Registration Dossier

Administrative data

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Objective of study:
other: hydrolysis in rat plasma
Test guideline
Qualifier:
no guideline required
Principles of method if other than guideline:
Investigation of the stability of Adipic acid, cyclic ester with diethyleneglycol in phosphate buffer, hydrochloric acid (pH 3.0), and in rat plasma.
GLP compliance:
yes (incl. certificate)
Remarks:
Experimental Toxicology and Ecolog y, BASF SE

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Adipic acid, cyclic ester with diethyleneglycol
Purity >90%
Test substance No.: 99/147-1
Appearence: white solid

Administration / exposure

Duration and frequency of treatment / exposure:
Sampling of the incubates 0, 1, 2 and 4 h after application of the test substance.
Doses / concentrations
Remarks:
Doses / Concentrations:
1 mg/ml in phosphate buffer (50 mM Na-phosphate, pH 7.1) and hydrochloric acid (pH 3.0) (nominal)
1 and 5 mg/ml in rat plasma (nominal)
Details on study design:
Plasma was prepared from two male Wistar rats.
All incubations were performed at 37°C.
Samples of the incubates were taken immediatelly after addition of Adipic acid, cyclic ester with diethyleneglycol (0 h) and after 1, 2 and 4 h.
1) Investigation of non-enzymatic hydrolysis in plasma:
stability of Adipic acid, cyclic ester with diethyleneglycol in phosphate buffer (50 mM Na-phosphate, pH 7.1) at a nominal concentration of 1 mg/ml.
2) Investigation of hydrolysis in stomach:
stability of Adipic acid, cyclic ester with diethyleneglycol in hydrochloric acid (pH 3.0) at a nominal concentration of 1 mg/ml.
3) Investigation of the hydrolysis of Adipic acid, cyclic ester with diethyleneglycol in freshly prepared rat plasma, nominal concentrations were 1 and 5 mg/ml.
Ahead of analysis the protein was precipitated by ZnSO4-solution and subsequently centrifuged.
Analytical determination of either the test substance itself or by formation of the hydrolytic product adipic acid by HPLC. Quantification in the samples was done by external calibration using stock solutions in aqua bidest.

Results and discussion

Any other information on results incl. tables

The limit of detection of adipic acid was about 4 µg/ml (27 nmol/ml; 27 µM). This means that hydrolysis of only 1% of Adipic acid, cyclic ester with diethyleneglycol could be detected with this method.

1)-2) After a 4h incubation of Adipic acid, cyclic ester with diethyleneglycol in phosphate buffer or hydrochloric acid, no hydrolysis of could be detected . Therefore a non-enzymatic hydrolysis either at neutral pH or at a low pH-value simulating conditions in the stomach can not be anticipated.

3) Hydrolysis of Adipic acid, cyclic ester with diethyleneglycol with corresponding formation of adipic acid could be demonstrated at both concentrations tested. The hydrolysed portion increased with the duration of the incubation and the concentration used.

Table: Enzymatic hydrolysis of Adipic acid, cyclic ester with diethyleneglycol in rat plasma.

Nominal conc. [µg/ml]

Time [h]

Mean adipic acid [µg/ml]; n=2

Corresponding % adipic acid, cyclic ester with diethyleneglycol hydrolysed

1068

0               

21.5

2.98

 

1

99.8

13.81

 

2

119.4

16.52

 

4

131.4

18.18

5335

0               

73.1

2.02

 

1

1552.5

43

 

2

2224.6

62.62

 

4

3268.3

90.53

 

In control experiments it could be shown that Adipic acid, cyclic ester with diethyleneglycol was considerably bound to plasma proteins. The fact that enzymatic ester hydrolysis is more pronounced at higher concentrations may therefore result from saturation of protein binding and a consequently higher free plasma concentration.

Applicant's summary and conclusion