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Endpoint summary

Description of key information

Hydrolysis: 79 ± 3% (4 h)
Formation of isostearic acid: 62 ± 7 (4 h) / 78 ± 1 (24 h) 

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Additional information

Hypothesis for the analogue approach

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, through the use of alternative methods, for example, in vitro methods or qualitative or quantitative structure-activity relationship models or from information from structurally related substances (grouping or read-across).” According 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 and toxicological are likely to be similar or follow a regular pattern as a result of structural similarity. 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 and toxicological profiles. There is convincing evidence that these chemicals lie in the overall common profile of this analogue approach. The key points that the target and source substances share are:

·                    Common functional groups:Both, source and target are fatty acid amides. Most of the components of the target substance have alkyl chain ranging between C16 and C18, while the source substance is predominantly C22.

·                    Similar physico-chemical properties:For the purpose of read-across of (eco)toxicity data, the most relevant physico-chemical parameters are physical state (appearance), vapour pressure, octanol/water partition coefficient and water solubility. Both substances are solid and have in common a low water solubility (<0.0191 mg/L), high partition coefficient (log Pow >6.62) and a low vapour pressure (< 3 Pa at 25 °C).

·                    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 free long-chain, saturated or unsaturated fatty acids (C16, C18 and C22). 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.

·                    Common properties for environmental fate & eco-toxicological profile of the target and source substance: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 available results demonstrate no acute or chronic toxicity of the substances up to the limit of water solubility. Based on the study results both substances are considered not toxic to aquatic organisms. Experimental data evaluating the toxicity of the target and source substance to soil/sediment organisms are not available. Since the substances are poorly water soluble an exposure of sediment organisms is considered unlikely.The Guidance on information requirements and chemical safety assessment, Chapter R7.b (ECHA, 2014) 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, 2014). Thus, discharged concentrations of this substance (if at all) into the aqueous/sediment and soil compartment are likely to be low. Based on the available information, toxicity to soil and sediment organisms is expected to be low. Evaporation into air and the transport through the atmospheric compartment are not expected since the target substance and the source substance are not volatile based on the low vapour pressure. Moreover, bioaccumulation is assumed to be low based on the results of the bioaccumulation assessment; the substances are not expected to bioaccumulate. Available data for the target and the source substance showed that the substances are not toxic 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). The target 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.

·                    Common levels and mode of human health related effects: The available data indicate that the target and source substance 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 substance are not mutagenic or clastogenic and have no toxic effects on reproduction or intrauterine development.

Analogue approach justification

Mammalian Toxicity

Possible ways for the uptake of the target substance (Amides, C18, branched and linear) and the source substance (erucamide) are the oral and the dermal route due to their use as slipping agents in the production of plastic articles and films, which might also be used as beverage containers. Therefore, the substances might enter the body as a result of foodstuff contamination, although they represent only a small fraction of the plastic. Upon systemic uptake, fatty acid amides were demonstrated to be degraded in simulated gastrointestinal fluids after 4 hours at 37 °C in the presence of bile salts; the products formed to a great extent were the corresponding fatty acids. There are sufficient toxicity data for the resulting fatty acids showing that the metabolites have little or no potential to cause toxic effects.

Amides, C18, branched and linear will be able to penetrate biomembranes like its fatty acid derivative due to their similar characteristics. Free fatty acids can penetrate plasma membranes due to their poor water solubility and high fat solubility.

Stearamide (C18 fatty acid amide) is one of the elements of the human meibomian gland secretion, which is a complex mixture of triglycerides, free fatty acids, diesters, cholesterol and wax esters, free cholesterol, hydrocarbons and polar lipids that prevents evaporation and assists in the maintenance of a stable tear film, which protects the surface of the eye.

Fatty acid amides represent a large and diverse class of lipid transmitters; magnitude and duration of their signalling in vivo are controlled by enzymatic hydrolysis. Stearamide, e.g., is one of the substrates of the fatty acid amide hydrolase (FAAH), originally named oleamide hydrolase, which is able to hydrolyse a wide range of fatty acid amides. Lately, a second enzyme with FAAH activity has been discovered which has been termed FAAH-2, referring to the original enzyme as FAAH-1. These enzymes exhibit overlapping but distinct tissue distribution, substrate selectivity, and inhibitor sensitivity profiles. Remarkably, the FAAH-2 gene is present in primates, as well as in a variety of distantly related vertebrates, but not in murids (mice and rats), suggesting even more possibilities for the metabolisation of fatty acid amides in humans. Whereas only FAAH-1 is expressed in brain, small intestine and testis, only FAAH-2 is expressed in the heart. Both enzymes are expressed in kidney, liver, lung and prostate. The expression of both enzymes in tissues known to be closely associated with absorption, metabolism and excretion of chemicals in the body, like small intestine, liver and kidney, makes it reasonable to assume their contribution also in the metabolism of fatty acid amides to their respective metabolites, which are fatty acids.

Due to the presence of adequate and effective metabolic pathways in the mammalian and especially the human organism, and the characteristics of the molecules themselves, a rapid and effective absorption, distribution and metabolism of the target substance, as well as the source substances, is very likely. The metabolites stearic acid and palmitic acid (in case of the target substance) are among the most common saturated fatty acids and are present in nearly all naturally occurring animal and vegetable oils and fats, which are common elements of our daily food uptake. Stearic and palmitic acid are included to a considerable extent (7.0 and 22.1%, respectively) even in human milk. A considerable amount of dietary saturated C18 fatty (stearic) acid was demonstrated to be oxidised to the unsaturated C18 fatty (oleic) acid in humans, which is the most abundant fatty acid in the human body as well in the daily diet.

The target and the source substances, as well as C16- or C18-fatty acids, are likely to be metabolised as any dietary fat; most of the absorbed triglycerides are utilised by muscle and fat tissue. As first step the fats are absorbed in the gastrointestinal tract. Fatty acids are separated from triglycerides by lipases. Free fatty acids can penetrate the plasma membranes due to their poor water solubility and their high fat solubility. They are transported into the mitochondria where they enter the catabolic pathway of beta oxidation which breaks down the fatty acid chain to acetyl-CoA in subsequent passages of the cycle. Acetyl-CoA enters the citric acid cycle where it is oxidised to carbon dioxide and used for the chemical reduction of NAD⁺and FAD. These in turn enter oxidative phosporylation where they are used for the production of ATP. The latter serves as universal energy source for the cells of the organism.

On the contrary, acetyl-CoA can also be used for the endogenous synthesis of fats which serve as energy reservoirs that can be mobilised in times of low carbohydrate availability. C18 and C16 saturated fatty acids were shown to be abundant in human adipose tissue.

Stearic acid might also be used for anabolic processes like formation of endogenous products like liver, bile and plasma lecithins (sphingolipids) and is a common element of intracellular messenger molecules of the phosphoinositide cascade, which is relevant for the conversion of extracellular signals into intracellular ones.

Considering the presence of effective metabolic pathways, the function of the metabolites as educts for the most effective processes of energy production, storage and signal transduction, and the abundant endogenous availability of the metabolites even in the human body, adverse effects after application of Amides, C18, branched and linear are not to be expected.

Based on the available data, Amides, C18, branched and linear as well as erucamide were not found to be harmful to the mammalian organism in any aspect including acute toxicity, irritation, sensitisation, repeated dose toxicity, and genetic toxicity. Table: Data matrix mammalian toxicity

	Target Chemical	Source Chemical
Name/CAS #	Amides, C18, branched and linear	Erucamide/112-84-5
In vitro skin irritation	Experimental result: EPISKIN, not irritating	Not required, in vivo data available
In vitro eye irritation	Experimental result: BCOP, not irritating	Not required, in vivo data available
In vivo skin irritation	Not required, data on acute dermal toxicity and in vitro skin irritation do not indicate irritation or corrosion	Experimental result: rabbit, not irritating
In vivo eye irritation	Not required, data on in vitro eye irritation do not indicate irritation or corrosion	Experimental result: rabbit, not irritating
Skin sensitization	Experimental result: LLNA, not sensitizing	Experimental result: LLNA, not sensitizing
In vitro gene mutation in bacteria	Experimentalresult: Ames, negative	Experimental result: Ames, negative
In vitro cytogenicity study in mammalian cells	Experimental result: Chromosomal aberration, negative	Experimental result: Chromosomal aberration, negative
In vitro gene mutation study in mammalian cells	Not required, in vivo data available	Experimental result: Mouse lymphoma, negative
In vivo cytogenicity study in mammalian cells	Experimental result: Mammalian Erythrocyte Micronucleus Test, negative	--
Acute oral route	Experimental result: rat LD50 >2500 mg/kg bw	Experimental result: rat LD50 >5000 mg/kg bw
Acute inhalation route	Data waiving: no route of exposure	Experimental result: rat LC50 > 2.8 mg/L
Acute dermal route	Experimental result: rat LD50 >2000 mg/kg bw	Experimental result: rat LD50 >2000 mg/kg bw
Short-term repeated dose toxicity (28 days)	Experimental result: rat, NOEL = 300 mg/kg bw/d; NOAEL≥1000 mg/kg bw/d	Experimental result: rat, NOAEL = 100000 ppm in feed
Sub-chronic toxicity study (90-day)	RA from Source Chemical: rat, NOAEL≥ 1000 mg/kg bw/d	Experimental result: rat, NOAEL≥ 1000 mg/kg bw/d
Chronic toxicity study (2 years)	Not required	Not indicated
Reproduction toxicity: screening study	Data waiving: Not required, RA from pre-natal developmental toxicity study from Source Chemical	Data waiving: Not required, pre-natal developmental toxicity study available
Reproduction toxicity: extended one-generation reproductive toxicity study	Data waiving: Not indicated based on result of RA from sub-chronic toxicity study from Source Chemical	Data waiving: Not indicated based on result of sub-chronic toxicity study.
Pre-natal developmental toxicity study	RA from Source Chemical: rat, NOAEL (maternal/developmental toxicity)≥ 1000 mg/kg bw/d	Experimental result: rat, NOAEL (maternal/developmental toxicity)≥ 1000 mg/kg bw/d
Assessment of the toxicokinetic behaviour	Expert statement: degradation to fatty acid and ammonia, no adverse effects	Expert statement: degradation to fatty acid and ammonia, no adverse effects

Toxicokinetics

Two reliable hydrolysis studies with Amides, C18, branched and linear are available.

A key hydrolysis study with Amides, C18, branched and linear using simulated intestinal fluid was conducted following the test protocols given in EFSA 'Note for Guidance' 30/7/2008 (Cooper, I., Krebs, A., 2015). The hydrolysis potential of the test material as well as the formation of isostearic acid was addressed in the study using GC/MS analysis. Therefore, a dose of 11.0 mg/mL of the test material was incubated with the simulant for 4 h (loss of the test material) as well as 4 and 24 h (formation of isostearic acid) and analyzed via GC/MS. The concentration of hydrolyzed test material was calculated to be 79 ± 3% after 4 h incubation with simulated intestinal fluid based on the starting concentration of 11.0 mg/mL. The formation of isostearic acid was considered to be 62 ± 7% (4 h) and 78 ± 1% (24 h).

 

A supporting hydrolysis study with Amides, C18, branched and linear using simulated intestinal fluid was conducted following the test protocols given in EFSA 'Note for Guidance' 30/7/2008 (Cooper, I., 2014). The hydrolysis potential of the test material as well as the formation of isostearic acid was addressed in the study using GC/MS analysis. Therefore, a dose of 5.75 mg/mL of the test material was incubated with the simulant for 1, 2 and 4 h to determine the hydrolysis of the test material. In addition, 11.5 mg/mL of the test material were incubated with the simulant for 1, 2 and 4 h for analysis of isostearic acid formation. The concentration of hydrolysed test material was calculated to be 85.5 ± 0.6% (1 h), 90.9 ± 0.7% (2 h) and 92.2 ± 1.6% (4 h). The formation of isostearic acid was considered to be 26 ± 2% (1 h), 38 ± 5% (2 h) and 56 ± 2% (4 h), respectively.