N,N-bis(2-hydroxyethyl)dodecanamide

Endpoint summary

Description of key information

Based on the available weight of evidence experimental studies, the test substance is expected to be have a moderate to high absorption potential through oral route and a low to moderate absorption potential via dermal route. Based on experimental data and QSAR predictions, the test substance will undergo aliphatic hydroxylation as the first metabolic reaction and will be primarily excreted via urine. Further, based on the MW and key physico-chemical properties it is likely to have low bioaccumulation potential.

Key value for chemical safety assessment

Bioaccumulation potential:

low bioaccumulation potential

Absorption rate - oral (%):

50

Absorption rate - dermal (%):

10

Absorption rate - inhalation (%):

100

Additional information

ABSORPTION:

Oral absorption

Based on physicochemical properties:

According to REACH guidance document R7.C (May 2014), oral absorption is maximal for substances with molecular weight (MW) below 500. Water-soluble substances will readily dissolve into the gastrointestinal fluids; however, absorption of hydrophilic substances via passive diffusion may be limited by the rate at which the substance partitions out of the gastrointestinal fluid. Further, absorption by passive diffusion is higher at moderate log Kow values (between -1 and 4). If signs of systemic toxicity are seen after oral administration (other than those indicative of discomfort or lack of palatability of the test substance), then absorption has occurred.

The test substance C12 DEA is a non-ionic surfactant which belongs to a diethanolamine - (DEA) derived fatty acid alkanolamide category. It is a mono-constituent containing majorly C12 alkyl chain length and having a molecular weight 287.45 g/mol. The purified form of the substance appears as an off-white solid. While a new experimental water solubility is ongoing, the test substance was predicted to have a low to moderate water solubility with values determined at 49.91 and 620.04 mg/L using WSKOWWIN v1.42 and WATERNT v1.01 programs methods respectively. The log Kow values were predicted to be moderate and in the range of 2.89 to 3.88 (mean = 3.48) using three QSAR models: KOWWIN v.1.69, ALOGPS 2.1 and Molinspiration v.2018.10.

Based on the R7.C indicative criteria, and the fact that non-ionic surfactants have a higher permeating potential, the test substance can be expected to have a low to moderate absorption potential from the gastrointestinal tract.

Based on (Q)SAR predictions:  

The “Lipinski’s rule OASIS” profiler of the OECD QSAR Toolbox v.4.4.1, which describes the molecular properties important for a drug’s pharmacokinetics in the human body, predicted the major constituents with C12 alkyl chains (present at ca. 90%) to be ‘bioavailable’. Therefore, the test substance can be considered to have moderate absorption and bioavailability potential.

Based on experimental data on test substance:

A study was conducted to evaluate the absorption, distribution, metabolism and excretion of the radiolabelled test substance, N,N-bis(2-hydroxyethyl)dodecanamide (C12 DEA, also known as LDEA) in F344 rats. Three male rats were administered a single dose of (14C) C12 DEA at 1000 mg/kg bw by oral gavage. Urine was collected 6 to 24 h post-dosing to isolate metabolites. Tissue to blood ratios (TBR) was also determined by collecting adipose tissue, blood, kidney and liver 72 h post-dosing. The results of the investigation showed that C12 DEA was well absorbed and mostly excreted in urine as two polar metabolites. Approximately 60 and 80% of the dose was recovered in urine after 24 and 72 h, respectively, and 9% of the dose was recovered in faeces after 72 h. The metabolites were isolated and characterized as the half-acid amides of succinic and of adipic acid. The TBRs were highest in the adipose and liver tissues, with values of approximately 50. Under the study conditions, the substance was well absorbed and mostly excreted in urine as two polar metabolites (Mathews, 1996).

Based on the results of the study, the test substance is considered to be well absorbed.

Based on ‘other toxicity’ studies:

According to REACH guidance document R7.C (ECHA, 2017), other toxicity studies can be helpful to get information on occurrence of absorption without any specification of the extent or amount. For example: if signs of systemic toxicity are present in acute or repeated dose studies, then absorption has occurred. Also coloured urine and/or internal organs can provide evidence that a coloured substance has been absorbed.

A 90-day repeated dose oral toxicity of the test substance, C12 DEA, showed relative increase of kidneyweight in all test groups except at 0.1% in females and at 0.1 and 0.5% in males, and increases in the relative liver weight in females on the two highest levels. There were also effects on haematological parameters at 1 and 2% dose levels. Serum levels of glutamic-oxaloacetic transaminase were elevated at 0.5% and above in females but only at 0.5 % in males (Gaunt, 1965). Based on the above information, the test substance can be considered to have more likelihood of absorption. 

Conclusion: Overall, based on the available weight of evidence information, the test substance can be expected to overall have a high to moderate absorption potential through the oral route. Therefore, as a conservative approach, a value of 100% has been considered for the risk assessment.

Dermal absorption

Based on physicochemical properties:  

According to REACH guidance document R7.C (ECHA, 2017), dermal absorption is maximal for substances having MW below 100 together with log Kow values ranging between 2 and 3 and water solubility in the range of 100-10,000 mg/L. Substances with MW above 500 are considered to be too large to penetrate skin. Further, dermal uptake is likely to be low for substances with log P values <0 or <-1, as they are not likely to be sufficiently lipophilic to cross the stratum corneum (SC). Similarly, substances with water solubility below 1 mg/L are also likely to have low dermal uptake, as the substances must be sufficiently soluble in water to partition from the SC into the epidermis. 

The test substance is a solid, with a MW exceeding 100 g/mol, low to moderate water solubility and moderate log Kow exceeding 3, suggesting a low to moderate absorption potential. However, given that the test substance is a non-ionic surfactant, which have a better permeating potential through skin due to their lower CMC, higher solubilisation capability (Ullah et al., 2019; Som et al., 2012) compared to other types of surfactants, the test substance can be considered to have a moderate absorption potential.

Based on (Q)SAR predictions:  

The two well-known parameters often used to characterise percutaneous penetration potential of substances are the dermal permeability coefficient (Kp[1]) and maximum flux (Jmax). Kp reflects the speed with which a chemical penetrates across SC and Jmax represents the rate of penetration at steady state of an amount of permeant after application over a given area of SC. Out of the two, although Kp is more widely used in percutaneous absorption studies as a measure of solute penetration into the skin. However, it is not a practical parameter because for a given solute, the value of Kp depends on the vehicle used to deliver the solute. Hence, Jmax i.e., the flux attained at the solubility of the solute in the vehicle is considered as the more useful parameter to assess dermal penetration potential as it is vehicle independent (Robert and Walters, 2007).  

In the absence of experimental data, Jmax can be calculated by multiplying the estimated water solubility with the Kp values from DERMWIN v2.02 application of EPI Suite v4.11. The calculated Jmax value for the major constituent was 1.96 μg/cm2/h. As per Kroes et al., 2004 and Shen et al. 2014, the default dermal absorption for substances with Jmax between >0.1 to ≤10 μg/cm2/h can be considered to be less than 40%. Based on the predicted Jmax value, the test substance can be considered to have moderate absorption potential through the dermal route.  

Based on experimental data on test substance:  

A study was conducted to evaluate the dermal absorption of the (14C) radiolabelled test substance N,N-bis(2-hydroxyethyl)dodecanamide (C12 DEA, also known as LDEA) in F344 rats. Four male rats were exposed to a single dose of 25 or 400 mg/kg bw of the substance placed on a 1x1 inch surface of previously clipped skin, covered with a non-occlusive patch. After 72 h of exposure, gauzes and aliquots of urine, feces, dermal rinse solutions and digested skin samples (in 2N ethanolic sodium hydroxide) were collected and analysed for radiochemical content by liquid scintillation counting. Absorption was moderate; approximately 25 to 30% of the applied dose penetrated the skin in 72 h, with 3 to 5% remained associated with the dose site. No significant differences were observed between doses in the absorption and elimination of the substance when calculated on a percentage of dose basis, but a higher mass of substance was absorbed at the higher dose (Mathews, 1996).

A study was conducted to evaluate the dermal absorption of the (14C) radiolabelled test substance C12 DEA or LDEA in B6C3F1 mice. Four male mice were exposed to a single dose of 50, 100, 200 or 800 mg/kg bw of the substance placed on a 1x1 inch surface of previously clipped skin, covered with a non-occlusive patch. After 72 h of exposure, gauzes and aliquots of urine, faeces, dermal rinse solutions and digested skin samples (in 2N ethanolic sodium hydroxide) were collected and analysed for radiochemical content by liquid scintillation counting. After 72 h of exposure, 50 to 70% of the applied doses were absorbed and there were no statistically significant differences in absorption across the range of doses. The disposition of substance in the tissues was similar across the four dose levels (Mathews, 1996).

The difference in absorption rates between animals and human skin has been investigated and reported by The European Center for Ecotoxicology and Toxicology of Chemicals (ECETOC, 1993) as well as by the European Commission (EC, 2004). Both reports state that available in vivo and in vitro data demonstrate that all animal skin are more permeable than human skin and in particular rat skin is much more permeable than human skin by a factor 3-7. Based on this consideration, the test substance can be considered to have a low absorption potential. 

Based on ‘other toxicity’ studies:

According to REACH guidance document R7.C (ECHA, 2017), other toxicity studies can be helpful to get information on occurrence of absorption without any specification of the extent or amount. For example: if signs of systemic toxicity in dermal studies indicate that absorption has occurred. Also, if the substance has beenidentified as a skin sensitizer then, provided the challenge application was to intact skin, some uptake must have occurred although it may only have been a small fraction of the applied dose.

Subchronic and chronic dermal repeated dose toxicity studies available with the test substance in rats and mice showed effects on kidney and/or liver weights at doses ≥100 mg/kg bw/day, suggesting a likelihood of absorption.

Conclusion: Overall, based on all the available weight of evidence information, the test substance can be expected to overall have a moderate absorption potential through the dermal route. Therefore, as a conservative approach, a value of 50% has been considered for the risk assessment.

Inhalation absorption

Based on physicochemical properties:   

According to REACH guidance document R7.C (ECHA, 2017), inhalation absorption is maximal for substances with VP >25 KPa, particle size (<100 μm), low water solubility and moderate log Kow values (between -1 and 4). Very hydrophilic substances may be retained within the mucus and not available for absorption. 

The test substance exists in the solid physical state under ambient conditions and a relatively low vapour pressure of 0.0015 Pa at 20 °C. Hence, it is not expected to be available as either particles or vapours for inhalation under ambient conditions. In case of spraying applications, only coarse droplets would be an exposure potential resulting in very low respiratory fraction. Of the inhalable fraction, due to the low to moderate water solubility, the test substance will be retained in the mucus to an extent and the amount reaching the deeper lungs will be reduced. The larger deposited droplets from the upper respiratory tract will be subsequently transported to the pharynx and swallowed via the ciliary-mucosal escalator. The absorption potential of this fraction of the test substance can be considered to be similar to the oral route.

Conclusion: Based on all the available weight of evidence information, the test substance can be expected to have moderate to high absorption through the inhalation route. Therefore, as a conservative approach, a default value of 100% has been considered for the risk assessment.

 

METABOLISM:

Based on identified literature:

A study was conducted to evaluate the in vitro metabolism of the test substance, N,N-bis(2-hydroxyethyl)dodecanamide (C12 DEA, also known as LDEA), in liver or kidney microsomes from rat to: 1) determine the extent of its hydroxylation, 2) identify the products formed and 3) examine whether treatment with the cytochrome P4504A inducer and peroxisome proliferator diethylhexyl phthalate(DEHP) would affect hydroxylation rates. Liver and kidney microsomes from DEHP-treated and control rats were incubated with 100 µM C12 DEA for 30 min at 37°C in a shaking water bath. The metabolites were then separated and analysed by GC-MS.  97% of the hydroxylated products were identified as two major substances: 11- hydroxyl and 12-hydroxy derivatives of C12 DEA. The specific activities for C12 DEA 11- and 12-hydroxylation in microsomes prepared from control rats were 2.23±0.40 and 0.71±0.17 nmol/min/mg protein, respectively. Treatment of rats with DEHP increased the C12 DEA 12-hydroxylation specific activity 5-fold to 3.50 ± 0.48 nmol/mm/mg protein, whereas the C12 DEA 11-hydroxylase activity remained unchanged. Incubating liver microsomes from DEHP-treated rats with a polyclonal anti-rat 4A inhibited the formation of 12-OH-C12 DEA by 80% (3.98±0.10 vs. 0.80±0.08 nmol/min/mg protein), compared with the pre-immune serum, but had no inhibitory effect on the rate of 11-OH-C12 DEA formation (1.93±0.09 vs. 2.20± 0.11 nmol/min/mg protein). Rat kidney microsomes also resulted in hydroxylation of C12 DEA at its 11- and 12-carbon atoms, with specific activities of 0.05±0.01 and 0.28±0.02 nmol/min/mg protein, respectively. In conclusion, under the study conditions, the test substance was rapidly converted into 11- and 12-hydroxy derivatives in rat liver and kidney microsomes (Merdink, 1996).

Based on (Q)SAR predictions:  

(Q)SAR modelling tools such as the OECD QSAR Toolbox v 4.4.1 and FAME/FAME 2/ FAME 3 (Kirchmair et al., 2013; Sicho et al., 2017; Sicho et al., 2019) allow the identification and prioritisation of Phase I metabolic pathways, which in turn allow in relative terms an assessment whether chemically similar substances follow similar or different metabolic pathways.

The OECD QSAR Toolbox was used to predict the first metabolic reaction, as two metabolic simulators (in vivo rat metabolism simulator and rat liver S9 metabolism simulator) take into account amide hydrolysis as a Phase I metabolic reaction. The results were compared with the output generated from FAME 3 model, which represents the third generation of the FAst MEtabolizer program. FAME (FAst MEtabolizer) is a fast and accurate predictor of sites of metabolism (SoM) which is based on a collection of random forest models trained on diverse chemical data sets of more than 20000 molecules annotated with their experimentally determined SoMs. It is not limited to a specific enzyme family or species. Besides a global model, dedicated models are available for human, rat, and dog metabolism; specific prediction of phase I and II metabolism is also supported (Kirchmair et al., 2013). FAME3 allows the prediction of both phase 1 and phase 2 SoMs (Sicho et al., 2019).

Based on predictions from the two simulators of the OECD QSAR Toolbox, FAME and expert judgement, ω and/or ω-1 aliphatic hydroxylation was predicted to be the first metabolic reaction for the representative structure of the test substance (see below Table). There was an additional oxidation reaction predicted at the hydroxy group of the DEA. Hydrolysis of the amide bond to release the free alkanolamine does not seem to be a preferred metabolism path.

 

Majorconstituent	Rat liver S9 metabolism simulator/ in vivo rat metabolism simulator / FAME 3
N N,N-bis(2-hydroxyethyl) dodecanamide (C12 DEA	ωand/orω-1 aliphatic hydroxylations

 

Overall, the results from the published literature and the QSAR models are in agreement. That is, none of them predicts hydrolysis of the amide bond to release free DEA and fatty acid to be a major metabolic pathway for the test substance.

DISTRIBUTION   

Based on physico-chemical properties:  

According to REACH guidance document R7.C (ECHA, 2017), the smaller the molecule, the wider the distribution. Small water-soluble molecules and ions will diffuse through aqueous channels and pores, although the rate of diffusion for very hydrophilic molecules will be limited. Further, if the molecule is lipophilic (log P >0), it is likely to distribute into cells and the intracellular concentration may be higher than extracellular concentration particularly in fatty tissues.

The test substance is a surfactant which has a higher permeating potential due to its non-ionic nature. Combined with its physico-chemical information (i.e., MW, moderate lipophilicity and low to moderate water solubility), it suggests that test substance could be distributed to the tissues to an extent, once absorbed and bioavailable. However, based on the predicted BCF value of 12 L/kg ww using Arnot-Gobas method of EPI Suite^TMv.4.1, the bioaccumulation potential of the substance is expected to be low.

Based on ‘other toxicity’ studies:

According to REACH guidance document R7.C (ECHA, 2017), identification of the target organs in repeated dose studies can be indicative of the extent of distribution.  

As discussed in section 5.6, sub-chronic and chromic repeated dose oral and dermal toxicity studies with test substance in rodents, showed adverse effects in liver and/or kidney, suggesting a distribution potential to certain extent for the test substance.

Conclusion: Based on all the available weight of evidence information, the test substance is expected to have a distribution potential, but it is not likely to bioaccumulate.    

EXCRETION:   

Based on physicochemical properties:  

According to REACH guidance document R7.C (ECHA, 2017), the characteristics favourable for urinary excretion are low molecular weight (below 300 in the rat), good water solubility, and ionization of the molecule at the pH of urine (4.5 to 8).

Given the low water solubility of the test substance itself and its MW just exceeding 300 g/mol, it is likely to be excreted primarily via faeces. Nevertheless, there is also urinary elimination equally likely following formation of water-soluble conjugates or metabolites via Phase II reactions.

Based on experimental data on test substance:  

The results of the investigation in the oral study with C12 DEA (Mathews, 1996), which is discussed above under absorption, showed that C12 DEA was well absorbed and mostly excreted in urine as two polar metabolites. Approximately 60 and 80% of the dose was recovered in urine after 24 and 72 h, respectively, and 9% of the dose was recovered in faeces after 72 h. 

Conclusion: Based on all the available weight of evidence information, the test substance is expected to be primarily excreted via urine. 

References not included in the reference list:

European Center for Ecotoxicology and Toxicology of Chemicals (ECETOC), 1993. Percutaneous absorption. Monograph No. 20. http://www.ecetoc.org/wp-content/uploads/2014/08/MON-020.pdf. European Commission (EC), DG SANCO (2004). Guidance document on dermal absorption.SANCO/222/2000 rev. 7. https://ec.europa.eu/food/sites/food/files/plant/docs/pesticides_ppp_app-proc_guide_tox_dermal-absorp-2004.pdf.

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

Kirchmairet al., 2013.FAst MEtabolizer (FAME): A rapid and accurate predictor of sites of metabolism in multiple species by endogenous enzymes. J. Chem. Inf. Model. 53(11):2896-2907.

Kroes Ret al., 2007. Application of the threshold of toxicological concern (TTC) to the safety evaluation of cosmetic ingredients. Food Chem. Toxicol. 45(12):2533-2562.

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Sichoet al., 2017. FAME 2: Simple and effective machine learning model of Cytochrome P450 regioselectivity. J. Chem. Inf. Model. 57(8):1832-1846.

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