2,2,2',2'-tetrakis(hydroxymethyl)-3,3'-oxydipropan-1-ol

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Objective of study:

absorption

Qualifier:

no guideline available

Principles of method if other than guideline:

The study investigated the absorption of the submission sustance in an in vitro everted rodent intestinal sac model.

GLP compliance:

no

Radiolabelling:

no

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

Intestinal sacs were prepared from male Han Wistar Rats, approximately 8-12 weeks of age. Rats were obtained from Harlan, Bicester, UK.

Route of administration:

other: in vitro

Vehicle:

other: 'Fed State' Simulated Intestinal Fluid (FeSSIF)

Details on exposure:

IN VITRO APPLICATION

- Concentration of test material: 50 mM
- Cell culture medium characteristics (temperature, pH): TC-199 tissue culture media, N,N-bis-trimethylsilyl-trifluoroacetamide (BSTFA) and pyridine (Sigma-Aldrich, UK)
- Incubation temperature: 37 °C
- Number of replicates (if more than one is used per run): sacs from two rats were incubated in triplicate
- Time points: 1 hour incubation time

Duration and frequency of treatment / exposure:

Absorption was assessed over a period of one hour

Dose / conc.:

50 other: mM

No. of animals per sex per dose / concentration:

The sacs from two rats were used

Control animals:

no

Positive control reference chemical:

Not required

Details on study design:

The everted intestinal sacs were prepared by gently everting a freshly excised rat proximal small intestine over a glass stirring rod, rinsing with TC-199 media and filling the everted intestine with oxygenated FeSSIF medium at 37°C and dividing it into sacs approximately 2.5 cm in length using braided suture silk.

Details on dosing and sampling:

After 1 hour the individual sacs were removed, washed with running water and blotted dry. The sacs were cut open and the serosal fluid drained into small tubes. Each tube was weighed before and after collection of the serosal fluid to accurately calculate the volume of medium collected from inside the sac.

Statistics:

Calculation of mean and standard deviation: no further statistical analyses required.

Details on absorption:

The calculated concentrations of Di-penta in the serosal fluid from the intestinal sac incubations were 6.148 mM (±3.791) from Rat 1 and 7.700 mM (±2.083) from Rat 2.

Metabolites identified:

not measured

Table 1: Calculated concentration of Di-penta in serosal fluid. 

Sample	Calculated concentration of Di-penta in serosal fluid (mM)	Mean (mM)	±SD
Serosal fluid 1 Rat 1	5.713
Serosal fluid 2 Rat 1	2.593
Serosal fluid 3 Rat 1	10.137	6.148	3.791
Serosal fluid 1 Rat 2	5.519
Serosal fluid 2 Rat 2	9.670
Serosal fluid 3 Rat 2	7.909	7.700	2.083
External media 60 min	35.895

 

Calculated amount of Di-penta absorbed into serosal fluid

 

Sample	Calculated amount of Di-penta absorbed into serosal fluid (µmoles/400µL)	Mean (µmoles)	±SD
Serosal fluid 1 Rat 1	2.285
Serosal fluid 2 Rat 1	1.037
Serosal fluid 3 Rat 1	4.055	2.459	1.516
Serosal fluid 1 Rat 2	2.208
Serosal fluid 2 Rat 2	3.868
Serosal fluid 3 Rat 2	3.164	3.080	0.833
External media 60 min	14.358

 

Conclusions:

The results of the study indicate that di-Penta is absorbed in the everted rodent intestinal sac model.

Executive summary:

In an absorption study, 50 mM of Di-pentaerythritol was administered in the serosal fluid from the intestinal sac of two male Han Wistar rats. The sacs from the two rats were incubated in triplicate at 37 °C for 1 hour. Afterwards the test item was extracted and derivatized from the media and the concentration was analysed by GC-FID. The concentrations of the test material in the serosal fluid were 6.148 mM (±0.626) from rat 1 and 7.700 mM (±2.083) from rat 2. From these results it can be concluded that Di-pentaerythritol was absorbed into the intestinal sacs.

The results of the study indicate that di-Penta is absorbed in the everted rodent intestinal sac model.

Reference 2

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Objective of study:

metabolism

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Determination the rat hepatocyte metabolism of Di-pentaerythritol and identification of formed metabolites
- Short description of test conditions: Incubation of hepatocytes with two concentrations of the test item for 60 minutes at 37 °C
- Parameters analysed / observed: Analysis of the incubated hepatocyte samples by GC-FID and identification of metabolites by accurate mass metabolite identification

GLP compliance:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material:
Perstorp, batch no.: 613060270
- Purity, including information on contaminants, isomers, etc.: 97.0%

Radiolabelling:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

IN VITRO TEST SYSTEM

• If purchased
- Commercial source:
In-Vitro Technologies GmbH (Leipzig, Germany)
- Strain: Sprague-Dawley Rat

Route of administration:

other: in vitro

Vehicle:

other: acetonitrile

Details on exposure:

IN VITRO APPLICATION

- Concentration of test material and reference chemical: 50 µM and 10 µM
- Method of preparation of stock solution(s) of test material and reference chemical: Stock concentrations of the test item were prepared in acetonitrile at concentrations of 5 mM and 1 mM, for a 1% (v/v) addition (50 µL) into 5 mL hepatocyte suspensions. Final incubation concentrations were 50 µM and 10 µM, respectively
- Cell culture medium characteristics (temperature, pH): Leibowitz CL15 culture medium containing 10% fetal calf serum
- Incubation temperature: 37 °C
- Cell density in suspension for incubation (nominal and measured): 10^6 viable cells/mL
- Number of replicates (if more than one is used per run): Hepatocyte incubations were performed in duplicate with one set for GC-FID determination of loss of the test item and the other set was for determination of possible metabolite formation by LC-time of Flight Mass Spectrometry
- Time points: 0, 30, 60, 90, 120 and 240 minutes

Duration and frequency of treatment / exposure:

Metabolism was assessed 0, 30, 60, 90, 120 and 240 minutes after incubation

Dose / conc.:

10 other: µM

Dose / conc.:

50 other: µM

No. of animals per sex per dose / concentration:

not applicable

Control animals:

not relevant

Positive control reference chemical:

Ethoxycoumarin (7-EC, 100 µM)

Details on dosing and sampling:

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled (delete / add / specify): hepatocyte samples
- Time and frequency of sampling: 0, 30, 60, 90, 120 and 240 minutes
- Method type(s) for identification (e.g. GC-FID, GC-MS, HPLC-DAD, HPLC-MS-MS, HPLC-UV, Liquid scintillation counting, NMR, TLC): GC-FID (concentration measurement of the test item), LC-TOF (Accurate Mass Metabolite Identification)

ANALYTICAL METHOD
- Gas Chromatography Flame Ionisation Detection conditions
• Instrument: Varian 3800
• Column: Substituted siloxane polymer (95% methyl: 5% phenyl) (30 m length x 0.53 mm megabore internal diameter)
• Stationary phase: DB-5 (1.5um film thickness)
• Carrier gas: Hydrogen 5 mL/min
• Air: 300mL/min
• Injector: Varian CP-8400 Autosampler
• Splitless valve closed 0-1.5 min then split 50:1 (40:1 split for the 10 µM samples)
• Injection volume: 1 µL (autosampler syringe)
• Injector temperature: 300°C
• Column temperature: Initial 100°C (6 minutes) then programmed at 10 °C/min to 300 °C (2 minutes) total run time 28 minutes. Detector: Flame ionization
• Detector temperature: 300°C
• Data processing: Varian Galaxie Chromatography Workstation Version 1.8.501.1

- Accurate Mass Metabolite Identification by LC-ToF
• Analysis of selected samples was performed on the LCT Premier mass spectrometer using accurate mass determination and Metabolynx software for identification of any Phase 1 and Phase 2 metabolites produced. A total ion scan was performed with a mass range (m/z 50 – 1000). The neutral monoisotopic masses and the +ve ion monoisotopic masses used for the test item was 254.1366 and 255.1444, respectively.

- HPLC conditions for mass spectrometric analysis on the LCT Premier
• The HPLC column: Phenomenex Kinetex C18, 2.61m, 100x4.6mm.
• Mobile phase A: 0.1% formic acid in water; mobile phase B 0.1% formic acid in acetonitrile with 0.8 mL/min flow rate. 101, of each sample injected.
• Gradient conditions see table 1 in box “Any other information on materials & methods incl. tables”

- Data Analysis
• Samples analysed on the LCT Premier mass spectrometer were processed using Metabolynx software to determine the appearance of the possible metabolites

Statistics:

Calculation of mean and standard deviation: no further statistical analyses required.

Type:

metabolism

Results:

The results from the LC-ToF analysis have not detected any peaks. The analysis of the hepatocyte incubations with 7-ethoxycoumarin has shown the expected metabolism to produce the hydroxy, glucuronide and sulphate metabolites.

Table 2: Calculated remaining concentration of Di-Pentaerythritol in incubated rat hepatocyte samples

Timepoint (min)	Calculated concentration of Di-Pentaerythritol in incubation (µM)
	10 µM	50 µM
0	10.00	50.00
30	10.83	52.94
60	10.45	49.90
90	10.37	53.50
120	10.83	52.29
240	11.31	47.36

Conclusions:

The test item was not metabolised by rat cryopreserved hepatocytes and no detectable metabolites were produced.

Executive summary:

In a metabolism study, 50 µM and 10 µM of Di-pentaerythritol was administered to hepatocyte suspensions of male and female Sprague Dawley rats. The suspension was incubated in duplicate at 37 °C for 240 min with one set for GC-FID determination of loss of the test items and the other set was for determination of possible metabolite formation by CL-Time of Flight Mass Spectrometry. Samples were removed at time points 0, 30, 60, 90, 120 and 240 minutes and analysed.

 

The GC-FID analysis of the rat hepatocyte incubations with the test item has shown that there was negligible or no loss of the compounds in the incubations with rat hepatocytes suggesting that here was no rat hepatic metabolism of these compounds. The analysis of the GC-FID samples for 50 µM TMP has shown no loss of the compound in the incubated samples. The analysis of the 10 µM TMP incubated samples by GC-FID was deemed not to be sensitive enough to suitably integrate the TMP analyte.

The LC-ToF analysis of the rat hepatocyte incubations with the test item did not detect any metabolite formation.

The analysis of the hepatocyte incubations with 7-elhoxycoumarin has shown the expected metabolism to produce the hydroxy, glucuronide and sulphate metabolites.

From these results it can be concluded that Di-pentaerythritol was not metabolised by rat hepatocytes and that no detectable metabolites were produced.

Description of key information

A theoretical assessment of the toxicokinetics of di-pentaerythritol is made, based on the molecular structure and information from toxicity studies. The theoretical assessment is supported by the results of the in vitro studies of absorption and metabolism.  

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

50

Absorption rate - dermal (%):

50

Absorption rate - inhalation (%):

100

Additional information

A theoretical assessment of the toxicokinetics of di-pentaerythritol is made, based on the molecular structure and information from toxicity studies. The theoretical assessment is supported by the results of the in vitro studies of absorption and metabolism.

Theoretical assessment indicates rapid and extensive absorption and distribution; rapid metabolism and excretion are likely to limit systemic exposure and toxicity. No bioaccumulation is predicted.

 

Absorption

Oral absorption of di-Penta is predicted based on the substance molecular size, solubility, chemical structure and on the basis of experience with other polyol substances. The molecule satisfies Lipinski's rule of 5 (OECD QSAR Toolbox). Studies of oral toxicity do not provide any direct indication of gastrointestinal absorption; however non-specific signs of toxicity were observed in the acute oral toxicity study at the limit dose. A 14-day range-finding study in the rat reports pale faeces at dose levels of 1000 and 1500 mg/kg bw/d; findings may be consistent with incomplete oral absorption at these high gavage dose levels. An OECD 422 screening study similarly reports observations of pale faeces at the highest dose level of 1000 mg/kg bw/d. The results of an in vitro study performed using everted rat intestinal sacs reports that di-Penta is absorbed; however the extent of absorption is less than seen for other polyol compounds. 

In the absence of data, absorption following inhalation exposure is assumed to be extensive.

The dermal absorption of di-Penta is likely; the extent of dermal absorption is likely to be less than oral absorption.  In the absence of any data, the extent of oral and dermal absorption is assumed to be comparable.

Distribution

No data are available; findings from repeated dose toxicity studies do not indicate any specific targets of toxicity. Rapid and extensive distribution of absorbed di-Penta and its metabolites can be predicted based on knowledge for related substances. Di-Penta is likely to be rapidly absorbed and subject to extensive hepatic metabolism resulting in the renal excretion of water-soluble metabolites.  The systemic distribution of di-Penta is therefore likely to be limited by its metabolism and excretion.

Metabolism

Sequential oxidative metabolism of the six hydroxy groups present in the di-Penta molecule is predicted, based on known metabolic reactions and the elucidated pathways for other alcohol compounds; hydrolysis of the central ether linkage to yield pentaerythritol can also be predicted. There are no additional chemical groups known to be susceptible to mammalian metabolism. Rapid hepatic metabolism is indicated, which will facilitate excretion and therefore act to limit systemic exposure and toxicity. OECD QSAR Toolbox hepatic metabolism simulator identifies a total of seven potential metabolites arising from ether hydrolysis and/or oxidation.

In the in vitro metabolism study, using rat hepatocytes as metabolism model, no metabolites have been detected for di-Penta.

 

Excretion

Rapid and extensive renal excretion of di-pentaerythritol and/or its metabolites is likely, with no potential for bioaccumulation based on chemical properties. Findings are consistent with the low toxicity seen in the repeated-dose toxicity studies.