1,2,4-Benzenetricarboxylic acid, mixed lauryl and octyl triesters

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19-May-2016 to 09-Sept-2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Objective of study:

bioaccessibility (or bioavailability)

toxicokinetics

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

Version adopted 22-July-2010

Deviations:

yes

Remarks:

For toxicokinetic arm of study, only two animals of each gender were studied rather than the four stipulated in the test guidelines.

Principles of method if other than guideline:

N/A

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Linplast 812 TM

IUPAC Name:

Linplast 812 TM

Test material form:

liquid

Remarks:

Colorless

Details on test material:

Storage conditions: Room temperature, avoid light, dry.

Expiration date: Jan., 2017.

Specific details on test material used for the study:

N/A

Radiolabelling:

no

Remarks:

An analytical method was developed to measure the unlabelled test material.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

SPF Grade.

Details on species / strain selection:

The rat was selected for this study because it is recognized as a species commonly used in toxicology studies. The rat is recommended by the Ministry of Environmental Protection of P. R. China "The Guidelines for the Testing of Chemicals - Health Effects (2nd edition)".

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Beijing WeiTongLiHua Test Animal Technology Co. Ltd
- Age: 6-8 weeks.
- Weight at end of quarantine period: Males 301.9 to 378.9 g. Females 232.3 to 275.6 g.
- Housing: Animals were housed in groups in transparent polycarbonate boxes and each cage rack contained 3-5 animals. Study number, test substance number, animal code, group, sex, and dose were labelled on the cage cards. Cages and bedding were replaced twice a week.
- Diet: Ad libitum. Standard extruded food for test animals (source: Beijing KeaoXieli Food Co. Ltd).
- Water: Ad libitum. Municipal water. Drinking water quality was monitored.
- Quarantine: All animals were quarantined for seven (7) days. During this period, no abnormalities of animals were observed. Clinical Verterinarian checked the animals and issued quarantine inspection report before the animals were used on-study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 25
- Humidity (%): 40 - 70
- Photoperiod: 12 hrs dark /12 hrs light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The appropriate amount of the test sample plus corn oil volume, mix, dubbed with 1000 mg/mL suspension for animal exposure. The volume of administration was 10 mL/kg. Animals were fasted for at least 12 h prior to administration.

Duration and frequency of treatment / exposure:

A single dose of test substance was administered.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 mg/kg bw/day

Remarks:

iv adminstration

Doses / concentrations 2

Dose / conc.:

5 mg/kg bw/day

Remarks:

iv administration

Doses / concentrations 3

Dose / conc.:

10 mg/kg bw/day

Remarks:

iv administration

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw (total dose)

Remarks:

Toxicokinetics arm of study.

Doses / concentrations 5

Dose / conc.:

50 mg/kg bw (total dose)

Remarks:

Digestive residue arm of study.

No. of animals per sex per dose / concentration:

3 males for iv administration

2 males and 2 females for toxicokinetics arm of study.

8 males and 8 females for digestive residue arm of study.

Control animals:

no

Positive control reference chemical:

N/A

Details on study design:

DOSE SELECTION

The dose selection was based on three preliminary experiments, details of which are provided in the pertinent RESULTS AND DISCUSSION section below.

ANALYTICAL METHOD

A LC-MS/MS method for LINPLAST 812 TM (m/z 547.4→305) was established as part of the study.

The method was validated for use in rat plasma anticoagulated by heparin in the range 50-500 ng/mL LINPLAST 812 TM.

The method was validated for use with digestive tract homogenate in the range 50-500 ng/mL LINPLAST 812 TM.

Details on dosing and sampling:

TOXICOKINETIC ARM OF STUDY

Dosing details have already been provided.

Following dosing, approximately 0.2 mL of blood was collected via the orbital vein and was added with anticoagulant heparin solution. Blood samples were collected at 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 24 h following the dose administration and before dose administration.

Blood samples should be pretreatment and analysed as soon as possible.

DIGESTIVE RESIDUE ARM OF STUDY

Dosing details have already been provided.

After the exposure of animals placed in metabolic cages for 30 min, 2 h, 6 h, and 24 h, each time two male and two female animals were euthanized, whichever digestive tract and faeces collected after administration, the digestive tract and feces combined weighed, then immediately added 0.01 M PBS at a weight ratio of 1:5 homogenate, homogenate vortex mix, 50 μL was added to 500 μL acetonitrile and then measured.

Statistics:

TOXICOKINETIC ARM OF STUDY
DAS2.0 pharmacokinetic program was used to analyse data and calculate the main pharmacokinetic parameters: AUC, Cmax, F, Tmax, MRT, t1/2 and so on.

DIGESTIVE RESIDUE ARM OF STUDY
Microsoft Excel was used for data processing, computing the gastrointestinal remaining amount.

Results and discussion

Preliminary studies:

PRELIMINARY EXPERIMENT 1

Two SD male rats were administered LINPLAST 812 TM at 20 or 1000 mg/kg by oral gavage, one male rat was administered LINPLAST 812 TM at 5 mg/kg by intravenous (iv) injection, obtained plasma to determine (plasma sample treatment: samples treated at 13000rpm, at 4 °C Centrifugal for 10min). Preliminary test results are shown in Table 1. The results indicate that LINPLAST 812 TM eliminates slowly in iv injection treatment group and can’t absorb in oral gavage group.

PRELIMINARY EXPERIMENT 2

Two SD male rats were administered LINPLAST 812 TM at 20 or 1000 mg/kg by oral gavage, one male rat was administered LINPLAST 812 TM at 5 mg/kg by intravenous (iv) injection, obtained blood to determine (sample treatment: 50 μL of rat whole blood was added into 500 μL acetonitrile). Preliminary test results are shown in Table 2. The result indicates that LINPLAST 812 TM eliminates slowly in iv injection treat group; and can’t absorb in oral gavage, without different plasma result.

PRELIMINARY EXPERIMENT 3

Two SD male rats were administered LINPLAST 812 TM at 1 or 10 mg/kg by intravenous (iv) injection, obtained plasma to determine (sample treatment: samples treated at 13000rpm, at 4 °C Centrifugal for 10min). Preliminary test results are shown in Table 3. The result indicates that LINPLAST 812 TM eliminates slowly in all iv injection treat group, and exposure level did not increase with the increase of dose.

Main ADME results

Main ADME results 1

Type:

other: Bioavailability

Results:

After oral gavage, LINPLAST 812 TM prototype was undetectable in the plasma of SD rats.

Main ADME results 2

Type:

other: Digestive residue

Results:

Less than 1% of the administered LINPLAST 812 TM was recovered in digestive tract and feces.

Toxicokinetic / pharmacokinetic studies

Details on absorption:

N/A

Details on distribution in tissues:

N/A

Details on excretion:

N/A

Metabolite characterisation studies

Metabolites identified:

not measured

Details on metabolites:

N/A

Enzymatic activity

Enzymatic activity measured:

N/A

Bioaccessibility (or Bioavailability)

Bioaccessibility (or Bioavailability) testing results:

TOXICOKINETIC ARM OF STUDY

Four SD rats were administered LINPLAST 812 TM at 1000 mg/kg by oral gavage. The concentration time-course data for LINPLAST 812 TM in plasma is shown in Table 4. LINPLAST 812 TM prototype was undetectable in plasma.

DIGESTIVE RESIDUE ARM OF STUDY

Sixteen (16) rats were administered LINPLAST 812 TM at 50 mg/kg by oral gavage. The concentration time course data for LINPLAST 812 TM in plasma, digestive track, feces are shown in Table 5 to Table 7. The recovery rate time course data for LINPLAST 812 TM in plasma, digestive tract, feces are shown in Table 8 and Table 9.

At different times after oral gavage, the recovery rate of LINPLAST 812 TM in digestive tract and feces is very low, less than 1%, and LINPLAST 812 TM prototype was undetectable in plasma.

Any other information on results incl. tables

Table 1  Plasma concentration of LINPLAST 812 TM following administration in SD rats

J01 means intravenous administration, R01 and R02 means oral gavage administration, NA means not detected, BLLOQ below the low limit of quantization.

Time (h)	Concentration of LINPLAST 812 TM in plasma (ng/mL)
	J01 (5 mg/kg, iv)	R01 (20 mg/kg, ig)	R02 (1000 mg/kg, ig)
0.0166667	199.73	N/A	N/A
0.0833333	189.88	BLLOQ	BLLOQ
0.25	165.83	BLLOQ	BLLOQ
0.5	165.00	BLLOQ	BLLOQ
1	151.14	BLLOQ	BLLOQ
2	138.23	BLLOQ	BLLOQ
4	119.85	BLLOQ	BLLOQ
6	111.89	BLLOQ	BLLOQ
8	BLLOQ	BLLOQ	BLLOQ
12	BLLOQ	BLLOQ	BLLOQ
24	BLLOQ	BLLOQ	BLLOQ

 

Table 2  The whole blood concentration of LINPLAST 812 TM following administration in SD rats

J01 means intravenous administration, R01 and R02 means oral gavage administration, NA means not detected, BLLOQ below the low limit of quantization.

Time (h)	Concentration of LINPLAST 812 TM in whole blood (ng/mL)
	J01 (5 mg/kg, iv)	R01 (20 mg/kg, ig)	R02 (1000 mg/kg, ig)
0.0166667	126.31	N/A	N/A
0.0833333	108.99	BLLOQ	BLLOQ
0.25	106.89	BLLOQ	BLLOQ
0.5	BLLOQ	BLLOQ	BLLOQ
1	101.81	BLLOQ	BLLOQ
2	100.01	BLLOQ	BLLOQ
4	101.60	BLLOQ	BLLOQ
6	BLLOQ	BLLOQ	BLLOQ
8	BLLOQ	BLLOQ	BLLOQ
12	BLLOQ	BLLOQ	BLLOQ
24	BLLOQ	BLLOQ	BLLOQ

 

Table 3  Plasma concentration of LINPLAST 812 TM following administration in SD rats

J03, J04 means intravenous administration, BLLOQ below low limit of quantization.

Time (h)	Concentration of LINPLAST 812 TM in plasma (ng/mL)
	J03 (1 mg/kg, iv)	J04 (10 mg/kg, iv)
0.0166667	79.25	394.48
0.0833333	84.43	376.48
0.25	80.44	351.89
0.5	78.57	348.58
1	72.30	299.75
2	74.07	243.01
4	59.91	255.68
6	57.16	178.23
8	52.22	168.81
12	BLLOQ	130.88
24	BLLOQ	89.17

 

Table 4  Concentration time-course data for 1000 mg/kg LINPLAST 812 TM in plasma

BLLOQ means below the low limit of quantitation (50 ng/mL).

Sampling time	Concentration of LINPLAST 812 TM in plasma (ng/mL)
	FEMALE		MALE
	R01	R02	R03	R04
Predose	BLLOQ	BLLOQ	BLLOQ	BLLOQ
15 min	BLLOQ	BLLOQ	BLLOQ	BLLOQ
30 min	BLLOQ	BLLOQ	BLLOQ	BLLOQ
1 h	BLLOQ	BLLOQ	BLLOQ	BLLOQ
2 h	BLLOQ	BLLOQ	BLLOQ	BLLOQ
4 h	BLLOQ	BLLOQ	BLLOQ	BLLOQ
8 h	BLLOQ	BLLOQ	BLLOQ	BLLOQ
24 h	BLLOQ	BLLOQ	BLLOQ	BLLOQ

 

Table 5  Testing result for 50 mg/kg LINPLAST 812 TM in plasma

BLLOQ means below the low limit of quantitation (50 ng/mL).

Time (h)	Animal No.	Gender	LINPLAST 812 TM (ng/mL)
0.5	D01	F	BLLOQ
0.5	D02	F	BLLOQ
0.5	D03	M	BLLOQ
0.5	D04	M	BLLOQ
2	D05	F	BLLOQ
2	D06	F	BLLOQ
2	D07	M	BLLOQ
2	D08	M	BLLOQ
6	D09	F	BLLOQ
6	D10	F	BLLOQ
6	D11	M	BLLOQ
6	D12	M	BLLOQ
24	D13	F	BLLOQ
24	D14	F	BLLOQ
24	D15	M	BLLOQ
24	D16	M	BLLOQ

 

Table 6  Testing result for 50 mg/kg LINPLAST 812 TM in digestive tract homogenate

BLLOQ means below the low limit of quantitation (50 ng/mL).

Time (h)	Animal No.	Gender	LINPLAST 812 TM (ng/mL)	LINPLAST 812 TM (ng/g)
0.5	D01	F	111.48	668.88
0.5	D02	F	110.24	661.44
0.5	D03	M	91.57	549.42
0.5	D04	M	146.65	879.9
2	D05	F	92.03	552.18
2	D06	F	112.92	677.52
2	D07	M	118.26	709.56
2	D08	M	123.20	739.20
6	D09	F	79.08	474.48
6	D10	F	77.81	466.86
6	D11	M	134.61	807.66
6	D12	M	109.67	658.02
24	D13	F	BLLOQ	BLLOQ
24	D14	F	BLLOQ	BLLOQ
24	D15	M	BLLOQ	BLLOQ
24	D16	M	BLLOQ	BLLOQ

 

Table 7  Testing result for 50 mg/kg LINPLAST 812 TM in feces

BLLOQ means below the low limit of quantitation (50 ng/mL).

Time (h)	Animal No.	Gender	LINPLAST 812 TM (ng/mL)	LINPLAST 812 TM (ng/g)
0~6	D10	F	BLLOQ	BLLOQ
0~6	D12	M	BLLOQ	BLLOQ
6~24	D13	F	118.86	713.16
6~24	D14	F	212.64	1275.84
6~24	D15	M	189.42	1136.52
6~24	D16	M	303.93	1823.58

 

Table 8  Residue and recovery for 50 mg/kg LINPLAST 812 TM in digestive tract

BLLOQ means below the low limit of quantitation (50 ng/mL).

Time (h)	Animal No.	Exposure Amount (mg)	Digestive Tract Weight (g)	Digestive Tract Residues (mg)	Recovery rate (%)	Average Recovery (%)
0.5	D01	13.0	25.580	0.0171	0.132	0.128
0.5	D02	13.0	28.263	0.0187	0.144
0.5	D03	18.5	33.301	0.0183	0.099
0.5	D04	18.0	28.244	0.0249	0.138
2	D05	14.5	36.977	0.0204	0.141	0.141
2	D06	12.5	25.145	0.0170	0.136
2	D07	19.5	39.462	0.0280	0.144
2	D08	17.5	33.953	0.0251	0.143
6	D09	13.0	28.265	0.0134	0.103	0.103
6	D10	12.5	19.742	0.0092	0.074
6	D11	18.0	29.170	0.0236	0.131
6	D12	16.5	26.404	0.0174	0.105
24	D13	13.5	17.835	BLLOQ	0	0
24	D14	14.0	19.586	BLLOQ	0
24	D15	17.0	18.739	BLLOQ	0
24	D16	20.0	23.098	BLLOQ	0

 

Table 9  Residue and recovery for 50 mg/kg LINPLAST 812 TM in feces

BLLOQ means below the low limit of quantitation (50 ng/mL).

Time (h)	Animal No.	Exposure Amount (mg)	Feces Weight (g)	Feces Excretion (mg)	Recovery rate (%)	Average Recovery (%)
0~6	D10	12.5	0.932	BLLOQ	0	0
0~6	D12	16.5	1.808	BLLOQ	0
6~24	D13	13.5	3.001	0.0021	0.016	0.019
6~24	D14	14	2.927	0.0037	0.027
6~24	D15	17	0.998	0.0011	0.007
6~24	D16	20	2.721	0.0050	0.025

 

Applicant's summary and conclusion

Conclusions:

The absorption kinetics characteristics of LINPLAST 812 TM in rats by oral gavage is:

LINPLAST 812 TM prototype wasn’t found in plasma following gavage administration, it may be metabolized and decomposed in digestive tract and/or there has first pass effect in liver.

Executive summary:

An LC-MS/MS analytical method for LINPLAST 812 TM in rat biological samples was established. The method was based on quantifying the mass transition m/z 547.4→305. The identity of the precursor and fragment ions are not provided in the report and it is not clear whether the method measures one, more, or all of the constituents in the test material.

 

The method was validated for use with rat plasma. Specificity was confirmed and linearity was established over a range of 50-500 ng/mL, with the lower bound representing the LLOQ for the method. Inter- and intra-assay precision and accuracy were satisfactory. Recovery was approximately 75%. Sample and extracted sample stability was determined.

 

Linear range, precision, accuracy, recovery rate and matrix effects were also confirmed partially for the alimentary canal of rats.

 

Preliminary test results showed that LINPLAST 812 TM has slow elimination in rats administered the substance by intravenous injection. Moreover, plasma exposure did not proportionally increase with increasing dose.

 

After oral administration of LINPLAST 812 TM by oral gavage (1000 mg/kg), LINPLAST 812 TM prototype was undetectable in plasma. Moreover, after oral gavage administration of 50 mg/kg LINPLAST 812 TM, the recovery rate in the digestive tract and feces was less than 1%. LINPLAST 812 TM may be metabolized and decomposed in the digestive tract and/or subject to significant first-pass hepatic metabolism.