Dimethyl (p-methoxybenzylidene)malonate

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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)

Version / remarks:

/ EC No. 440/2008 Method C.7

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

no

Analytical monitoring:

yes

Details on sampling:

- Sampling intervals for the parent products:
For the preliminary test, samples were taken at test start (0 h) and test end (120 h).
For the advanced test, samples were taken at test start (0 h) and at a minimum of 7 spaced points for pH 4, pH 7 and 9, normally between 10 and 90 % of hydrolysis, at each test temperature. The bath temperature was checked automatically once in an hour and at least once per day manually.

Buffers:

Test system
Preliminary test: Sterile buffer solutions with pH values 4, 7 and 9 (50 °C).
Advanced test: Sterile buffer solution with pH values 4, 7 and 9 (20, 30 and 50 °C).

Buffers were prepared from chemicals with analytical grade or better quality following the composition guidance given in “KÜSTER-THIEL, Rechentafeln für die Chemische Analytik” and the OECD Guideline No. 111, respectively, by direct weighing of the buffer components. Buffers were purged with nitrogen for 5 min and then the pH was checked to a precision of at least 0.1 at the test temperatures. Buffers were sterilised by filtration through 0.2 µm.

Buffer solution pH 4 0.18 g of NaOH and 5.7555 g of mono potassium citrate were dissolved in 500 mL double distilled water.

Buffer solution pH 7 0.7358 g of NaOH and 4.3012 g of KH2PO4 were dissolved in 500 mL double distilled water.

Buffer solution pH 9 0.426 g NaOH, 1.8638 g KCl and 1.5458 g H3BO3 were dissolved in 500 mL double distilled water.

Reason for the selection These buffer systems were selected according to the guidelines. The buffer systems were suitable for their pH values.

Details
Chemical Origin Batch number Purity [%]
NaOH VWR 14G150013 ≥99.7
H3BO3 VWR 13J180014 100.1
KCl ROTH 475237252 ≥ 99.5
KH2PO4 VWR 14K030026 99.8
KH2 Citrate FLUKA BCBH3957V ≥ 98
Double distilled water ROTH 335233316 conductivity: ≤ 2.0 μS/cm
415234632 conductivity: ≤ 2.0 μS/cm
086239961 conductivity: ≤ 2.0 μS/cm

Details on test conditions:

TEST SYSTEM
- Type, material and volume of test flasks, other equipment used: HPLC vials, volume 4 mL
- Measures taken to avoid photolytic effects:Photolytic effects were avoided by using opaque water baths.
- Measures to exclude oxygen: None
- 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: The test item was dissolved in acetonitrile. 1.98 mL of buffer solutions were spiked with 0.02 mL test item solution at 1000 mg/L in acetonitrile to a test item concentration of 10 mg/L in the test containers. After the vials were sealed they were transferred into the thermostat. The time between test item application and transfer to thermostat as well as analysis of the start valuesdid not exceed 30 min for all test conditions.

- Incubation:
preliminary test: 2016-03-24 to 2016-03-29 (pH 4, 7 and 9; 50 °C)
advanced test: 2016-05-18 to 2016-06-17 (pH 4 20 and 30 °C; pH 7 20 and 30 °C)
2016-05-18 to 2016-06-08 (pH 4 50; pH 9 20 °C)
2016-05-18 to 2016-05-27 (pH 7 50 °C)
2016-05-18 to 2016-05-23 (pH 9 30 °C)
2016-05-30 to 2016-05-31 (pH 9 50 °C)

- Temperatures:
Preliminary test: 50 ± 0.5 °C
Advanced test: 20, 30 and 50 ± 0.5 °C

- Sterility:
The sterility of the test solutions was checked by colony forming units (CFU)-determination with Water Plate Count Agar from additional samples at test end by incubation at 36 ± 1 °C for 48 hours and at 22 ± 1 °C for 72 hours.

- Renewal of test solution: None

Duration:

720 h

pH:

4

Temp.:

20 °C

Initial conc. measured:

10.4 mg/L

Duration:

719 h

pH:

4

Temp.:

30 °C

Initial conc. measured:

10.4 mg/L

Duration:

509 h

pH:

4

Temp.:

50 °C

Initial conc. measured:

10.4 mg/L

Duration:

720 h

pH:

7

Temp.:

20 °C

Initial conc. measured:

10.4 mg/L

Duration:

719 h

pH:

7

Temp.:

30 °C

Initial conc. measured:

10.4 mg/L

Duration:

216 h

pH:

7

Temp.:

50 °C

Initial conc. measured:

10.4 mg/L

Duration:

510 h

pH:

9

Temp.:

20 °C

Initial conc. measured:

10.1 mg/L

Duration:

128 h

pH:

9

Temp.:

30 °C

Initial conc. measured:

10.1 mg/L

Duration:

24.5 h

pH:

9

Temp.:

50 °C

Initial conc. measured:

10.5 mg/L

Number of replicates:

One sampling at test start and a minimum of 7 spaced points (actual 10 for pH 4 20 °C and 30 °C, pH 7 20, 30 and 50 °C, pH 9 20 and 30 °C as well as 9 for pH 4 50 °C and pH 9 50 °C).

Positive controls:

no

Negative controls:

yes

Remarks:

buffer solutions (pH 4, 7 and 9)

Preliminary study:

In the preliminary test more than 10% of the test item degraded after 120 hours at pH 4, 7 and 9.

Degradation [%] at 50 °C after 120 Hours
 
Hydrolysis Time Degradation [%]
[hours] pH 4 pH 7 pH 9
120 53.7 73.3 100

Test performance:

CHRONOLOGICAL TEST DESCRIPTION
- Method validation
- Preparation of the test solutions (experimental starting)
- Thermostatisation of the test solutions
- Analysis of samples
- Calculation of reaction rate constants and half-lives for the test item

Transformation products:

not specified

Key result

pH:

4

Temp.:

25 °C

Hydrolysis rate constant:

0 s-1

DT50:

43.2 d

Type:

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

Key result

pH:

7

Temp.:

25 °C

Hydrolysis rate constant:

0 s-1

DT50:

31 d

Type:

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

Key result

pH:

9

Temp.:

25 °C

Hydrolysis rate constant:

0 s-1

DT50:

3.15 d

Type:

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

Details on results:

TEST CONDITIONS
- pH, sterility, temperature, and other experimental conditions maintained throughout the study: Yes

Hydrolysis Results

Hydrolysis Results forthe Test Item at pH 4 and 20 °C

Hydrolysis Time [h]	Concentration [mg/L]	Ln Concentration
0.00	10.4	2.35
4.72	10.0	2.31
25.1	9.79	2.28
47.7	9.81	2.28
127	9.58	2.26
176	9.43	2.24
217	9.24	2.22
337	8.84	2.18
456	8.32	2.12
552	8.02	2.08
720	7.62	2.03

Hydrolysis Results forthe Test Item at pH 4 and 30 °C

Hydrolysis Time [h]	Concentration [mg/L]	Ln Concentration
0.00	10.4	2.35
4.48	9.99	2.30
24.9	9.62	2.26
47.4	9.59	2.26
75.9	9.60	2.26
126	8.77	2.17
217	7.92	2.07
337	7.03	1.95
456	6.10	1.81
552	5.56	1.72
719	4.63	1.53

Hydrolysis Results forthe Test Item at pH 4 and 50 °C

Hydrolysis Time [h]	Concentration [mg/L]	Ln Concentration
0.00	10.4	2.35
4.25	9.85	2.29
24.6	8.59	2.15
29.7	8.37	2.12
47.2	7.55	2.02
52.4	7.28	1.99
75.6	6.23	1.83
126	4.57	1.52
217	2.65	0.975
337	1.26	0.234
509	0.434	-0.835

Last value < LOQ, not included in calculation

Hydrolysis Results forthe Test Item at pH 7 and 20 °C

Hydrolysis Time [h]	Concentration [mg/L]	Ln Concentration
0.00	10.4	2.34
4.88	9.94	2.30
25.0	9.63	2.26
47.7	9.72	2.27
126	9.23	2.22
176	9.27	2.23
217	8.89	2.18
337	8.62	2.15
457	7.76	2.05
552	7.34	1.99
720	6.84	1.92

Hydrolysis Results forthe Test Item at pH 7 and 30 °C

Hydrolysis Time [h]	Concentration [mg/L]	Ln Concentration
0.00	10.4	2.34
4.65	9.91	2.29
24.7	9.62	2.26
47.5	9.25	2.22
76.2	8.93	2.19
126	8.44	2.13
217	7.07	1.96
337	6.17	1.82
456	4.76	1.56
552	4.17	1.43
719	3.24	1.18

Hydrolysis Results forthe Test Item at pH 7 and 50 °C

Hydrolysis Time [h]	Concentration [mg/L]	Ln Concentration
0.00	10.4	2.34
4.45	9.69	2.27
24.4	7.79	2.05
30.2	7.29	1.99
47.2	6.12	1.81
52.9	5.82	1.76
76.0	4.47	1.50
126	2.59	0.952
148	2.11	0.748
176	1.60	0.471
216	1.03	0.0263

Hydrolysis Results forthe Test Item at pH 9 and 20 °C 

Hydrolysis Time [h]	Concentration [mg/L]	Ln Concentration
0.00	10.1	2.31
5.02	9.78	2.28
6.85	9.56	2.26
24.9	8.85	2.18
31.6	8.58	2.15
47.7	7.80	2.05
76.6	6.94	1.94
126	5.43	1.69
217	3.66	1.30
337	2.08	0.731
510	0.825	-0.193

 

Hydrolysis Results forthe Test Item at pH 9 and 30 °C 

Hydrolysis Time [h]	Concentration [mg/L]	Ln Concentration
0.00	10.1	2.31
4.82	9.06	2.20
6.68	8.99	2.20
24.7	6.62	1.89
29.0	5.87	1.77
31.4	5.89	1.77
47.5	4.38	1.48
50.7	4.26	1.45
54.2	4.02	1.39
76.4	2.75	1.01
127	1.06	0.0588

Hydrolysis Results forthe Test Item at pH 9 and 50 °C

Hydrolysis Time [h]	Concentration [mg/L]	Ln Concentration
0.00	10.5	2.35
0.483	9.75	2.28
0.833	9.19	2.22
1.32	8.72	2.17
1.97	7.60	2.03
2.98	6.68	1.90
4.25	5.18	1.64
6.23	4.13	1.42
8.45	2.93	1.08
10.6	2.20	0.787
24.5	0.222	-1.50

 

Last value < LOQ, not included in calculation

 Kinetic Considerations

 

For all test conditions, the ln concentration vs. time plots have regression graphs with slopes significantly non zero. Therefore first order reaction kinetics was applied for data computation. A confirmation of pseudo first order reaction kinetics with coefficients of determination > 0.8 could be obtained for all test conditions.

 

Reaction Rate Constants and Half-Lives at pH 4

	pH 4
	20 °C	30 °C	50 °C
Slope of regression graph	-4.04 · 10^-4	-1.10 · 10^-3	-6.20 · 10^-3
Correlation factor [r^2]	0.980	0.996	1.000
Reaction rate constant kobs[1/s]	1.12 · 10^-7	3.05 · 10^-7	1.72 · 10^-6
Half-lifeT½[h]	1715	632	112
Confidence interval of half-lifeT½[h]	1559 to 1910	610 to 655	110 to 114
Half-lifeT½[d]	71.5	26.3	4.66
Confidence interval of half-lifeT½[d]	65.0 to 79.6	25.4 to 27.3	4.58 to 4.75

Reaction Rate Constants and Half-Lives at pH 7 

	pH 7
	20 °C	30 °C	50 °C
Slope of regression graph	-5.38 · 10^-4	-1.58 · 10^-3	-1.06 · 10^-2
Correlation factor [r^2]	0.976	0.997	1.000
Reaction rate constant kobs[1/s]	1.50 · 10^-7	4.42 · 10^-7	2.94 · 10^-6
Half-lifeT½[h]	1287	436	65.4
Confidence interval of half-lifeT½[h]	1176 to 1428	423 to 449	64.4 to 66.4
Half-lifeT½[d]	53.6	18.2	2.73
Confidence interval of half-lifeT½[d]	49.0 to 59.5	17.6 to 18.7	2.68 to 2.77

Reaction Rate Constants and Half-Lives at pH 9 

	pH 9
	20 °C	30 °C	50 °C
Slope of regression graph	-4.81 · 10^-3	-1.73 · 10^-2	-1.49 · 10^-1
Correlation factor [r^2]	0.999	0.999	0.998
Reaction rate constant kobs[1/s]	1.34 · 10^-6	4.81 · 10^-6	4.14 · 10^-5
Half-lifeT½[h]	144	40.0	4.66
Confidence interval of half-lifeT½[h]	140 to 148	39.1 to 40.9	4.52 to 4.80
Half-lifeT½[d]	6.01	1.67	0.194
Confidence interval of half-lifeT½[d]	5.83 to 6.17	1.63 to 1.70	0.188 to 0.200

pH-Value of the Test Systems

measured before start of hydrolysis

Test	Preliminary	Advanced
Intended pH-value	Measured pH‑value at 50 °C	Measured pH‑value at 20 °C	Measured pH‑value at 30 °C	Measured pH‑value at 50 °C
4.0 ± 0.1	3.946	4.044	4.047	4.046
7.0 ± 0.1	6.977	7.016	7.021	7.025
9.0 ± 0.1	8.926	9.063	9.055	9.055

 n.a.: not applicable, only tested at 50 °C in preliminary testing

Temperature of the Test System

measured every hour

Test	Intended Temperature	Measured Temperature
		Mean ± SD	Min. / Max.
Preliminary	50.0 ± 0.5	50.1 ± 0.2	49.8 / 51.5^1
Advanced pH 4	20.0 ± 0.5	20.0 ± 0.03	20.0 / 20.1
	30.0 ± 0.5	30.0 ± 0.03	29.9 / 30.0
	50.0 ± 0.5	50.0 ± 0.06	49.8 / 50.2
Advanced pH 7	20.0 ± 0.5	20.0 ± 0.03	20.0 / 20.1
	30.0 ± 0.5	30.0 ± 0.03	29.9 / 30.0
	50.0 ± 0.5	50.0 ± 0.1	49.8 / 50.2
Advanced pH 9	20.0 ± 0.5	20.0 ± 0.03	20.0 / 20.1
	30.0 ± 0.5	30.0 ± 0.03	29.9 / 30.0
	50.0 ± 0.5	50.0 ± 0.06	50.0 / 50.1

                  SD = Standard deviation

                   1    = temperature was > 50.5 °C for 6 h (not contigued); due to the performance of the advanced test, the deviation was accepted

              

Validity criteria fulfilled:

yes

Conclusions:

The test item hydrolysed slowly (half-life t1/2 > 30 d) for pH 4 and 7 at 20 °C, and a moderately (2.4 h ≤ t1/2 ≤ 30 d) for the remaining test conditions (pH 4 at 30 and 50 °C, pH 7 at 30 and 50 °C as well as for pH 9 at 20, 30 and 50 °C).

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 from 2016 -03 -24 to 2016 -06 -30 at Noack Laboratorien GmbH, Sarstedt, Germany.

 

Analyses of the test item were performed via LC‑DAD on a reversed phase analytical column using the test item as external standard. The analytical method was validated with satisfactory results with regard to linearity, accuracy, precision and specificity.

 

The preliminary test was conducted with a test item concentration of 10 mg/Lin buffer solutions at pH 4, 7 and 9 and 50 °C. For each pH‑value (pH 4, 7 and 9) the advanced test was performed, as a significant reduction (> 10 %) of the test item concentration was observed in the preliminary test.

 

The advanced test was conducted with a test item concentration of 10 mg/Lin buffer solution of pH 4, 7 and 9 containing 1% acetonitrile at temperatures of 20, 30 and 50 °C (pH 4, 7 and 9), respectively. Samples were taken at test start (0 h) and at 9 to 10 spaced points until test end. Pure test systems, buffer solutions containing 1% acetonitrile, were analysed at test start and test end and there was no analytical interference with the test item.

 

Reaction rate constants and half-lives were calculated from the analysed samples based on a first order reaction kinetics model and are presented below.

 

The test item showed a long hydrolysis half-life (t_1/2> 30 d) for pH 4 and 7 at 20 °C, and a moderate hydrolysis half-life (2.4 h ≤ t_1/2≤ 30 d) for the remaining test conditions (pH 4 at 30 and 50 °C, pH 7 at 30 and 50 °C as well as for pH 9 at 20, 30 and 50 °C), with the shorter half-lives at alkaline conditions.

Reaction Rate Constants and Half-Lives at pH4

	pH 4
	20 °C	30 °C	50 °C	25 °C^1)
Reaction rate constant kobs[1/s]	1.12 · 10^-7	3.05 · 10^-7	1.72 · 10^-6	1.86 · 10^-7
Half-lifeT½[h]	1715	632	112	1037
Half-lifeT½[d]	71.5	26.3	4.66	43.2
Number of data points	11	11	10	EA= 7.16 · 10^4J * mol^-1
Slope of regression graph	significantly non zero

1)     = values calculated via Arrhenius equation

E_A    = activation energy

Reaction Rate Constants and Half-Lives at pH7

	pH 7
	20 °C	30 °C	50 °C	25 °C^1)
Reaction rate constant kobs[1/s]	1.50 · 10^-7	4.42 · 10^-7	2.94 · 10^-6	2.59 · 10^-7
Half-lifeT½[h]	1287	436	65.4	743
Half-lifeT½[d]	53.6	18.2	2.73	31.0
Number of data points	11	11	11	EA= 7.80 · 10^4J * mol^-1
Slope of regression graph	significantly non zero

1)     = values calculated via Arrhenius equation

E_A    = activation energy

Reaction Rate Constants and Half-Lives at pH9 

	pH 9
	20 °C	30 °C	50 °C	25 °C^1)
Reaction rate constant kobs[1/s]	1.34 · 10^-6	4.81 · 10^-6	4.14 · 10^-6	2.55 · 10^-6
Half-lifeT½[h]	144	40.0	4.66	75.6
Half-lifeT½[d]	6.01	1.67	0.194	3.15
Number of data points	11	11	10	EA= 8.98 · 10^4J * mol^-1
Slope of regression graph	significantly non zero

1)     = values calculated via Arrhenius equation

E_A    = activation energy

Description of key information

The submission substance hydrolysed slowly (half-life t1/2 > 30 d) for pH 4 and 7 at 20 °C, and moderately (2.4 h ≤ t1/2 ≤ 30 d) for the remaining test conditions (pH 4 at 30 and 50 °C, pH 7 at 30 and 50 °C as well as for pH 9 at 20, 30 and 50 °C) (OECD 111, 2016)

Key value for exposure assessment: 102 days at 12°C, pH 7 (calculated acc. to ECHA guidance R.16)

Key value for chemical safety assessment

Half-life for hydrolysis:

102 d

at the temperature of:

12 °C

Additional information

In a reliable key study, hydrolysis as a function of pH was determined according to OECD 111 and in compliance with GLP (RL1). As a significant reduction (> 10 %) was observed in the preliminary test, the advanced test was conducted in buffer solution of pH 4, 7 and 9 containing 1% acetonitrile at temperatures of 20, 30 and 50 °C (pH 4, 7 and 9), respectively. Samples were taken at test start (0 h) and at 9 to 10 spaced points until test end. Hydrolysis reaction rate constants and half-lives of the submission substance were calculated from analysed samples based on a first order reaction kinetics model. Half-lives were shorter in alkaline conditions (higher pH). The test was considered reliable and adequate for the environmental fate assessment of the submission substance.