Phthalaldehyde

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-05-30 to 2019-02-13

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)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

no

Analytical monitoring:

yes

Details on sampling:

- Sampling intervals for the parent/transformation products: Samples were taken and measured after 0, 5, 22, 28, 93, 100 and 116 h in the preliminary test. For details see Table 1 below.
- Sampling method: not reported
- Sampling methods for the volatile compounds, if any: n/a
- Sampling intervals/times for sterility check: not reported
- Sample storage conditions before analysis: no storage, directly measured (within a few minutes)
- Other observation, if any (e.g.: precipitation, color change etc.): The colour of the solutions at pH 9 changed to dark yellow.

Buffers:

- Preparation of the buffer solutions
The buffer solutions used for pH 4, 7 and 9 were:
pH 4: acetic acid (0.05 mol/L) / sodium acetate (0.01 mol/L) 1:1 v/v
pH 7: solution A: potassium dihydrogen phosphate (0.07 mol/L), solution B: di-potassium hydrogen phosphate (0.07 mol/L), buffer solution: 306 mL solution B + 194 mL solution A
pH 9: ammonia 25 % (14 mol/L) / ammonium chloride (0.05 mol/L) 1:1 v/v (adjusted to the pH with HCl (1 mol/L))
Alternative buffer solutions with pH 7 and 9: For the alternative buffer solutions at pH 7 and 9 one stock buffer solution was prepared with 0.1 mol/L KCl and 0.1 mol/L H3BO3. For the two pH values, 0.1 M NaOH and 1 M HCl were added, respectively:
pH 7: 125 mL stock buffer solution + 6.5 mL NaOH, filled up to 250 mL with double distilled water (adjusted to the pH with HCl (1 mol/L))
pH 9: 125 mL stock buffer solution + 53.3 mL NaOH, filled up to 250 mL with double distilled water
The pH value of each buffer solution was checked with a calibrated pH meter to a precision of 0.1.

Details on test conditions:

TEST SYSTEM
The test item is dissolved in sterile aqueous buffer solutions of different pH values (pH 4, 7 and 9) and incubated in the dark under controlled laboratory conditions (at constant temperatures).
After appropriate time intervals, the buffer solutions are analyzed for the test item.
This test method is designed as a multi-level concept. The individual steps of the test are based on the results from the previous stage.
Since the hydrolysis behavior of the test item is initially unknown, a preliminary test (Tier 1) is conducted at a temperature of 50 °C. Kinetic tests on a higher level may be carried out at least at three temperatures (which include the test at 50 °C) if the test item – as determined in the tests at Tier 1 – behaves unstable. A temperature range of 10–70 °C is recommended (where possible at least one of the temperatures should be below 25 °C).
The preliminary test (Tier 1) is carried out over a period of 5 days, the test at a higher level (Tier 2) shall be carried out until six data points between 10 and 90 % hydrolysis of the test item are collected or the time exceeds 30 days (whichever is reached first).

TEST DESCRIPTION
The study was performed in measuring flasks (dried at 200 °C for 16 hours) under dark and sterile conditions (under inert atmosphere) in temperature-controlled water baths. The test item was dissolved in acetonitrile (stock solution with approx. 10–13 g/L) and added to the different buffer solutions (pH 4, pH 7, pH 9) under nitrogen atmosphere.
For the determination of the concentrations of the test item in the different measuring flasks at different time intervals, samples were taken under nitrogen atmosphere and measured undiluted by means of HPLC with UV-detection.
Whenever samples were measured also standard solutions freshly prepared from the test item in acetonitrile and blank buffer solutions were measured for verification of the analytical method.

Duration:

116 h

pH:

4

Temp.:

50 °C

Initial conc. measured:

ca. 10.3 mg/L

Remarks:

nominal concentration of 103.3 mg/L; initial concentration given as mean value of two measurements after 0.1 h

Duration:

116 h

pH:

7

Temp.:

50 °C

Initial conc. measured:

ca. 17.06 mg/L

Remarks:

nominal concentration of 103.3 mg/L; initial concenctration given as mean value of two measurements after 0.1 h

Duration:

3.8 h

pH:

9

Temp.:

50 °C

Initial conc. measured:

ca. 23.8 mg/L

Remarks:

nominal concentration of 103.3 mg/L; initial concentration given as mean value of two measurements after 0.1 h; after 3.8 h no test item could be measured in the dilutions and the test was stopped.

Number of replicates:

2 replicates

Positive controls:

no

Negative controls:

no

Preliminary study:

Five preliminary studies were conducted due to difficulties with the substance. Details are reported below in the section "Details on results".

Transformation products:

not measured

Details on hydrolysis and appearance of transformation product(s):

- pH 4: At the first measurement only 10 % of the nominal concentration were detected via HPLC. The chromatogram shows, that the substance reacted with water. The remaining 10 % of the substance are stable until the end of the experiment (i.e. for 116 h). This suggests an equilibrium reaction with water instead of a continuous hydrolysis.
- pH 7: Similar observations were made for pH 7. A strong and fast reaction in the beginning (20 % of the parent substance were detected at test start) and no further reaction over a period of 5 days was observed.
- pH 9: No test item could be detected after approximately 4 hours. The solution of the test item was dark yellow indicating fast degradation by hydrolysis.

pH:

9

Temp.:

50 °C

Remarks on result:

not determinable

Remarks:

Not determinable due to fast reaction and insufficient number of data points

pH:

7

Temp.:

50 °C

Remarks on result:

not determinable

Remarks:

Due to the high degree of reaction at the start of the test, and due to the fact that no further reaction process over 5 days could be detected, a kinetic calculation of the reaction is not possible. For pH 4 and 7, it can be assumed that the test item shows an equilibrium reaction with water, where the equilibrium lays on the side of the reaction products.

pH:

4

Temp.:

50 °C

Remarks on result:

not determinable

Remarks:

Due to this high degree of reaction at the start of the test, and due to the fact that no further reaction process over 5 days could be detected, a kinetic calculation of the reaction is not possible. For pH 4 and 7, it can be assumed that the test item shows an equilibrium reaction with water, where the equilibrium lays on the side of the reaction products.

Details on results:

First and second preliminary tests (Tier 1) – mobile Phase in the HPLC: 100 % acetonitrile:
The first preliminary test showed, that the nominal start concentrations of the test item in the buffer solutions could not be confirmed. The measurements of the start concentrations were carried out directly after diluting the stock solution in the buffer solutions within a few minutes. The signals of the test item showed a tailing and a second signal could be observed at a retention time of approx. 1 minute, which gives evidence on a potential reaction of the test item with water (e.g. chromatogram of the test item in pH 7 buffer solution, directly after diluting, see Figure 1).
After these measurements, the concentrations of freshly prepared standards of the test item in ACN could also not be confirmed. Since, no explanation for the wrong measured concentrations was available, the complete preliminary test was repeated. The repetition showed similar results.

Third preliminary test (Tier 1) – mobile Phase in the HPLC: 100 % acetonitrile:
The signals of the test item in the buffers were tailing, the start concentrations could not be confirmed, and the freshly prepared standard solutions of the test item in ACN showed no signals anymore.

Fourth preliminary test (Tier 1) – mobile Phase in the HPLC: 100 % acetonitrile:
The measured start concentrations of the test item in the buffer solutions were twice as high as they should have been. Additional water blanks were measured in the HPLC. Every measured water blank showed two signals in the chromatogram, at retention times of approx. 1 and 2.4 min (see Figure 2). Thus, the whole HPLC apparatus was purged with isopropanol/water and the HPLC column was changed. Purging the apparatus and measuring further water blanks, a lot of test item could be measured in the blanks.

Fifth preliminary test (Tier 1) – mobile Phase in the HPLC: 75:25 % acetonitrile/water:
HPLC measurements of the three buffer solutions without test item showed no signal of the test item. The measurements of the test item solutions, directly after diluting the stock solution in the buffer solutions showed that only about 10 % of the nominal concentration could be found. Additionally, to the signal of the test item at approx. 3.8 min, a second signal could be observed at approx. 2.7 min (see Figure 3, Figure 4 and Figure 5).
The preliminary test at pH 4, 7 and 9 was performed at a temperature of 50 °C. Samples were taken and measured after 0, 5, 22, 28, 93, 100 and 116 h. The measured data at the time points are summarized in Tables 1 - 3. The nominal start concentration of the test item was 103.3 mg/L. Chromatograms of buffer blanks and the c0-measurements at each pH value are show in Figure 3 to 8.
In this fifth preliminary test, it can be observed, that the test item reacted with water by approx. 90 % at pH 4, by approx. 82 % at pH 7 and by approx. 73 % at pH 9, directly after diluting the stock solutions in the different buffer solutions. At pH 4 and 7, no further reaction was observed over a period of 5 days. At pH 9, no test item could be measured in the dilutions after 4 hours. The colour of the solutions at pH 9 changed to dark yellow.
Due to this high degree of reaction at the start of the test, and due to the fact that no further reaction process over 5 days could be detected, a kinetic calculation of the reaction is not possible. For pH 4 and 7, it can be assumed that the test item shows an equilibrium reaction with water, where the equilibrium lays on the side of the reaction products.
At pH 9, a full degradation by hydrolysis is likely as reaction with buffer components could be excluded by testing in different buffer systems. The hydrolysis rate for pH 9 cannot be estimated, because too few data points are available up to 90 % degree of hydrolysis. For a calculation of the reaction kinetics, the concentration of the test item has to be measured at six times at least, which cannot be performed for such a fast reaction.


Measurement of standard solutions:
The HPLC-method was checked several times on every day that samples of the tests were taken and measured. The control standards were freshly prepared with the test item in ACN. Table 4 shows all standard measurements during the fifth Tier 1.
Except for one measurement with a recovery rate of 88.7 %, the recovery rates of the standard solutions in acetonitrile ranged from 90.0 % to 103.7 % and show a sufficient accuracy of the analytical method.

Tests with pre-cooled buffers:
Due to the high degree of hydrolysis, bad recovery of the test item in the buffer solutions, directly after dilution the stock solution in the three buffer solutions, three further tests were performed with pre-cooled buffer solutions. The nominal concentration of the test item in the buffer solutions was also 103.3 mg/L and the concentrations were determined twice, directly after dilution. Table 5 shows the results of these tests.
These measurements show that the pre-cooling of the buffer solutions has only small influence on the degree of reaction.

Tests on the solubility in water:
The HPLC measurements of the flasks of the water solubility study show that the test item reacted with water. Separating the signal of the test item at 3.7 minutes from the other signals, a concentration of the test item in water of 6.9 g/L was determined.
The measurement with a dilution factor of 10000 is shown in Figure 9. Here, the result of the water solubility is about 67 g/L, which seems to be very high.
Two further solubility tests of the test item, dissolved and diluted in water, were carried out.
In all measurements it was observed, that the test item has changed in water, because it did not show its single signal at 3.7 minutes, like it does in pure ACN.
The calculation of the concentration of the test item in of one of these solutions (standard D, nominal concentration = 105 mg/L) shows a recovery rate of the test item in water of <10 %.

Tests in other buffer systems (boric acid – buffers):
The two additional tests in other buffer systems show that the test item reacts in a similar way (see Figure 10 and Figure 11). The chromatogram of the test item in pH 9 shows a much better signal of the test item, but the recovery rate, directly after dilution of the test item in the buffer is also at approx. 33 % and at pH 7 at 12 %.
Thus, it can be assumed that the type of buffer has only small influence on the reaction of the test item with the aqueous system.
Due to the fact that the nominal start concentration of the test item in the buffer solutions could not be confirmed in the HPLC measurements no further kinetic tests (Tier 2) at other temperatures were carried out.

Results with reference substance:

n/a

Table 1: Preliminary test for the hydrolysis at pH 4 and 50 °C

pH	sample no.	time [h]	measured concentration [mg/L]	degree of reaction* [%]
4	2a-0	0.1	10.79	90
4	2b-0	0.1	9.72	91
4	2a-1	4.8	10.63	90
4	2b-1	4.8	10.36	90
4	2a-2	21.9	10.71	90
4	2b-2	21.9	10.88	90
4	2a-3	27.6	10.57	90
4	2b-3	27.6	10.60	90
4	2a-4	93.1	10.87	90
4	2b-4	93.1	10.60	90
4	2a-5	99.6	10.94	89
4	2b-5	99.6	10.80	90
4	2a-6	116.6	11.01	89
4	2b-6	116.6	11.06	89

*referring to the nominal concentration of 103.3 mg/L

 

Table 2: Preliminary test for the hydrolysis at pH 7 and 50 °C

pH	sample no.	time [h]	measured concentration [mg/L]	degree of reaction* [%]
7	3a-0	0.1	15.71	85
7	3b-0	0.1	18.41	82
7	3a-1	4.3	17.01	84
7	3b-1	4.3	20.37	80
7	3a-2	21.4	16.64	84
7	3b-2	21.4	20.13	81
7	3a-3	27.1	16.67	84
7	3b-3	27.1	19.76	81
7	3a-4	92.6	15.03	86
7	3b-4	92.6	18.75	82
7	3a-5	99.1	16.40	84
7	3b-5	99.1	19.91	81
7	3a-6	116.1	16.24	84
7	3b-6	116.1	19.43	81

*referring to the nominal concentration of 103.3 mg/L

Table 3: Preliminary test for the hydrolysis at pH 9 and 50 °C

pH	sample no.	time [h]	measured concentration [mg/L]	degree of reaction* [%]
9	4a-0	0.1	27.58	73
9	4b-0	0.1	20.02	81
9	4a-1	3.8	--	100
9	4b-1	3.8	--	100

*referring to the nominal concentration of 103.3 mg/L

Table 4: Standard solutions during the fifth Tier 1

measuring date	c (nominal) [mg/L]	c (measured) [mg/L]	recovery rate [%]
20.09.18	66.96	67.21	100.4
		69.44	103.7
		61.78	92.3
21.09.18	53.22	47.88	90.0
		48.20	90.6
		47.21	88.7
		48.17	90.5
24.09.18	49.32	47.98	97.3
		48.90	99.1
		46.66	94.6
		48.79	98.9
25.09.18	73.14	71.43	97.7
		74.60	102.0

Table 5: Tests with pre-cooled buffer solutions

pH	measured concentration [mg/L]	degree of reaction* [%]
4	15.71 / 11.53	87
7	13.43 / 16.14	86
9	41.18 / 35.03	62

*referring to the mean measured concentration and the nominal concentration of 103.3 mg/L

Validity criteria fulfilled:

not applicable

Conclusions:

The study on hydrolysis of the test item according to OECD 111 and EC 440/2008, method C.7 could not be performed properly. HPLC measurements of the test item, dissolved in acetonitrile showed good analytical results, a calibration with the test item could be prepared and the analytical method was validated.
As soon as the test item was in contact with water or aqueous buffer solutions, the UV chromatograms of the test item (one sharp signal) changed (compared to test item dissolved in acetonitrile). Depending on the pH of the buffer solutions, at least one additional signal could be observed. Therefore, it is concluded that the test item undergoes a pH dependent reaction with water.
The preliminary tests (Tier 1) in buffers with pH 4, 7 and 9 show that immediately after dissolution in the buffers the measured concentrations of the test item were considerably lower than nominal. This observation was made in different buffer systems and clearly indicates a pH dependent reaction with water.
Immediate reduction of analytically determinable test item concentration by 90 % at pH 4 and approximately 80 % at pH 7 was determined, but no further reaction could be observed over a period of five days, i.e. approximately 10 % (pH 4) and 20 % (pH 7) of the test item, respectively, remained stable. This behaviour indicates that the reaction of the test item with water is not an irreversible hydrolytic reaction but rather an equilibrium reaction.
A pH induced addition reaction of water to the carbonyl carbon with a potential subsequent cyclisation reaction with the adjacent aldehyde functionality of the test item could be a possible reaction.
At pH 9 no test item could be detected anymore after approximately 4 hours. The solution of the test item in pH 9 buffer was dark yellow. This observation indicates fast degradation by hydrolysis. Potential reactions of test item with buffer components were excluded by using different buffer systems.
In conclusion, for pH 4 and 7, no degradation kinetics (hydrolysis rate) could be determined. At pH 9, a full degradation by hydrolysis is likely as reaction with buffer components could be excluded by testing in different buffer systems. The hydrolysis rate for pH 9 cannot be estimated, because too few data points are available up to 90 % degree of hydrolysis. For a calculation of the reaction kinetics, the concentration of the test item has to be measured at six times at least, which cannot be performed for such a fast reaction.

Executive summary:

Hydrolysis of ortho-phthalaldehyde at 103.3 mg/L (nominal) was studied in the dark at 50 °C in sterile aqueous buffered solutions at pH 4 (acetic acid/sodium acetate), pH 7 (potassium dihydrogen phosphate/ di-potassium hydrogen phosphate) and pH 9 (ammonia/ammonium chloride) for 5 days (measurements with pH 9 ended after 3.8 hours as the parent substance was completely transformed). For the alternative buffer solutions at pH 7 and 9 one stock buffer solution was prepared with 0.1 mol/L KCl and 0.1 mol/L H3BO3. For the two pH values, 0.1 M NaOH and 1 M HCl were added, respectively.

The experiment was conducted in accordance with the OECD guideline 111 and in compliance with GLP. Samples were analysed at 0.1, 4.8, 21.9, 27.6, 93.1, 99.6 and 116.6 hours for pH 4, at 0.1, 4.3, 21.4, 27.1, 92.6, 99.1, and 116.1 h for pH 7, and at 0.1 and 3.8 h for pH 9, respectively. The test material was analysed by HPLC with UV-detection. At test termination, the concentration of the parent compound was stable until the end of the test for pH 4 and 7, and completely transformed within 3.8 h at pH 9.

The hydrolysis rate for pH 9 cannot be estimated, because too few data points are available up to 90 % degree of hydrolysis. For a calculation of the reaction kinetics, the concentration of the test item has to be measured at six times at least, which cannot be performed for such a fast reaction. A reaction with buffer components could be excluded by testing in different buffer systems.

Description of key information

No degradation kinetics (hydrolysis rate) hence nor any half-lives could be determined, due to immediate reaction of the substance with water.

The test results suggest that the reaction of the test item with water at pH 4 and pH 7 is not an irreversible hydrolytic reaction but rather an equilibrium reaction, as immediate reaction with water was reported reaching a stable status.

The half-life for pH 9 cannot be estimated. For a calculation of the reaction kinetics, the concentration of the test item has to be measured at six times at least, which was not possible for the present case.