[2S-[2α,5α,6β(S*)]]-6-[[[[(4-ethyl-2,3-dioxopiperazin-1-yl)carbonyl]amino]phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16/05/2018 – 13/09/2018

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:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

no

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Individual replicate test samples (a minimum of duplicate samples contained in separate reaction vessels) were removed from the incubator and the contents analysed at each of at least 6 sampling times.
- Sampling intervals/times for measurements:
- Tier I samples were measured at day 0 and after 5 days;
- Tier II: The test was conducted until 90% hydrolysis was observed: At pH 4 (20ºC), the amount of parent compound was measured at 0, 4, 25, 73, 145, 168, and 192 h after the start of the test; at pH 4 (30ºC), it was measured at 0, 1, 18, 25, 43, 91, 163, 187 and 214 h; at pH 4 (50ºC), it was measured at 0, 4, 5, 22, 26, 27, and 47 h. At pH 7 (20ºC), it was measured at 0, 3, 21, 48, 96 and 168 h; at pH 7 (30ºC), at 0, 17, 24, 42, 90, 162, 714 h; at pH 7 (50ºC), at 0, 4, 5, 22, 27, 28 and 143 h. At pH 9 (20ºC), it was measured at 0, 3, 21, 28, 46, and 50 h; at pH 9 (30ºC), at 0, 18, 21, 22 h; and at pH 9 (50ºC), at 0, 1, 3, and 4 h after the start of the test.
- Tier III: sampling was performed after 24 hours of hydrolysis at 50 ºC, in all the pH tested (4, 7, 9).

Buffers:

- pH: 4.0, 7.0, 9.0 (commercial buffers, no further details).

Details on test conditions:

TEST SYSTEM
- Type, material and volume of test flasks, other equipment used: glass flasks fitted with glass stoppers, incubator, magnetic stirrer, pH-meter.
- Measures taken to avoid photolytic effects: preliminary tests were performed in the dark incubator.
- If no traps were used, is the test system: closed.
- Is there any indication of the test material adsorbing to the walls of the test apparatus? no.

TEST MEDIUM
- Volume used/treatment
- Kind and purity of water: water for HPLC (Milipore)
- Preparation of test medium: BUFFER
- Renewal of test solution: no.
- Identity and concentration of co-solvent:

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

Number of replicates:

2

Positive controls:

no

Negative controls:

no

Statistical methods:

The statistical calculations and the graphs were performed using SigmaPlot 9.0 software of SYSTAT Software, Inc., USA purchased from GAMBIT CoiS Ltd, Poland.

Preliminary study:

- Measurements in buffer solution at pH 4.0: The determined rate constant after 5 days at 50ºC was 0.048 h-1 and the half-life period was 14.5h (>12h). About 96.5% of the test item had been hydrolyzed after 5 days.
- Measurements in buffer solution at pH 7.0: The determined rate constant after 5 days at 50ºC was 0.033 h-1 and the half-life period was 21.2h (>12h). About 96.7% of the test item had been hydrolyzed after 5 day.
- Measurements in buffer solution at pH 9.0: The determined rate constant after 5 days at 50ºC was 0.544 h-1 and the half-life period was 1.27h (<12h), so it was deemed hydrolytically unstable. About 93.1% of the test item had been hydrolyzed after 5 days.
- On the basis of these results, Tier 2 and 3 studies were performed.

Test performance:

No abnormal observations or deviations reported.

Transformation products:

yes

No.:

#1

No.:

#2

No.:

#3

No.:

#4

Key result

pH:

4

Temp.:

20 °C

Hydrolysis rate constant:

0.009 h-1

DT50:

80.24 h

Type:

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

Remarks on result:

other: experimental

Key result

pH:

4

Temp.:

25 °C

Hydrolysis rate constant:

0.01 h-1

Type:

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

Remarks on result:

other: calculated

Key result

pH:

4

Temp.:

30 °C

Hydrolysis rate constant:

0.01 h-1

DT50:

64.77 h

Type:

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

Remarks on result:

other: experimental

Key result

pH:

4

Temp.:

50 °C

Hydrolysis rate constant:

0.048 h-1

DT50:

14.5 h

Type:

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

Remarks on result:

other: experimental

Key result

pH:

7

Temp.:

20 °C

Hydrolysis rate constant:

0.004 h-1

DT50:

175 h

Type:

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

Remarks on result:

other: experimental

Key result

pH:

7

Temp.:

25 °C

Hydrolysis rate constant:

0.005 h-1

Type:

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

Remarks on result:

other: calculated

Key result

pH:

7

Temp.:

30 °C

Hydrolysis rate constant:

0.005 h-1

DT50:

149 h

Type:

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

Remarks on result:

other: experimental

Key result

pH:

7

Temp.:

50 °C

Hydrolysis rate constant:

0.033 h-1

DT50:

21.19 h

Type:

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

Remarks on result:

other: experimental

Key result

pH:

9

Temp.:

20 °C

Hydrolysis rate constant:

0.043 h-1

DT50:

16.15 h

Type:

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

Remarks on result:

other: experimental

Key result

pH:

9

Temp.:

25 °C

Hydrolysis rate constant:

0.066 h-1

Type:

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

Remarks on result:

other: calculated

Key result

pH:

9

Temp.:

30 °C

Hydrolysis rate constant:

0.097 h-1

DT50:

7.14 h

Type:

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

Remarks on result:

other: experimental

Key result

pH:

9

Temp.:

50 °C

Hydrolysis rate constant:

0.544 h-1

DT50:

1.27 h

Type:

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

Remarks on result:

other: experimental

Other kinetic parameters:

Activation energy, E = 46.859 kJ/mol (pH 4), 58.329 kJ/mol (pH 7), 67.107 kJ/mol (pH 9).

Details on results:

TEST CONDITIONS
- pH, sterility, temperature, and other experimental conditions maintained throughout the study: Yes, pH and temperature were set and maintaned at various fixed values.
- Anomalies or problems encountered: no.

MAJOR TRANSFORMATION PRODUCTS
At pH4:
- Transformation products observed were 2-(2-{2-[(4-Ethyl-2,3-dioxo-piperazine-1-carbonyl)-amino]-2-phenyl-acetylamino}-vinylamino)-3,3-dimethyl-butyric acid; (5,5-Dimethyl-thiazolidin-2-yl)-{2-[(4-ethyl-2,3-dioxo-piperazine-1-carbonyl)-amino]-2-phenyl-acetylamino}-acetic acid; and 2-({2-[(4-Ethyl-2,3-dioxo-piperazine-1-carbonyl)-amino]-2-phenyl-acetylamino}-methyl)-5,5-dimethyl-thiazolidine-4-carboxylic acid.

At pH7:
- Transformation products observed were 2-(2-{2-[(4-Ethyl-2,3-dioxo-piperazine-1-carbonyl)-amino]-2-phenyl-acetylamino}-vinylamino)-3,3-dimethyl-butyric acid; (5,5-Dimethyl-thiazolidin-2-yl)-{2-[(4-ethyl-2,3-dioxo-piperazine-1-carbonyl)-amino]-2-phenyl-acetylamino}-acetic acid; and 2-({2-[(4-Ethyl-2,3-dioxo-piperazine-1-carbonyl)-amino]-2-phenyl-acetylamino}-methyl)-5,5-dimethyl-thiazolidine-4-carboxylic acid.

At pH9:
- The transformation product observed was: 2-(Carboxy-{2-[(4-ethyl-2,3-dioxo-piperazine-1-carbonyl)-amino]-2-phenyl-acetylamino}-methyl)-5,5-dimethyl-thiazolidine-4-carboxylic acid.

INDICATION OF UNSTABLE TRANSFORMATION PRODUCTS: no.

PATHWAYS OF HYDROLYSIS
- Figures of chemical structures attached: Yes

Table 1. Preliminary study results.

pH		4.0		7.0		9.0
Time (d) at 50ºC		0	5	0	5	0	5
Concentration (mg/L)	R1	668.7	23.5	2487.4	83.4	5289.5	372.0
	R2	679.7	23.2	2519.1	82.2	5678.5	381.0
Average conc. (mg/L)		674.2	23.4	2503.3	82.8	5484.0	376.5
Concentration loss (%)		96.5		96.7		93.1
pH, measured		4.00	4.02	6.98	6.97	8.65	8.67

Table 2. Full hydrolysis study, summary of results: Hydrolysis rate, half-life period and activation energy.

temperature, ºC	pH	4	7	9
20 ºC (exp)		kobs= 0.00864 h^-1 t0.5= 80.24 h	kobs= 0.00396 h^-1 t0.5= 175.0 h	kobs= 0.0429 h^-1 t0.5= 16.15 h
25 ºC (calculated)		kcalc= 0.0101 h^-1	kcalc= 0.00460 h^-1	kcalc= 0.0657
30 ºC (exp)		kobs= 0.0107 h^-1 t0.5= 64.77 h	kobs= 0.00465h^-1 t0.5= 149.0 h	kobs= 0.0971 h^-1 t0.5= 7.14 h
50 ºC (exp)		kobs= 0.0478 h^-1 t0.5= 14.50 h	kobs= 0. 0327 h^-1 t0.5= 21.19 h	kobs= 0.544 h^-1 t0.5= 1.27 h
Activation energy E, kJ/mol		46.859	58.329	67.107

Table 3. Quantification of transformation products.

pH	Temp. ºC	Initial conc. of PA, mg/L	Final conc. of PA, mg/L	Final time, hours	Percentage of the detected substances, %
					PA	#1	#2	#3	#4	#5
4	20	506.7	50.4	192	17.36	2.34	0.79	9.22	64.07	5.53
4	30	476.3	46.5	214	16.25	3.23	1.50	2.67	69.90	6.45
4	50	506.8	50.5	47	15.22	1.09	2.03	0.40	73.82	7.44
7	20	523.0	15.6	694	10.54	2.00	9.32	1.84	68.42	7.89
7	30	496.6	19.1	714	13.18	0.33	1.19	3.98	67.98	13.33
7	50	490.5	4.5	143	14.90	2.43	1.20	4.03	61.75	15.68
9	20	453.5	51.9	50	9.64	0.37	22.39	17.70	49.81	0.09
9	30	463.6	26.4	22	10.22	0.77	26.23	18.35	39.86	4.56
9	50	402.6	47.7	4	7.14	1.50	27.10	16.70	43.37	3.98

Validity criteria fulfilled:

yes

Conclusions:

The substance was found to be hydrolitically unstable only at pH 9 (≥ 30ºC) with a half-life < 12h. The major hydrolysis products were identified as 2-(2-{2-[(4-Ethyl-2,3-dioxo-piperazine-1-carbonyl)-amino]-2-phenyl-acetylamino}-vinylamino)-3,3-dimethyl-butyric acid, 5,5-Dimethyl-thiazolidin-2-yl-{2-[(4-ethyl-2,3-dioxo-piperazine-1-carbonyl)-amino]-2-phenyl-acetylamino}-acetic acid and 2-({2-[(4-Ethyl-2,3-dioxo-piperazine-1-carbonyl)-amino]-2-phenyl-acetylamino}-methyl)-5,5-dimethyl-thiazolidine-4-carboxylic acid at pH 4 and pH 7; and as 2-(Carboxy-{2-[(4-ethyl-2,3-dioxo-piperazine-1-carbonyl)-amino]-2-phenyl-acetylamino}-methyl)-5,5-dimethyl-thiazolidine-4-carboxylic acid at pH 9.

Executive summary:

An hydrolysis study was conducted on the test item, according to OECD TG 111 / EU C.7 (Hydrolysis as a function of pH), under GLP conditions. A validated HPLC method was used for determining the content of the test substance in the samples. A preliminary Tier 1 test was conducted at pH 4.0, 7.0 and 9.0 and 50ºC, which showed that the test item is hydrolitically unstable at pH 9, and >90% of the substance was degraded after 5 days, so further testing was necessary. Based on this data, Tier 2 assays were performed at 20, 30 and 50ºC and at three pH values (4.0, 7.0 and 9.0) in order to determe the hydrolysis rate constants and half-life periods for the test item. Finally, a Tier 3 assay was conducted to identify the major hydrolysis products by UPLC-MS. The results obtained are as follows:

Temperature, ºC	pH	4	7	9
20 ºC (exp)		kobs= 0.00864 h^-1 t0.5= 80.24 h	kobs= 0.00396 h^-1 t0.5= 175.0 h	kobs= 0.0429 h^-1 t0.5= 16.15 h
25 ºC (calculated)		kcalc= 0.0101 h^-1	kcalc= 0.00460 h^-1	kcalc= 0.0657
30 ºC (exp)		kobs= 0.0107 h^-1 t0.5= 64.77 h	kobs= 0.00465h^-1 t0.5= 149.0 h	kobs= 0.0971 h^-1 t0.5= 7.14 h
50 ºC (exp)		kobs= 0.0478 h^-1 t0.5= 14.50 h	kobs= 0. 0327 h^-1 t0.5= 21.19 h	kobs= 0.544 h^-1 t0.5= 1.27 h

The substance was found to be hydrolitically unstable only at pH 9 (≥ 30ºC) with a half-life < 12h. The major hydrolysis products were identified as #1: 2-(2-{2-[(4-Ethyl-2,3-dioxo-piperazine-1-carbonyl)-amino]-2-phenyl-acetylamino}-vinylamino)-3,3-dimethyl-butyric acid, #2: 5,5-Dimethyl-thiazolidin-2-yl-{2-[(4-ethyl-2,3-dioxo-piperazine-1-carbonyl)-amino]-2-phenyl-acetylamino}-acetic acid, #3: 2-({2-[(4-Ethyl-2,3-dioxo-piperazine-1-carbonyl)-amino]-2-phenyl-acetylamino}-methyl)-5,5-dimethyl-thiazolidine-4-carboxylic acid at pH 4 and pH 7; and as #4: 2-(Carboxy-{2-[(4-ethyl-2,3-dioxo-piperazine-1-carbonyl)-amino]-2-phenyl-acetylamino}-methyl)-5,5-dimethyl-thiazolidine-4-carboxylic acid at pH 9. Based on the quantification results, the main hydrolysis product is #4 (40-60%).

Description of key information

Key study. Method according to OECD 111, GLP Study. The substance is hydrolytically unstable at pH 9 (30 and 50ºC), with a half-life <12h. The major hydrolysis products were identified as #1: 2-(2-{2-[(4-Ethyl-2,3-dioxo-piperazine-1-carbonyl)-amino]-2-phenyl-acetylamino}-vinylamino)-3,3-dimethyl-butyric acid, #2: 5,5-Dimethyl-thiazolidin-2-yl)-{2-[(4-ethyl-2,3-dioxo-piperazine-1-carbonyl)-amino]-2-phenyl-acetylamino}-acetic acid, #3: 2-({2-[(4-Ethyl-2,3-dioxo-piperazine-1-carbonyl)-amino]-2-phenyl-acetylamino}-methyl)-5,5-dimethyl-thiazolidine-4-carboxylic acid at pH 4 and pH 7; and #4 (main): 2-(Carboxy-{2-[(4-ethyl-2,3-dioxo-piperazine-1-carbonyl)-amino]-2-phenyl-acetylamino}-methyl)-5,5-dimethyl-thiazolidine-4-carboxylic acid at pH 9.

Key value for chemical safety assessment

Half-life for hydrolysis:

1 h

at the temperature of:

25 °C

Additional information

An hydrolysis study was conducted on the test item, according to OECD TG 111 / EU C.7 (Hydrolysis as a function of pH), under GLP conditions. A validated HPLC method was used for determining the content of the test substance in the samples. A preliminary Tier 1 test was conducted at pH 4.0, 7.0 and 9.0 and 50ºC, which showed that the test item is hydrolitically unstable at pH 9, and >90% of the substance was degraded after 5 days, so further testing was necessary. Based on this data, Tier 2 assays were performed at 20, 30 and 50ºC and at three pH values (4.0, 7.0 and 9.0) in order to determe the hydrolysis rate constants and half-life periods for the test item. Finally, a Tier 3 assay was conducted to identify the major hydrolysis products by UPLC-MS. The results obtained are as follows:

Temperature, ºC	pH	4	7	9
20 ºC (exp)		kobs= 0.00864 h^-1 t0.5= 80.24 h	kobs= 0.00396 h^-1 t0.5= 175.0 h	kobs= 0.0429 h^-1 t0.5= 16.15 h
25 ºC (calculated)		kcalc= 0.0101 h^-1	kcalc= 0.00460 h^-1	kcalc= 0.0657
30 ºC (exp)		kobs= 0.0107 h^-1 t0.5= 64.77 h	kobs= 0.00465h^-1 t0.5= 149.0 h	kobs= 0.0971 h^-1 t0.5= 7.14 h
50 ºC (exp)		kobs= 0.0478 h^-1 t0.5= 14.50 h	kobs= 0. 0327 h^-1 t0.5= 21.19 h	kobs= 0.544 h^-1 t0.5= 1.27 h

The substance was found to be hydrolitically unstable only at pH 9 (≥ 30ºC) with a half-life < 12h. The major hydrolysis products were identified as 2-(2-{2-[(4-Ethyl-2,3-dioxo-piperazine-1-carbonyl)-amino]-2-phenyl-acetylamino}-vinylamino)-3,3-dimethyl-butyric acid, 5,5-Dimethyl-thiazolidin-2-yl)-{2-[(4-ethyl-2,3-dioxo-piperazine-1-carbonyl)-amino]-2-phenyl-acetylamino}-acetic acid and 2-({2-[(4-Ethyl-2,3-dioxo-piperazine-1-carbonyl)-amino]-2-phenyl-acetylamino}-methyl)-5,5-dimethyl-thiazolidine-4-carboxylic acid at pH 4 and pH 7; and as 2-(Carboxy-{2-[(4-ethyl-2,3-dioxo-piperazine-1-carbonyl)-amino]-2-phenyl-acetylamino}-methyl)-5,5-dimethyl-thiazolidine-4-carboxylic acid at pH 9. Based on the quantification results, the main hydrolysis product is #4 (40-60%).