acid cycle, sugar synthesis and lipid synthesis. Ethylenediamine, a further 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 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 physicochemical properties, environmental fate and environmental and human health hazards. Thus, where applicable, environmental and human health effects are predicted from adequate and reliable data for the source substance, by interpolation to the target substance (read-across approach), applying the group concept in accordance with Annex XI, Item 1.5, of Regulation (EC) No 1907/2006. In particular, for each specific endpoint the source substance 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 the 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 fatty acids and ethylendiamine.

(ii) Similar structural features: They have two aliphatic hydrocarbon chains bound to ethylendiamine through an amide bond.

(iii) Similar physico-chemical properties: Similar appearance; comparable melting point; similar vapour pressure; both substances have a high log Pow; both substances are insoluble in water.

(iv) Common properties for environmental fate & eco-toxicological profile: Considering the low water solubility and the high potential for adsorption to organic soil and sediment particles, the main compartment for environmental distribution is expected to be soil and sediment. The Guidance on information requirements and chemical safety assessment, Chapter R7.b (ECHA, 2012) states that once insoluble chemicals enter a standard STP, they will be extensively removed in the primary settling tank and fat trap, and thus, only limited amounts will get in contact with activated sludge organisms. Nevertheless, once this contact takes place, these substances are expected to be removed from the water column to a significant degree by adsorption to sewage sludge (Guidance on information requirements and chemical safety assessment, Chapter R.7a, (ECHA, 2012). Thus, discharged concentrations of this 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 vapor pressure. Moreover, bioaccumulation is assumed to be low based on the results of the bioaccumulation study. 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 and chronic studies up to the limit of water solubility (fish, aquatic invertebrates and algae). Target and source substances did not exhibit any effects on aquatic microorganisms. Therefore, effects on the microorganism community and the degradation process in sewage treatment plants is not anticipated.

(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 ethylenediamine as well as free long-chain, saturated or unsaturated fatty acids (C16, C18 or C20). 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. They are either re-esterified into triacylglycerols and stored in adipose tissue, or enzymatically degraded in order to generate energy, primarily via β-oxidation and the subsequent catabolic pathways citric acid cycle and oxidative phosphorylation. Unsaturated fatty acids require additional isomerization prior to entering the β-oxidation cycle. Ethylenediamine, 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 and kidney. Rapid and major excretion of ethylenediamine occurs via urine, with N-acetylethylenediamine being identified as main urinary metabolite. To a minor extent, ethylenediamine is also exhaled as CO₂ and excreted via faeces.

(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 skin sensitisation. Furthermore, the target and source substances are not mutagenic or clastogenic and have no toxic effects on reproduction or intrauterine development.

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 is a function of the potential of 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 Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis and the source substance Amides, C16-C18 (even), N,N'-ethylenebis is higher than 500 g/mol, indicating that both 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 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 Amides, C16-C18 (even), N,N'-ethylenebis consistently showed LD50 > 5000 mg/kg bw and no systemic effects in rats of different strains (Lilja, 1981; Til and Spanjers, 1983; Nham and Harrison, 1986). Moreover, data on the oral repeated dose toxicity in rat is available for the target substance Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis, indicating that dietary application of Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis did not result in any adverse effects up to and including the highest dose tested (731.8 and 834.5 mg/kg bw/day for males and females, respectively) (Rutten and Til, 1988). Furthermore, prenatal developmental toxicity studies conducted with the source substance Amides, C16-C18 (even), N,N'-ethylenebis via the oral route did not indicate any maternal toxicity and any adverse effects on development in rat up to the currently applied limit dose value of 1000 mg/kg bw/day (Liberti, 2008; Liberti, 2009).

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 different metabolites, identified to be the corresponding saturated or mono-unsaturated long-chain fatty acids and the primary amine ethylenediamine after full hydrolysis, as well as the monosubstituted fatty acid amides of ethylenediamine with the corresponding fatty acid after partial hydrolysis.

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 substances oleamide and anandamide (arachidonylethanolamide) to show that primary and secondary 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, 2002; Boger, 2000; Wei, 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, 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.

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 substance Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis is anticipated to be enzymatically hydrolysed to long-chain saturated and mono-unsaturated fatty acids (C16, C18 and C20) as well as ethylenediamine in the organism.

After hydrolysis, the free fatty acids are readily absorbed by the intestinal mucosa. 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, ethylenediamine, has been demonstrated to be rapidly absorbed after oral and intravenous administration to mice (Leung, 2000; OECD, 2001).

In conclusion, based on the available information, the physicochemical properties and molecular weight of the target substance Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis and the source substance Amides, C16-C18 (even), N,N'-ethylenebis 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 is also relevant. Regarding the available information, the absorption rate of the hydrolysis products is considered to be high.

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 Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis and the source substance Amides, C16-C18 (even), N,N'-ethylenebis are solids that are almost insoluble in water and have a molecular weight exceeding 500 g/mol, thus indicating a low dermal absorption potential (ECHA, 2012). The log Pow is > 10, which means that the uptake into the stratum corneum is likely to be slow and the rate of transfer between the stratum corneum and the epidermis will be slow (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 animal data on the source substance Amides, C16-C18 (even), N,N'-ethylenebis show that no significant skin irritation occurred, which excludes enhanced penetration of the substance due to local skin damage (Leist, 1981; Lina, 1981; Rinehart, 1978; Lilja, 1981), and the source substance was demonstrated to be not sensitising via the skin (Stitzinger, 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 Amides, C16-C18 (even), N,N'-ethylenebis, resulting in an dermal LD50 > 2000 mg/kg bw in rabbit. 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 Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis is predicted to be low.

Inhalation

As the vapour pressure of the target substance Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis and the source substance Amides, C16-C18 (even), N,N'-ethylenebis is very low (< 2.9E-02 Pa at 25 °C and 2.74 Pa at 25 °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 0.1% of particles with an aerodynamic diameter < 500 µm (Croda Europe Limited, 2012). 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, ethylenediamine and long-chain fatty acids (C16, C18 and C20), available for absorption via the respiratory epithelium.

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 studies on acute inhalation toxicity of the source substance Amides, C16-C18 (even), N,N'-ethylenebis demonstrate several clinical signs of toxicity due to local effects after inhalation exposure in rat (Siglin, 1987; Rusch, 1979). In one of the studies, clinical signs were clearly attributable to treatment-related local irritant effects as observed by the reversible occurrence of mucoid and red nasal discharge as well as dry rales during the 14-day observation period. In addition, 4/10 animals from this study also showed discolouration of the lungs (Rusch, 1979). Likewise, rales and dark material around the nose and mouth were also observed in the study by Siglin (1987), being indicative of discharge induced by the test substance, presumably due to treatment-related local irritant effects.

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

Thus, due to the information available (low volatility and no inhalable particle size fraction), absorption via the inhalation route is assumed to be identical compared to the oral route in a worst case approach 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 the 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, ethylenediamine and long-chain fatty acids (C16, C18 and C20) are the most relevant components to assess for the target and source substance. Both classes of hydrolysis products are expected to be widely distributed in the body.

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 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).

Stored fatty acids underlie a continuous turnover as they are permanently metabolised for energy generation and excreted as CO₂. Bioaccumulation of fatty acids takes place if their intake exceeds the caloric requirements of the organism.

Ethylenediamine is a small molecule with high water solubility, thus suggesting ready distribution in the body and absorption by either passive diffusion or active transport via cell membranes (ECHA, 2012). A study on the pharmacokinetics and metabolism of radiolabelled ethylenediamine in mice via the oral and intravenous route demonstrated that radioactivity was distributed throughout the body, with the liver and kidney attaining the highest concentration among the major organs (liver, kidney, lung and brain) assessed (Leung, 2000). However, the potential for accumulation was considered to be very low, since most of the absorbed material was rapidly eliminated via urine (Leung, 2000; OECD, 2001).

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 ranging from C16 to C20 and ethylenediamine. 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 acid requires an additional isomerisation step. Further oxidation via the citric acid cycle leads to the formation of H₂O and CO₂ (Lehninger, 1993). Alternative pathways for long-chain fatty acids include the omega-oxidation at high concentrations (WHO, 1999). The fatty acids can also be conjugated (with e.g. glucuronides, sulphates) to form more polar products that are easily excreted in the urine.

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. Depending on the fatty acid moieties linked to the primary amine groups of ethylenediamine, up to 148 hepatic metabolites and up to 12 dermal metabolites were predicted for the target substance. The amide bond is partially cleaved in both the liver and skin, and the hydrolysis products (monosubstituted fatty acid amide and the corresponding free fatty acid) may be further metabolised. Besides hydrolysis, the resulting liver and skin metabolites are mainly the product of omega and beta-oxidation of fatty acids. In the case of omega-oxidation the formation of an alcohol is followed by further oxidation to the aldehyde, which is then oxidised to the corresponding carboxylic acid. In general, the hydroxyl groups render the substances more water-soluble and susceptible to metabolism by phase II-enzymes. The metabolites formed in the skin are expected to enter the blood circulation and have the same fate as the hepatic metabolites. Depending on the fatty acid moieties linked to the primary amine groups of ethylenediamine, up to 148 metabolites were predicted to result from all kinds of microbiological metabolism of the substance. In the case of microbial metabolism, full hydrolysis of the amide bond was also observed, resulting in the occurrence of free ethylenediamine.

A study on the pharmacokinetics and metabolism of radiolabelled ethylenediamine showed that the major metabolite identified in urine was N-acetylethylenediamine (Leung, 2000), a phase II metabolite mainly resulting from N-acetylation in liver.

Furthermore, the available data from the source substance Amides, C16-C18 (even), N,N'-ethylenebis 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 (Jagannath, 1978; Wright, 2006; Verspeek-Rip, 2010).

Excretion

The saturated and unsaturated 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 excreted via exhaled air as CO₂ or stored as described above.

Data on the pharmacokinetics and metabolism of the second hydrolysis product ethylenediamine revealed that most of the radiolabelled substance (> 70% of the applied dose) was eliminated from the body within the first 24 hours after application (Leung, 2000; OECD, 2001). Approximately 56-72% of the applied dose was recovered in urine, with N-acetylethylenediamine being the major urinary metabolite identified. Within the 48-hour study period, 10% of the applied dose of ethylenediamine was exhaled as CO₂ and 5-14% was excreted via faeces, whereas only 5-7% of the dose remained in the carcass (OECD, 2001).

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. Thus, no hazard regarding cumulative effects after repeated administration of the target and source substance is expected.

 

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 Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis and source substance Amides, C16-C18 (even), N,N'-ethylenebis 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 ethylenediamine as well as free long-chain, saturated or unsaturated fatty acids (C16, C18 or C20). Based on the common metabolic fate, which is irrespective of the fatty acid chain length and saturation, the target and source substance show no acute oral, dermal or inhalative toxicity, no potential for skin and eye irritation, no skin sensitisation properties, and no systemic toxicity after repeated oral exposure. Furthermore, they are neither mutagenic nor clastogenic, and indicate no potential for reproduction and developmental toxicity.

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

Mammalian toxicity

Chemical Name	Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis (a)	Amides, C16-C18 (even) , N,N'-ethylenebis (b)
Acute toxicity Oral	WoE: RA: Amides, C16-C18 (even) , N,N'-ethylenebis	WoE: Experimental result: LD50 > 5000 mg/kg bw
Acute toxicity Dermal	RA: Amides, C16-C18 (even) , N,N'-ethylenebis	Experimental result: LD50 > 2000 mg/kg bw
Acute toxicity Inhalation	RA: Amides, C16-C18 (even) , N,N'-ethylenebis	Experimental result: LC50 > 6.3 mg/L
Skin irritation	WoE: RA: Amides, C16-C18 (even) , N,N'-ethylenebis	WoE: Experimental result: not irritating
Eye irritation	WoE: RA: Amides, C16-C18 (even) , N,N'-ethylenebis	WoE: Experimental result: not irritating
Skin sensitisation	RA: Amides, C16-C18 (even) , N,N'-ethylenebis	Experimental result: not sensitising
Repeated dose toxicity oral	Experimental result: NOAEL (rat): 834.5 (males) and 731.8 (females) mg/kg bw/day (90-day study) RA: Amides, C16-C18 (even) , N,N'-ethylenebis	Experimental result: NOAEL (rat) ≥ 2500 mg/kg bw/day (2-year study)
Genetic Toxicity in vitro: gene mutation in bacteria	RA: Amides, C16-C18 (even) , N,N'-ethylenebis	Experimental result: not mutagenic
Genetic Toxicity in vitro: cytogenicity in mammalian cells	RA: Amides, C16-C18 (even) , N,N'-ethylenebis	Experimental result: not clastogenic
Genetic Toxicity in vitro: gene mutation in mammalian cells	RA: Amides, C16-C18 (even) , N,N'-ethylenebis	Experimental result: not mutagenic
Toxicity to reproduction - Fertility	Waiving	Waiving
Toxicity to reproduction – Developmental toxicity	RA: Amides, C16-C18 (even) , N,N'-ethylenebis	Experimental result: NOAEL ≥1000 mg/kg bw/day

(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 Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis is available. Therefore, read-across based on the analogue approach from the structurally related source substance Amides, C16-C18 (even), N,N'-ethylenebis is performed to cover this endpoint.

Three studies are available investigating the acute oral toxicity of Amides, C16-C18 (even), N,N'-ethylenebis in rat, all showing no systemic effects and resulting in LD50 values > 5000 mg/kg bw. Similarly, no acute toxicity was observed in two inhalation toxicity studies with the source substance, resulting in LC50 values of > 6.3 mg/L in rat. 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 the structurally related source substance, the target substance Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis is considered to be not toxic via the oral, dermal and inhalation route, either.

Skin and eye irritation / corrosion

There are no data available on skin and eye irritation of the target substance Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis. Therefore, hazard assessment is conducted by means of read-across from the structurally related source substance Amides, C16-C18 (even), N,N'-ethylenebis.

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 Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis does not have a skin or eye irritating potential, either.

Skin sensitisation

No study investigating the skin sensitisation potential of the target substance Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis is available. Therefore, read-across based on the analogue approach from the structurally related source substance Amides, C16-C18 (even), N,N'-ethylenebis 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 data on the source substance provide strong evidence that the target substance Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis is not sensitising to skin, either.

Repeated dose toxicity oral

A subchronic (90-day) oral repeated dose toxicity study in rats was performed with the target substance Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis, 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 ≥ 731.8 and 834.5 mg/kg bw/day for males and females, respectively.

Since adequate and reliable data are available for the target substance, no read-across is necessary to cover this endpoint. However, there is also supporting data available from the structurally related source substance Amides, C16-C18 (even), N,N'-ethylenebis. In a chronic (2-year) feeding study with the source substance, no adverse effects were observed in rats up to dietary dose levels of 2500 mg/kg bw/day. However, in light of the extremely high NOAEL ≥ 2500 mg/kg bw/day observed in the chronic study with the structural analogue, the use of a NOAEL of 731.8 mg/kg bw/day for males from the subchronic study with the target substance was considered more appropriate and sufficiently conservative for hazard assessment.

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 Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis is available. Therefore, read-across based on the analogue approach from the structurally related source substance Amides, C16-C18 (even), N,N'-ethylenebis 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 Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis 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 Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis.

However, a subchronic oral repeated dose toxicity study with the target substance and a chronic oral repeated dose toxicity study with the structurally related source substance did not indicate adverse effects on reproductive organs or tissues at dose levels near to or well above the currently applied limit dose value of 1000 mg/kg bw/day. In addition, the available developmental toxicity studies with the structurally related source substance did not indicate adverse effects on fertility up to and including the highest dose tested (1000 mg/kg bw/day).

Therefore, the available data on repeated dose toxicity provide sufficient weight of evidence to conclude that the target substance Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis is not toxic to reproduction, either.

Developmental toxicity

No study investigating the developmental toxicity/teratogenicity of the target substance Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis is available. Therefore, read-across based on the analogue approach from the structurally related source substance Amides, C16-C18 (even), N,N'-ethylenebis is performed to cover this endpoint.

The prenatal developmental toxicity studies conducted with the source substance via the oral route did not show any adverse effects on development in rat. The NOAEL derived from these studies was greater than the currently applied limit dose value of 1000 mg/kg bw/day, thus indicating no hazard for developmental toxicity.

Based on the results for the structurally related source substance, there is no developmental toxicity expected to occur for target substance Amides, C16 and C18-C20 (even numbered, unsaturated), N,N’-ethylenebis, either.

Classification

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