Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
basic toxicokinetics
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 February 2019 - 13 May 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was conducted according to OECD 417 and under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2020

Materials and methods

Objective of study:
absorption
distribution
excretion
metabolism
toxicokinetics
Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 417 (Toxicokinetics)
Version / remarks:
The Guidelines for the Testing of Chemicals Health Effects, China Environmental Science Press, 2013
Deviations:
yes
Remarks:
The relative humidity in this experiment is within the range of 33.19%~72.17%RH, which is deviated from the range of 30%~70%RH of the reference standard relative humidity. The deviation was thus judged having no influence on the test results.
Principles of method if other than guideline:
Not relevant
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Nonanoic acid, mixed diesters, with oxybis[propanol] and dodecanoic acid
Cas Number:
2166089-27-4
Molecular formula:
Non applicable (UVCB)
IUPAC Name:
Nonanoic acid, mixed diesters, with oxybis[propanol] and dodecanoic acid
Test material form:
liquid
Details on test material:
- Appearance: Limpid liquid
- Storage condition of test material: Keep container tightly closed. Preferably store in the original packaging. Store at room temperature, protect from humidity.
Radiolabelling:
no

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
SD rats are commonly used rodents in toxicokinetic tests. Rat is an appropriate experimental animal model with advantages of plenty of historical data, showing relatively stable response to test substances, small inter-individual variation, small size and sample-saving, and easily to be handled.
To meet the requirements of study objectives and the testing guideline, a minimum number of animals have been used.
Both male and female animals were used to test gender difference related to the experiment regimen.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Beijing Vital River Laboratories Animal Technology Co. Ltd. (Permit No.: SCXK (Jing) 2016-0006; Valid from 2016-08-31 to 2021-08-31; Issued by Beijing Municipal Science & Technology Commission)
- Age at study initiation: 6-9 weeks old
- Weight at study initiation: Females 169.7~222.3 g; Males 209.7~244.4 g.
- Housing: polypropylene plastic boxes with the cage size of 423 mm × 269 mm × 192 mm. Two animals of the same gender were group-housed in one cage.
- Diet (e.g. ad libitum): Ad libitum, certified laboratory diets for SPF animals were supplied by the manufacturer Sibeifu
- Water (e.g. ad libitum): Ad libitum, treating municipal tap water with a primary filter column, followed by filtration with reverse-osmosis filter. Drinking water were filled into autoclaved bottle and given to animals.
- Acclimation period: 6-8 days before study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.93~24.55 ºC
- Humidity (%): 33.19~72.17% RH
- Air changes (per hr): At least 15 times of air exchanges per hour.
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
other: Intravenous and oral gavage
Vehicle:
other: The test item is insoluble in water. Dimethyl sulfoxide (solubilizer) and polyoxyethylene castor oil (protectant) for the intravenous formulation. Sodium carboxymethyl cellulose (CMC-Na) for the oral formulation.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS IN VIVO:
- Intravenous formulation
Test formulations were prepared every time prior to use as follows: accurately weighed an appropriated amount of sample, dissolved in DMSO, added the same volume of polyoxyethylene castor oil, and mixed by vortex mixer. Saline was added gradually and the final solution was prepared by a volumetric flask. The final formulation contained 5% DMSO and 5% polyoxyethylene castor oil.
- Oral formulation
The formulations were prepared every time prior to use as follows: accurately weighed an appropriate amount of sample, suspended in 0.5% CMC-Na solution in volumetric flasks, and mixed by vortex mixer.

PREPARATION OF DOSING SOLUTIONS IN VITRO:
- Protein Binding Test Solution
The solution was prepared every time prior to use as follows:
Accurately weighed 0.0203 g and 0.0201 g of test item, dissolved in acetonitrile with a volumetric flask of 10 mL to obtain a stock solution of 2.03 mg/mL and 2.01 mg/mL. Measured 0.1 mL of the above solution and added into 9.9 mL of acetonitrile to make a second stock standard solution of 20.30 µg/mL and 20.10 µg/mL. A series working solutions were then prepared at concentrations of 10.15, 5.08, and 2.54 µg/mL, respectively, by 2 times dilutions with acetonitrile.
- Liver Microsomal Metabolism Test Solution
Accurately weighed 0.0212 g of test item, dissolved in acetonitrile with a volumetric flask of 10 mL to obtain a stock solution of 2.12 mg/mL, followed by dilution with acetonitrile to make the solution of 212 μg/mL. The working solution at the concentration of 4.24 μg/mL was further prepared with phosphate buffer (PBS). Prepared before use.
- Recombinant CYP 450 Enzymes Metabolism Test Solution
Accurately weighed 0.0199 g of test item, dissolved in acetonitrile with a volumetric flask of 10 mL to obtain a stock solution of 1.99 mg/mL, followed by dilution with acetonitrile to make the solution of 199 μg/mL. The working solution at the concentration of 3.98 μg/mL was further prepared with phosphate buffer (PBS). Prepared before use.

HOMOGENEITY AND STABILITY OF TEST MATERIAL:
Formulation analysis was performed and reported.
Duration and frequency of treatment / exposure:
Single intravenous and oral dose and repeated oral dose (see Table 1.1)
Doses / concentrationsopen allclose all
Dose / conc.:
5 mg/kg bw/day (nominal)
Remarks:
Group 1 - Single intravenous dose at a volume of 10 mL/kg
Group 7 - Bile excretion group at a volume of 10 mL/kg
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Group 2 - Low single oral dose (Combined as initial dosing group) at a volume of 10 mL/kg
Group 4 - Repeated oral dose (Trough group) at a volume of 10 mL/kg
Group 5 - Repeated oral dose (Last dosing group) at a volume of 10 mL/kg
Group 6 - Feces and urine excretion group at a volume of 10 mL/kg
Group 8 - Tissue distribution group at a volume of 10 mL/kg
Group 9 - Gastrointestinal residual group at a volume of 10 mL/kg
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 3 - High single oral dose at a volume of 10 mL/kg
No. of animals per sex per dose / concentration:
Four SD rats (2M & 2F) were used in Groups #1~7. Eight animals (4M & 4F) were used in digestive tract group (group #9) and twelve animals (6M & 6F) were used in tissue distribution (group #8).
Control animals:
no
Positive control reference chemical:
Not relevant
Details on study design:
PILOT STUDY
- Plasma concentration curve by single intravenous dosing
Two SD rats (1F, 1M) were intravenously administrated with 5 mg/kg test substance.
- Plasma concentration curve by single oral dosing
Four SD rats (2F, 2M) were orally administrated with the test substance at 1000 mg/kg as high dose level, or 5 mg/kg as low dose level.
- Distribution and excretion
Four male rats were used to explore the excretion in feces and gastrointestinal (GI) contents. Animals were adapted in the metabolic cage for 24 h before dosing.

IN VIVO STUDY DESIGN (see Table 1.1 below)
Before blood sample collection, animals in Groups #2 and #3 were fasted overnight (12~16 h), and resumed feeding within 1~2 h after administration. Animals drink freely during fasting.
Cage-side observations, clinical observation and body weight were recorded as below.
All animals before grouping and animals enrolled in the study were observed once daily for death, appearance, fur, activity, reaction, breathing and posture.
All animals were observed once before grouping. Animals enrolled in the study were observed before and after administration by taking out of the cage to examine neck, head (including eyes, ears, mouth and nose), low abdomen, anus, perineum, skin color, muscles and any other abnormalities, if any.
All animals were weighted with electronic balance upon arrival and group assignment. Animals enrolled in the study were weighed before initial dosing.
Once all samples were collected, animals were euthanatized with 100% carbon dioxide (CO2).
Once morbid or moribund animals are found, SD and veterinarian will be informed immediately. Necropsy will be conducted for any pathological change, for potential cause of animal death. Once animals for bile sample collections were found dead, bile samples will be collected immediately. The time of death found, gender and animal number were recorded.

IN VITRO STUDY DESIGN
- Plasma Protein Binding Test
A series of working solutions (5 μL) of test substance at different concentrations were added into plasma (245 μL) collected from SD rats, dogs and human, respectively. The final concentrations in the reaction solution were 50, 100 and 200 ng/mL, respectively. Three parallels were conducted for each concentration level. Phosphate buffer (PBS) was used as the blank control.
Testing solutions were then added to the front and back side of the equilibrium dialysis respectively. The equilibrium dialysis device was incubated at 37 ℃ for 6 hours. In the end of incubation, 0.1 mL of the equilibrium dialysis solution of each side was taken out for measurement of C12-C12, C9-C12 and C9-C9.
- Liver Microsome Metabolism Test
The working solution (100 μL, 4 μg/mL) was mixed with liver microsome (80 μL) of SD rat, dog or human and NADPH regeneration solution (20 μL) to make a final volume of 0.2 mL. The reaction system was incubated at 37 ℃ for 0, 15, 30, 60, and 90 mins, respectively. In the end of incubation, samples were immediately placed on the ice, followed by adding the termination solution (200 μL) and mixed with vortex for 2 mins.
- Recombinant CYP450 Enzyme Metabolism Test
The working solution (100 μL, 4 μg/mL) of the test substance was mixed with NADPH regeneration solution (50 μL) and six recombinant CYP450 enzymes subtypes (50 μL) of CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 or CYP3A4 respectively, to make a final volume of 0.2 mL. The testing solutions were incubated at 37 ℃ for 90 mins, followed by adding the termination solution (0.2 mL).
Details on dosing and sampling:
PILOT STUDY
- Plasma concentration curve by single intravenous dosing (2 rats/intravenous/5 mg/kg test substance)
Blood samples (~0.3 mL/time point) were collected via orbital venous plexus at time points of pre-dose, 1 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h and 8 h (nine in total). Anti-coagulant plasma with heparin sodium were obtained by centrifuging (4℃, 2006 g) for 10 min and kept -20℃ until transfer to the Testing Site. Plasma concentrations were analyzed by LC-MS/MS.
- Plasma concentration curve by single oral dosing (4 rats/oral gavage/1000 or 5 mg/kg test substance)
Blood samples (~0.3 mL/time point) were collected via orbital venous plexus at time points of pre-dose, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h and 8 h (eight in total). Heparin sodium anti-coagulant plasma were obtained by centrifuging (4℃,2006 g) for 10 min and kept -20℃ until transfer to the Testing Site. Plasma concentrations were analyzed by LC-MS/MS.
- Distribution and excretion (4 male rats/oral gavage/20 mg/kg test substance)
To explore the excretion in feces and gastrointestinal (GI) contents, animals were adapted in the metabolic cage for 24 h before dosing. Feces and gastrointestine including its contents were collected at 2 h, 4 h, 8 h and 24 h, respectively. Samples were weighed upon collection and kept at -20℃ until transferring to the testing site. Results were shown in table 5.2.3-4.

IN VIVO STUDY DESIGN (see Table 1.1 below)
- Plasma sample collection
Animals were anesthetized with mixed gas (70% CO2 + 30% O2) via inhalation before blood sampling. Blood was collected from the orbital venous plexus and heparin sodium was used for anti-coagulation. At each scheduled time point, about 0.3 mL blood sample was collected, and placed on wet ice immediately after collection. Samples were centrifuged at 4 ℃ (2006 g, 10 min) within 2 h of sampling and supernatant plasma was collected and stored at -20 ℃. Samples were transferred on ice to the subcontracting Test Site for further analysis. After preparation of the plasma samples, the concentration of C12-C12, C9-C12, C9-C9 and the peak area of metabolites were analyzed by LC-MS/MS.
The collection time points of the test group (group #1~5) are described below:
Single intravenous dose (Group #1): Pre-dose, 1 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h, 36 h and 48 h post-dose (12 time points in total).
Single oral dose group (Group #2 and #3): Pre-dose, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h, 36 h and 48 h post-dose (11 time points in total).
Trough repeated oral dose group (Group #4): Once daily before each administration for 6 days and at 24 h after the last administration on the 6th day (7 time points in total).
Last for repeated oral dose group (Group #5): pre-dose, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 24 h, 36 h and 48 h post-dose on the 7th day (11 time points in total).
- Feces and Urine Sample Collection
Animals in Group #6 were placed in a metabolic cage after administration. Feces and urine samples were continuously collected for 0~4 h, 4~8 h, 8~12 h, 12~24 h, 24~36 h, 36~48 h, 48~60 h and 60~72 h. The collected urine volume was recorded. The collected feces were weighed. Before sample collection, the animal were adapted in the metabolic cage for at least 24 h. Samples were stored at -20℃ immediately upon collection. Samples were transferred on ice to the testing site for further analysis. After preparation of the samples, the concentration of C12-C12, C9-C12, C9-C9 and the peak area of metabolites were analyzed by LC-MS/MS.
- Bile Sample Collection
Animals in Group #7 underwent bile duct tubing surgery after anesthesia. Animals were intravenously administrated with the test substance while awake. Sample were collected depending on animal’s survival length and as follows:
25/M&27/F: 0~2 h, 2~4 h, 4~8 h, 8~12 h, 12~24 h, 24~36 h, 36~43 h;
26/M: 0~2 h, 2~4 h, 4~8 h, 8~12 h, 12~24 h, 24~36 h, 36~48 h;
28/F: 0~2 h, 2~4 h, 4~8 h, 8~12 h, 12~24 h, 24~32 h.
The volume of bile was recorded upon collection. Sample were stored at -20℃ and then transferred on ice to the Testing Site for further analysis. After preparation of the samples, the concentration of C12-C12, C9-C12, C9-C9 and the peak area of metabolites were analyzed by LC-MS/MS.
- Tissue Sample Collection
Tissues were collected from animals in Group #8 at 0.5 h, 2 h and 24 h after administration. Four animals (2M and 2F) were used at each time point. After animals were anesthetized, blood, heart, liver, spleen, lung, kidney, digestive tract and its contents, testicle or ovary, epididymis or uterus, muscle, eye, brain, thyroid, fat were collected and weighed. Samples were stored at -20 ℃ immediately, and then were transferred on ice to the Testing Site for further analysis. After preparation of the plasma samples, the concentration of C12-C12, C9-C12, C9-C9 and the peak area of metabolites were analyzed by LC-MS/MS.
- Digestive Tract Collection
Digestive tract and its content were collected from animals in Group #9 at 0.5 h, 2 h, 4 h, 8 h and 24 h after administration. Four animals (2M and 2F) were used at each time point. After the animal was euthanized by 100% CO2, the digestive tract and its contents were taken and weighed. Samples were stored at -20 ℃ immediately, and then were transferred on ice to the Testing Site for further analysis. After preparation of the plasma samples, the concentration of C12-C12, C9-C12, C9-C9 and the peak area of metabolites were analyzed by LC-MS/MS.

IN VITRO STUDY DESIGN
- Plasma Protein Binding Test
Plasma protein binding rate (%) was calculated as follows: (1-Cf/Cp)×100%, where Cf is the concentration of free substance (the front side of the membrane) and Cp is the concentration of total (the back side of the membrane).
- Liver Microsome Metabolism Test
Samples were collected for analysis. Phosphate buffer (PBS) was used as the blank control. Three parallels were conducted for each treatment. Samples were measured with the same LC-MS/MS method on plasma.
- Recombinant CYP450 Enzyme Metabolism Test
Samples were collected for analysis. For initial incubation solution (0 min), the termination solution (0.2 mL) was added first, followed by adding 50 μL preheated NADPH regeneration solution. Phosphate buffer (PBS) was used as the blank control. Three parallels were conducted for each treatment. Samples were measured with the same LC-MS/MS method on plasma.

ANALYTICAL METHOD
Bio-analytical methods of Liquid Chromatography-Tandem Mass Spectrometer (LC-MS/MS) were developed and validated to determine the test substance in biological samples of SD rats. The LC-MS/MS analytical methods were established to analyze three components of C12-C12, C9-C12 and C9-C9 of the test substance in biological matrixes of SD rats. Full validation was conducted on plasma matrix.
Statistics:
DAS 2.0 software was used to conduct data fitting of time-course plasma concentration to calculate toxicokinetic parameters. Values of mean and standard deviation were calculated and plotted using Microsoft Office 2016. Data were expressed as mean ± standard deviation.

Results and discussion

Preliminary studies:
The test substance is composed of three components of C12-C12 at 26.43%, C9-C12 at 50.82%and C9-C9 at 22.75%.
Validation results showed that parameters on C12-C12 have fulfilled the acceptance criteria and the bio-analytical methods established can be used for quantitative analysis of C12-C12 components in all the matrixes tested. Parameters of component C9-C12 have partially met validation criteria, and the quantitative data obtained with the method was used as a reference for data explanation. Parameters of component C9-C9 did not show satisfied validation results, and the data obtained was used for qualitative description in this study.

- Plasma concentration curve by single intravenous dosing
Three metabolites (M1-M3) were identified in one animal (#2) and the half-lives (t1/2) were 0.881 h for the test item, 0.978 h for M1, 0.830 h for M2, and 2.437 h for M3, respectively.

- Plasma concentration curve by single oral dosing
Three metabolites (M1-M3) were identified correspondingly in two animal (#3 & #6). Moreover, results showed that, when animals were orally administrated with 5 mg/kg test substance, neither C12-C12 nor its 3 metabolites were detectable in plasma. When animals were given with 1000 mg/kg of test substance, C12-C12 and its 3 metabolites were all detectable at certain time periods post-administration (1~4 h for the test item, 15 min~8 h for M1-M3). Data fitting results showed that the half-lives (t1/2) were 9.24 h for M1, 36.556 h for M2, and 21.849 h for M3. Fitting calculation was not conducted on C12-C12 due to limited data available.

- Distribution and excretion
Results showed that, the test substance was detected at all sampling time points in GI content with peak level at 2 h, whereas it was only detected in feces at 8 h and 24 h. The total recovery rate of the test substance during 0-2 h in feces and GI content was 28.88%.
Main ADME resultsopen allclose all
Type:
absorption
Results:
Single oral dose: small amount of C12-C12 and C9-C12 was absorbed into the blood. Repeated-dose oral: no accumulation occurred.
Type:
distribution
Results:
Absorbed into circulation and rapidly metabolized in the digestive tract.
Type:
metabolism
Results:
In vitro studies: Liver microsomes not involved in the metabolism of C12-C12 and C9-C12, slightly metabolized by CYP2E1 of C12-C12 and almost completely bounded to proteins in plasma of SD rat, dog and human at the binding rate of 100% for the both.
Type:
metabolism
Results:
Multiples In vivo metabolites detected after intravenous injection. After oral administration, no hepatic first-pass effect. Taking together with the metabolite candidates observed, these two components were rapidly metabolized in the digestive tract.
Type:
excretion
Results:
Oral: [%C12-C12]+ [%C9-C12] in feces within 72-h = 3.88% and 1.04% of the dose administrated. [%C12-C12]+ [%C9-C12] in feces at 0.5h = 15.92% and 13.62% of the dose administrated.They were rapidly eliminated in the GI tract.
Type:
excretion
Results:
Intravenous: 95% of the original substance in the body has been eliminated after 4.59 hrs (i.e. 4.5 times of the t1/2).
Type:
distribution

Toxicokinetic / pharmacokinetic studies

Details on absorption:
Toxicokinetic parameters showed that the test substance has low bioavailability and exhibited inter-individual difference.
After single-dose oral exposure of low and high dose level in rats (Group 2&3), only a small amount of C12-C12 was absorbed into the blood; C9-C12 was barely absorbed into circulation, and C9-C9 was absorbed into the circulation. After a repeated-dose oral exposure in rats (Group 4&5), there was a slight increase of C12-C12 absorption, but it was still limited and there was no accumulation as well as with the C9-C12 content.
Details on distribution in tissues:
DIGESTIVE TRACT
After animals were orally administrated with a single-dose at 50 mg/kg, the average concentrations of three components in digestive tract and its content at different time points (0.5, 2, 4, 8 and 24 h) and the percentages of exposure dose (Group 9) showed that C12-C12 and C9-C12 were detected in digestive tract including contents in rats within 24 h, but showed a time-course decrease in its average concentration and the percentage to dose administrated. The percentage of C12-C12 and C9-C12 at 0.5 h was only 15.92% and 13.62% of the dose administrated, indicating that only limited residual stayed in the GI tract. Taking together the results of limited absorption of C12-C12 and C9-C12 after oral administration and limited in vitro metabolism by liver microsomes, it was speculated that C12-C12 and C9-C12 were metabolized in the digestive tract. The other component of C9-C9 was also detected in the most samples, and its concentration showed gradually decreased.
TISSUES
After animals were orally administrated with a single-dose of 50 mg/kg, the average concentrations at different time points of three components in different tissues (Group 8) showed that C12 was detected in the liver, eyes, brain and thyroid gland, and C9-C12 was detected in eyes, brain and thyroid gland. In another animal (#34) at 2 h, C12-C12 was only detected in the liver and C9-C12 was detected in the brain at low levels close to LOQ. In the other 2 animals, C12-C12 and C9-C12 were only detected in digestive tract and its contents, indicating that they were not absorbed into the circulation and mainly distributed in digestive tract contents. C9-C9 was detected in several tissues of almost all animals, indicating that it was absorbed into circulation to some degree.
Details on excretion:
In rats administrated intravenously with the test substance, the Apparent Volume of Distribution (Vz) of C12-C12 was 0.24 +/- 0.040 L/kg, and the elimination half-life (t1/2z) of C12-C12 was 1.02 +/- 0.18 h. C9-C12 in plasma was also rapidly eliminated in rats.
After single-dose oral exposure, C12-C12 and C9-C12 was detected in feces, but not in urine. The cumulative percentage of C12-C12 and C9-C12 in feces increased rapidly within 12 h, but the 72-h cumulative excretion only accounted for 3.88% and 1.04% of the dose administrated, indicating that only limited amount of C12-C12 and C9-C12 were excreted via feces in the original form. The third component of C9-C9 was detected in feces and urine, indicating that it was excreted via feces and urine in its original form.
After single-dose intravenous exposure, C12-C12 and C9-C9 were detected in bile of 2 animals at several time points. C9-C12 was not detected. These data indicated that C12-C12, C9-C12 and C9-C9 hardly passed through the bile in the original form (Group 6&7).
Toxicokinetic parameters
Test no.:
#1
Toxicokinetic parameters:
half-life 1st: 1.02 ± 0.18 hours
Remarks:
Group 1 (Single-dose Intravenous Administration of 5mg/kg test substance)

Metabolite characterisation studies

Metabolites identified:
yes
Details on metabolites:
IN VITRO METABOLISM RESULTS
In the study of the Liver microsomal Metabolism, there were no obvious differences in the concentrations of C12-C12 and C9-C12 after incubation with liver microsomes from different species, indicating that liver microsomes were not involved in the metabolism of C12-C12 and C9-C12.
During the study of the Recombinant CYP 450 Metabolism, the test item was incubated with 6 subtypes of recombinant cytochrome P450 enzyme (CYP450) individually for 90 mins. No obvious changes of C12-C12 was observed excepted in the incubation system with CYP2E1, which showed minor decreases after incubation (78.08%), suggesting that C12-C12 was slightly metabolized by CYP2E1.
There was no significant change of the mean concentration of C9-C12 after 90-min incubation with six types of CYP 450 enzymes, indicating that CYP 450 enzymes were not involved in metabolism of C9-C12.
During the study of the plasma protein binding determined by equilibrium dialysis at concentrations of 50~200 ng/mL, protein binding rates of different species were all 100%, indicating that C12-C12 and C9-C12 were almost completely bound to plasma proteins and mainly existed in the conjugated form.

IN VIVO METABOLISM RESULTS
Three metabolites (M1-M3) were identified in the preliminary studies.
- Intravenous exposure
In rats intravenously administrated with a single dose of 5 mg/kg test substance, three potential metabolites with mass-to-charge Ratio (m/z) of 321.4→89.1, 343.2→173.5 and 365.5→133.3 were detected in plasma by full ion scanning. These metabolites were detected at high level since 1~5 mins post exposure, indicating test material was rapidly metabolized into different products in plasma. However, three other metabolites with m/z of 179.2→151.1, 273.0→124.0 and 287.0→138.0 were detected in bile since 0~2 hours post exposure.
The test item was metabolized into multiple metabolites and excreted via bile after intravenous injection. Moreover, differed metabolites observed in bile and plasma indicated that metabolic pathway of the test item was complicated in rats.

- Oral exposure
After single or repeated oral administration of test substance in SD rats, no metabolite peaks were identified in plasma by full ion scanning, possible metabolites m/z 521.7→321.1 and m/z 479.7→321.3 were found in urine and possible metabolites m/z 479.7→321.3 was found in feces. The results showed that C12-C12 and C9-C12 had no first-pass effect of liver.
In the tissue distribution test, C9-C9 was almost detected in individual tissues of every animal, indicating that C9-C9 might be absorbed and metabolized in rats.
Through full ion scanning of the digestive tract and its contents and the tissue distribution samples, two other possible metabolites m/z 280.2→221.1 and m/z 521.7→321.1 were found, indicating that C12-C12, C9-C12 and C9-C9 could be metabolized in the digestive tract and indicated that C9-C9 was distributed and metabolized in various tissues of rats, and metabolized into the same products in tissues as in digestive tract and its contents.
As metabolites found in this study varied greatly in molecular weights, it is speculated that the diester bond of the test substance is easily broken into fragments, and new esters will be formed in the digestive tract and body by binding to fatty acids of various carbon chain lengths.

Applicant's summary and conclusion

Conclusions:
This toxicokinetic study (OECD 417) in Sprague-Dawley rats revealed:
- a low bioavailability, no accumulation and an inter-individual difference.
- C12-C12 and C9-C12 only detected in GI tract. C9-C9 detected in several tissues of almost all animals and absorbed into circulation to some degree.
- rapid elimination and metabolization in the digestive tract of rats.
Executive summary:

ADME properties of the test substance (CAS: 2166089-27-4), dodecanoic acid mixed diesters with dipropylene glycol and nonanoic acid was tested in the Sprague-Dawley rats based on OECD guideline 417. Bio-analytical methods (LC-MS/MS) were developed and validated to determine the test substance in biological samples of SD rats. Potential metabolites of the test substance were identified using full ion scanning mode. A total of nine experimental groups (Groups #1~9) were designed in the in vivo studies as follows: a single intravenous dose group (#1), a low single oral dose group combined with initial exposure (#2), a high single oral dose group (#3), a trough repeated oral dose group (#4), a repeated oral dose group with last exposure (#5), a feces and urine excretion group (#6), a biliary excretion group (#7), a tissue distribution group (#8) and a gastrointestinal residual group (#9). In addition, three in vitro studies were conducted on the test item, including plasma protein binding test, liver microsomal metabolism test, and recombinant CYP 450 enzymes metabolism test.

The test substance is composed of three components of C12 -C12 at 26.43%, C9 -C12 at 50.82%and C9 -C9 at 22.75%. In rats administrated intravenously with the test substance, the Apparent Volume of Distribution (Vz) of C12 -C12 was 0.24±0.040 L/kg, and the elimination half-life(t1/2z) of C12 -C12 was 1.02±0.18 h. C9 -C12 in plasma was also rapidly eliminated in rats. After single-dose oral exposure tothe test substance, only a small amount of C12 -C12 was absorbed into the blood. C9 -C12 was barely absorbed into circulation, and C9 -C9 was absorbed into the circulation. After single-dose oral exposure of the test substance, the 72-h cumulative excretion of C12-C12 and C9-C12 in feces only accounted for 3.88% and 1.04% of the dose administrated, and the percentage of C12 -C12 and C9 -C12 at 0.5 h was only 15.92% and 13.62% of the dose administrated, indicating that C12 -C12 and C9 -C12 were rapidly eliminated in the digestive tract. Results of in vitro studies showed that C12 -C12 and C9 -C12 were not metabolized by liver microsomes and recombinant CYP450 enzymes. C12 -C12 and C9 -C12 was almost completely bound to plasma protein with binding rate of 100%. The test substance was rapidly metabolized in the digestive tract of rats. As metabolites found in this study varied greatly in molecular weights via different administration routes, it is speculated that the diester bond of the test substance is easily broken into fragments, and new esters will be formed in the digestive tract and body by binding to fatty acids of various carbon chain lengths.

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