Chlorothalonil

Hydrolysis

Link to relevant study record(s)

Reference

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 Dec 2011 to 03 Mar 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Version / remarks:

adopted 13th April 2004

Deviations:

yes

Remarks:

This deviations were deemed to have no impact on the overall study results. For details see Any other information on materials and methods incl tables

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.2120 (Hydrolysis of Parent and Degradates as a Function of pH at 25°C)

Version / remarks:

October 2008

Qualifier:

according to guideline

Guideline:

other: Regulation (EC) No. 1107/2009

Version / remarks:

21 October 2009

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

yes

Analytical monitoring:

yes

Details on sampling:

- The definitive sampling regimes are summarised in Table 4 in “Any other information on materials and methods incl tables”.

Buffers:

- Hydrolysis was performed at pH 4, 7 and 9. The buffer solutions were used can be fouond in Table 1 in “Any other information on materials and methods incl tables”.

Details on test conditions:

STUDY DESIGN
- The experimental design is summarised in Table 2 in “Any other information on materials and methods incl tables”.

PREPARATION OF TEST SYSTEMS
To minimize the process of microbial degradation during incubation, the buffer solutions were filter sterilised to 0.2 µm. Appropriate equipment (such as volumetric flasks, glass jars and hydrolysis incubation vessels) was autoclaved before use. Other materials such as vial caps were rinsed with isopropanol before use. Sterilized material was kept in a sterile bench until use. All treatments were performed in a sterile fumehood under laminar flow conditions. Buffer solution (ca 10 g, except for the first preliminary study where ca 5 g was used) was dispensed into each of the autoclaved hydrolysis vessels.

RADIOCHEMICAL PURITY
The radiochemical purity of the treatment solutions was determined by HPLC and TLC.

PREPARATION OF TREATMENT SOLUTION
Due to a limited amount [14C]-substance, a stock solution of [14C]-substance was prepared from stock solutions prepared. The stock solution was transferred to a 25 mL volumetric flask and made to volume with acetonitrile. Six 10 µL aliquots from the stock solution were analysed by LSC to determine radioactive content and homogeneity. The stock solution concentration was 0.878 mg/mL (Stock Solution 1). A second stock solution of [14C]-substance was prepared by dissolving 1.64 mg in 20 mL acetonitrile. Six 10 µL aliquots were analysed by LSC to determine radioactive content and homogeneity. This yielded a stock solution with concentration 0.130 mg/mL (Stock Solution 2). Further dilutions were carried out as detailed in Table 3 in “Any other information on materials and methods incl tables”.

APPLICATION OF 14C-SUBSTANCE
- Test vessels were sterilised by autoclaving. Treatment was conducted in an aseptic environment under reduced lighting. The total weight of each test vessel was recorded. Sterilised buffer (10 mL) was added to 96 vessels for each pH (16 for each pH and temperature, including 2 contingency samples) and the headspace purged with argon gas. An aliquot of test solution was added to each sample to give a final nominal test concentration of 0.4 mg/L in buffer. The actual amount of test item added to each test vessel was determined by taking aliquots of the treatment solution into volumetric flasks (mock doses) at the start and at regular intervals throughout the dosing period. Each flask was made to volume using acetonitrile and triplicate aliquots taken to assess radioactive concentration by LSC. Control samples were used for each pH to test sterility and to allow pH measurement at the beginning and end of the incubation period. Two flasks for each buffer and label were analysed immediately following treatment and remaining flasks were incubated in a water bath at 50, 60 or 70ºC for pH 7 samples and 15, 25 or 35ºC for pH 9 samples, for 0, 1, 3, 7, 14, 21 or 30 days (28 days for 60ºC).

INCUBATION, MONITORING OF TEMPERATURE AND PH MEASUREMENT
- Samples were placed in a water bath maintained at 15, 25, 35, 50, 60 or 70ºC, in the dark. The temperature was monitored using a digital thermometer with values periodically recorded throughout the incubation period and recorded manually. The pH values for control samples were periodically checked using a Jenway 3510 pH meter.

DEGRADATION RATE (DEGT50 AND DEGT90) FOR SUBSTANCE
-The degradation rate (DegT50 and DegT90) of substance was determined using non-linear regression and a single first order kinetic model (SFO, CAKE, Version 1.4). Appropriate administrative and procedural controls were implemented to assure quality and integrity of the data. The amount of substance remaining was calculated using HPLC data generated from analysis of representative samples at each pH and temperature.

Duration:

30 d

pH:

7

Temp.:

50 °C

Initial conc. measured:

0.412 mg/L

Duration:

30 d

pH:

7

Temp.:

60 °C

Initial conc. measured:

0.411 mg/L

Duration:

30 d

pH:

7

Temp.:

70 °C

Initial conc. measured:

0.4 mg/L

Duration:

30 d

pH:

9

Temp.:

15 °C

Initial conc. measured:

0.448 mg/L

Duration:

30 d

pH:

9

Temp.:

25 °C

Initial conc. measured:

0.436 mg/L

Duration:

30 d

pH:

9

Temp.:

35 °C

Initial conc. measured:

0.421 mg/L

Number of replicates:

2

Positive controls:

no

Negative controls:

yes

Remarks:

sterility controls

Statistical methods:

The kinetic evaluations and the statistical calculations for the quality checks were implemented using the numerical software routines from R version 2.12 via CAKE version 1.4. The Single first-order model was used.

Preliminary study:

The preliminary tests showed that the substance was unstable at pH 7 and 9.

Transformation products:

yes

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

IDENTIFICATION OF DEGRADATES
- M15 and M16: Analysis of the extracted ion chromatogram for m/z = 244.9082 in the samples shows the presence of a minor component with this mass with retention time ca 27.2 min. This is postulated to be a positional isomer of M4 and is observed in the radio chromatograms of several samples. The full scan MS spectra for this component in the samples were consistent with that of M4. Typical accurate mass measurement of the samples corresponded well (mass error 3.7 ppm) for the molecular formula C8HCl3N2O. Analysis of the extracted ion chromatogram for m/z = 244.9082 in the samples shows the presence of a minor component with this mass with retention time ca 32.9 min. This is postulated to be a M16 and is observed in the radio chromatograms of several samples. The full scan MS spectra for this component in the samples were consistent with that of M4. Typical accurate mass measurement of the samples corresponded well (mass error 2.0 ppm) for the molecular formula C8HCl3N2O. M16 has the hydroxyl group ortho to a cyano group and is likely to exhibit hydrogen bonding between these groups. It is postulated that this isomer would have a similar polarity to the reference standard M4 and elute in a similar region of the chromatogram. M15, where the hydroxyl group is meta to a cyano group, would likely have more polar properties which is shown by the earlier elution than M4 and M16.
- M14: Analysis of the extracted ion chromatogram for m/z = 300.9097 in the samples shows the presence of a component with this mass with retention time ca 16.7 min. This is postulated to be a M14 and is observed in the radio chromatograms of several samples. Typical accurate mass measurement of the samples corresponded well (mass error 2.7 ppm) for the molecular formula C8H4Cl4N2O2.

DEGRADATION PATHWAY
- The principal route of degradation was by hydrolysis of a cyano group to yield the amide (M13) with subsequent oxidation/hydrolysis to form the M14. Substitution of chlorine for oxygen occurred to form M4 and 2 of its positional isomers.
- Key Study Dates: A list of key study dates was recorded.

% Recovery:

98.3

St. dev.:

3.5

pH:

7

Temp.:

50 °C

Duration:

30 d

Remarks on result:

other: Based on total mass balance in the definitive study

% Recovery:

98.6

St. dev.:

1.8

pH:

7

Temp.:

60 °C

Duration:

30 d

Remarks on result:

other: Based on total mass balance in the definitive study

% Recovery:

100

St. dev.:

2.4

pH:

7

Temp.:

70 °C

Duration:

30 d

Remarks on result:

other: Based on total mass balance in the definitive study

% Recovery:

98

St. dev.:

2.4

pH:

9

Temp.:

15 °C

Duration:

30 d

Remarks on result:

other: Based on total mass balance in the definitive study

% Recovery:

99.8

St. dev.:

1.9

pH:

9

Temp.:

25 °C

Duration:

30 d

Remarks on result:

other: Based on total mass balance in the definitive study

% Recovery:

96.7

St. dev.:

2.4

pH:

9

Temp.:

35 °C

Duration:

30 d

Remarks on result:

other: Based on total mass balance in the definitive study

Key result

pH:

7

Temp.:

25 °C

DT50:

1 386 d

Type:

other: Arrhenius equation

pH:

9

Temp.:

25 °C

DT50:

15 d

Type:

other: Arrhenius equation

pH:

7

Temp.:

50 °C

DT50:

29.7 d

Type:

(pseudo-)first order (= half-life)

pH:

7

Temp.:

60 °C

DT50:

6.9 d

Type:

(pseudo-)first order (= half-life)

pH:

7

Temp.:

70 °C

DT50:

2.1 d

Type:

(pseudo-)first order (= half-life)

pH:

9

Temp.:

15 °C

DT50:

83.5 d

Type:

(pseudo-)first order (= half-life)

pH:

9

Temp.:

25 °C

DT50:

11.5 d

Type:

(pseudo-)first order (= half-life)

pH:

9

Temp.:

35 °C

DT50:

3.9 d

Type:

(pseudo-)first order (= half-life)

Details on results:

RADIOCHEMICAL PURITY
The radiochemical purity of the 14C- labeled substance in the treatment solutions was determined by HPLC and TLC before and after application and found to be ≥ 96.7%.

TREATMENT RATE
- The test concentrations for pH 7: 50, 60 and 70C experiments were 0.412-0.413, 0.411 and
0.400-0.410 mg/L, respectively. The test concentrations for pH 9: 15, 25 and 35ºC experiments were 0.448, 0.436 and 0.421 mg/L, respectively.

TEMPERATURE AND PH MEASUREMENTS
- Measured pH values were ± 0.1 of the nominal value in the majority of the samples, however occasionally samples were outside of this limit. No value was greater than ± 0.4 of nominal in any of these samples. There was deemed to be no impact on the study results. Throughout the study the measured temperatures were within ± 0.5ºC of 50ºC for the Tier 1 test. The following average temperatures (1) were recorded for the definitive tests:
- pH 7, 50ºC; 50.0 ± 0.6ºC(2)
- pH 7, 60ºC; 59.8 ± 1.7ºC(2)
- pH 7, 70ºC; 69.9 ± 0.7ºC(3)
- pH 9, 15ºC; 15.0 ± 0.8ºC(2)
- pH 9, 25ºC; 25.0 ± 0.5ºC
- pH 9, 35ºC; 35.0 ± 0.6ºC(2)
(1) Actual temperatures. Max/min temperatures showed a wider range due to variances associated with sampling days and temperature checking.
(2)Temperature out with ± 0.5ºC guideline requirement. Where temperatures were out with acceptance remedial action was taken to bring temperature back to nominal values (eg water bath levels were adjusted). This guideline deviation was deemed to have no impact on the overall study results.
(3) Actual temperature 71.7ºC recorded on 1 occasion and water bath levels were adjusted. This was deemed to have no impact on the study results.

STERILITY
- The absence of turbidity in sterility test samples was visually confirmed showing that the test system remained sterile throughout each incubation (pH and temperature).

MASS BALANCE
The mass balance for each incubation group is summarised in Table 1 in “Any other information on results incl tables”. Full details are provided in Table 2 and Table 3 in “Any other information on results incl tables”.

RADIOACTIVE RESIDUES IN HYDROLYSIS SAMPLES
- HPLC analysis: Samples were submitted for HPLC analysis. Results from HPLC analysis, presented as percent applied radioactivity (% AR), are provided in Table 2 and Table 3 in “Any other information on results incl tables”.
- TLC analysis: Confirmation of identification by co-chromatography was carried out by analysis of selected samples by TLC. A comparison of HPLC versus TLC results shows that data corresponds well between the two analytical techniques.
- pH 7 buffer 50ºC: At zero time, substance represented 102.1% applied radioactivity (%AR) which declined to 50.4% AR after 30 days (all values are the mean value across 2 replicates). The hydrolysis product M4 was detected at zero time, accounting for 2.3% AR and increased to 14.2% AR after 30 days. An isomer of this degradate (labelled as M16) was detected after 14 days (0.5% AR) and accounted for 1.7% AR after 30 days. A degradate identified as M13 was detected after 1 day, accounting for 1.2% AR and increased to 30.4% AR after 30 days. A degradate identified as a M14 was detected accounting for 1.0% AR after 30 days.
- pH 7 buffer 60ºC: At zero time, substance represented 99.9% applied radioactivity (%AR) which declined to 4.2% AR after 28 days. The hydrolysis product M4 was detected at zero time, accounting for 1.7% AR and increased to 29.2% AR after 30 days. An isomer of this degradate (labelled as M15) was detected after 1 day, accounting for 2.6% AR and accounted for 5.0% AR after 21 days, but was not detectable at 30 days. A second isomer (M16) was detected after 3 days (0.8% AR) and accounted for 4.3% AR after 28 days. A degradate identified as M13 was detected after 1 day, accounting for 5.5% AR and increased to 47.8% AR after 28 days. A degradate identified as a M14 was detected after 7 days (1.0% AR) and accounted for 10.8% AR after 28 days. Unidentified components accounted for no greater than 3.4% AR at any timepoint.
- pH 7 buffer 70ºC: At zero time, substance represented 102.9% applied radioactivity (%AR) which declined to 0.3% AR after 14 days and was not detected thereafter. The hydrolysis product M4 was detected at zero time, accounting for 2.1% AR and increased to 31.3% AR after 30 days. An isomer of this degradate (labelled as M16) was detected after 1 day, accounting for 1.5% AR and accounted for 5.1% AR after 30 days. A degradate identified as M13 was detected after 1 day, accounting for 17.2% AR and increased to 46.9% AR after 7 days and then declined to 27.8% after 30 days. A degradate identified as a M14 was detected after 3 days (2.2% AR) and accounted for 37.4% AR after 30 days. An unidentified component accounted for 0.4% AR after 14 days.
- pH 9 buffer 15ºC: At zero time, substance represented 99.0% applied radioactivity (%AR) which declined to 74.1% AR after 30 days. The hydrolysis product M4 was detected at zero time, accounting for 3.6% AR, increasing to 8.3% AR after 21 days and accounting for 7.1% AR after 30 days. An isomer of this degradate (M15) was detected in one replicate after 7 days and accounted for 0.3% AR. A degradate identified as M13 was detected after 1 day, accounting for 0.8% AR and increased to 14.4% AR after 30 days. Unidentified components accounted for no greater than 1.1% AR at any timepoint.
- pH 9 buffer 25ºC: At zero time, substance represented 101.4% applied radioactivity (%AR) which declined to 17.6% AR after 30 days. The hydrolysis product M4 was detected at zero time, accounting for 1.1% AR and increased to 16.6% AR after 30 days. An isomer of this degradate (labelled as isomer 1of R182281) was detected at zero time accounting for 0.3% AR and accounted for 5.8% AR after 30 days. A second isomer (M16) was detected after 3 days (0.8% AR), increasing to 10.2% AR after 21 days and accounted for 2.0% AR after 30 days.A degradate identified as M13 was detected after 1 day, accounting for 4.6% AR and increased to 47.7% AR after 30 days. A degradate identified as a M14 was detected after 7 days (0.3% AR) and accounted for 6.2% AR after 30 days. Unidentified components accounted for no greater than 4.3% AR at any timepoint.
- pH 9 buffer 35ºC: At zero time, substance represented 98.0% applied radioactivity (%AR) which declined to 1.3% AR after 30 days. The hydrolysis product M4 was detected at zero time, accounting for 3.0% AR and increased to 29.8% AR after 30 days. An isomer of this degradate (labelled as M15) was detected in the 7 days sample, accounting for 5.4% AR. M15 was not detected at any other timepoint. A second isomer (M16) was detected after 7 days (2.3% AR), increasing to 3.3% AR after 21 days and accounted for 3.1% AR after 30 days. A degradate identified as M13 was detected after 1 day, accounting for 7.3% AR, increasing to 47.3% AR after 21 days and accounted for 43.4% AR after 30 days. A degradate identified as a M14 was detected after 7 days (3.0% AR) and accounted for 19.3% AR after 30 days. Unidentified components accounted for no greater than 3.6% AR at any timepoint.

MS RESULTS
- Analysis of standards: Retention times, accurate masses, formulae and mass errors for reference standards are recorded.
- CONFIRMATION OF STANDARDS
- Substance: Substance was present in samples with retention time ca 31.3 minutes, however it is thought that the concentration of substance present and possible suppression of ionisation from components in the matrix have resulted in the radical anion for substance not being observable in this instance. Positive confirmation of substance was confirmed via co-chromatography by HPLC and TLC.
- M4: M4 was confirmed in all samples at a retention time of ca 32.2 min. The extracted ion chromatograms (EIC) of the standard were compared against a typical sample with retention times being consistent at ca 31.9 min. The full scan MS and MS2 spectra for the samples were consistent with that of the standard. Typical accurate mass measurement of the samples corresponded well (mass error 0.0 ppm) for the molecular formula C8HCl3N2O.
- M13: M13 was confirmed in samples at a retention time of ca 26.4 min. The extracted ion chromatograms (EIC) of the standard were compared against a typical sample with retention times being consistent at ca 26.1 min. The full scan MS spectra for the samples were consistent with that of the standard. Typical accurate mass measurement of the samples corresponded well (mass error 4.3 ppm) for the molecular formula C8H2Cl4N2O.

HYDROLYTIC DEGT50 OF SUBSTANCE
- The degradation rate (DegT50) of the parent was determined using non-linear regression and a single first order kinetic model (CAKE, Version 1.4). Two replicate data points for each sampling interval were used, thereby generating a single DegT50 estimate for each pH and temperature. Results are provided in Table 4 “Any other information on results incl tables”.
Using the Arrhenius equation, at 25ºC the half-life for substance would be 1386 days at pH 7 and 15 days at pH 9.

Table 1. Mass balance Range

pH	Temperature (ºC)	Mass Balance Range
pH 7	50	Range 94.4 to 107.3% of applied radioactivity - Average 98.3%
	60	Range 95.3 to 102.2% of applied radioactivity - Average 98.6%
	70	Range 97.3 to 106.3% of applied radioactivity - Average 100.2%
pH 9	15	Range 93.9 to 103.3% of applied radioactivity – Average 98.0%
	25	Range 98.1 to 105.9% of applied radioactivity - Average 99.8%
	35	Range 92.9 to 101.3% of applied radioactivity - Average 96.7%

Table 2. Recovery and Characterisation / Identification of Radioactivity from pH 7 Buffer Solutions Incubated in the Definitive Study.

Values expressed as percent of applied radioactivity:

Fraction	Rep.	Incubation time (days)
		0	1	3	7	14	21	30
Incubated at50°C
Substance	A	99.2	93.4	87.0	82.1	70.2	59.3	51.6
	B	104.9	92.7	92.0	82.3	74.3	55.9	49.1
	mean	102.1	93.1	89.5	82.2	72.3	57.6	50.4
M14	A	ND	ND	ND	ND	ND	ND	0.8
	B	ND	ND	ND	ND	ND	ND	1.1
	mean	NA	NA	NA	NA	NA	NA	1.0
M13	A	ND	1.4	6.7	8.7	15.1	22.9	29.4
	B	ND	0.9	7.0	8.3	15.5	27.6	31.3
	mean	NA	1.2	6.9	8.5	15.3	25.3	30.4
M4	A	2.1	2.3	4.7	5.2	8.1	11.5	13.9
	B	2.4	3.0	4.2	6.0	8.2	12.7	14.5
	mean	2.3	2.7	4.5	5.6	8.2	12.1	14.2
M16	A	ND	ND	ND	ND	1.0	1.2	1.7
	B	ND	ND	ND	ND	ND	1.7	1.6
	mean	NA	NA	NA	NA	0.5	1.5	1.7
Total (mass balance)	A	101.3	97.1	98.4	96.0	94.4	94.9	97.4
	B	107.3	96.6	103.2	96.6	98.0	97.9	97.6
Mean ± SD		98.3 ± 3.5
Incubated at60°C
Substance	A	99.0	84.5	69.7	51.8	27.2	13.4	4.8
	B	100.7	88.8	64.7	44.2	25.3	13.7	3.6
	mean	99.9	86.7	67.2	48.0	26.3	13.6	4.2
M14	A	ND	ND	ND	0.7	3.9	5.5	10.4
	B	ND	ND	ND	1.2	3.6	6.1	11.1
	mean	NA	NA	NA	1.0	3.8	5.8	10.8
M13	A	ND	5.6	16.9	27.4	40.6	42.8	46.2
	B	ND	5.4	15.4	25.2	39.7	45.0	49.3
	mean	NA	5.5	16.2	26.3	40.2	43.9	47.8
M15	A	ND	3.6	ND	ND	ND	6.2	ND
	B	ND	1.6	9.8	7.2	3.9	3.7	ND
	mean	NA	2.6	4.9	3.6	2.0	5.0	NA
M4	A	1.9	4.0	10.0	15.8	23.4	24.4	28.6
	B	1.5	3.8	9.2	14.9	22.0	24.3	29.8
	mean	1.7	3.9	9.6	15.4	22.7	24.4	29.2
M16	A	ND	ND	1.5	1.8	2.9	3.3	4.0
	B	ND	ND	ND	1.8	2.8	3.1	4.5
	mean	NA	NA	0.8	1.8	2.9	3.2	4.3
Unidentified Radioactivity (1)	A	ND	ND	ND	ND	ND	4.3	1.3
	B	ND	ND	ND	1.3	1.7	2.5	ND
	mean	NA	NA	NA	0.7	0.9	3.4	0.7
Total (mass balance)	A	100.9	97.7	98.1	97.5	98.0	99.9	95.3
	B	102.2	99.6	99.1	95.8	99.0	98.4	98.3
Mean ± SD		98.6 ± 1.8
Incubated at70°C
Substance	A	104.1	72.7	40.5	10.0	ND	ND	ND
	B	101.7	69.4	39.2	11.6	0.6	ND	ND
	mean	102.9	71.1	39.9	10.8	0.3	NA	NA
M14	A	ND	ND	2.0	8.0	23.8	28.6	36.6
	B	ND	ND	2.4	7.7	22.8	28.0	38.2
	mean	NA	NA	2.2	7.9	23.3	28.3	37.4
M13	A	ND	17.0	35.4	47.0	41.9	36.4	27.8
	B	ND	17.4	35.8	46.7	41.4	38.3	27.8
	mean	NA	17.2	35.6	46.9	41.7	37.4	27.8
M4	A	2.2	9.0	18.4	28.1	29.7	28.5	32.3
	B	2.0	9.7	18.7	29.3	28.6	30.1	30.2
	mean	2.1	9.4	18.6	28.7	29.2	29.3	31.3
M16	A	ND	1.1	3.2	4.2	4.4	4.6	4.9
	B	ND	1.8	3.0	4.5	3.6	4.1	5.2
	mean	NA	1.5	3.1	4.4	4.0	4.4	5.1
Unidentified Radioactivity (2)	A	ND	ND	ND	ND	ND	ND	ND
	B	ND	ND	ND	ND	0.7	ND	ND
	mean	NA	NA	NA	NA	0.4	NA	NA
Total (mass balance)	A	106.3	99.8	99.5	97.3	99.8	98.1	101.6
	B	103.7	98.3	99.1	99.8	97.7	100.5	101.4
Mean ± SD		100.2 ± 2.4

(1)       No individual component present >4.3% AR

(2)       No individual component present >0.7% AR

ND      Notdetected

NA       Notapplicable

 

 

Table 3. Recovery and Characterisation / Identification of Radioactivity from pH 9 Buffer Solutions Incubated in the Definitive Study

Values expressed as percent of applied radioactivity:

Fraction	Rep.	Incubation time (days)
		0	1		3		7		14		21		30
Incubated at15°C
Substance	A	101.6	90.1		86.0		88.7		85.7		75.7		72.7
	B	96.3	94.2		86.2		88.4		85.5		78.6		75.4
	mean	99.0	92.2		86.1		88.6		85.6		77.2		74.1
M13	A	ND	1.5		4.2		4.1		8.4		12.7		14.2
	B	ND	ND		4.0		4.3		8.1		12.5		14.5
	mean	NA	0.8		4.1		4.2		8.3		12.6		14.4
M15	A	ND	ND		ND		ND		ND		ND		ND
	B	ND	ND		ND		0.6		ND		ND		ND
	mean	NA	NA		NA		0.3		NA		NA		NA
M4	A	1.7	4.5		6.0		3.9		4.7		8.4		6.2
	B	5.4	1.8		6.2		3.8		4.8		8.2		8.0
	mean	3.6	3.2		6.1		3.9		4.8		8.3		7.1
Unidentified Radioactivity (1)	A	ND	ND		ND		0.8		ND		1.5		0.8
	B	ND	ND		ND		ND		ND		ND		1.4
	mean	NA	NA		NA		0.4		NA		0.8		1.1
Total (mass balance)	A	103.3	96.1		96.2		97.5		98.8		98.3		93.9
	B	101.7	96.0		96.4		97.1		98.4		99.3		99.3
Mean ± SD		98.0 ± 2.4
Incubated at pH25°C
Substance	A	98.7		92.0		82.3		67.5	40.3		27.7		16.5
	B	104.1		91.8		83.8		65.9	42.0		29.5		18.7
	mean	101.4		91.9		83.1		66.7	41.2		28.6		17.6
M14	A	ND		ND		ND		0.5	2.2		2.8		6.1
	B	ND		ND		ND		ND	1.5		1.7		6.3
	mean	NA		NA		NA		0.3	1.9		2.3		6.2
M13	A	ND		4.7		10.6		22.1	36.7		40.5		46.4
	B	ND		4.4		11.0		21.9	31.2		33.1		48.9
	mean	NA		4.6		10.8		22.0	34.0		36.8		47.7
M15	A	ND		ND		0.9		2.1	3.6		3.7		7.3
	B	0.6		ND		0.8		2.9	4.7		3.5		4.3
	mean	0.3		NA		0.9		2.5	4.2		3.6		5.8
M4	A	1.0		1.8		4.2		6.7	13.1		14.3		16.5
	B	1.2		2.5		4.0		6.1	12.0		12.2		16.6
	mean	1.1		2.2		4.1		6.4	12.6		13.3		16.6
M16	A	ND		ND		1.5		0.5	2.3		6.7		2.0
	B	ND		ND		ND		0.9	5.0		13.7		2.0
	mean	NA		NA		0.8		0.7	3.7		10.2		2.0
Unidentified Radioactivity (2)	A	ND		ND		ND		0.7	1.1		2.4		5.3
	B	ND		ND		ND		0.8	3.1		6.1		3.2
	mean	NA		NA		NA		0.8	2.1		4.3		4.3
Total (mass balance)	A	99.7		98.5		99.5		100.1	99.3		98.1		100.1
	B	105.9		98.7		99.6		98.5	99.5		99.8		100.0
Mean ± SD		99.8 ± 1.9
Incubated at pH35°C
Substance	A	97.6	81.3		56.1		31.2		12.6	5.3		1.2
	B	98.3	82.5		55.2		20.0		13.7	5.3		1.4
	mean	98.0	81.9		55.7		25.6		13.2	5.3		1.3
M14	A	ND	ND		ND		2.7		8.1	13.1		19.0
	B	ND	ND		ND		3.2		7.6	11.9		19.5
	mean	NA	NA		NA		3.0		7.9	12.5		19.3
M13	A	ND	7.5		23.2		37.0		46.6	47.9		42.6
	B	ND	7.0		21.4		40.3		47.7	46.7		44.1
	mean	NA	7.3		22.3		38.7		47.2	47.3		43.4
M15	A	ND	ND		ND		3.0		ND	ND		ND
	B	ND	ND		ND		7.8		ND	ND		ND
	mean	NA	NA		NA		5.4		NA	NA		NA
M4	A	3.0	5.3		13.9		18.0		23.1	25.4		31.0
	B	3.0	4.3		14.6		18.1		22.9	27.5		28.6
	mean	3.0	4.8		14.3		18.1		23.0	26.5		29.8
M16	A	ND	ND		ND		1.9		2.8	3.2		2.8
	B	ND	ND		ND		2.6		2.0	3.3		3.4
	mean	NA	NA		NA		2.3		2.4	3.3		3.1
Unidentified Radioactivity (3)	A	ND	1.0		1.4		2.7		1.5	2.8		1.9
	B	ND	ND		1.7		4.5		2.0	2.1		1.3
	mean	NA	0.5		1.6		3.6		1.8	2.5		1.6
Total (mass balance)	A	100.6	95.1		94.6		96.5		94.7	97.7		98.5
	B	101.3	93.8		92.9		96.5		95.9	96.8		98.3
Mean ± SD		96.7 ± 2.4

(1)       No individual component present >1.5% AR

(2)       No individual component present >4.2 % AR

(3)       No individual component present >2.6% AR

ND      Notdetected

NA       Notapplicable

 

Table 4. Summary of DegT50 Values

Temperature (ºC)	SFO
	Half-Life [days]	DegT90 [days]	K	χ2	R2	Prob > t
pH 7
50	29.7	98.7	0.0233	2.411	0.9715	2.28E-10
60	6.9	23.0	0.1002	3.935	0.9915	3.21E-11
70	2.1	7.1	0.3243	2.499	0.9975	6.67E-9
pH 9
15	83.5	277.3	0.0083	2.801	0.8228	5.19E-6
25	11.5	38.3	0.0601	1.781	0.9962	2.48E-14
35	3.9	13.0	0.1770	4.957	0.9927	2.67E-10

 

Validity criteria fulfilled:

yes

Conclusions:

Based on the findings, the DT50 values in pH 7 buffer were calculated to be 29.7, 6.9 and 2.1 days following incubation at 50, 60 and 70ºC, respectively. The DT50 values in pH 9 buffer were 83.5, 11.5 and 3.9 days following incubation at 15, 25 and 35ºC, respectively. Using the Arrhenius equation, at 25ºC the half-life for substance would be 1386 days at pH 7 and 15 days at pH 9.

Executive summary:

The hydrolysis of [14C]-substance dissolved at a nominal concentration of 0.4 mg/L was investigated at pH 4 (potassium phthalate monobasic), pH 7 (potassium phosphate monobasic) and pH 9 (boric acid) in the dark under sterile conditions at 50ºC for 5 days. Following these preliminary tests substance was shown to be unstable at pH 7 and 9. Further investigations were carried out at pH 7 at 50, 60, and 70ºC and at pH 9 at 15, 25 and 35ºC for 30 days. In each test, duplicate samples were taken for analysis at up to 7 intervals during incubation.

 The radioactivity balance in the definitive test was in a range of 98.0 to 100.2% for all the tests, indicating no loss of volatile products from the test system. At pH 7 substance degraded from 102.1 to 50.4% of the applied radioactivity at 50ºC, 99.9 to 4.2% of the applied radioactivity at 60ºC and from 102.9 to 0% of the applied radioactivity at 70ºC from incubation to test termination. At pH 9 substance degraded from 99.0 to 74.1% of the applied radioactivity at 15ºC, 101.4 to 17.6% of the applied radioactivity at 25ºC and from 98.0 to 1.3% of the applied radioactivity at 35ºC from incubation to test termination. In this study, the major degradates of substance was found to be M13 which reached a maximum of 7.8% of applied radioactivity at test termination for pH 7.0 incubated at 60°C and 47.7% of applied radioactivity at test termination for pH 9.0 incubated at 25°C. In addition, a number of unknown degradates (up to a total of 4.3% of the applied radioactivity in one replicate) were also observed, primarily at pH 9.0. At pH 7, the significant transformation products detected were a M14, M4 and 2 positional isomers of M4 (assigned as M15 and M16). The M14 reached a maximum of 37.4% of the applied radioactivity at test termination. M4 reached a maximum of 31.3% of the applied radioactivity at test termination. M15 reached a maximum concentration of 5.0% of the applied radioactivity after 21 days incubation whilst M16 reached a maximum of 5.1% of the applied radioactivity at test termination. At pH 9, the significant transformation products detected were the same as those observed at pH 7. The M14 reached a maximum of 19.3% of the applied radioactivity observed at test termination. M4 reached a maximum of 29.8% of the applied radioactivity observed at the test termination. M15 reached a maximum of 5.8% of the applied radioactivity at test termination whilst M16 reached a maximum of 10.2% of the applied radioactivity observed after 21 days incubation.

This study demonstrated that the test substance is hydrolytically stable under acidic conditions, but prone to hydrolysis under neutral or basic conditions, especially at higher temperatures. The principal route of degradation was by hydrolysis of a cyano group to yield the M13 with subsequent oxidation/hydrolysis to form the M14. Substitution of chlorine for oxygen occurred to form M4 along with 2 of its positional isomers.

Based on the findings, the DT50 values in pH 7 buffer were calculated to be 29.7, 6.9 and 2.1 days following incubation at 50, 60 and 70ºC, respectively. The DT50 values in pH 9 buffer were 83.5, 11.5 and 3.9 days following incubation at 15, 25 and 35ºC, respectively. Using the Arrhenius equation, at 25ºC the half-life for substance would be 1386 days at pH 7 and 15 days at pH 9.

Description of key information

Hydrolytic DT50 at pH 7at 25 °C = 1386 days (calculated using the arrhenius plot), OECD TG 111, Wicksted et. al 2014

Key value for chemical safety assessment

Half-life for hydrolysis:

1 386 d

at the temperature of:

25 °C

Additional information

The hydrolysis of [14C]-substance dissolved at a nominal concentration of 0.4 mg/L was investigated at pH 4 (potassium phthalate monobasic), pH 7 (potassium phosphate monobasic) and pH 9 (boric acid) in the dark under sterile conditions at 50ºC for 5 days. Following these preliminary tests substance was shown to be unstable at pH 7 and 9. Further investigations were carried out at pH 7 at 50, 60, and 70ºC and at pH 9 at 15, 25 and 35ºC for 30 days. In each test, duplicate samples were taken for analysis at up to 7 intervals during incubation.

The radioactivity balance in the definitive test was in a range of 98.0 to 100.2% for all the tests, indicating no loss of volatile products from the test system. At pH 7 substance degraded from 102.1 to 50.4% of the applied radioactivity at 50ºC, 99.9 to 4.2% of the applied radioactivity at 60ºC and from 102.9 to 0% of the applied radioactivity at 70ºC from incubation to test termination. At pH 9 substance degraded from 99.0 to 74.1% of the applied radioactivity at 15ºC, 101.4 to 17.6% of the applied radioactivity at 25ºC and from 98.0 to 1.3% of the applied radioactivity at 35ºC from incubation to test termination. In this study, the major degradates of substance was found to be M13 which reached a maximum of 7.8% of applied radioactivity at test termination for pH 7.0 incubated at 60°C and 47.7% of applied radioactivity at test termination for pH 9.0 incubated at 25°C. In addition, a number of unknown degradates (up to a total of 4.3% of the applied radioactivity in one replicate) were also observed, primarily at pH 9.0. At pH 7, the significant transformation products detected were a M14, M4 and 2 positional isomers of M4 (assigned as M15 and M16). The M14 reached a maximum of 37.4% of the applied radioactivity at test termination. M4 reached a maximum of 31.3% of the applied radioactivity at test termination. M15 reached a maximum concentration of 5.0% of the applied radioactivity after 21 days incubation whilst M16 reached a maximum of 5.1% of the applied radioactivity at test termination. At pH 9, the significant transformation products detected were the same as those observed at pH 7. The M14 reached a maximum of 19.3% of the applied radioactivity observed at test termination. M4 reached a maximum of 29.8% of the applied radioactivity observed at the test termination. M15 reached a maximum of 5.8% of the applied radioactivity at test termination whilst M16 reached a maximum of 10.2% of the applied radioactivity observed after 21 days incubation.

This study demonstrated that the test substance is hydrolytically stable under acidic conditions, but prone to hydrolysis under neutral or basic conditions, especially at higher temperatures. The principal route of degradation was by hydrolysis of a cyano group to yield the M13 with subsequent oxidation/hydrolysis to form the M14. Substitution of chlorine for oxygen occurred to form M4 along with 2 of its positional isomers.

Based on the findings, the DT50 values in pH 7 buffer were calculated to be 29.7, 6.9 and 2.1 days following incubation at 50, 60 and 70ºC, respectively. The DT50 values in pH 9 buffer were 83.5, 11.5 and 3.9 days following incubation at 15, 25 and 35ºC, respectively. Using the Arrhenius equation, at 25ºC the half-life for substance would be 1386 days at pH 7 and 15 days at pH 9.