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Physical & Chemical properties

Water solubility

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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 [r2]

0.956

Reaction rate constantk[1/s]

5.01 ∙ 10-4

Half-life[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 [r2]

0.923

Reaction rate constantk[1/s]

3.24 ∙ 10-3

Half-life[h]

0.0594

Half-life[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

0.3651)

52813611)

15.51)

91)

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 [r2]

0.997

Reaction rate constantk[1/s]

7.49 ∙ 10-4

Half-life[h]

0.257

Half-life[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 [r2]

0.977

Reaction rate constantk[1/s]

2.70 ∙ 10-4

Half-life[h]

0.712

Half-life[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

Key value for chemical safety assessment

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.