Diphosphoric acid, compd. with piperazine (1:1)

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 March 2004 to 15 April 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Version / remarks:

May 1981

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

no

Analytical monitoring:

yes

Details on sampling:

The concentration of T-1063FM in the study samples was determined in duplicate at t = 0 and t = 5 days for each pH value.
The sample vials (10 ml) were filled with hydrolysis solutions with a minimum of headspace and closed with a teflon-coated crimpcap closure.

Buffers:

-pH 4: 0.1 mol/L monopotassium citrate + 0.1 mol/L sodium hydroxide:
Two solutions were prepared, containing 11.48 g monopotassium citrate in 500 ml Milli-Q water and 4.08 g sodium hydroxide in 1L Milli-Q water, respectively. To 500 ml citrate solution, 90 ml 0.1 ml 0.1 mol/L NaOH was added. The resulting solution was made up to 1L with Milli-Q water. The pH of this solution was 4.07.
-pH 7: 0.1 mol/l monopotassium phosphate + 0.1 mol/l sodium hydroxide:
Two solutions were prepared, containing 6.84 g monopotassium phosphate in 500 ml Milli-Q water and 4.08 g sodium hydroxide in 1L Milli-Q water, respectively. To 500 ml phosphate solution, 295 ml 0.1 ml 0.1 mol/l NaOH was added. The resulting solution was made up to 1L with Milli-Q water. The pH of this solution was 7.04.
-pH 9: 0.1 mol/l boric acid in 0.1 mol/l potassium chloride + 0.1 mol/l sodium hydroxide:
Two solutions were prepared, containing 3.77 g KCL and 3.08 g H3B03 in 500 ml Milli-Q water and 4.08 g sodium hydroxide in 1L Milli-Q water, respectively. To 500 ml boric acid in KCl solution, 215 ml 0.1 ml 0.1 mol/l NaOH was added. The resulting solution was made up to 1L with Milli-Q water. The pH of this solution was 9.03.

Details on test conditions:

The procedure of this study is based on the draft update OECD guideline for testing of chemicals No. 111 (2002).

TEST SYSTEM
- Type, material and volume of test flasks, other equipment used: The test was carried out at 50 ± 1 °C using a thermostatically controlled enclosure (stove). Sample vials (glass tubes) were filled with approximately 10 ml of hydrolysis solution and closed with a teflon coated crimpcap.
- Sterilisation method: In order to exclude the occurrence of biodegradation of the test substance, glassware was sterilised at 120 °C for 30 minutes before use. All hydrolysis solutions were sterilised by filtration over a 0.22 μm filter.
- Lighting: dark, to avoid photolytic interference
- Measures to exclude oxygen: bubbling nitrogen (ca. 5 min) through the hydrolysis solution
No sterility test was carried out (as the test substances was considered hydrolytically stable).

PREPARATION STUDY SAMPLES
Study samples (hydrolysis solutions) containing approximately 400 mg T -1063FM/l were prepared as follows:
- hydrolysis solution pH 4: 39.2 mg T-1063FM was dissolved in 100 ml of buffer solution of pH 4
- hydrolysis solution pH 7: 39.5 mg T-1063FM was dissolved in 100 ml of buffer solution of pH 7
- hydrolysis solution pH 9: 40.0 mg T-1063FM was dissolved in 100 ml of buffer solution of pH 9

Duration:

5 d

Temp.:

50 °C

Initial conc. measured:

39.2 - 40 mg/L

Positive controls:

not specified

Negative controls:

not specified

Transformation products:

no

% Recovery:

100 - 103

pH:

4

Temp.:

50 °C

Duration:

5 d

% Recovery:

105 - 108

pH:

7

Temp.:

50 °C

Duration:

5 d

% Recovery:

99.8 - 101

pH:

9

Temp.:

50 °C

Duration:

5 d

Key result

pH:

4

Temp.:

50 °C

Remarks on result:

other: Analytical problems were encountered with the determinations at pH 4.

Key result

pH:

7

Temp.:

25 °C

DT50:

> 1 yr

Type:

(pseudo-)first order (= half-life)

Key result

pH:

9

Temp.:

25 °C

DT50:

> 1 yr

Type:

(pseudo-)first order (= half-life)

Details on results:

During the hydrolysis tests the pH of the incubated samples at all pH values remained within ±0.1 of the initial pH values.
The percentage hydrolysis after 5 days was 31.5 % for hydrolysis solutions at pH 4. This result was obtained on 6 April 2004 (t = 5 days) using the calibration graph for calculations (for details see attachment “T-1063FM - results hydrolysis.pdf”).
During the analysis on 6 April 2004, first all calibration solutions prepared in (diluted) buffer pH 4 were injected (2- 12 mg T -1063FM/l). The correlation coefficient (r) of the calibration graph was 0.997. Next the t = 5, pH 4 samples were injected. The mean T-1063FM concentration, determined as the piperazine ion, dropped to 68.5 % of its initial value on t = 0 days. However, the peak area for the next following calibration solution (10.09 mg T-1063FM/l diluted buffer pH 4, which is similar to the t = 0 concentration of the hydrolysis solutions) was only 78 % of its expected value. This indicated a distinct loss of sensitivity of the LC/MS system. If the t = 5, pH 4 value was calculated with help of the latter calibration standard the hydrolysis after 5 days was 12 %. If standard bracketing was used, the calculated percentage hydrolysis after 5 days was 24 %.
To obtain certainty, aliquots of the diluted hydrolysis solutions from t = 5 prepared on 6 April 2004 and stored at ambient temperature were reanalyzed on 8 April 2004. The peak area of these solutions was compared with the peak area (n = 2) of a calibration solution (10.09 mg T-1063FM/l diluted buffer pH 4) that was prepared on 6 April 2004 and stored similarly. The mean concentration of T-1063FM in the hydrolysis solutions stored for 5 days at 50 °C in a thermostatic stove and subsequently diluted 40 times with water and stored for two days at ambient temperature was 387 mg T-1063FM/l. This indicates a maximal hydrolysis of 7 % for the piperazine ion at pH 4 after 5 days.
With a mean value of 469 mg T-1063FM/l, while the initial value at t = 0 was only 412 mg test substance/l, certainly an overestimation occurred for the t = 5, pH 7 samples using the calibration graph prepared in buffer pH 7. Next following to these samples the t = 5 days QC samples were injected. The mean recovery of T-1063FM from these freshly prepared samples that were treated like a sample, was 119 % if the result from the table was corrected for this recovery the mean T-1063FM concentration in the t = 5, pH 7 samples was 394 mg/l, which results in a hydrolysis of the piperazine ion of T-1063FM of 1.7 %.
The percentage hydrolysis after 5 days was 1.5 %for hydrolysis solutions at pH 9 (for details see attachment “T-1063FM - results hydrolysis.pdf”).
The results for the additional QC-samples for the analysis of spare samples that were stored for 14 days at 50 °C in the thermostatic stove were very satisfactory. The percentage hydrolysis after 14 days was 12 % for hydrolysis solutions at pH 4. This result was obtained using the calibration graph for calculations.
There was no hydrolysis at pH 7 and the percentage hydrolysis after 14 days was 5.3 % for hydrolysis solutions at pH 9 (for details see attachment “T-1063FM - results hydrolysis.pdf”).
The t = 0 samples contained exactly 392, 395 and 400 mg T-1063FM /L buffer of pH 4, pH 7 and pH 9, respectively (see section 3.3 of this report). Using these prepared concentrations for calculations instead of the measured concentrations, the percentage hydrolysis after 14 days was 6.6 %, 0 % ( + 4%) and 2.5 % for pH 4, pH 7 and pH 9, respectively.
On the basis of the t = 14, pH 4 result an assessment was made of the percentage hydrolysis after 5 days. Assuming a pseudo first order behavior hydrolysis an assessment of the percentage hydrolysis at t = 5, pH 4 was made. This resulted in a value well below 10% .
Since the decline of T-1063FM in all buffer solutions was less than 10 % after incubation at 50 °C for 5 days, the piperazine ion of the test substance T-1063FM was considered to be hydrolytically stable at pH 4, pH 7 and pH 9 (t 0.5 25°C > 1 year) and no additional testing is required. There was no need to perform a sterility test.

Validity criteria fulfilled:

yes

Conclusions:

Since in the buffer solutions in question less than 10% of the hydrolysis reaction was observed after 5 days at 50 °C, the piperazine ion of T-1063FM was considered hydrolytically stable at pH 7 and pH 9 (t0.5 25°c > 1 year). Analytical problems were encountered with the determinations at pH 4. Ultimately, the conclusion was drawn that the piperazine ion of T-1063FM was hydrolytically stable at pH 4. No additional testing is required.

Description of key information

dissipation half-life > 1 year at 25°C (OECD guideline 111)

Additional information

half-life for hydrolysis > 1 year at 25 °C