1-(4-METHOXYPHENYL)-4-(4-NITROPHENYL)PIPERAZINE

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 March 2016 - 10 June 2016

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)

Deviations:

yes

Remarks:

the t° variation (1.1°C) of the stove (20°C) fell outside the criterion (0.5°C) in the Tier 2 testing. The study integrity was not adversely affected by the deviation.

Qualifier:

according to guideline

Guideline:

EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.2120 (Hydrolysis of Parent and Degradates as a Function of pH at 25°C)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: I15FB2768
- Expiration date of the lot/batch: 01 July 2017 (retest date)
- Purity: 100% (based on base titration assay)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: no data

Radiolabelling:

no

Analytical monitoring:

yes

Details on sampling:

- Sampling intervals for the parent/transformation products:
* Tier 1: at the beginning and after 5 days
* Tier 2: at the beginning and at several sampling points after t=0

- Sampling method:
* Tier 1: The samples taken at t=5 days were cooled to room temperature using running tap water. The samples were diluted in a in a 1:1 (v:v) ratio with acetonitrile and analysed.
* Tier 2: Test samples were prepared in 0.01 M buffer solutions and treated similarly as during the preliminary test. Spiking of samples in buffer pH 7 to be used in the study at 50°C was repeated, as t=0 analysis revealed inaccurate preparation of the test solutions.

- Sampling methods for the volatile compounds, if any: not applicable
- Sampling intervals/times for pH measurements:
* Tier 1: The pH of each of the test solutions (except for the blanks) was determined at each sampling time.
* Tier 2: The pH of each of the test solutions (except for the blanks) was determined at least at the beginning and at the end of the test.
- Sampling intervals/times for sterility check: not applicable
- Sample storage conditions before analysis: not applicable
- Other observation, if any: no data

Buffers:

- pH:
* Tier 1: 4.0, 7.0, and 9.0
* Tier 2: 4.0, 7.0, and 9.0
- Type, final molarity and composition of buffer:
* buffer pH 4, 0.1 M: solution of 16.7% 0.1 M sodium acetate in water and 83.3% 0.1 M acetic acid in water. The buffer contains 0.0009% (w/v) sodium azide.
* buffer pH 7, 0.1 M: solution of 0.1 M potassium di-hydrogenphosphate in water adjusted to pH 7 using 10 N sodium hydroxide. The buffer contains 0.0009% (w/v) sodium azide.
* buffer pH 9, 0.1 M: solution of 0.1 M boric acid in water and 0.1 M potassium chloride in water adjusted to pH 9 using 10 N sodium hydroxide. The buffer contains 0.0009% (w/v) sodium azide.
* buffer pH 4, 0.01 M: solution of 16.7% 0.1 M sodium acetate in water and 83.3% 0.01 M acetic acid in water. The buffer contains 0.0009% (w/v) sodium azide.
* buffer pH 7, 0.01 M: solution of 0.01 M potassium di-hydrogenphosphate in water adjusted to pH 7 using 1 N sodium hydroxide. The buffer contains 0.0009% (w/v) sodium azide.
* buffer pH 9, 0.01 M: solution of 0.01 M boric acid in water and 0.01 M potassium chloride in water adjusted to pH 9 using 1 N sodium hydroxide. The buffer contains 0.0009% (w/v) sodium azide.

Details on test conditions:

TEST SYSTEM
- Type, material and volume of test flasks, other equipment used: sterile vessel
- Sterilisation method: the solutions were filter-sterilised through a 0.2 µm FP 30/0.2 CA-S filter (Whatman, Dassel, Germany) and transferred into a sterile vessel.
- Lighting: in the dark
- Measures taken to avoid photolytic effects: vessels placed in the dark
- Measures to exclude oxygen: Nitrogen was passed through the solutions for about 5 minutes.
- Details on test procedure for unstable compounds: no data
- Details of traps for volatile, if any: not applicable
- If no traps were used, is the test system closed/open: under vacuum
- Is there any indication of the test material adsorbing to the walls of the test apparatus? no

TEST MEDIUM
- Volume used/treatment: 6 mL
- Kind and purity of water: Tap water purified by a Milli-Q water purification system (Millipore, Bedford, MA, USA)
- Preparation of test medium:
* Tier 1: Test item solutions were prepared in 0.1 M buffer solutions at a target concentration of 0.018 mg/L.
* Tier 2: Test samples were prepared in 0.01 M buffer solutions and treated similarly as during the preliminary test. Spiking of samples in buffer pH 7 to be used in the study at 50°C was repeated, as t=0 analysis revealed inaccurate preparation of the test solutions.
- Renewal of test solution: not applicable
- Identity and concentration of co-solvent: not applicable

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Dissolved oxygen: no data

Duration:

5 d

pH:

4

Temp.:

50 °C

Initial conc. measured:

0.018 - 0.019 mg/L

Remarks:

preliminary test

Duration:

5 d

pH:

7

Temp.:

50 °C

Initial conc. measured:

0.018 - 0.019 mg/L

Remarks:

preliminary test

Duration:

5 d

pH:

9

Temp.:

50 °C

Initial conc. measured:

0.016 - 0.017 mg/L

Remarks:

preliminary test

Duration:

722.9 h

pH:

4

Temp.:

20 °C

Initial conc. measured:

16.3 - 16.7 µg/L

Remarks:

main test

Duration:

744.08 h

pH:

4

Temp.:

50 °C

Initial conc. measured:

17 - 17.5 µg/L

Remarks:

main test

Duration:

26.05 h

pH:

4

Temp.:

60 °C

Initial conc. measured:

17.4 - 18.7 µg/L

Remarks:

main test

Duration:

748.22 h

pH:

7

Temp.:

20 °C

Initial conc. measured:

15.2 - 15.6 µg/L

Remarks:

main test

Duration:

745.17 h

pH:

7

Temp.:

40 °C

Initial conc. measured:

15.6 - 16 µg/L

Remarks:

main test

Duration:

765.4 h

pH:

7

Temp.:

50 °C

Initial conc. measured:

17.6 - 18.5 µg/L

Remarks:

main test

Duration:

722.03 h

pH:

9

Temp.:

20 °C

Initial conc. measured:

17.8 - 18.3 µg/L

Remarks:

main test

Duration:

743.57 h

pH:

9

Temp.:

50 °C

Initial conc. measured:

17 - 17.8 µg/L

Remarks:

main test

Duration:

26.82 h

pH:

9

Temp.:

60 °C

Initial conc. measured:

17.1 - 18.9 µg/L

Remarks:

main test

Number of replicates:

2

Positive controls:

no

Negative controls:

no

Statistical methods:

no data

Preliminary study:

During the preliminary hydrolysis tests, a degree of hydrolysis of ≥ 10% was observed at pH 4, pH 7 and pH 9 after 5 days (degree of hydrolysis > 56% for each pH). According to the guideline, the higher Tier test was required to determine the half-life time of the test item.

A response was observed in one of the pretreated samples from the blank mixture for pH 4. This may have been caused by contamination. There was no negative impact on the study since the presence of this response did not hamper clear interpretation of the preliminary test.

Transformation products:

not measured

% Recovery:

92

pH:

4

Temp.:

20 °C

Duration:

0 h

% Recovery:

96

pH:

4

Temp.:

50 °C

Duration:

0 h

% Recovery:

100

pH:

4

Temp.:

60 °C

Duration:

0 h

% Recovery:

86

pH:

7

Temp.:

20 °C

Duration:

0 h

% Recovery:

88

pH:

7

Temp.:

40 °C

Duration:

0 h

% Recovery:

100

pH:

7

Temp.:

50 °C

Duration:

0 h

% Recovery:

100

pH:

9

Temp.:

20 °C

Duration:

0 h

% Recovery:

97

pH:

9

Temp.:

50 °C

Duration:

0 h

% Recovery:

100

pH:

9

Temp.:

60 °C

Duration:

0 h

Key result

pH:

4

Temp.:

20 °C

DT50:

> 10 000 h

Type:

other: no pseudo-first order reaction kinetic

Key result

pH:

7

Temp.:

20 °C

DT50:

> 10 000 h

Type:

other: no pseudo-first order reaction kinetic

Key result

pH:

9

Temp.:

20 °C

DT50:

> 10 000 h

Type:

other: no pseudo-first order reaction kinetic

Key result

pH:

7

Temp.:

40 °C

DT50:

815 h

Type:

other: no pseudo-first order reaction kinetic

Key result

pH:

4

Temp.:

50 °C

DT50:

43 h

Type:

other: no pseudo-first order reaction kinetic

Key result

pH:

7

Temp.:

50 °C

DT50:

8.7 h

Type:

other: no pseudo-first order reaction kinetic

Key result

pH:

9

Temp.:

50 °C

DT50:

58 h

Type:

other: no pseudo-first order reaction kinetic

Key result

pH:

4

Temp.:

60 °C

DT50:

0.83 h

Type:

other: no pseudo-first order reaction kinetic

Key result

pH:

9

Temp.:

60 °C

DT50:

0.69 h

Type:

other: no pseudo-first order reaction kinetic

Other kinetic parameters:

At pH4, pH7 and pH9, for testing of pseudo-first order kinetics the mean logarithms of the relative concentrations between 10% and 90% were plotted against time. Relationships were not linear above room temperature. Reaction rates were not constant during the experiment. Accordingly the Arrhenius equation could not be used to determine the rate constant and half-life time at 25°C. Instead, values for the disappearance time 50 (DT50) were calculated according to the Gustafson and Holden Model or First-Order Multi-Compartment (FOMC) Model using Computer Aided Kinetic Evaluation (CAKE) Version 3.3, developed by Tessella Ltd, Abingdon, Oxfordshire, UK, sponsored by Syngenta. Confidence intervals of the DT50 were back calculated using the 90% Confidence Intervals of the α and β parameter as determined with CAKE. Confidence intervals of the DT50 were not calculated if the DT50 was >10000, as variability and trend of the corresponding data did not allow meaningful extrapolation.

Preliminary test -tier 1

A response was observed in one of the pretreated samples from the blank mixture for pH 4. This may have been caused by contamination. There was no negative impact on the study since the presence of this response did not hamper clear interpretation of the preliminary test.

Main study - Tier 2

pH 4

- A small response was observed in the pretreated samples from the blank mixture. This may have been caused by contamination.

- hydrolysis of the test item at pH 4 and 20°C:

Sampling time [hours]	Analysed concentration [µg/L]	Relative concentration [%]	Logarithm relative concentration
0.00	16.7	101	2.01
0.00	16.3	99	1.99
4.10	16.2	98	1.99
4.10	16.3	99	1.99
22.10	17.0	103	2.01
22.10	16.2	98	1.99
192.32	15.4	93	1.97
192.32	15.2	92	1.96
381.60	15.9	96	1.98
381.60	16.0	97	1.99
407.50	16.2	98	1.99
407.50	16.2	98	1.99
429.80	15.7	95	1.98
429.80	16.2	98	1.99
549.80	16.4	99	2.00
549.80	16.2	98	1.99
722.90	15.5	94	1.97
722.90	15.7	95	1.98

- hydrolysis of the test item at pH 4 and 50°C:

Sampling time [hours]	Analysed concentration [µg/L]	Relative concentration [%]	Logarithm relative concentration
0.00	17.0	99	1.99
0.00	17.5	101	2.01
23.45	9.12	53	1.72
23.45	9.85	57	1.76
25.42	9.45	55	1.74
25.42	9.82	57	1.76
26.28	9.04	52	1.72
26.28	9.56	55	1.74
26.80	9.10	53	1.72
26.80	8.64	50	1.70
49.47	8.88	51	1.71
49.47	8.43	49	1.69
53.63	9.33	54	1.73
53.63	8.78	51	1.71
71.75	7.73	45	1.65
71.75	8.06	47	1.67
191.78	6.50	38	1.58
191.78	6.57	38	1.58
364.88	5.15	30	1.47
364.88	5.32	31	1.49
744.08	3.65	21	1.33
744.08	3.60	21	1.32

- hydrolysis of the test item at pH 4 and 60°C:

Sampling time [hours]	Analysed concentration [µg/L]	Relative concentration [%]	Logarithm relative concentration
0.00	17.4	96	1.98
0.00	18.7	104	2.02
2.00	6.07	34	1.53
2.00	6.21	34	1.54
2.80	5.17	29	1.46
2.80	5.25	29	1.46
3.38	4.73	26	1.42
3.38	5.03	28	1.45
4.62	4.10	23	1.36
4.62	4.23	23	1.37
5.18	3.85	21	1.33
5.18	3.90	22	1.33
5.68	3.67	20	1.31
5.68	3.76	21	1.32
26.05	1.60	8.9	0.95
26.05	1.66	9.2	0.96

pH 7

- No test item was detected in the blank buffer solutions.

- hydrolysis of the test item at pH 7 and 20°C:

Sampling time [hours]	Analysed concentration [µg/L]	Relative concentration [%]	Logarithm relative concentration
0.00	15.2	99	1.99
0.00	15.6	101	2.01
5.75	15.8	103	2.01
5.75	15.9	104	2.02
6.33	15.8	103	2.01
6.33	16.0	104	2.02
23.75	15.5	101	2.00
23.75	15.8	102	2.01
170.13	14.5	94	1.97
170.13	15.0	98	1.99
362.13	14.0	91	1.96
362.13	14.2	92	1.97
527.55	13.8	90	1.95
527.55	14.0	91	1.96
748.22	14.8	96	1.98
748.22	13.8	90	1.95

- hydrolysis of the test item at pH 7 and 40°C:

Sampling time [hours]	Analysed concentration [µg/L]	Relative concentration [%]	Logarithm relative concentration
0.00	15.6	99	2.00
0.00	16.0	101	2.00
1.55	13.4	85	1.93
1.55	13.4	85	1.93
24.70	9.34	59	1.77
24.70	9.39	60	1.77
27.38	9.40	60	1.78
27.38	9.60	61	1.78
28.80	8.95	57	1.75
28.80	9.15	58	1.76
169.72	9.73	62	1.79
169.72	9.83	62	1.79
335.23	8.63	55	1.74
335.23	8.69	55	1.74
555.60	8.53	54	1.73
555.60	8.20	52	1.72
745.17	8.29	53	1.72
745.17	7.84	50	1.70

- hydrolysis of the test item at pH 7 and 50°C:

Sampling time [hours]	Analysed concentration [µg/L]	Relative concentration [%]	Logarithm relative concentration
0.00	18.5	102	2.01
0.00	17.6	98	1.99
0.95	13.0	72	1.86
0.95	13.2	73	1.86
1.55	12.1	67	1.83
1.55	12.6	70	1.84
1.95	11.7	65	1.81
1.95	11.8	65	1.81
2.47	12.1	67	1.83
2.47	12.1	67	1.83
2.98	11.6	64	1.81
2.98	11.7	65	1.81
4.30	11.3	63	1.80
4.30	11.4	63	1.80
18.78	9.09	50	1.70
18.78	8.85	49	1.69
49.03	4.74	26	1.42
49.03	4.77	26	1.42
189.95	4.37	24	1.38
189.95	4.08	23	1.35
355.47	3.25	18	1.26
355.47	3.40	19	1.28
575.83	2.80	16	1.19
575.83	2.78	15	1.19
765.40	2.62	15	1.16
765.40	2.17	12	1.08

pH 9

- A small response was observed in the pretreated samples from the blank mixture. This may have been caused by contamination.

- hydrolysis of the test item at pH 9 and 20°C:

Sampling time [hours]	Analysed concentration [µg/L]	Relative concentration [%]	Logarithm relative concentration
0.00	17.8	99	1.99
0.00	18.3	101	2.01
3.23	17.1	95	1.98
3.23	17.3	96	1.98
21.23	16.8	93	1.97
21.23	16.8	93	1.97
191.45	15.0	83	1.92
191.45	15.7	87	1.94
380.73	14.8	82	1.91
380.73	14.1	78	1.89
407.83	14.7	82	1.91
407.83	14.6	81	1.91
428.93	15.1	84	1.92
428.93	15.3	85	1.93
548.93	13.5	75	1.87
548.93	13.8	76	1.88
722.03	14.4	80	1.90
722.03	14.3	80	1.90

- hydrolysis of the test item at pH 9 and 50°C:

Sampling time [hours]	Analysed concentration [µg/L]	Relative concentration [%]	Logarithm relative concentration
0.00	17.0	98	1.99
0.00	17.8	102	2.01
23.35	10.1	58	1.76
23.35	10.4	60	1.78
24.90	9.93	57	1.76
24.90	9.79	56	1.75
25.77	9.46	54	1.74
25.77	9.75	56	1.75
26.28	9.73	56	1.75
26.28	9.90	57	1.76
50.17	9.04	52	1.72
50.17	9.31	54	1.73
53.12	9.06	52	1.72
53.12	9.30	53	1.73
71.23	8.60	49	1.69
71.23	9.51	55	1.74
191.27	6.99	40	1.60
191.27	7.26	42	1.62
364.37	6.45	37	1.57
364.37	6.24	36	1.55
743.57	5.08	29	1.47
743.57	5.21	30	1.48

-hydrolysis of the test item at pH 4 and 60°C:

Sampling time [hours]	Analysed concentration [µg/L]	Relative concentration [%]	Logarithm relative concentration
0.00	17.1	95	1.98
0.00	18.9	105	2.02
1.55	6.45	36	1.55
1.55	6.37	35	1.55
2.35	5.32	30	1.47
2.35	5.41	30	1.48
2.93	5.08	28	1.45
2.93	4.96	28	1.44
4.17	4.17	23	1.36
4.17	4.03	22	1.35
4.73	3.92	22	1.34
4.73	3.95	22	1.34
5.23	3.78	21	1.32
5.23	3.64	20	1.31
26.82	1.69	9.4	0.97
26.82	1.57	8.7	0.94

Validity criteria fulfilled:

yes

Remarks:

The mean recoveries of the buffer solutions fell within the criterion range of 90-110%. It demonstrated that the analytical method was adequate to support the hydrolysis study on the test item.

Conclusions:

During the prliminary test, a degree of hydrolysis of ≥ 10 % was observed at pH 4, pH 7 and pH 9 after 5 days. According to the guideline, the higher Tier test was required to determine the half-life time of the test item.
At pH 4, pH 7 and pH 9 no pseudo-first order reaction kinetic was observed above room temperature. Reaction rates were not constant during the experiment. Accordingly the Arrhenius equation could not be used to determine the rate constant and half-life time at 25°C. Instead, values for DT50 were calculated according to the Gustafson and Holden Model or First-Order Multi-Compartment (FOMC) Model using Computer Aided Kinetic Evaluation (CAKE). DT50 (90% CL) (hrs) were:
At 20°C: for pH 4, 7 and 9: > 10,000
At 40°C for pH 4, 7 and 9: -, 815 (0 to 38950) and -, respectively
At 50°C: for pH 4, 7 and 9: 43 (14 to 107), 8.7 (3.6-18) and 58 (19 to 149) respectively
At 60°C: for pH 4, 7 and 9: 0.83 (0.34 to 1.7), -, 0.69 (0.23 to 1.6) respectively

Description of key information

One study (Reingruber, 2016) was performed according to OECD guideline 111 and regarded as a key study (Klimisch score of 1). A higher tier test was required to determine the half-life of the test item, since a degree of hydrolysis of ≥ 10 % was observed at pH 4, pH 7 and pH 9 after 5 days during the preliminary test. At pH 4, pH 7 and pH 9 no pseudo-first order reaction kinetic was observed above room temperature. Reaction rates were not constant during the experiment. Accordingly the Arrhenius equation could not be used to determine the rate constant and half-life time at 25°C. Instead, values for DT50 were calculated according to the Gustafson and Holden Model or First-Order Multi-Compartment (FOMC) Model using Computer Aided Kinetic Evaluation (CAKE) Version 3.3, developed by Tessella Ltd, Abingdon, Oxfordshire, UK, sponsored by Syngenta. Confidence intervals of the DT50 were back calculated using the 90% Confidence Intervals of theαandβparameter as determined with CAKE.

At 20°C for pH 4, 7 and 9, DT50 was > 10,000.