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Diss Factsheets

Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 September 2010 to 23 September 2010
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Objective of study:
metabolism
Test guideline
Qualifier:
no guideline required
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
25084-89-3/9051-51-8
IUPAC Name:
25084-89-3/9051-51-8
Constituent 2
Reference substance name:
Reaction products of methyloxirane with formaldehyde, oligomeric reaction products with aniline and reaction products of methyloxirane with 2,2'-oxydiethanol
Molecular formula:
Not Applicable, UVCB substance.
IUPAC Name:
Reaction products of methyloxirane with formaldehyde, oligomeric reaction products with aniline and reaction products of methyloxirane with 2,2'-oxydiethanol
Details on test material:
Identity: DADPM/DEG PO
Chemical name: Reaction mass of 2,2′-oxydiethanol, propoxylated and formaldehyde, polymer with benzamine and 2-methyl oxirane
Appearance: Yellow viscous liquid
Storage conditions: Ambient temperature in the dark
Lot no.: RZBL 001367
Date received: 10 September 2010
Radiolabelling:
no

Test animals

Species:
human
Strain:
not specified
Sex:
not specified
Details on test animals or test system and environmental conditions:
Experimental Procedure
Chemicals
Test material DADPM/DEG PO was provided by Huntsman.
[14C]Testosterone (53 mCi/mmol) was supplied by GE Healthcare UK Ltd.
NADPH, UDPGA, alamethicin, saccharic acid 1,4-lactone and testosterone were purchased from the Sigma-Aldrich Company Ltd.
Acetonitrile, methanol, EDTA, magnesium chloride, potassium chloride, potassium di-hydrogen orthophosphate, di-sodium hydrogen orthophosphate, formic acid and tris (hydroxylmethyl)methylamine were obtained from Fisher Laboratory Supplies. PerkinElmer Life and Analytical Sciences supplied Ultima gold scintillation cocktail. Monoflow 3 was supplied by National Diagnostics. Super-pure water was produced in situ from an in-house Elga Option 4 water purification system.

IN-LIFE DATES: From: 13 September 2010 To: 23 September 2010

Administration / exposure

Route of administration:
other: in vitro
Vehicle:
other: acetonitrile
Details on exposure:
Incubation of DADPM/DEG PO with pooled human liver microsomes
DADPM/DEG PO was incubated with pooled human liver microsomes at final substrate concentrations of 50 and 200 mg/L. All reaction mixtures contained sodium/potassium phosphate buffer (100 mM, pH 7.4 containing 4 mM saccharic acid 1,4-lactone and 0.85 mM MgCl2), microsomal protein (either 0.5 or 2 mg/mL, pre-treated with alamethicin (50 µg/mg protein) on ice for 15 minutes), NADPH generating system (2 mM NADP+, 3.3 mM glucose 6-phosphate, 1 U/mL glucose 6-phosphate dehydrogenase) and UDPGA (3 mM) in a total volume of 1 mL. Following brief vortex mixing and pre-incubation for 5 minutes at 37ºC, the reaction in the samples was started by addition of substrate solution (either 5 or 20 mg/mL DADPM/DEG PO solution prepared in acetonitrile, 10 µL; final concentrations = 50 or 200 mg/L, respectively). Control incubations were performed in the absence of microsomal protein at both 50 and 200 mg/L of DADPM/DEG PO for the longest time point only. All samples were incubated for a further 0, 0.5, 1 or 2 hours before the reaction was terminated by the addition of chilled acetonitrile (1 mL) and standing on ice for at least 10 minutes. All incubations were conducted as single samples in microcentrifuge tubes.
Duration and frequency of treatment / exposure:
All reaction mixtures were kept on ice for 15 minutes. Following brief vortex mixing and pre-incubation for 5 minutes at 37ºC, the reaction in the samples was started by addition of substrate solution. Control incubations were performed in the absence of microsomal protein at both 50 and 200 mg/L of DADPM/DEG PO for the longest time point only. All samples were incubated for a further 0, 0.5, 1 or 2 hours before the reaction was terminated by the addition of chilled acetonitrile (1 mL) and standing on ice for at least 10 minutes. All incubations were conducted as single samples in microcentrifuge tubes.
Doses / concentrations
Remarks:
Doses / Concentrations:
see above.
No. of animals per sex per dose / concentration:
N/A
Control animals:
other: N/A
Positive control reference chemical:
[14C]Testosterone
Details on study design:
DADPM/DEG PO (50 and 200 mg/L) was incubated with pooled human liver microsomes (0.5 and 2.0 mg/mL), pre-treated with alamethicin and in the presence of NADPH and UDPGA, for up to 120 minutes. NADPH and UDPGA are the cofactors required for cytochrome P450- and UDP-glucuronosyltransferase (UGT)-catalysed reactions, respectively. The active site of the UGT enzymes resides in the lumen of the endoplasmic reticulum; alamethicin is a peptide that forms pores in the endoplasmic reticulum membrane and thus allows access of substrates to the UGT active site. The reaction was stopped by addition of acetonitrile. The samples were centrifuged and the supernatants taken for LC-MS analysis.

The LC-MS analysis of the human liver microsome incubation samples was carried out on a Waters Symmetry C18 column (150 × 3.9 mm) with 10 mM ammonium formate and acetonitrile gradient system. The TSQ7000 mass spectrometer was operated in positive ion mode.
Details on dosing and sampling:
See above.
Statistics:
N/A

Results and discussion

Main ADME results
Type:
metabolism
Results:
glucuronide conjugates

Metabolite characterisation studies

Metabolites identified:
not measured
Details on metabolites:
Glucuronide conjugates which are observed at masses 176 daltons above the mass of the test compounds.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): no bioaccumulation potential based on study results
From the present data there is clear evidence that the test compound mixture (DADPM/DEG-PO) does undergo biotransformation in incubations with human liver microsomes pre-treated with alamethicin to enable access of substrates to the UGT active site and in the presence of the enzyme cofactors NADPH (for oxidative metabolism) and UDPGA (for glucuronic acid conjugation), as far as the aromatic components in the mixture are concerned. The data derived from the 2 hour incubations at both microsomal protein concentrations show the presence of glucuronide conjugates of the main aromatic components of the test compound mixture.
Executive summary:

The objective of this study was to investigate if DADPM/DEG PO is metabolised in incubations of human liver microsomes.

DADPM/DEG PO (50 and 200 mg/L) was incubated with pooled human liver microsomes (0.5 and 2.0 mg/mL), pre-treated with alamethicin and in the presence of NADPH and UDPGA, for up to 120 minutes. NADPH and UDPGA are the cofactors required for cytochrome P450- and UDP-glucuronosyltransferase (UGT)-catalysed reactions, respectively. The active site of the UGT enzymes resides in the lumen of the endoplasmic reticulum; alamethicin is a peptide that forms pores in the endoplasmic reticulum membrane and thus allows access of substrates to the UGT active site. The reaction was stopped by addition of acetonitrile. The samples were centrifuged and the supernatants taken for LC-MS analysis.

The LC-MS analysis of the human liver microsome incubation samples was carried out on a Waters Symmetry C18 column (150 × 3.9 mm) with 10 mM ammonium formate and acetonitrile gradient system. The TSQ7000 mass spectrometer was operated in positive ion mode.

The LC-MS results indicate that the human liver microsomes cause biotransformation of the aromatic components in the DADPM/DEG PO test mixture. The products of biotransformation are glucuronide conjugates which are observed at masses 176 daltons above the mass of the test compounds.