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Hydrolysis

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Reference
Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 Jun 2016 to 28 Nov 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Version / remarks:
April, 2004
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
- Name of test material (as cited in study report): zinc bis(diethyldithiocarbamate)
- Batch number: 50724904
- Purity: 99.12%
- Date of reception in the test facility: 19 October 2015
- Expiration date: 31 October 2019
- Internal identification: 0066-15
- Appearance: White powder
- Water solubility: 1 mg/L according to the SDS
- Relative density: 1480 g/cm3 according to the SDS
- Storage conditions: Well-ventilated place, ambient temperature and obscurity
Radiolabelling:
no
Analytical monitoring:
yes
Details on sampling:
- Sampling intervals for the parent/transformation products: see tables in ‘any other information on results incl. tables’
- Sampling method: For each pH tested (pH7 and pH9), a freshly prepared solution at 10 mg/L of test item in pH medium was directly distributed in Head-space vials of 10 ml, with a volume of solution of 5 ml per vial. Each vial was hermetically sealed with septum plugs before being placed for hydrolysis in an oven (setpoint 50°C). After 24h at 50°C (for pH7) and 72h at 50°C (for pH9), samples were submitted to FI/MS, HS/GC/MS and GC/MS analysis. For each pH testing, a solution of the test substance in pH medium close to 10 mg/l was freshly prepared the day of analysis, corresponding to blank samples.
- Sampling intervals/times for pH measurements: 0 and 5 days (preliminary test); 0, 3.5, 21.5, 23.5, 26.2, 28.0, 44.9, 48.9, 51.9, 70.0, 76.0 hours (definite test, pH 7), 0, 3.0, 6.0, 21.5, 24.0, 30.0, 45.5, 48.0, 53.0, 71.0, 78.0. 95.0, 100.3, 167.0 197.0, 221.0, 245.0, 269.0, 341.0, 366.0, 436.0, 509.0, 581.0, 720.4 hours (definite test, pH 9)
Buffers:
The definitive test was carried at two pH values: 7 and 9. The corresponding buffer solutions were prepared using reagent grade chemicals and ultra-pure water as follows:
- pH 7: Potassium dihydrogen phosphate (20.42 g) was mixed with 1M sodium hydroxide (88.8 mL) and made to volume (3L) with ultra-pure water. The pH was measured as 7.0.
- pH 9: Boric acid (9.28 g) and potassium chloride (11.18 g) were mixed with 1M sodium hydroxide (63.9 mL) and made to volume (3L) with ultra-pure water. The pH was measured as 9.0.
Estimation method (if used):
- Calculations were performed with Microsoft Excel.
- Graphs of the common logarithm of the concentration versus time were plotted for each test and the rate constant and hal-life calculated as: kobs = -slope x 2.303 and Half-life (t1/2) = Ln(2) / kobs, using t1/2 = hydrolysis half-life (hour) and kobs = hydrolysis rate constant (hour-1).
- By plotting the natural logarithm of the rate constants against the reciprocal of the temperature (K), further estimates of the rate constant and half-lives at 25°C were obtained by interpolation using the Arrhenius equation: Ln (K) = f(1/T)
- The first order rate constant and the half-life were calculated from values where degradation is between about 10% and 90%.
Details on test conditions:
PROCEDURE
As the test item has a low water solubility and is known to be unstable in the test conditions, it falls into the category of a "difficult substance" as defined by the OECD Guidance Document No 23 (OECD, 2000); the method of preparation was thus based on the recommendations of this document

PREPARATION OF THE TEST SOLUTIONS (PRELIMINARY TEST, TIER I)
- The preliminary test was carried at three pH values: 4, 7 and 9.
- The test item was dissolved into each buffer solutions (which had been purged with nitrogen) as follows: 5.0 mg of test item were mixed in 500 mL volumetric flasks filled with each buffer (loading rate of 10 mg/L). The aqueous phases were kept under stirring during approximately 1 hour to achieve the saturation concentration. Non-dissolved particles were removed per filtration through a 0.45 μm membrane filter, then test solutions were diluted with buffer solutions to get the required test solutions for hydrolysis test (i.e. half the saturation concentration).
- Samples pH and incubation temperature were monitored over the period of the test.

PREPARATION OF THE TEST SOLUTIONS (DEFINITE TEST, TIER II)
- The test item was dissolved into pH 7 and 9 buffer solutions (which had been purged with nitrogen) as follows: 10.0 mg of test item were mixed in 1000 mL volumetric flasks filled with each buffer (loading rate of 10 mg/L). The aqueous phases were kept under stirring during approximately 1 hour to achieve the saturation concentration. Non-dissolved particles were removed per filtration through a 0.45 μm membrane filter, then test solutions were diluted with buffer solutions to get the required test solutions for hydrolysis test (i.e. half the saturation concentration).
- Two replicate flasks were prepared for each buffer and all test vessels (including a buffer control flask) were incubated at 15, 37 and 50°C: at each sampling time, flasks were removed from the oven and an aliquot of each flask was sampled then analysed

TEST SYSTEM
- Sterilisation method: The last analysed sample at each pH and temperature was checked for sterility with the help of a microscopic observation: no sample contained microorganisms and only small particles were observed.
- Measures taken to avoid photolytic effects: All test vessels were protected from light until sampling was required.
- Measures to exclude oxygen: the buffers were purged with nitrogen
- Details on test procedure for unstable compounds: The higher Tier test was performed at the pH values at which the test substance was found unstable as defined by the preliminary test. Most hydrolysis reactions follow apparent first order reaction rates and therefore, half-lives are independent of concentration. To study the first-order behaviour, each reaction solution should be analysed in time intervals which provide a minimum of six spaced data points (in duplicate) normally between 10% and 90% hydrolysis, with a maximum duration of 30 days. The definitive test was carried at two pH values: 7 and 9.
- If no traps were used, is the test system closed/open: Each vial was hermetically sealed with septum plugs before being placed for hydrolysis in an oven (setpoint 50°C).
Duration:
5 d
pH:
4
Temp.:
50 °C
Remarks:
Tier I, concentration lower than the Detection Limit of the analytical method
Duration:
5 d
pH:
7
Temp.:
50 °C
Initial conc. measured:
0.53 mg/L
Remarks:
Tier I
Duration:
5 d
pH:
9
Temp.:
50 °C
Initial conc. measured:
0.75 mg/L
Remarks:
Tier I
Duration:
51.9 h
pH:
7
Temp.:
15 °C
Initial conc. measured:
0.81 mg/L
Remarks:
Tier II
Duration:
48.9 h
pH:
7
Temp.:
37 °C
Initial conc. measured:
0.81 mg/L
Remarks:
Tier II
Duration:
44.9 h
pH:
7
Temp.:
50 °C
Initial conc. measured:
0.81 mg/L
Remarks:
Tier II
Duration:
720.4 h
pH:
9
Temp.:
15 °C
Initial conc. measured:
0.33 mg/L
Remarks:
Tier II
Duration:
341 h
pH:
9
Temp.:
37 °C
Initial conc. measured:
0.33 mg/L
Remarks:
Tier II
Duration:
100.3 h
pH:
9
Temp.:
50 °C
Initial conc. measured:
0.33 mg/L
Remarks:
Tier II
Number of replicates:
2
Positive controls:
no
Negative controls:
yes
Remarks:
buffer control
Preliminary study:
At pH 4 the test item was not detected at T0 and T5 days which indicates that test item is not soluble in this buffer or is very quickly hydrolysed. From these hypothesis and with agreement of the Study Monitor it was decided to not assess the test item hydrolysis at pH 1.2 as well as to not conduct the Tier II hydrolysis test at this pH. The mean loss of test item, compared to T0, after 5 days was determined to have changed by more than 10% for pH 7 and 9. This indicated that the test item was clearly hydrolytically unstable at these pH and significant hydrolysis had occurred. Definitive test was thus performed at pH 7 and 9.
Transformation products:
yes
No.:
#1
Details on hydrolysis and appearance of transformation product(s):
On the HS/GC/MS chromatographic profiles of the test substance after hydrolysis, the only hydrolysis product weakly detected in both pH 7 and pH 9 medium is carbon disulphide (CS2). No more hydrolysis product were found even when extracting characteristic ions from the mass spectrum of expected hydrolysis products (Dieethyldithiocarbamic acid; CAS 147-84-2, Carbon oxysulfide; CAS 463-58-1, (Iso-)thiocyanic acid; CAS 463-56-9 and N,N-diethylformamide; CAS 617-84-5).
Remarks on result:
not measured/tested
Key result
pH:
7
Temp.:
25 °C
Hydrolysis rate constant:
0.091 h-1
DT50:
7.61 h
Type:
(pseudo-)first order (= half-life)
Key result
pH:
9
Temp.:
25 °C
Hydrolysis rate constant:
0.005 h-1
DT50:
135.31 h
Type:
(pseudo-)first order (= half-life)
pH:
7
Temp.:
15 °C
Hydrolysis rate constant:
0.05 h-1
DT50:
14 h
Type:
(pseudo-)first order (= half-life)
pH:
7
Temp.:
37 °C
Hydrolysis rate constant:
0.118 h-1
DT50:
5.89 h
Type:
(pseudo-)first order (= half-life)
pH:
7
Temp.:
50 °C
Hydrolysis rate constant:
0.94 h-1
DT50:
0.74 h
Type:
(pseudo-)first order (= half-life)
pH:
9
Temp.:
15 °C
Hydrolysis rate constant:
0.002 h-1
DT50:
300.91 h
Type:
(pseudo-)first order (= half-life)
pH:
9
Temp.:
37 °C
Hydrolysis rate constant:
0.011 h-1
DT50:
65.42 h
Type:
(pseudo-)first order (= half-life)
pH:
9
Temp.:
50 °C
Hydrolysis rate constant:
0.044 h-1
DT50:
15.59 h
Type:
(pseudo-)first order (= half-life)
Details on results:
The determined concentrations of the test substance throughout the hydrolysis test at pH7 and pH 9 are provided in a data table in 'Any other information on results incl. tables'.

TEST CONDITIONS
- pH, sterility, temperature, and other experimental conditions maintained throughout the study: Yes

TRANSFORMATION PRODUCTS
Whatever the analytical technic used, the only hydrolysis product which was significantly detected in both pH 7 and pH 9 medium is carbon disulphide (CS2). CS2 quantification using HS/GC/MS results are presented in 'Any other information on results incl. tables'. If CS2 results are converted to test item equivalent (1 mole test substance _ 2 mole CS2), it can be concluded that, looking at results concerning freshly prepared samples, almost the totality ZDEC is hydrolysed at pH 7 or pH 9 during the equibration time of the Head-Space analyser.

Table: Definitive test at pH 7 at 15 °C: test substance hydrolysis

Sampling time (hours)

Determined conc. (mg/L)

*% loss a

*% loss b

*% loss mean

Buffer control

Replicate a

Replicate b

Mean

0

<DL

0.81

0.81

0.81

0

0

0

3.50

<DL

0.69

0.65

0.67

14.9

19.4

17.1

21.50

<DL

0.15

0.18

0.17

81.4

77.7

79.5

26.20

<DL

0.22

0.19

0.21

72.7

76.4

74.6

28.00

<DL

0.14

0.16

0.15

82.6

80.1

81.4

44.90

<DL

0.09

0.08

0.09

88.8

90.1

89.5

48.90

<DL

0.07

0.07

0.07

91.3

91.3

91.3

51.90**

<DL

0.1

0.05

0.08

87.6

93.8

90.7

*% loss: compared to the test item concentration measured at T0

ND Not Determined

< DL (1.0 μg/L): concentration lower than the Detection Limit of the analytical method

< QL (3.3 μg/L): concentration lower than the Quantification Limit of the analytical method

**: sampling time not retained for calculations

 

Table: Definitive test at pH 7 at 37 °C: test substance hydrolysis

Sampling time (hours)

Determined conc. (mg/L)

*% loss a

*% loss b

*% loss mean

Buffer control

Replicate a

Replicate b

Mean

0

<DL

0.81

0.81

0.81

0.0

0.0

0.0

3.50

<DL

0.19

0.21

0.20

76.4

73.9

75.2

21.50

<DL

0.05

0.04

0.05

93.8

95.0

94.4

23.50**

<DL

0.02

0.01

0.02

97.5

98.8

98.1

26.20**

<DL

0.02

0.01

0.02

97.5

98.8

98.1

28.00**

<DL

0.02

0.01

0.02

97.5

98.8

98.1

44.90**

<DL

0.02

0.01

0.02

97.5

98.8

98.1

48.90**

<DL

0.01

0.01

0.01

98.8

98.8

98.8

*% loss: compared to the test item concentration measured at T0

ND Not Determined

< DL (1.0 μg/L): concentration lower than the Detection Limit

< QL (3.3 μg/L): concentration lower than the Quantification Limit

**: sampling time not retained for calculations

 

Table: Definitive test at pH 7 at 50 °C: test substance hydrolysis

Sampling time (hours)

Determined conc. (mg/L)

*% loss a

*% loss b

*% loss mean

Buffer control

Replicate a

Replicate b

Mean

0

<DL

0.81

0.81

0.81

0.0

0.0

0.0

3.50

<DL

0.03

0.03

0.03

96.3

96.3

96.3

21.50**

<DL

<QL

<QL

NA

NA

NA

NA

23.50**

<DL

<QL

<QL

NA

NA

NA

NA

26.20**

<DL

<QL

<QL

NA

NA

NA

NA

28.00**

<DL

<QL

<QL

NA

NA

NA

NA

44.90**

<DL

<DL

<DL

NA

NA

NA

NA

*% loss: compared to the test item concentration measured at T0

ND Not Determined

< DL (1.0μg/L): concentration lower than the Detection Limit

< QL (3.3μg/L): concentration lower than the Quantification Limit

**: sampling time not retained for calculations

 

 

Table: Definitive test at pH 9 at 15 °C: test substance hydrolysis

Sampling time (hours)

Determined conc. (mg/L)

*% loss a

*% loss b

*% loss mean

Buffer control

Replicate a

Replicate b

Mean

0.0

<DL

0.33

0.33

0.33

0.0

0.0

0.0

3.0**

<DL

0.22

0.21

0.22

33.3

36.4

34.8

6.0

<DL

0.27

0.44

0.36

18.2

-33.3

-7.6

21.5

<DL

0.31

0.45

0.38

6.1

-36.4

-15.2

24.0

<DL

0.37

0.62

0.50

-12.1

-87.9

-50.0

95.0

<DL

0.20

0.31

0.26

39.4

6.1

22.7

100.3

<DL

0.22

0.34

0.28

33.3

-3.0

15.2

167.0

<DL

0.19

0.27

0.23

42.4

18.2

30.3

197.0

<DL

0.18

0.21

0.20

45.5

36.4

40.9

221.0

<DL

0.15

0.17

0.16

54.5

48.5

51.5

341.0

<DL

0.12

0.13

0.13

63.6

60.6

62.1

436.0

<DL

0.13

0.15

0.14

60.6

54.5

57.6

509.0

<DL

0.09

0.14

0.12

72.7

57.6

65.2

581.0

<DL

0.13

0.09

0.11

60.6

72.7

66.7

720.4

<DL

0.1

0.06

0.08

69.7

81.8

75.8

*% loss: compared to the test item concentration measured at T0

ND Not Determined

< DL (0.5 μg/L): concentration lower than the Detection Limit

< QL (1.6 μg/L): concentration lower than the Quantification Limit

**: suspected outlier: not used in further calculations.

 

Table: Definitive test at pH 9 at 37 °C: test substance hydrolysis

Sampling time (hours)

Determined conc. (mg/L)

*% loss a

*% loss b

*% loss mean

Buffer control

Replicate a

Replicate b

Mean

0.0

<DL

0.33

0.33

0.33

0.0

0.0

0.0

3.0

<DL

0.15

0.19

0.17

54.5

42.4

48.5

6.0

<DL

0.26

0.24

0.25

21.2

27.3

24.2

21.5

<DL

0.24

0.22

0.23

27.3

33.3

30.3

24.0

<DL

0.21

0.28

0.25

36.4

15.2

25.8

95.0

<DL

0.10

0.13

0.12

69.7

60.6

65.2

100.3

<DL

0.10

0.11

0.11

69.7

66.7

68.2

167.0

<DL

0.03

0.04

0.04

90.9

87.9

89.4

197.0

<DL

0.02

0.05

0.04

93.9

84.8

89.4

221.0

<DL

0.03

0.03

0.03

90.9

90.9

90.9

341.0**

<DL

0.01

0.02

0.01

97.3

93.9

95.6

*% loss: compared to the test item concentration measured at T0

ND Not Determined

< DL (0.5 μg/L): concentration lower than the Detection Limit

< QL (1.6 μg/L): concentration lower than the Quantification Limit

**: sampling time not retained for calculations

 

Table: Definitive test at pH 9 at 50 °C: test substance hydrolysis

Sampling time (hours)

Determined conc. (mg/L)

*% loss a

*% loss b

*% loss mean

Buffer control

Replicate a

Replicate b

Mean

0.0

<DL

0.330

0.330

0.33

0.0

0.0

0.0

3.0

<DL

0.270

0.240

0.26

18.2

27.3

22.7

6.0

<DL

0.250

0.270

0.26

24.2

18.2

21.2

21.5

<DL

0.250

0.210

0.23

24.2

36.4

30.3

24.0

<DL

0.140

0.100

0.12

57.6

69.7

63.6

45.5

<DL

0.050

0.040

0.05

84.8

87.9

86.4

48.0

<DL

0.050

0.050

0.05

84.8

84.8

84.8

71.0

<DL

0.015

0.011

0.01

95.5

96.7

96.1

78.0**

<DL

0.011

0.008

0.01

96.7

97.6

97.1

95.0**

<DL

0.004

0.003

0

98.8

99.1

98.9

100.3**

<DL

0.005

0.002

0

98.5

99.4

98.9

*% loss: compared to the test item concentration measured at T0

ND Not Determined

< DL (0.5 μg/L): concentration lower than the Detection Limit

< QL (1.6 μg/L): concentration lower than the Quantification Limit

**: sampling time not retained for calculations

Table: CS2 quantification results using HS/GC/MS 

Reference

CS2 (mg/L)

Test substance equivalent

pH 7

pH 9

pH 7

pH 9

Standard test substance 10 mg/L freshly prepared (ACN / Medium - 50/50)

5.6

3.8

13

9.0

Sample test substance after hydrolysis (medium / ACN - 50/50)

0.69

3.03

1.6

7.2

 

Validity criteria fulfilled:
yes

Description of key information

The hydrolysis of zinc bis(diethyldithiocarbamate) is fast, a half-life of 7.61 h (i.e. 0.3 d) at a pH of 7 and 25 ºC is used in the assessment.

Key value for chemical safety assessment

Half-life for hydrolysis:
7.61 h
at the temperature of:
25 °C

Additional information

The hydrolysis rate of the test substance as a function of pH was determined in a study according to OECD TG 111 and in compliance with GLP.

The hydrolysis of the test item was assessed in aqueous buffers at pH 4, 7 and 9 at 50°C. The mean loss of test item, compared to T0, after 5 days was determined to have changed by more than 10% for pH 7 and 9. This indicated that the test item was clearly hydrolytically unstable at all pH and significant hydrolysis had occurred. At pH 4 the test item was not detected at T0 and T5 days which indicates that test item is not soluble in this buffer or is very quickly hydrolysed. From these hypothesis it was decided to not assess the test item hydrolysis at pH 1.2 as well as to not conduct the Tier II hydrolysis test at this pH. The definitive test was thus conducted at pH 7 and 9 (15, 37 and 50°C).

In the definite test a loading rate of 10 mg/L test substance was dissolved in pH 7 and pH 9 buffer solutions at 15, 37 and 50 °C in the dark. Two replicate flasks were prepared for each treatment. At each sampling time, flasks were removed from the oven and an aliquot of each flask was sampled and analysed for determination of test item stability and screening of hydrolysis products (FI/MS, HS/GC/MS and GC/MS techniques). Sampling continued until 90 % hydrolysis of the test substance was observed or after 30 days (720 hours). The last analysed sample at each pH and temperature was checked for sterility with the help of a microscopic observation. The half-life values (hydrolysis rate constant) of the test item were determined to be 14.00 (0.050 h-1), 5.89 (0.118 h-1) and 0.74 (0.940 h-1) hours at 15, 37 and 50 °C, respectively, at pH 7 and 300.91 (0.002 h-1), 65.42 (0.011 h-1) and 15.59 (0.044 h-1) hours at 15, 37 and 50 °C, respectively, pH 9. Based on these findings, The half-life of the test item at pH 7.0 and 25°C was determined to be 7.61 hours (0.3 days). The half-life of the test item at pH 9.0 and 25°C was determined to be 135.31 hours (5.6 days).

Whatever the analytical technique used, the only hydrolysis product which was significantly detected in both pH 7 and pH 9 medium is carbon disulphide (CS2). Converting CS2results to test item equivalent in fresh samples and samples after hydrolysis, it can be concluded that almost the totality of the test substance is hydrolysed at pH 7 or pH 9 during the equilibration time of the Head-Space analyser.

It is well-known that dithiocarbamates exhibits a high propensity to chelate metal ions. The FI/MS and UPLC/MS results are proving that the dissociation of the test substance (and ligand exchanges) in solution can be very fast. But according to the high level of CS2 measured, showing that almost all test substance is rapidly converted, it is likely that the decrease of the test substance concentration at pH7 and pH9 along time, is reflecting not only the dissociation of the molecule in solution but also other chemical reactions.