N-[4-[[(2-amino-1,4,5,6,7,8-hexahydro-4-oxo-6-pteridinyl)methyl]amino]benzoyl]-L-glutamic acid

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-07-27 to

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Version / remarks:

adopted 13 April 2004

Deviations:

no

Principles of method if other than guideline:

Study was performed to determine the stability of the test substance in different aqueous media to clarify the feasibility and design of experimental studies regarding physico-chemical, environmental fate and ecotoxicological properties.

GLP compliance:

no

Analytical monitoring:

yes

Remarks:

Liquid chromatography and mass spectrometry

Details on sampling:

- Sample storage conditions before analysis: 100 mg/L test item was dissolved in buffer solution pH 4, 7 and 9, respectively and analysed directly.

Buffers:

- pH: 4, 7 and 9
- Type and final molarity of buffer: n/a
- Composition of buffer: n/a

Number of replicates:

2

Positive controls:

no

Negative controls:

no

Transformation products:

yes

No.:

#1

No.:

#2

Remarks on result:

not measured/tested

Key result

pH:

4

DT50:

0.059 h

Type:

not specified

Remarks on result:

other: Temperature and Hydrolysis rate constant not reported

Key result

pH:

7

DT50:

0.257 h

Type:

not specified

Remarks on result:

other: Temperature and Hydrolysis rate constant not reported

Key result

pH:

9

DT50:

0.712 h

Type:

not specified

Remarks on result:

other: Temperature and Hydrolysis rate constant not reported

Determination of Hydrolysis in Aqueous Phase – pH 4

Nominal concentration, test item: 100 mg/L

pH 4
Time [h]	Peak Area	ln Peak Area	%
0.000	33509948	17.3	-
0.183	675904	13.4	2
0.364	179466	12.1	0.5
0.547	43528	10.7	0.1

%          = percent of initial concentration, t₀set to 100%

Hydrolysis in Aqueous Phase – pH 4

pH 4
Slope of regression graph	-0.117
Correlation factor [r^2]	0.923
Reaction rate constantk[1/s]	3.24 ∙ 10^-3
Half-lifeT½[h]	0.0594
Half-lifeT½[min]	3.56

 

 Determination of Hydrolysis in Aqueous Phase – pH 7

Nominal concentration, test item: 100 mg/L

pH 7
Time [h]	Peak Area	ln Peak Area	%
0.000	56905440	17.9	-
0.183	29781291	17.2	52
0.365^1)	5281361^1)	15.5^1)	9^1)
0.736	7279096	15.8	13
0.919	4985612	15.4	9
1.10	3174099	15.0	6
1.28	1808882	14.4	3
1.47	1143522	14.0	2
1.65	600828	13.3	1
1.83	357862	12.8	1

% = percent of initial concentration, t₀set to 100%

1)= outlier, not taken into account

Hydrolysis in Aqueous Phase – pH 7

pH 7
Slope of regression graph	-2.70
Correlation factor [r^2]	0.997
Reaction rate constantk[1/s]	7.49 ∙ 10^-4
Half-lifeT½[h]	0.257
Half-lifeT½[min]	15.4

Determination of Hydrolysis in Aqueous Phase – pH 9

Nominal concentration, test item: 100 mg/L

pH 9
Time [h]	Peak Area	ln Peak Area	%
0.000	128982791	18.7	-
0.183	86149063	18.3	67
0.365	64018083	18.0	50
0.547	50905504	17.8	39
0.730	43442552	17.6	34
0.913	35485067	17.4	28
1.10	30533632	17.2	24
1.28	26036767	17.1	20
1.46	22208419	16.9	17
1.64	19819906	16.8	15
2.01	15356634	16.6	12
2.19	12085931	16.3	9
2.38	10140678	16.1	8

% = percent of initial concentration, t₀set to 100%

Hydrolysis in Aqueous Phase – pH 9

pH 9
Slope of regression graph	-0.974
Correlation factor [r^2]	0.977
Reaction rate constantk[1/s]	2.70 ∙ 10^-4
Half-lifeT½[h]	0.712
Half-lifeT½[min]	42.7

Validity criteria fulfilled:

not applicable

Conclusions:

The present study was conducted in order to provide information about the stability of Tetrahydrofolic acid in aqueous solutions. The water solubility could not be determined due to fast hydrolysis of the substance.The half-lifes were determined in 0.01 M CaCl2 solution, in buffer solutions at pH 4, 7 and 9 and in Algae Dilution Medium. T1/2 was 0.4 h in 0.01M CaCl2, and 0.0594 h for pH 4, 0.257 h for pH 7 and 0.712 h for pH 9 with LC-MS. Plots of the stability of Tetrahydrofolic acid in Algae Dilution Medium showed a decline of the parent substances and an increase of two hydrolysis products 2-amino-6-methylpteridin-4(1H)-one and (2S)-2-(4-aminobenzamido)pentanedioic acid.

Description of key information

- study on hydrolysis performed similar to OECD guideline 111, non-GLP test, half-lifes T1/2 was 0.4 h in 0.01M CaCl2, and 0.0594 h for pH 4, 0.257 h for pH 7 and 0.712 h for pH 9 with LC-MS, determination of two hydrolysis products 2-amino-6-methylpteridin-4(1H)-one and (2S)-2-(4-aminobenzamido)pentanedioic acid.

Additional information

The present study was conducted in order to provide information about the stability of Tetrahydrofolic acid in aqueous solutions. The water solubility could not be determined due to fast hydrolysis of the substance.The half-lifes were determined in 0.01 M CaCl2 solution, in buffer solutions at pH 4, 7 and 9 and in Algae Dilution Medium. T1/2 was 0.4 h in 0.01M CaCl2, and 0.0594 h for pH 4, 0.257 h for pH 7 and 0.712 h for pH 9 with LC-MS. Plots of the stability of Tetrahydrofolic acid in Algae Dilution Medium showed a decline of the parent substances and an increase of two hydrolysis products 2-amino-6-methylpteridin-4(1H)-one and (2S)-2-(4-aminobenzamido)pentanedioic acid.