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

Water solubility

Link to relevant study record(s)

Reference

Endpoint:

water solubility

Remarks:

Stability of the substance in aqueous solutions

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Principles of method if other than guideline:

no guideline followed. 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

Type of method:

other: Half-life of the substance was determined in 0.01 M aqueous calcium chloride solution (OECD TG 106), in buffered aqueous media at pH 4, 7 and 9 (OECD TG 111) and in algae dilution water (OECD TG 201) by LC-MS

Key result

Conc. based on:

test mat.

Loading of aqueous phase:

100 mg/L

Remarks on result:

other: Determination of stability in aqueous solutions

Remarks:

Water solubility was not determined due to hydrolytical instability

The stability of the test item is limited due to hydrolysis in aqueous environment. Decline rates and half-lives were determined in 0.01 M CaCl2 solution and in buffer solutions at pH 4, 7 and 9.

Hydrolysis in 0.01 M CaCl2

Two replicates were measured. The reaction rate constant k was determined to be 5.01E-4 1/s and half-life t1/2 was determined to be 0.4 h.

Hydrolysis in Buffer Solutions at pH 4, 7 and 9

The reaction rate constants k were determined to be 3.24 E-3 1/s for pH 4, 7.49 E-4 1/s for pH 7 and 2.70 E-4 1/s for pH 9. The half-life t1/2 was determined to be 0.0594 h for pH 4, 0.257 h for pH 7 and 0.712 h for pH 9.

Table3:      Determination of Hydrolysis in Aqueous Phase –0.01 M CaCl₂

Nominal concentration, test item: 10 mg/L

0.01 M CaCl2
Time [h]	Mean c [mg/L]	ln c	%
0.00	10.2	2.32	-
0.5	5.61	1.73	55
1	3.10	1.13	31
1.5	1.11	0.105	11
2	0.251	-1.38	2

Mean c = mean concentration of two test item replicates, internal standard taken into account

% = percent of initial concentration, t0 set to 100%

Table4:      Hydrolysis in Aqueous Phase –0.01 M CaCl₂

0.01 M CaCl2
Slope of regression graph	-1.80
Correlation factor [r^2]	0.956
Reaction rate constantk[1/s]	5.01 ∙ 10^-4
Half-lifeT½[h]	0.4

 

Table5:      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%

Table6:      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

 

Table7:      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

 

Table8:    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

Table9:    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%

 

Table10:    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

Conclusions:

The water solubility of Tetrahydrofolic acid could not be determined due to fast hydrolysis of the substance in aqueous solutions. 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

- non-GLP study, determination of stability of Tetrahydrofolic acid in aqueous solutions, 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, breakdown products: 2-amino-6-methylpteridin-4(1H)-one and (2S)-2-(4-aminobenzamido)pentanedioic acid

Additional information

The water solubility of Tetrahydrofolic acid could not be determined due to fast hydrolysis of the substance in aqueous solutions. 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. Based on these results Tetrahydrofolic acid is considered to be not stable in aqueous solutions and testing of water solubility is scientifically not justified.