1,3-Isobenzofurandione, hexahydro-, reaction products with epichlorohydrin

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

August 1993 to March 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Objective of study:

metabolism

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Determination of Initial Alkylation Potencies
The test compounds were dissolved in acetone/ethylene glycol (1+2 by volume) to yield concentrations of 0.4 mM and 2 mM. 2.5 ml of these solutions were mixed with 2.0 ml phosphate buffer (100 mM, pH 7.2) and 0.5 ml 0.12 M p-nitrobenzyl pyridine (NBP) in acetone. After different time periods, 0.8 ml were withdrawn and made alkaline by the addition of 0.2 ml triethylamine. The optical density at 580 nm was recorded immediately.

Determination of Chemical Hvdrolvsis
The test compounds, dissolved in acetone/ethylene glycol (1+2 by volume), were mixed with an equal amount of 0.2 N HCI or 0.02 N HCI, to give a final concentration of 2 mM. After different preincubation times at 37 °C aliquots of 530 pi of the hydrolysate were diluted with 530 pi acetone/ethylene glycol (1 +2 by volume) and neutralized by the addition of 110 pi 0.5 M phosphate buffer, pH 7.2, and 265 pi 0.2 N NaOH or 0.02 N NaOH. Control incubations were made in 100 mM phosphate buffer, pH 7.2. The alkylation activity of the neutralized hydrolysate was determined by incubation with 160 pi NBP-solution (0.12 M in acetone) for 1 hour.

Determination of Enzvmatic Hvdrolysis
The test compounds were dissolved in DMSO to give a final concentration of about 2 mM. 30 pi of these solutions were incubated with mouse liver homogenate (about 7 mg protein/ml) in a final volume of 0.6 ml. After different time periods the proteins were precipitated by the addition of 2 volumes acetone and by centrifugation. 200 pi of the supernatant was assessed for alkylating activity by incubation with 1 ml NPB (57.6 mM) in acetone/ethylene glycol/100 mM phosphate buffer, pH 7.2 (18 +41+41 by volume) for 24 hours.

GLP compliance:

no

Test material

Test material information

Constituent 1

Test material form:

liquid

Radiolabelling:

yes

Results and discussion

Metabolite characterisation studies

Metabolites identified:

not measured

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): bioaccumulation potential cannot be judged based on study results
Alkylation Potency of Glycidyl Compounds
The glycidyl compounds (epoxide concentration: 1 and 0.2mM) were incubated with NBP (12mM) for various time periods. A linear regression of the optical density at 580 nm vs. incubation time was used to calculate the increase of the optical density per minute. So the alkylation potency for the the test material was deteremined as increase of the optical density at 580 nm per minute.
-For test material (1 mM) the oD 580 nm/min was 6.12E-03
- For test material (0.2 mM) the oD 580 nm/min was 1.04E-03

Chemical Hydrolysis of Glycidyl Compounds
A preincubation of the glycidyl compounds in 0.1 N HCI reduced the initial alkylation activity due to hydrolysis of their epoxide moieties (Figure 3). This reduction of the alkylation activity, reflects the susceptibility of the test compounds to chemical hydrolysis. Preincubation of the test compounds in 0.01 N HCI did not substantially decrease the alkylation activities. Under the assumption of a first order kinetics for the chemical hydrolysis, a linear regression of the logarithmically transformed data allowed the calculation of the half-life times of the epoxide moieties in 0.1 N HCI.
- For test material: Chemical Half Life in minutes: 12.9 +/- 0.7

Enzvmatic Hydrolysis of Glycidyl Compounds
Upon preincubation with mouse liver homogenates the alkylation activities were also reduced. The time course of the inactivation of the glycidyl compounds is presented. A logarithmic presentation of the data suggests a first order kinetics. This allowed the calculation of the half-life times for the test compounds upon enzymatic hydrolysis. The test material was inactivated relatively slowly by mouse liver homogenates.
- For test material: Enzymatic Half Life in minutes: 54.8 +/- 13.2