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Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
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
Reason / purpose:
reference to other study
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 N,N-bis(2-hydroxyethyl)dodecanamide (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 LDEA 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 LDEA. The specific activities for LDEA 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 LDEA 12-hydroxylation specific activity 5-fold to 3.50 ± 0.48 nmol/mm/mg protein, whereas the LDEA 11-hydroxylase activity remained unchanged. Incubating liver microsomes from DEHP-treated rats with a polyclonal anti-rat 4A inhibited the formation of 12-OH-LDEA 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-LDEA formation (1.93±0.09 vs. 2.20± 0.11 nmol/min/mg protein). Rat kidney microsomes also resulted in hydroxylation of LDEA 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:
experimental study
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
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 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:
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 radiolabelled N,N-bis(2-hydroxyethyl)dodecanamide (LDEA) in F344 rats. Three male rats were administered a single dose of (14C) LDEA 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 LDEA 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:
experimental study
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
Qualifier:
equivalent or similar to
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 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 (14C) radiolabelled N,N-bis(2-hydroxyethyl)dodecanamide (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:
experimental study
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
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
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 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 (14C) radiolabelled N,N-bis(2-hydroxyethyl)dodecanamide (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

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

Basic toxicokinetics

In vivo testing conducted in rats with the read-across substance N,N-bis(2-hydroxyethyl)dodecanamide (C12 DEA) suggests that it is well absorbed via the oral route, then relatively rapidly metabolised to polar metabolites and excreted principally in urine (Matthews, 1996). In vitro metabolism studies with rat liver and kidney microsomes show conversion of the substance into 11- and 12-hydroxy derivatives (Merdink, 1996). Overall, based on the data, moderate to high oral absorption, relatively rapid metabolisation and low bioaccumulation of the test substance is expected. For risk assessment purposes, a default value of 50% oral absorption is therefore retained.

Dermal absorption

In vivo dermal absorption studies conducted with the read-across substance N,N-bis(2-hydroxyethyl)dodecanamide (C12 DEA) in rats showed that uptake 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. Similar studies in mice indicated higher uptake: 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 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. Hence, for risk assessment purposes, a value of 10% is retained.

Inhalation route

For the inhalation route, a default assumption of 100% absorption is taken for risk assessment.

References

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.