(6R-trans)-7-amino-8-oxo-3-[[(1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-1,2,4-triazin-3-yl)thio]methyl]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

Hydrolysis

Administrative data

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Only the preliminary test at 50 °C was performed.

Cross-reference

Reason / purpose for cross-reference:

other: Analytical method.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Version / remarks:

2004

Deviations:

yes

Remarks:

Only the preliminary test at 50 °C was performed.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

yes

Remarks:

Only the preliminary test at 50 °C was performed.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(6R-trans)-7-amino-8-oxo-3-[[(1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-1,2,4-triazin-3-yl)thio]methyl]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

EC Number:

261-491-7

EC Name:

(6R-trans)-7-amino-8-oxo-3-[[(1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-1,2,4-triazin-3-yl)thio]methyl]-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

Cas Number:

58909-56-1

Molecular formula:

C12H13N5O5S2

IUPAC Name:

7-amino-3-{[(2-methyl-5,6-dioxo-1,2,5,6-tetrahydro-1,2,4-triazin-3-yl)sulfanyl]methyl}-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

Test material form:

solid: particulate/powder

Details on test material:

Test item: 7-ACT
Chemical name: 7-Amino-3-(2-methyl-5,6-dioxo-1,2,5,6-tetrahydro-[1,2,4]triazin-3-ylsulfanylmethyl)-8-oxo-5-thia-1-aza-bicyclo[4.2.0]oct-2-ene-2-carboxylic acid
Batch No.: B364630
Active ingredient content: >99.0 %
CAS No.: 58909-56-1
Appearance: White, weakly odorous powder
Manufacturing date: 15 May 2016
Retest date: 17 May 2017
Storage: Refrigerator (2-8ºC), protected from light

Radiolabelling:

no

Study design

Analytical monitoring:

yes

Details on sampling:

Samples were twofold diluted with the mobile phase (0.5 mL → 1 mL) and analysed by HPLC method.

Buffers:

Buffer solutions:
pH 4.0: 1 mL 0.2 M Sodium hydroxide and 125 mL 0.2M Potassium hydrogen phthalate were diluted to 500 mL with ultrapure water
pH 7.0: 74 mL 0.2 M Sodium hydroxide and 125 mL 0.2 M Potassium dihydrogen phosphate were diluted to 500 mL with ultra-pure water
pH 9.0: 53.5 mL 0.2 M Sodium hydroxide and 125 mL 0.2 M Boric acid and Potassium chloride were diluted to 500 mL with ultra-pure water

Details on test conditions:

Principle of the Method
Sterile aqueous buffer solutions of different pH values are treated with the test item and incubated in the dark for five days at 50±0.5 °C. Concentration of the test substance is determined at the start, after one and five days. If the concentration of the test substance is lower than 10 % of the initial concentration after 1 day, the study will be stopped at this pH value.

Test Conditions:
Hydrolysis was examined at three different pH values: 4.0, 7.0 and 9.0, at 50 °C in the dark.

The hydrolysis reaction was carried out using a dark thermostat to avoid photolytic effects. Nitrogen was bubbled into the water for five minutes before the preparation of the solutions in order to exclude oxygen.

Performance of the Test:
Preparation of the test solutions:
In case of pH 4.0 25 mL of the Stock Solution was diluted to a total volume of 250 mL with the buffer solution.
In case of pH 7.0 and pH 9.0 test solution, about 5 mg of test item was weighted to the nearest 0.01 mg into a volumetric flask and dissolved in the respective buffer solutions to a total volume of 50 mL. This solution was tenfold diluted with the respective buffer solution.
The pH of each test solution was checked with a calibrated pH meter to a precision of 0.001.
Test solutions were transferred into 20 mL headspace–vials under sterile circumstances, under a laminar flow hood. The tubes were entirely filled with the solution. From each test solution three tubes were prepared.
The tubes were thermostated at 50±0.5°C.

Number of replicates:

3

Positive controls:

no

Results and discussion

Preliminary study:

At pH 4 the concentration of the test item was decreased by about 98.5 %, at pH 7 by 91.9 % and at pH 9 the concentration of the test item was decreased by about 99.5 % after 5 days at 50°C.

Transformation products:

not measured

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

pH 4: The concentration of the test item decreased at 50 °C to 9 % of the start concentration after 1 day.
pH 7: The concentration of the test item decreased at 50 °C to 30 % of the start concentration after 1 day and to 15 % after 5 days.
pH 9: The concentration of the test item decreased at 50 °C to 64 % of the start concentration after 1 day and to 27 % after 5 days.

Dissipation DT50 of parent compound

Dissipation DT50 of parent compound 1

Key result

pH:

4

Temp.:

50 °C

DT50:

< 1 d

Type:

not specified

Dissipation DT50 of parent compound 2

Key result

pH:

7

Temp.:

50 °C

DT50:

< 1 d

Type:

not specified

Dissipation DT50 of parent compound 3

Key result

pH:

9

Temp.:

50 °C

DT50:

< 5 d

Type:

not specified

Applicant's summary and conclusion

Conclusions:

pH 4: The concentration of the test item decreased at 50 °C to 9 % of the start concentration after 1 day.
pH 7: The concentration of the test item decreased at 50 °C to 30 % of the start concentration after 1 day and to 15 % after 5 days.
pH 9: The concentration of the test item decreased at 50 °C to 64 % of the start concentration after 1 day and to 27 % after 5 days.

Executive summary:

Hydrolysis of the test item in different pH buffer solutions was determined at 50 °C ± 0.5 °C.

Results:

pH 4: The concentration of the test item decreased at 50 °C to 9 % of the start concentration after 1 day.

pH 7: The concentration of the test item decreased at 50 °C to 30 % of the start concentration after 1 day and to 15 % after 5 days.

pH 9: The concentration of the test item decreased at 50 °C to 64 % of the start concentration after 1 day and to 27 % after 5 days.

Based on the results obtained, the test item can be considered as hydrolytically unstable at pH 4, pH 7 and pH 9 under the conditions investigated.