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Administrative data

Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-11-30 to 2011-12-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study, no deficiencies

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Dihydro-3-(tripropenyl)furan-2,5-dione
EC Number:
295-556-6
EC Name:
Dihydro-3-(tripropenyl)furan-2,5-dione
Cas Number:
92077-08-2
Molecular formula:
R-C4H3O3 , whereas R=C8-C10-alkyl-(branched, unsaturated)
IUPAC Name:
3-[(1E)-2-methyloct-1-en-1-yl]oxolane-2,5-dione
Details on test material:
- Name of test material (as cited in study report): Genopur ASA
- Physical state: Liquid, clear brown
- Analytical purity: 100 %
- Purity test date: 2011-09-10
- Lot/batch No.: ESD0010823
- Expiration date of the lot/batch: 2012-12-01
- Stability under test conditions: Aim of this study
- Storage condition of test material: Room temperature, protected from light, in the original container
Radiolabelling:
no

Study design

Analytical monitoring:
yes
Details on sampling:
- Sampling intervals for the parent/transformation products:
For pH 1.2, 4, 7 and 9, samples were taken at regular intervals depending on the hydrolysis results.
The time between test item application and transfer to the temperature controlled sample compartment of the HPLC autosampler did not exceed 3 min, except test start of pH 4 , 35 °C (5 min) and pH 7, 15 °C (11 min) due to technical reasons.
Due to the rapid transformation, replicates were sampled subsequently and automatically by employing an HPLC autosampler system with temperature controlled sample compartment. Injections were performed at test start (0 h) and at a minimum of 7 spaced points, normally between 10 and 90 % of hydrolysis except for pH 9 because of rapid transformation. For details see below.
All test item containing samples were analysed immediately (max. 1 % of total incubation time until start of analyses) via HPLC and UV (DAD) detection. The incubation temperature was measured every minute.

Buffers:
Buffer solution pH 1.2 250 mL of 0.2 mol/L KCl and 425 mL of 0.2 mol/L HCl.

Buffer solution pH 4 90 mL of 0.1 mol/L NaOH was mixed with 500 mL 0.1 mol/L mono potassium citrate and diluted to 1000 mL with double distilled water.

Buffer solution pH 7 296.3 mL of 0.1 mol/L NaOH was mixed with 500 mL 0.1 mol/L KH2PO4 and diluted to 1000 mL with
double distilled water.

Buffer solution pH 9 213.0 mL of 0.1 mol/L NaOH was mixed with 500 mL 0.1 mol/L H3BO3 in 0.1 mol/L KCl and diluted to
1000 mL with double distilled water.
Details on test conditions:
TEST SYSTEM
- Type, material and volume of test flasks, other equipment used: Sterile amber HPLC vials, volume: 2 mL
- Measures taken to avoid photolytic effects: Photolytic effects were avoided by exclusion of direct light
- Measures to exclude oxygen: Buffers were purged with nitrogen for 5 min, permanent nitogen flow during incubation
- Is there any indication of the test material adsorbing to the walls of the test apparatus? No
TEST MEDIUM
- Volume used/treatment: 2 mL
- Preparation of test medium: 300 mg/L in respective buffer solution
- Renewal of test solution: None
Duration of testopen allclose all
Duration:
0 h
pH:
9
Temp.:
15 °C
Initial conc. measured:
52.261 mg/L
Duration:
84.5 min
pH:
1.2
Temp.:
37.3 °C
Initial conc. measured:
35.401 mg/L
Duration:
229 min
pH:
4
Temp.:
14.9 °C
Initial conc. measured:
52.61 mg/L
Duration:
166 min
pH:
4
Temp.:
24.6 °C
Initial conc. measured:
28.141 mg/L
Duration:
65.3 min
pH:
4
Temp.:
34.8 °C
Initial conc. measured:
42.187 mg/L
Duration:
178 min
pH:
7
Temp.:
15 °C
Initial conc. measured:
44.641 mg/L
Duration:
121 min
pH:
7
Temp.:
24.5 °C
Initial conc. measured:
32.313 mg/L
Duration:
45.5 min
pH:
7
Temp.:
34.7 °C
Initial conc. measured:
22.772 mg/L
Number of replicates:
One replicate at each sampling interval (sequential sampling)
Positive controls:
no
Negative controls:
yes
Remarks:
buffer solutions (pH 1.2, 4, 7 and 9)

Results and discussion

Test performance:
Chronological Test Description:
- Method validation
- Preparation of the sterile test solutions (experimental starting)
- Thermostatisation of the test solutions
- Analysis of samples
- Evaluation of reaction rate constants and half lives for the test
item
Transformation products:
yes
Identity of transformation products
No.:
#1
Reference
Reference substance name:
Unnamed
IUPAC name:
Tripropenyl succinic acid
CAS number:
147384-84-7
Molecular formula:
R-C4H6O4 , whereas R=C8-C10-alkyl-(branched, unsaturated)
Molecular weight:
> 228 - < 256
Details on hydrolysis and appearance of transformation product(s):
The transformation product of the registration substance is Tripropenyl succinic acid, CAS No. 147384-84-7. This was verified analytically using a prprepared standard.
Dissipation DT50 of parent compoundopen allclose all
pH:
9
Temp.:
15 °C
DT50:
0.1 h
Type:
(pseudo-)first order (= half-life)
pH:
7
Temp.:
15 °C
DT50:
1.5 h
Type:
(pseudo-)first order (= half-life)
pH:
4
Temp.:
15 °C
DT50:
1.4 h
Type:
(pseudo-)first order (= half-life)
pH:
1.2
Temp.:
37.3 °C
DT50:
0.5 h
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: physiological condition of stomach in mammals
Details on results:
TEST CONDITIONS
- pH, sterility, temperature, and other experimental conditions maintained throughout the study: Yes





Any other information on results incl. tables

Reaction Rate Constants and Half Lives of Genopur ASAat pH 1.2

Temperature

37.3 °C

Reaction rate constant kobs
[1/s]

4.03 x 10-4

Half life T½ [h]

0.478

Half life T½ [min]

28.7

Reaction Rate Constants and Half Lives of Genopur ASAat pH 4

Temperature

14.9 °C

24.6 °C

34.8 °C

Reaction rate constant kobs
[1/s]

1.26 x 10-4

2.45 x 10-4

4.72 x 10-4

Half life T½ [h]

1.53

0.786

0.408

Half life T½ [min]

91.8

47.2

24.5

Reaction Rate Constants and Half Lives of Genopur ASAat pH 7

Temperature

15.0 °C

24.5 °C

34.7 °C

Reaction rate constant kobs
[1/s]

1.42 x 10-4

2.51 x 10-4

7.68 x 10-4

Half life T½ [h]

1.36

0.767

0.251

Half life T½ [min]

81.6

46.0

15.1

Due to the rapid transformation of Genopur ASAat pH 9, 15 °C reaction rate constants and half lives could not be evaluated.
The half live is determined to be 0.1 h ( 6 min) due to the result of the analysis of the sample at test start (0 h).

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Reaction rate constants and half are presented in Table 16 to Table 18. The test item Genopur ASA is rapidly hydrolyzed at all tested conditions, with increasing instability to higher pH and higher temperature. The Half-life at 15 deg C is 0.1h (pH 9), 1.5h (pH 7), 1.4h (pH 4) and 0.5h (pH 1.2, 37 deg Cphysiologically condition in stomach of mammals).
Executive summary:

Hydrolysis as a function of pH was determined according to OECD Guideline No. 111 and Council Regulation (EC) No. 440/2008, Method C.7 for the test item Genopur ASA (batch number: ESD0010823) from 2011-11-30 to 2011-12-05 at Dr.U.Noack-Laboratorien, Sarstedt, Germany.

The definitive test was conducted under the following nominal conditions: Test item concentration 300 mg/L in buffer solutions at pH 1.2 (37 ± 0.5 °C), 4, 7 (15, 25 and 35 ± 0.5 °C) and 9 (15 ± 0.5 °C).

Due to technical reasons the test temperatures could not be set to the exact nominal values. Therefore the real mean test temperature was given as hydrolysis temperature throughout the report. Temperature deviations from the real mean test temperature were significantly below ± 0.5 °C.

Analyses of the test item were performed via HPLC-DAD on a reversed phase column against external standard. The method was validated with satisfactory results in regard to linearity, repeatability of injections, accuracy, precision and specificity for the test item Genopur ASA.

For the pH 1.2 (37.3 °C), 4 (34.8 °C) and 7 (15.0 °C) test conditions eleven, for the pH 4 (14.9 and 24.6 °C) test conditions nine, for the pH 7 (24.5 °C) test condition ten and for pH 7 (34.7 °C) test condition seven replicates were analysed. For the pH 9 (15 °C) test condition only one replicate could be analysed, due to the rapid transformation and no kinetic evaluation could be performed for the this test condition. Buffer solutions as control samples were analysed at test end and no analytical interference with the test item occurred.

Reaction rate constants and half lives were calculated from the analysed concentrations. The test item Genopur ASA is seen to be hydrolytically instable at all tested conditions.

Reaction Rate Constants and Half Lives of Genopur ASA

pH 1.2

pH 4

pH 7

pH 9 1)

37.3 °C

14.9 °C

24.6 °C

34.8 °C

15.0 °C

24.5 °C

34.7 °C

15.0 °C

Reaction rate constant kobs
[1/s]

4.03 x  10-4

1.26 x  10-4

2.45 x  10-4

4.72 x  10-4

1.42 x  10-4

2.51 x  10-4

7.68 x  10-4

n.a.

Half life T½ [h]

0.478

1.53

0.786

0.408

1.36

0.767

0.251

0.1

Half life T½ [min]

28.7

91.8

47.2

24.5

81.6

46.0

15.1

6