Reaction mass of N-(2-hydroxypropyl)decan-1-amide and N-(2-hydroxypropyl)octanamide

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Read across study

Justification for type of information:

Refer to Section 13 for details of the read-across justification.

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

read-across source

Objective of study:

metabolism

Principles of method if other than guideline:

Liver and kidney microsomes from DEHP-treated and control rats were incubated with 100 µM test substance for 30 min at 37°C in a shaking water bath. The metabolites were then separated and analysed by GC-MS. 

GLP compliance:

no

Radiolabelling:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on exposure:

Diethyl hexyl pthalate (DEHP) and control treated liver and kidney microsomes were incubated with 100 µM of test substance for 30 min at 37°C in a shaking water bath according to the method of Okita et al, 1990 .

Duration and frequency of treatment / exposure:

30 min

Dose / conc.:

100 other: µM

Details on dosing and sampling:

METABOLITE CHARACTERISATION STUDIES:
- Method of identification: Mass spectral identification (GC/MS).
- Because LDEA contains a 12-carbon side chain, LDEA hydroxylation rates were compared with the hydroxylation rates for lauric acid.

Metabolites identified:

yes

Details on metabolites:

The test substance was metabolised by rat liver microsomes to two major products that were identified by GC/MS to be the 11- hydroxyl and 12-hydroxy derivatives. The specific activities for 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 the cytochrome P4504A inducer and peroxisome proliferator, diethylhexyl phthalate (DEHP) increased the test substance 12-hydroxylation rate to 3.50 ± 0.48 nmol/mm/mg protein, a 5-fold increase in specific activity, whereas the 11-hydroxylase activity remained unchanged.

The specific activities of 11- and 12-hydroxylation reactions in DEHP treated rats were 1.7-fold and 3.2-fold greater than the 11- and 12-hydroxylation rates, respectively.

Incubating liver microsomes from DEHP-treated rats with a polyclonal anti-rat 4A inhibited the formation of 12-OH-test substance by 80% (3.98±0.10 vs. 0.80±0.08 nmol/min/mg protein), compared with the preimmune serum, but had no inhibitory effect on the rate of 1 1-OH-test substance formation (1.93±0.09 vs. 2.20± 0.11 nmol/min/mg protein).

Rat kidney microsomes also resulted in hydroxylation of the test substance 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.

A 5.1-fold increase in specific activity was observed for the test substance l2-hydroxylation reaction after DEHP treatment, whereas the rate for 11-hydroxylation was similar in microsomes from control and DEHP-treated rats.

Other studies: Human liver microsome results: LDEA was also metabolised to 11- and 12-hydroxy derivatives by human liver microsomes at specific activities of 0.22±0.06 and 0.84±0.26 nmol/min/mg protein, respectively.

Conclusions:

Under the study conditions, the test substance was rapidly converted into 11- and 12-hydroxy derivatives in rat liver and kidney microsomes.

Executive summary:

A study was conducted to evaluate the in vitro metabolism of the read across 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 1 1-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).

Reference 2

Endpoint:

basic toxicokinetics in vivo

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Read across study

Justification for type of information:

Refer to Section 13 for details of the read across justification.

Reason / purpose for cross-reference:

read-across source

Objective of study:

toxicokinetics

Principles of method if other than guideline:

Three male rats were administered a single dose of (14C) test substance 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) were determined by collecting adipose tissue, blood, kidney and liver 72 h post-dosing.

GLP compliance:

not specified

Radiolabelling:

yes

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc. (Raleigh, NC)
- Age at study initiation: 81 to 87 d
- Housing: Individual glass metabolism chambers, which allowed separate collection of carbon dioxide, urine, and feces.
- Individual metabolism cages: Yes
- Diet: Purina Rodent Chow (no. 5002), ad libitum
- Water: Ad libitum

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

ORAL DOSE FORMULATION: 16 to 18 µCi radiolabel per dose, an appropriate amount of unlabeled LDEA and water

DOSE VOLUME: 5 mL/kg bw

Duration and frequency of treatment / exposure:

Duration of treatment: 72 h
Frequency of treatment: Single dose

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose / concentration:

Three

Control animals:

not specified

Details on study design:

ANESTHESIA
- Identity: Sacrificed by overdosing with sodium pentobarbital (300 mg/kg bw) through intracardiac route

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: Urine, faeces, blood, adipose tissue, liver and kidney
- Time and frequency of sampling: 72 h after dosing
- Method type(s) for identification: Radioactivity was determined using a Packard Tricarb 1500 Liquid Scintillation Analyzer (Packard Instrument Company, Downers Grove, IL).
- Brief description on method of analysis: Digested samples of tissues, feces, and blood in Soluene-350 (Packard Instrument Company, Meriden, CT) overnight, were bleached with perchloric acid/hydrogen peroxide before addition of scintillation cocktail (Ultima Gold).


METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: Urine
- Time and frequency of sampling: 6 to 24 h after dosing
- From how many animals: samples were pooled from 3 animals
- Method type(s) for identification: Lyophilised samples of urine were analysed for metabolites using HPLC reversed- phase, further purified using cation and anion-exchange chromatography and identification of metabolites were done using mass spectrophotometry; The trimethylsilyl derivative of LDEA were prepared and analyzed by GC/MS with chemical ionisation (Thomas et.al., 1990).

Statistics:

Values for test groups were compared by ANOVA followed by Dunnett’s test.

Details on absorption:

The test substance was readily absorbed

Details on distribution in tissues:

Tissue to blood ratio (TBR) was highest in adipose tissue and liver, which had TBRs of about 50.

Details on excretion:

The radiolabelled test substance was excreted mostly in urine as two polar metabolites. Approximately 60 and 80% of the dose was recovered in the urine after 24 and 72 h, respectively, and 9% of the dose was recovered in feces after 72 h.

Metabolites identified:

yes

Details on metabolites:

Urine chromatographed on a reverse phase column resulted in 2 peaks. The mass spectrum of Peak 1 was assigned as the half-acid amide of succinate and DEA (loss of 8 carbons) and Peak 2 as the adipate (loss of 6 carbons) half-acid amide.

Other examinations: Metabolism in rat and human liver slices: LDEA partitioned well into liver slices, and about 70% of the radioactive LDEA was absorbed into the slices within 4h.The absorbed radioactivity was present mostly as parent compound. About 20 and 43% of the radioactivity present in media from the un-induced and DEHP-induced rats respectively were comprised of metabolites. About 30% of the radioactivity in the media of the human liver slice incubations was in the form of metabolites.

Analytes present in the incubation media from human and rat liver slices include the half-acid amides identified as metabolites in vivo, parent LDEA, and perhaps three other metabolites that have been identified as products of ω - and ω-1 to 4 hydroxylation (Merdink et al., 1996).

Conclusions:

Under the study conditions, the substance was well absorbed and mostly excreted in urine as two polar metabolites.

Executive summary:

A study was conducted to evaluate the absorption, distribution, metabolism and excretion of the radiolabelled read across 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).

Reference 3

Endpoint:

dermal absorption in vivo

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Read across study

Justification for type of information:

Refer to Section 13 for details of the read-across justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 427 (Skin Absorption: In Vivo Method)

Deviations:

not specified

GLP compliance:

not specified

Radiolabelling:

yes

Species:

mouse

Strain:

B6C3F1

Sex:

male

Details on test animals or test system and environmental conditions:

- Source: Charles River Laboratories, Inc. (Raleigh, NC)
- Age at study initiation: 72 to 75 d
- Housing: Individual glass metabolism chambers, which allowed separate collection of carbon dioxide, urine, and feces.
- Individual metabolism cages: Yes
- Diet: Purina Rodent Chow (no. 5002), ad libitum
- Water: Ad libitum

Type of coverage:

open

Vehicle:

ethanol

Duration of exposure:

72 h

Doses:

- Actual doses: 50, 100, 200 and 800 mg/kg bw
- Dose volume: 50 µL of 50, 100 and 200 mg/kg bw and 30 µL of 800 mg/kg bw

No. of animals per group:

Four

Control animals:

no

Details on study design:

DOSE FORMULATION: 2 to 17 µCi radiolabel, an appropriate amount of unlabelled LDEA and 95% ethanol for a total volume of about 50 µL per dose

VEHICLE
- Concentration (if solution): 95%

TEST SITE
- Preparation of test site: Application site had been clipped of hair the previous day
- Area of exposure: 1 x 1 inch
- Type of cover / wrap if used: A non-occlusive protective appliance, glued over the dose area with cyanoacrylate adhesive.

SITE PROTECTION / USE OF RESTRAINERS FOR PREVENTING INGESTION: No

REMOVAL OF TEST SUBSTANCE: No

SAMPLE COLLECTION: Dose site skin was excised and thoroughly rinsed with ethanol, then gently wiped with cotton gauzes soaked with soapy water. Gauzes and aliquots of urine, dermal rinse solutions, feces, tissues (liver and kidney) and digested skin samples (in 2N ethanolic sodium hydroxide) were collected and analysed for radiochemical content by adding to vials containing scintillation cocktail (Ultima Gold, Packard Instrument Company).

ANALYSIS
- Method type(s) for identification: Liquid scintillation counting

Signs and symptoms of toxicity:

not examined

Dermal irritation:

not examined

Absorption in different matrices:

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.

Total recovery:

Approximately 91, 85, 88 and 85% at 50, 100, 200 and 800 mg/kg bw, respectively.

Key result

Time point:

72 h

Dose:

50 mg/kg bw

Parameter:

percentage

Absorption:

ca. 49.1 %

Key result

Time point:

72 h

Dose:

100 mg/kg bw

Parameter:

percentage

Absorption:

ca. 66.8 %

Key result

Time point:

72 h

Dose:

200 mg/kg bw

Parameter:

percentage

Absorption:

ca. 69.1 %

Key result

Time point:

72 h

Dose:

800 mg/kg bw

Parameter:

percentage

Absorption:

ca. 50.2 %

Conversion factor human vs. animal skin:

No data

Conclusions:

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.

Executive summary:

A study was conducted to evaluate the dermal absorption of the (14C) radiolabelled read across substance N,N-bis(2-hydroxyethyl)dodecanamide (C12 DEA, also know as 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).

Reference 4

Endpoint:

dermal absorption in vivo

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Read across study

Justification for type of information:

Refer to Section 13 for details of the read-across justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 427 (Skin Absorption: In Vivo Method)

Deviations:

not specified

GLP compliance:

not specified

Radiolabelling:

yes

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc. (Raleigh, NC)
- Age at study initiation: 81 to 87 d
- Housing: Individual glass metabolism chambers, which allowed separate collection of carbon dioxide, urine, and feces.
- Individual metabolism cages: Yes
- Diet: Purina Rodent Chow (no. 5002), ad libitum
- Water: Ad libitum

Type of coverage:

other:

Vehicle:

ethanol

Duration of exposure:

72 h

Doses:

- Actual doses: 25 and 400 mg/kg bw
- Dose volume: 200 and 100 µL of 25 and 400 mg/kg bw respectively

No. of animals per group:

Four

Control animals:

no

Details on study design:

DOSE FORMULATION: 16 to 18 µCi radiolabel, an appropriate amount of unlabelled LDEA and 95% ethanol for a total volume of about 200 µL per dose

VEHICLE
- Concentration (if solution): 95%

TEST SITE
- Preparation of test site: Application site had been clipped of hair the previous day
- Area of exposure: 1 x 1 inch
- Type of cover / wrap if used: A non-occlusive protective appliance, glued over the dose area with cyanoacrylate adhesive.

SITE PROTECTION / USE OF RESTRAINERS FOR PREVENTING INGESTION: No

REMOVAL OF TEST SUBSTANCE: Dose site skin was excised and thoroughly rinsed with ethanol, then gently wiped with cotton gauzes soaked with soapy water.

SAMPLE COLLECTION: Gauzes and aliquots of urine, dermal rinse solutions, feces, tissues (liver and kidney) and digested skin samples (in 2N ethanolic sodium hydroxide) were collected and analysed for radiochemical content by adding to vials containing scintillation cocktail (Ultima Gold, Packard Instrument Company).

ANALYSIS
- Method type(s) for identification: Liquid scintillation counting

Signs and symptoms of toxicity:

not examined

Dermal irritation:

not examined

Absorption in different matrices:

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

Total recovery:

95 and 92% recovery at 25 and 400 mg/kg bw, respectively

Key result

Time point:

72 h

Dose:

25 mg/kg bw

Parameter:

percentage

Absorption:

ca. 26.3 %

Key result

Time point:

72 h

Dose:

400 mg/kg bw

Parameter:

percentage

Absorption:

ca. 29.2 %

Conversion factor human vs. animal skin:

No data

TBRs (tissue to blood ratio) were higher in liver (30-40) and kidney (ca.20) than adipose tissue (6-7) and non-dose site skin (ca.2).

Results of jugular vein-cannulated rats, dermally dosed at 100 mg/kg bw of LDEA: 

- Only LDEA and the half-acid amide metabolites were detected in plasma, and their levels were near maximal at about 24 h after dosing, with no marked changes in the profiles thereafter. 

- Most of the circulating LDEA equivalents were comprised of the two metabolites, with about 15% present as LDEA. (See Figure 1 in the attached document).

Conclusions:

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.

Executive summary:

A study was conducted to evaluate the dermal absorption of the (14C) radiolabelled read across 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, faeces, 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).

Key value for chemical safety assessment

Bioaccumulation potential:

low bioaccumulation potential

Absorption rate - oral (%):

50

Absorption rate - dermal (%):

50

Absorption rate - inhalation (%):

100

Additional information

Oral absorption

Based on physicochemical properties:

According to REACH guidance document R7.C (May 2017), 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 C8-10 MIPA is a non-ionic surfactant which belongs to a monoisopropanolamine- (MIPA) derived fatty acid alkanolamide category. It is an UVCB with majorly C8 and C10 alkyl chain lengths and molecular weight ranging from 173.3 to 257.4 g/mol (weighted average = 212.49 g/mol). The purified form of the substance appears as white or pale straw solid. While a new experimental water solubility is ongoing, the test substance can be considered to have high water solubility potential, based on an experimental value of 3877 mg/L at 20°C. The measured log Kow values were determined to be >2.6 at 20°C indicating a moderate lipophilicity.

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 high 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 all the constituents to be ‘bioavailable’. Therefore, the test substance can be overall considered to have a high absorption and bioavailability potential.

Based on experimental data on read across substances:

A study was conducted to evaluate the absorption, distribution, metabolism and excretion of the radiolabelled read across 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 read across study, which was contained only C12 alkyl chains, and given the shorter (C8-10) and wider carbon chain distribution of the test substance, which is an UVCB, a higher absorption potential is expected.

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 50% 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, high water solubility and moderate log Kow not exceeding 3, all of which suggest a likelihood of absorption potential through the dermal route. Further, 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 high to 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 values for the different carbon chains of the UVCB substance was determined to be range between 1.81 to 66.3 μg/cm2/h, leading to a weighted average value of 16.80 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 and can be considered to be less than 40% and for those with Jmax exceeding ≥10 μg/cm2/h, the default dermal absorption can be considered to be no more than 80%. Based on the Jmax values, the test substance can be considered to have high to moderate absorption potential through the dermal route.  

Based on experimental data on read across substances:  

A study was conducted to evaluate the dermal absorption of the (14C) radiolabelled read across 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 read across substance N,N-bis(2-hydroxyethyl)dodecanamide (C12 DEA, also known as 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 the results of the read across study, which was contained only C12 alkyl chains, and given the shorter (C8-10) and wider carbon chain distribution of the test substance, which is an UVCB, a higher absorption potential is expected.

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 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 relatively low vapour pressure of 2 Pa at 20 °C. Hence, it is not expected to be available as 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 droplet size and the high-water solubility almost all droplets are likely to be retained in the mucus and will not be available to reach the deeper lungs. The deposited droplets in the upper respiratory tract are expected to be absorbed at a relatively slower rate compared to the deeper lungs due to differences in vascularity. Some amount of these deposited droplets is also expected to be transported to the pharynx and swallowed via the ciliary-mucosal escalator. Therefore, the systemic uptake of the test substance via respiratory route can be considered to be similar or slightly higher compared 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 read across 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 1 1-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 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.Based on predictions from the two simulators of the OECD QSAR Toolbox and expert judgement, ω and ω-1 aliphatic hydroxylation was predicted to be the first metabolic reactions for the representative structure of the test substance (see below Table). There was an additional oxidation reaction predicted at the hydroxy group of the MIPA.Hydrolysis of the amide bond to release the free alkanolamine does not seem to be a preferred metabolism path.

Representative constituent	Rat liver S9 metabolism simulator/ in vivo rat metabolism simulator
N-(2-hydroxypropyl) octanamide (C8 MIPA	ω and/or ω-1 Aliphatic hydroxylations

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 high water solubility), it suggests that test substance could be distributed to many tissues, once absorbed and bioavailable. However, based on the predicted BCF values, which ranges from 1.1-30 L/kg ww (weighted average = 7 L/kg ww) using Arnot-Gobas method of EPI Suite^TMv.4.1, the bioaccumulation potential of the substance is expected to be low.

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 high water solubility, MW just exceeding 300 g/mol, the test substance is expected to be primarily excreted via urine.

Based on experimental data on read across substances:  

The results of the investigation in the read across 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.

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