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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).

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).

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).

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/kgbw 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, 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).

Description of key information

Based on the available weight of evidence experimental studies, the test substance is expected to be have a low to moderate absorption potential through oral route and a low absorption potential via dermal route. Based on QSAR predictions, it is likely to 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 C8-18 and C18-unsatd. MEA is a non-ionic surfactant which belongs to a monoethanolamine (MEA) derived fatty acid alkanolamide category. It is an UVCB with majorly C18, C10, C12, C14, C16, C18 and C18-unsatd. alkyl chain lengths and molecular weight ranging from 187.3 to 327.6 g/mol (weighted average = 257.32 g/mol). The purified form of the substance appears as a yellow solid. While a new experimental water solubility is ongoing, the test substance can be considered to have low water solubility potential, based on a predicted value of 7.5 mg/L at 20°C using WATERENT v.1.01 QSAR model. The log Kow values were predicted to be moderate and in the range of 3.71 to 4.73 (mean = 4.24) 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 constituents with C8-C14 alkyl chains (present at ca.85%) to be ‘bioavailable’ and the constituents with C16-18 and C18-unsatd. alkyl chains (present at ca. ca. 15-20%) to be ‘less bioavailable’. Therefore, the test substance can be overall considered to have a low to moderate 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 wide carbon chain distribution of the test substance, which is an UVCB, a lower absorption potential is expected.

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.

Two acute toxicity studies available with the test substance, C8-18 and C18-unsatd. MEA, in rats did not reveal any mortality or clinical signs at doses up to 3000 mg/kg bw(see section 5.2 of the CSR for further details),suggesting a less likelihood of absorption. 

Conclusion: Overall, based on the available weight of evidence information, the test substance can be expected to overall have a low 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, low water solubility and high log Kow exceed 3, suggesting a lower 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 different constituents were determined to be 0.055 to 24.6 μg/cm2/h leading to a weighted average value of 3.5 μg/cm2/h. Based on the principles Kroes et al., 2004 and/or 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% and for Jmax below ≤0.1 μg/cm2/h, the default dermal absorption can be considered to be no more than 10%. Based on the individual or weighted average Jmax value, the test substance can be considered to have low 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 wide carbon chain distribution of the test substance, which is an UVCB, a lower absorption potential is expected.

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 been identified 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.

An acute dermal toxicity study conducted with C8-18 and C18-unsatd. MEA in rabbits did not show any clinical signs throughout the observation period at doses u. Similar absence of clinical signs and sensitisation response was also observed in a skin sensitisation study available with C8-18 and C18-unsatd. MEA in guinea pigs (see section 5.2.1.3 and 5.5.1.1 of the CSR for further details). Based on this information, the test substance is expected to have low absorption potential.

Conclusion: Overall, based on all the available weight of evidence information, the test substance can be expected to overall have a low absorption potential through the dermal route. Therefore, a value of 10% 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 substance exists in the solid physical state under ambient conditions and exhibits a relatively low vapour pressure of 1.5E-5 Pa at 20 °C. Hence it will not 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 water solubility, the test substance will not be retained in the mucus and hence will reach the deeper lungs for absorption. 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 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 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 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 Toolbox, FAME 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). Hydrolysis of the amide bond to release the free alkanolamine does not seem to be a preferred metabolism path.

Representative structure

 

Rat liver S9 metabolism simulator/ in vivo rat metabolism simulator / FAME 3

N-(2-hydroxyethyl) dodecanamide (C12 MEA)

 

ωand/orω-1 Aliphatic hydroxylations

Overall, the results of the literature and the different metabolism simulators used 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 water solubility), this suggests that test substance could be distributed to the tissues to an extent, once absorbed and bioavailable. However, based on the predicted BCF values, which ranges from 1.58 to 217.4 L/kg ww (weighted average = 45 L/kg ww) using Arnot-Gobas method of EPI SuiteTMv.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.  

No target organs could be identified based on available repeated dose toxicity studies with the read across substance C12-18 and C18-unsatd. MEA (see section 5.6.1.1 of the CSR for further details).

Conclusion: Based on all the available weight of evidence information, the test substance is expected to have a low distribution and bioaccumulation potential.

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 physicochemical properties of the test substance, MW just exceeding 300 g/mol, the test substance 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 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.

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.

OECD, 2020. The OECD QSAR toolbox for grouping chemicals into categories, version 4.4.1., http://toolbox.oasis-lmc.org/?section=download (accessed March 2020).

Roberts MS and Walters KA, 2007. Dermal absorption and toxicity assessment. CRC Press; 2007 December 14.

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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.

Šícho, Met al., 2019. FAME 3: Predicting the Sites of metabolism in synthetic compounds and natural products for phase 1 and phase 2 metabolic enzymes. J. Chem. Inf. Model. 59 (8):3400-3412.

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