pymetrozine (ISO); (E)-4,5-dihydro-6-methyl-4-(3-pyridylmethyleneamino)-1,2,4-triazin-3(2H)-one

Hydrolysis

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

21 Mar 1994 to 10 Feb 1995

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Biologische Bundesanstalt für Land- und Forstwirtschaft (BBA, Germany) Pamphlet No. 55: Testing of the Behaviour of Plant Treatment Agents in Water

Version / remarks:

October 1980

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA Guideline Subdivision N 161-1 (Hydrolysis)

Version / remarks:

October 1982

Deviations:

no

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

14C-labelled at postition 5 of pyridine ring

Analytical monitoring:

yes

Remarks:

pH, LSC, HPLC, MS, GC, TLC

Details on sampling:

- pH 1: 0, 0.5, 1, 2, 3, 6, 24, 48 hours
- pH 5: 0, 3, 6, 10, 15, 20, 25, 30 days
- pH 7: 0, 3, 6, 10, 14, 17, 21, 24, 27, 30 days
- pH 9: 0, 3, 6, 10, 14, 17, 21, 24, 27, 30 days
Duplicate vessels were taken for analysis at each time. Two additional tubes of treated buffer solution were taken for microbiological examination at the start and at the completion of each incubation.

- Storage of samples: Radioassay of each test solution, measurement of pH and quantitative HPLC analysis were all carried out on the day of sampling. Quantitative two-dimensional analysis of selected solutions was also carried out on the day of sampling. Subsequent HPLC and TLC co-chromatography with reference substances, and mass spectrometry, were carried out up to 10 months after sampling. When not being analysed, test solutions were stored at <-20°C. The unlabelled reference standards were also stored, as neat compounds, at < -20°C during the course of the study. In addition to spectroscopic analysis towards the end of the study, their stability was also assessed by chromatographic analysis during the course of the study: in most cases essentially only one HPLC UV peak or TLC UV spot was observed, although by the end of the study M3 also contained a significant proportion of the corresponding acid. The presence of the acid was also apparent from the mass spectrum.

Buffers:

- 0.1M pH 1 buffer solution: 0.1M hydrochloric acid was used.
- 0.01M pH 5 buffer solution: Acetic acid (0.6 mL) was mixed with ca 900 mL water and the pH of the solution adjusted to 5.0 by the addition of sodium hydroxide solution. The solution was made up to a volume of 1 litre with water. The pH of the final solution was checked.
- 0.01M pH 7 buffer solution: Sodium dihydrogen orthophosphate (1.561 g) was dissolved in ca 900 mL water and the pH of the solution adjusted to 7 .0 by the addition of sodium hydroxide solution. The solution was made up to a volume of 1 litre with water. The pH of the final solution was checked.
- 0.01M pH 9 buffer solution: Boric acid (0.618 g) was dissolved in ca 900 mL water and the pH of the solution adjusted to 9.0 by the addition of sodium hydroxide. The solution was made up to a volume of 1 litre with water. The pH of the final solution was checked.

Details on test conditions:

STERILITY PRECAUTIONS
All test vessels, glassware and buffer solutions were sterilised by autoclaving at 121 °C for 15 minutes. Each buffer solution was divided into two portions of equal volume which were autoclaved separately. After cooling, the pH of one portion was measured. No further adjustment of pH was found to be necessary and the other portion of each buffer solution was used for the study. Addition of the radiolabelled test substance solution to the buffer solutions and dispensing of treated buffer solutions into test vessels were carried out in a laminar flow cabinet. Samples of treated buffer solutions were taken for microbiological examination immediately and at the end of the incubation period.

ADDITION OF THE TEST SUBSTANCE TO THE BUFFER SOLUTIONS
The radiolabelled test substance was used as supplied, without any radiodilution. A stock solution of radiolabelled test substance in methanol was prepared at a concentration of 1.229 mg/mL. The radiochemical purity of the radiolabelled test substance in the solution was measured by HPLC prior to addition to the buffer solutions, and was greater than 97 % in each case. Aliquots of the radiolabelled test substance stock solution were added to portions of pH 1.0, pH 5.0, pH 7.0 and pH 9.0 buffer solutions. The concentration of the radiolabelled test substance was accurately determined by the radioassay of triplicate 100 μL aliquots of treated buffer solution. The concentration of the methanol co-solvent was 0.4% by volume. Further aliquots (10 mL) of treated buffer solutions were transferred to pre-weighed glass tubes of 20 mL capacity fitted with Teflon-lined screw caps. The tubes were reweighed. Twenty-two tubes were established for solutions buffered to pH 1 or pH 5, and twenty-six tubes for those buffered to pH 7 or pH 9. Each tube was labelled, using waterproof ink, with the study schedule number, the buffer pH, the date of incorporation of the test substance, and a unique identification number.

INCUBATION OF TEST SOLUTIONS
With the exception of those tubes taken for immediate analysis or microbiological examination, tubes containing treated buffer solution were placed on a rotary shaker either in an incubator (pH 1 experiment) or in a temperature-controlled room (pH 5, pH 7 and pH 9 experiments). The tubes were incubated in darkness while being agitated, at ca 25 °C. Temperatures were recorded at regular intervals (generally daily) using a thermometer located in the room or incubator. Throughout the entire study the mean recorded temperature was 25.1 ° C (± 0.6 ° C). The temperature range was 23.0 - 27.5°C, although only four readings were outside the range 24.5 - 26.0°C. The temperature range in the pH 5 experiment was 24.5 - 25.5°C.

pH MEASUREMENTS
Measurements of the pH of test solutions were made using a model 3030 pH meter. The meter was calibrated prior to each measurement using standard buffer solutions of the appropriate pH, or in the case of pH 1 measurements, 0.1M hydrochloric acid.

Duration:

48 h

pH:

1

Temp.:

25 °C

Initial conc. measured:

5 mg/L

Duration:

30 d

pH:

5

Temp.:

25 °C

Initial conc. measured:

5 mg/L

Duration:

30 d

pH:

7

Temp.:

25 °C

Initial conc. measured:

5 mg/L

Duration:

30 d

pH:

9

Temp.:

25 °C

Initial conc. measured:

5 mg/L

Number of replicates:

Duplicate vessels were taken for analysis at each time.

Positive controls:

no

Negative controls:

no

Test performance:

- Recoveries: Total recoveries of applied radioactivity from incubated test solutions were in almost all cases in the range 97.4 - 100.6%. Each recovery was calculated as the ratio of the radioactivity concentration in the test solution to that in the original treated buffer solution. Changes in the weights of vessels during the incubation periods were negligible (< 1 %) and were not accounted for. The only low recovery of radioactivity (93% AR) was observed in one of the pH 1 solutions taken for analysis after 0.5 hours incubation.
- pH: In all cases the pH of the incubated test solution had not changed significantly from that of the original treated buffer solution (< 0.1 pH unit).
- Temperatures: Recorded temperatures generally did not deviate by more than 1 °C from 25°C (four values were outside these limits, in the range 23.0 - 27.5°C). No microbial contamination was observed in any test vessel.

Transformation products:

yes

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

Both HPLC and TLC analysis of incubated test solutions resolved a single radioactive component in addition to the test substance. This component was present in all incubated solutions and corresponded, in both chromatographic systems, to the reference substance M3. Levels of M3 in incubated test solutions rose to 3 - 4 % (pH 7 and 9), 63 % (pH 5) and 77 % (pH 1) of the initial test solution radioactivity after 30 days. Regions of the HPLC chromatograms apart from the test substance and M3 did not each generally contain more than 3 % test solution radioactivity at any time. Co-chromatographic analyses with reference substances using HPLC and TLC were made up to 10 months after the day of sampling. It was noticed that freezer storage of the test solutions affected the degradate profiles in that there was some reversion of the hydrolysis product back to the test substance. Consequently, chromatograms generated in co-chromatographic analysis differ quantitatively, but not qualitatively, from those produced on the day of sampling.

Chromatographic correspondence of both the test substance and M3 with the radioactive constituents of selected pH 5 10-day and 30-day incubated solutions was confirmed by HPLC. (Although some degradation of the reference standard M3 was also observed during storage resulting in a significant amount of M10, enough M3 was intact to confirm the identity of the degradate by co-chromatography of the test solution with the reference standard). Similarly, correspondence of the test substance with the major radioactive constituent in selected pH 7 and pH 9 30-day incubated solutions was also confirmed. The identities of both the test substance and M3 in a pH 5 incubated solution were further confirmed by mass spectrometry. The spectra of the isolated products were essentially similar to those of the respective reference substances. In each case the formation of M3 mirrored the decline of the test substance.

% Recovery:

98.29

St. dev.:

1.68

pH:

1

Temp.:

25 °C

Duration:

48 h

Remarks on result:

other: % Recovery and St.Dev calculated by registrant

Remarks:

see 'Any other information on results incl. tables' for individual recoveries

% Recovery:

99.74

St. dev.:

0.59

pH:

5

Temp.:

25 °C

Duration:

30 d

Remarks on result:

other: % Recovery and St.Dev calculated by registrant

Remarks:

see 'Any other information on results incl. tables' for individual recoveries

% Recovery:

99.41

St. dev.:

0.53

pH:

7

Temp.:

25 °C

Duration:

30 d

Remarks on result:

other: % Recovery and St.Dev calculated by registrant

Remarks:

see 'Any other information on results incl. tables' for individual recoveries

% Recovery:

99.64

St. dev.:

0.53

pH:

9

Temp.:

25 °C

Duration:

30 d

Remarks on result:

other: % Recovery and St.Dev calculated by registrant

Remarks:

see 'Any other information on results incl. tables' for individual recoveries

Key result

pH:

1

Temp.:

25 °C

DT50:

2.8 h

Type:

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

Remarks on result:

other: Data were fitted to the non-linear exponential function

Key result

pH:

5

Temp.:

25 °C

DT50:

5 d

Type:

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

Remarks on result:

other: Data were fitted to the non-linear exponential function

Key result

pH:

7

Temp.:

25 °C

DT50:

800 d

Type:

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

Remarks on result:

other: Half-life calculated from linear regression of the derived equation: lnC = lnC0 - k*t

Remarks:

These kinetic data must be regarded with caution due to the relatively short duration of the incubations

Key result

pH:

9

Temp.:

25 °C

DT50:

510 d

Type:

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

Remarks on result:

other: Half-life calculated from linear regression of the derived equation: lnC = lnC0 - k*t

Remarks:

These kinetic data must be regarded with caution due to the relatively short duration of the incubations

Other kinetic parameters:

Not reported

Details on results:

HYDROLYSIS OF THE TEST SUBSTANCE
Each test solution was analysed by HPLC on the day of sampling. The method used provided a good separation of the test substance from its single degradation product. In the pH 1 experiment, the pH of the incubated solution was immediately adjusted to ca. pH 7 prior to analysis, in order to halt the expected rapid hydrolysis of the test substance at this pH. The mean proportions of the test substance remaining in test solutions at each pH, as determined by HPLC, are shown in ‘Any other information on results incl. tables’. The rate of disappearance of the test substance was compared to first-order reaction kinetics. At pH values of 7 and 9, the test substance was essentially hydrolytically stable, with losses of only 4 - 5 % the test substance over the 30-day incubation periods. With decreasing pH, hydrolysis was more rapid, with initial rates equivalent to half-lives of 5.0 days at pH 5 and 2.8 hours at pH 1. At these two pH values, the rates of hydrolysis were linear only during the initial period of each incubation and the rates eventually declined to zero, presumably due to the establishment of an equilibrium between the test substance and its initial hydrolysis products, M3 and M2. The equilibrium position varied with pH: at ca 36% the test substance at pH 5 and ca 21 % at pH 1. These results are in good agreement with those obtained in a study of the hydrolysis of the test substance labelled in its triazine ring, albeit in this study the equilibrium position in the pH 5 experiment was reached slightly earlier. Selected pH 1 and pH 5 test solutions were also analysed by two-dimensional TLC. These estimates of the test substance were in reasonable agreement with those obtained by HPLC, although TLC consistently gave slightly higher values (by about 5 % test solution radioactivity) for the test substance (and correspondingly lower values for the degradation product).

Table: Combined results of recovery, measured pH, mean proportions (in %AR and mg/L) at 25°C for pH 1 in duplicate

Time (hours)	Recovery (%)	Measured pH	Proportion of test substance in solution (expressed as % test solution radioactivity)	Proportion of test substance in solution (expressed as mg/L (ppm))
0	NM	0.96(a)	96.7	4.69
	NM	0.96(a)
0.5	99.1	0.97	87.1	4.22
	92.7	1.00
1	98.8	1.02	81.6	3.96
	99.0	0.97
2	99.1	1.00	68.8	3.34
	99.2	0.95
3	98.9	1.07	57.3	2.78
	98.9	1.08
6	99.2	1.02	36.8	1.78
	98.5	0.99
24	98.5	1.01	21.5	1.04
	98.4	1.04
48	98.3	0.93	21.3	1.03
	97.4	0.95

a) pH value of the treated buffer solution

NM = Not Measured

 

Table: Combined results of recovery, measured pH, mean proportions (in %AR and mg/L) at 25°C for pH 5 in duplicate

Time (days)	Recovery (%)	Measured pH	Proportion of test substance in solution (expressed as % test solution radioactivity)	Proportion of test substance in solution (expressed as mg/L (ppm))
0	98.9	5.04	98.1	4.71
	98.6	5.05
3	99.2	5.06	77.7	3.76
	98.9	5.07
6	99.6	5.08	62.1	2.98
	99.8	5.05
10	100.5	5.07	50.9	2.44
	99.4	5.07
15	100.2	5.10	41.5	1.99
	100.1	5.10
20	100.1	5.08	37.5	1.80
	100.1	5.07
25	99.8	4.98	36.5	1.76
	99.9	5.01
30	100.2	5.00	36.1	1.73
	100.6	5.03

 

Table: Combined results of recovery, measured pH, mean proportions (in %AR and mg/L) at 25°C for pH 7 in duplicate

Time (days)	Recovery (%)	Measured pH	Proportion of test substance in solution (expressed as % test solution radioactivity)	Proportion of test substance in solution (expressed as mg/L (ppm))
0	99.1	7.09	98.1	4.70
	99.1	7.06
3	99.0	7.07	97.4	4.67
	98.8	7.06
6	99.7	7.03	97.3	4.66
	99.8	7.07
10	99.4	7.08	96.7	4.63
	99.6	7.08
14	100.0	7.04	96.4	4.62
	99.4	7.03
17	98.4	7.06	96.4	4.62
	98.5	7.06
21	100.2	7.06	96.4	4.62
	98.8	7.06
24	99.2	7.10	95.8	4.59
	99.8	7.07
27	100.2	7.07	95.7	4.58
	99.8	7.01
30	99.7	7.07	95.1	4.56
	99.7	7.08

 

Table: Combined results of recovery, measured pH, mean proportions (in %AR and mg/L) at 25°C for pH 9 in duplicate

Time (days)	Recovery (%)	Measured pH	Proportion of test substance in solution (expressed as % test solution radioactivity)	Proportion of test substance in solution (expressed as mg/L (ppm))
0	99.6	9.02	97.8	4.78
	100.1	8.93
3	98.8	9.00	97.3	4.76
	98.9	9.06
6	100.2	8.98	96.8	4.73
	99.9	9.06
10	100.0	8.99	96.2	4.71
	99.4	8.96
14	99.3	8.96	95.7	4.68
	99.7	9.01
17	98.8	9.01	95.2	4.66
	99.2	8.96
21	99.4	9.01	95.1	4.65
	99.1	8.97
24	100.6	9.06	94.3	4.62
	99.7	9.00
27	99.5	9.06	94.2	4.61
	99.8	9.05
30	100.3	9.06	93.9	4.59
	100.4	8.98

 

Table: Calculated half-lives for the decline of the test substance in various pHs

 

pH	Half-life	Correlation coefficient	Chi-squared
1(a)	2.8 hours	-	1.42
5(a)	5.0 days	-	0.70
7(b,c)	800 days	-0.970	-
9(b,c)	510 days	-0.993	-

a) Data were fitted to the non-linear exponential function C = C0*e^(-k*t)

b) Half-life calculated from linear regression of the derived equation: lnC = lnC0- k*t

c) These kinetic data must be regarded with caution due to the relatively short duration of the

incubations

 

Table: Mean Proportions of radioactive components in pH 1 test solutions stored at 25°C after addition of the radiolabelled test substance at a nominal concentration of 5 mg/L (Results are expressed as % test solution radioactivity),

Component	Time after application (hours)
	0	0.5	1	2	3	6	24	48
M3	0.6	10.1	16.3	29.2	40.7	61.6	76.2 (71.8)	77.1 (69.5)
Test substance	96.7	87.1	81.6	68.8	57.3	36.8	21.5 (25.9)	21.3 (26.2)
Diffuse radioactivity (a)	2.7	2.9	2.2	2.1	2.1	1.7	2.4	1.8

a) Radioactivity not associated with either M3 or the test substance. No other discrete radioactive components were apparent in the chromatogram

Values in parentheses were obtained by two-dimensional TLC

 

Table: Mean Proportions of radioactive components in pH 5 test solutions stored at 25°C after addition of the radiolabelled test substance at a nominal concentration of 5 mg/L (Results are expressed as % test solution radioactivity)

Component	Time after application (days)
	0	3	6	10	15	20	25	30
M3	0.8	20.2	35.8	47.9	57.1	60.7	62.3 (55.8)	62.8 (56.1)
Test substance	98.1	77.7	62.1	50.9	41.5	37.5	36.5 (40.8)	36.1 (41.5)
Diffuse radioactivity (a)	1.2	2.1	2.2	1.3	1.5	1.9	1.3	1.2

a) Radioactivity not associated with either M3 or the test substance. No other discrete radioactive components were apparent in the chromatogram

Values in parentheses were obtained by two-dimensional TLC

 

Table: Mean Proportions of radioactive components in pH 7 test solutions stored at 25°C after addition of the radiolabelled test substance at a nominal concentration of 5 mg/L (Results are expressed as % test solution radioactivity)

Component	Time after application (days)
	0	3	6	10	14	17	21	24	27	30
M3	0.6	0.7	1.4	1.3	1.7	1.8	2.1	2.3	2.5	2.8
Test substance	98.1	97.4	97.3	96.7	96.4	96.4	96.4	95.8	95.7	95.1
Diffuse radioactivity (a)	1.3	1.9	1.4	2	2	1.9	1.6	2.1	1.9	2.1

a) Radioactivity not associated with either M3 or the test substance. No other discrete radioactive components were apparent in the chromatogram

 

Table: Mean Proportions of radioactive components in pH 9 test solutions stored at 25°C after addition of the radiolabelled test substance at a nominal concentration of 5 mg/L (Results are expressed as % test solution radioactivity)

Component	Time after application (days)
	0	3	6	10	14	17	21	24	27	30
M3	0.5	1	2	1.8	2.6	2.7	3.2	3.5	3.7	4.3
Test substance	97.8	97.3	96.8	96.2	95.7	95.2	95.1	94.3	94.2	93.9
Diffuse radioactivity (a)	1.7	1.8	1.3	2	1.7	2.1	1.9	2.2	2.2	1.9

a) Radioactivity not associated with either M3 or the test substance. No other discrete radioactive components were apparent in the chromatogram

Validity criteria fulfilled:

yes

Remarks:

Recoveries ranged from 93-100.6%; see 'Test performance'

Conclusions:

The test substance is hydrolysed in aqueous solution at 25°C at rates dependent upon solution pH. The rate of hydrolysis increased in the order pH 7 ≈ pH 9 < pH 5 < pH 1. At pH values of 7 and 9, the test substance was essentially stable, whereas at pH 5 and pH 1, the test substance was hydrolysed at initial rates equivalent to half-lives of 5.0 days and 2.8 hours respectively. At acid pHs, the test substance reached an equilibrium with its hydrolysis products; the position of this equilibrium varied with pH.

Only one degradation product was formed in incubated solutions, and this was found at all pH values. This was identified, chromatographically (HPLC and TLC) and spectroscopically, as M3.

Executive summary:

The hydrolytic stability of the test substance was studied in aqueous solutions buffered to pH values of 1, 5, 7 or 9 and incubated at a temperature of ca 25°C in accordance with OECD TG 111 and in compliance with GLP-criteria. The test substance was labelled with carbon-14 in its pyridine ring. The approximate concentration of the test substance in the hydrolysis media was 5 mg/L, which is below its water solubility of 270 mg/L. Test solutions were incubated under sterile conditions, in darkness. At each pH, samples were analysed at zero time and at a number of further times, up to 48 hours (pH 1) or 30 days (pH 5, 7 and 9). Total recoveries of the applied radioactivity were in almost all cases in the range 97 - 101 %. Incubated solutions were analysed by high performance liquid chromatography (and for selected samples by thin-layer chromatography) to determine the relative proportions of the test substance and its degradation products. The rate of hydrolysis was dependent upon pH. At pH values of 7 and 9, the test substance was essentially stable, with losses of 4 - 5 % over the 30-day incubation periods. At pH 5, the initial rate was equivalent to a half-life of 5.0 days, while at pH 1 the initial half-life was shortened to 2.8 hours. At both pH 1 and pH 5, the rates of hydrolysis declined to zero, presumably due to an equilibrium between the test substance and its hydrolysis products. The position of the equilibrium varied with pH. Hydrolysis of the test substance yielded only one product with a pyridine moiety. This product, which was formed at all pH values, corresponded to M3 in the two chromatographic systems. The identities of this hydrolysis product and of unchanged test substance in incubated solution were further confirmed by mass spectrometry. At each pH, the formation of M3 mirrored the decline of the test substance, with levels increasing to 3 -4% (pH 7 and 9) and 63 % (pH 5) of the initial radioactivity after 30 days and 77 % of the initial radioactivity after 48 hours at pH 1.

Reference 2

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

19 Aug 1992 to 26 Jul 1995

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Biologische Bundesanstalt für Land- und Forstwirtschaft (BBA, Germany) Pamphlet No. 55: Testing of the Behaviour of Plant Treatment Agents in Water

Version / remarks:

October 1980

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA Guideline Subdivision N 161-1 (Hydrolysis)

Version / remarks:

October 1982

Deviations:

no

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

14-C labelled at position 6 of the triazine ring

Analytical monitoring:

yes

Details on sampling:

- pH 1 at 25 °C: 0, 0.5, 1, 2*, 3, 6, 24, 48 hours (duplicate)*
- pH 1 at 50 °C: 0, 0.5, 1, 2, 3, 6, 24 hours
- pH 1 at 70 °C: 0, 0.5, 1, 2, 3, 6, 24 hours

- pH 5 at 25 °C: 0, 5, 10, 15, 20, 25, 30 days (duplicate; 32 days instead of 30 days for the last duplicate sample)*
- pH 5 at 50 °C: 0, 0.25, 1, 2, 4, 7, 10 days
- pH 5 at 70 °C: 0, 0.5, 1, 3, 6, 24, 48 hours

- pH 7 at 25 °C: 0, 5, 9, 15, 20, 25, 30 days
- pH 7 at 50 °C: 0, 5, 10, 15, 20, 25, 30 days
- pH 7 at 70 °C: 0, 5, 10, 15, 20, 25, 30 days

- pH 9 at 25 °C: 0, 5, 9, 15, 20, 25, 30 days
- pH 9 at 50 °C: 0, 5, 10, 15, 20, 25, 30 days
- pH 9 at 70 °C: 0, 1, 3, 7, 10, 15, 20 days
* Additional incubations were carried out to generate samples for the investigation of the pattern of formation of hydrolysis products

- Storage of samlples: Radioassay of each test solution and measurement of pH were carried out on the day of sampling. Quantitative HPLC analysis (method 1) of test solutions (pH adjusted as necessary) was also generally carried out on the day of sampling. Some pH7 and pH9 solutions incubated at 25°C were analysed on the following day, and some pH1 solutions incubated at 25°C (pH-adjusted) were analysed two days after sampling. Those additional test solutions (pH1 and pH5, at 25°C) were analysed by HPLC method 2 (pH1) or HPLC method 4 (pH5) on the day of sampling. Two-dimensional TLC analysis of a selected solution from the additional pH5/25 °C incubation was also carried out on the day of sampling. HPLC and TLC co-chromatography with reference substances was also, unless stated otherwise, carried out on the day of sampling. When not being analysed, test solutions were stored at -20°C. The unlabelled reference standards were also stored, as neat compounds, at -20°C during the course of the study. In addition to spectroscopic analysis towards the end of the study, their stability was also assessed chromatographically during the course of the study: in all cases essentially only one HPLC UV peak or TLC UV spot was observed. Their stability was further confirmed by HPLC at the end of the study.

Buffers:

- 0.1M pH 1 buffer solution: 0.1M hydrochloric acid was used.
- 0.01M pH 5 buffer solution: Acetic acid (0.6 mL) was mixed with ca 900 mL water and the pH of the solution adjusted to 5.0 by the addition of sodium hydroxide solution. The solution was made up to a volume of 1 litre with water. The pH of the final solution was checked and adjusted to 5.0
with further hydroxide solution if necessary.
- 0.01M pH 7 buffer solution: Potassium dihydrogen orthophosphate (1.361 g) was dissolved in ca 900 mL water and the pH of the solution adjusted to 7.0 by the addition of potassium hydroxide solution. The solution was made up to a volume of 1 litre with water. The pH of the final solution was checked and adjusted to 7 .0 with further hydroxide solution if necessary
- 0.01M pH 9 buffer solution: Boric acid (0.618 g) was dissolved in ca 900 mL water and the pH of the solution adjusted to 9.0 by the addition of sodium hydroxide. The solution was made up to a volume of 1 litre with water. The pH of the final solution was checked and adjusted to 9.0 with further hydroxide solution if necessary.

Details on test conditions:

STERILITY PRECAUTIONS
All test vessels, glassware and buffer solutions were sterilised by autoclaving at 121 °C for 20 minutes. Each buffer solution was divided into two portions of equal volume which were autoclaved separately. After cooling, the pH of one portion was measured. No further adjustment of pH was found to be necessary and the other portion of each buffer solution was used for the study. Addition of the radiolabelled test substance solution to the buffer solutions and dispensing of treated buffer solutions into test vessels were carried out in a laminar flow cabinet. Samples of treated buffer solutions were taken for microbiological examination immediately and at the end of the incubation period.

ADDITION OF THE TEST SUBSTANCE TO THE BUFFER SOLUTIONS
The radiolabelled test substance was used as supplied, without any radiodilution. Stock solutions of the radiolabelled test substance in methanol were prepared at concentrations of 0.5 - 2.6 mg/mL. The radiochemical purity of the radiolabelled test substance in each of these solutions was measured, by TLC and/or HPLC, prior to addition to buffer solution, and was generally greater than 97% in each case. Aliquots of the radiolabelled test substance stock solutions were added to portions of pH 1.0, pH 5.0, pH 7.0 and pH 9.0 buffer solutions. The concentrations of the test substance were accurately determined by the radioassay of triplicate 50 μL aliquots of treated buffer solution. The concentration of the methanol co-solvent was 0.2 - 0.9% by volume. Further aliquots (10 mL) of treated buffer solutions were transferred to preweighed glass tubes of 20 mL capacity fitted with Teflon-lined screw caps. The tubes were re-weighed. Twenty tubes (main incubations) were established for each buffer to be incubated at 25°C and twelve for each buffer to be incubated at 50°C or 70°C.

INCUBATION OF TEST SOLUTIONS
With the exception of those tubes taken for immediate analysis or microbiological examination, tubes containing treated buffer solution were placed either on a rotary shaker in an incubator (pH5/25°C experiments) or in a water bath fitted with a shaking tray (all other experiments). The tubes were incubated in darkness while being agitated, at temperatures of ca 25°C, 50°C or 70°C. Temperatures were recorded at regular intervals (generally daily) using a thermometer located in the water bath or incubator. Throughout the entire study, mean recorded temperatures were 25.2°C (±0.4°C), 50.0°C (±0.2°C) and 69.8°C (±0.4°C). The temperature ranges were 24.0 - 26.0°C, 49.0 - 50.5°C and 69.0 - 70.0°C.

PH MEASUREMENTS
Measurements of the pH of test solutions were made using a model 3030 pH meter. The meter was calibrated prior to each measurement using standard buffer solutions of the appropriate pH, or in the case of pH 1 measurements, 0.1 M hydrochloric acid.

Duration:

48 h

pH:

1

Temp.:

25 °C

Initial conc. measured:

5 mg/L

Duration:

24 h

pH:

1

Temp.:

50 °C

Initial conc. measured:

5 mg/L

Duration:

24 h

pH:

1

Temp.:

70 °C

Initial conc. measured:

5 mg/L

Duration:

30 d

pH:

5

Temp.:

25 °C

Initial conc. measured:

5 mg/L

Duration:

10 d

pH:

5

Temp.:

50 °C

Initial conc. measured:

5 mg/L

Duration:

48 h

pH:

5

Temp.:

70 °C

Initial conc. measured:

5 mg/L

Duration:

30 d

pH:

7

Temp.:

25 °C

Initial conc. measured:

5 mg/L

Duration:

30 d

pH:

7

Temp.:

50 °C

Initial conc. measured:

5 mg/L

Duration:

30 d

pH:

7

Temp.:

70 °C

Initial conc. measured:

5 mg/L

Duration:

30 d

pH:

9

Temp.:

25 °C

Initial conc. measured:

5 mg/L

Duration:

30 d

pH:

9

Temp.:

50 °C

Initial conc. measured:

5 mg/L

Duration:

20 d

pH:

9

Temp.:

70 °C

Initial conc. measured:

5 mg/L

Number of replicates:

For experiments at 25°C duplicate vessels were taken for analysis at each time. Single vessels were taken for analysis in experiments conducted at 50°C and 70°C. Additional tubes of treated buffer solution (two at 25°C; one at 50°C and 70°C) were taken for microbiological examination at the start and at the completion of each incubation.

Positive controls:

no

Negative controls:

no

Test performance:

- Recoveries: Total recoveries of applied radioactivity (AR) from incubated test solutions were in almost all cases in the range 96.6 - 103.5%. Each recovery was calculated as the ratio of the radioactivity concentration in the test solution to that in the original treated buffer solution. Changes in the weights of test vessels during the incubation periods were in most cases negligible (< 1 %) and were not accounted for (it was also demonstrated that empty vessels, when similarly incubated in a water bath, increased in weight to a similar extent to those containing test solutions). The only low recoveries of radioactivity (93 % AR) were observed in pH7 and pH9 solutions taken for analysis after 30 days incubation at 50°C. Concentrations of radioactivity in spare vessels were similar.
- pH: In all cases the pH of the incubated test solution had not changed significantly from that of the original treated buffer solution.
- Microbial contamination: Corresponding (additional) vessels taken for microbiological examination revealed bacterial contamination, especially at pH7. No microbial contamination was observed in any other test vessels.
- Temperatures: Temperatures did not deviate by more than 1 °C during each incubation.

Transformation products:

yes

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

CHROMATOGRAPHIC ANALYSIS OF TEST SOLUTIONS
HPLC analysis of test solutions using method 1 resolved a single radioactive component in addition to the test substance. Selected solutions were also analysed using TLC (system A) which showed, however, that this 'component' consisted of a number of degradation products. While this TLC system provided insufficient resolution of the hydrolysis products to allow for accurate quantification by the TLC linear analyser, the analyses did reveal an essentially similar degradate profile at each pH. Quantification of the degradation products in additional test solutions buffered to pH5 and incubated at 25°C was achieved by using HPLC method 4. This separated five components in these solutions in addition to the test substance itself. A 6-day incubated solution also analysed using the two-dimensional TLC system (system D). The major radioactive hydrolysis product in these solutions, in addition to the test substance, corresponded in both chromatographic systems to the reference substance M2. Levels of M2 in these solutions rose to 48 % of the initial test solution radioactivity after 32 days. Another product, which represented up to about 3 % test solution radioactivity, was similarly identified, as M11. None of the three other products resolved by the HPLC method represented more than about 1 % test solution radioactivity. The presence of unchanged test substance in incubated solution was also confirmed chromatographically.

A minor component which was resolved by the two-dimensional TLC system corresponded to the reference substance M5. In the HPLC system however, this reference substance chromatographed very close to the main degradate peak (ie M2). Where M5 in incubated solution (pH5, 25°C) was quantified, using TLC, it did not represent more than about 1 % test solution radioactivity. Quantification of the degradation products in additional test solutions buffered to pH 1 and incubated at 25°C was also attempted, using HPLC method 2. This separated three to four components in addition to the test substance. Using this method, however, the major radioactive component (representing up to about 47% applied radioactivity after 48 hours) co-eluted with both of the reference substances M2 and M11. As both of these substances have been identified as hydrolysis products of the test substance, their relative proportions in these solutions could not be determined. It may be presumed though, that M2 was the major hydrolysis product at pH 1, as was the case at pH5 (above). A minor component, representing up to about 3 % test solution radioactivity, corresponded in this system to M5. A third component (HT1; Unknown 1) representing up to about 14 % of the initial test' solution radioactivity, was shown, after isolation of the eluate solution and re-chromatography (TLC using system C), to be comprised of a number of components. In a 30-day incubated solution, in which the proportion of HT1 was the greatest, none of these represented more than about 4 % of the initial radioactivity. The identities of the test substance, M2 and M11 in incubated solutions were further confirmed by mass spectrometry. The formation of M2 mirrored the decline of the test substance.

Remarks on result:

other: See 'Any other information on results incl. tables' for the individual recoveries

Key result

pH:

1

Temp.:

25 °C

DT50:

>= 2.7 - <= 2.9 h

Type:

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

Key result

pH:

1

Temp.:

50 °C

DT50:

0.5 h

Type:

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

Key result

pH:

1

Temp.:

70 °C

DT50:

0.1 h

Type:

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

Key result

pH:

5

Temp.:

25 °C

DT50:

>= 9.7 - <= 13.2 d

Type:

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

Key result

pH:

5

Temp.:

50 °C

DT50:

2.2 d

Type:

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

Key result

pH:

5

Temp.:

70 °C

DT50:

13 h

Type:

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

Key result

pH:

7

Temp.:

25 °C

DT50:

616 d

Type:

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

Key result

pH:

7

Temp.:

50 °C

DT50:

79 d

Type:

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

Key result

pH:

7

Temp.:

70 °C

DT50:

16 d

Type:

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

Key result

pH:

9

Temp.:

25 °C

DT50:

1 210 d

Type:

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

Key result

pH:

9

Temp.:

50 °C

DT50:

44 d

Type:

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

Key result

pH:

9

Temp.:

70 °C

DT50:

6.2 d

Type:

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

Details on results:

HYDROLYSIS OF THE TEST SUBSTANCE
Each test solution was analysed by HPLC. Method 1 provided a good separation of the test substance from its degradation products, although these themselves were not resolved. Method 4 did separate a number of degradation products and was used to investigate the pattern of their formation in an additional series of test solutions buffered to pH5 and incubated at 25°C. In some experiments (those at pH1, and pH5/70°C), the pH of the incubated solution was immediately adjusted to ca. pH7, in order to halt the (expected) rapid hydrolysis of the test substance under these conditions. The proportions of the test substance remaining in test solutions incubated at 25°C, 50°C and 70°C, as determined by HPLC, are shown in ‘Any other information on results incl. tables’. The rate of disappearance of the test substance was compared to first-order reaction kinetics.

At 25°C, the test substance was essentially hydrolytically stable at pH7 and pH9, with loss of only 2 - 3% test substance over the 30-day incubation periods. Closer examination of the data possibly indicated a slightly greater stability at pH9. In comparison, the test substance was hydrolysed much more rapidly at pH5 with a half-life of 9.7 days. The rates of hydrolysis at pH5, pH7 and pH9 were predictably greater at 50°C and again at 70°C. At these higher temperatures, pH7 was clearly the pH at which the test substance was hydrolytically the most stable. Calculated half-lives at pH5 shortened to 2.2 days at 50°C and 13 hours at 70°C. At pH9, half-lives at 50°C and 70°C were 44 and 6.2 days respectively, while at pH7 the corresponding values were 79 days (50°C) and 16 days (70°C).

Hydrolysis of the test substance at pH1 was even more rapid, and again, more so with increasing temperature. However, the rates of hydrolysis were linear only during the initial period of each incubation. At all three temperatures the hydrolysis rate declined to zero, presumably due to the establishment of an equilibrium between the test substance and its initial hydrolysis products, M2 and M3. The position of the equilibrium varied with temperature: at about 20 - 25% test substance at 25°C; 10% at 50°C and 7 - 8% at 70°C. There was also some evidence of these equilibria being approached in the pH5 test solutions. In certain incubations, 50% degradation of the test substance had not occurred over the calculated half-life period. This phenomenon, which was most noticeable at pH5/25°C, was due to a relatively fast initial decline (upon which the DT 50 was calculated) followed by a fall in the rate of degradation of the test substance, caused by the test substance/product equilibrium. The activation energies for the hydrolysis of the test substance at pHs 1, 5, 7 and 9 were calculated as 64, 53, 69 and 100 kJ/mol respectively.

Table: Calculated half-lives for the decline of the test substance at various temperatures and pHs

Temperature	pH	Half-life	Correlation coefficient (r)	Chi-squared
25 °C	1(a)	2.7 hours	-	1.69
	1(a,d)	2.9 hours	-	0.47
	5(a)	9.7 days	-	0.35
	5(a,d)	13.2 days	-	0.29
	7(b,e)	616 days	-0.994	-
	9(b,e)	1210 days	-0.953	-
50 °C	1(c)	0.5 hours	-	26.2
	5(a)	2.2 days	-	0.82
	7(b)	79 days	-0.995	-
	9(b)	44 days	-0.986	-
70 °C	1 (c)	0.1 hours	-	0.21
	5 (b)	13 hours	-0.989	-
	7(b)	16 days	-0.998	-
	9(b)	6.2 days	-0.994	-

a) Data were fitted to the non-linear exponential function

b) Half-life calculated from linear regression of the derived equation

c) Data were fitted to the bi-exponential function

d) Derived from additional vessels established to investigate the pattern of formation of hydrolysis products

e) These kinetic data must be regarded with caution due to the relatively short duration of the incubations

 

PH 1 RESULTS

Table: Combined results of recovery, measured pH, mean proportions (in %AR and mg/L) at 25°C for pH 1

Time (hours)	Recovery (%)	Measured pH	Proportion of test substance in solution (expressed as % test solution radioactivity)	Proportion of test substance in solution (expressed as mg/L (ppm))
0	NM	1.00(a)	96.8	4.82
	NM	1.00(a)
0.5	99.5	0.96	90.1	4.49
	98.6	0.99
1	99.4	0.96	82.3	4.10
	100.1	0.96
3	99.5	0.98	56.8	2.83
	99.3	1.00
6	99.5	0.95	37.3	1.86
	99.7	0.94
24	99.7	1.01	22.8	1.14
	99.7	1.02
48	99.8	1.03	21.5	1.07
	99.3	1.03

a) pH value of the treated buffer solution

NM – Not measured

 

Table: Combined results of recovery, measured pH, mean proportions (in %AR and mg/L) at 25°C for the additional vesselsto investigate the pattern of formation of degradation productspH 1

Time (hours)	Recovery (%)	Measured pH	Proportion of test substance in solution (expressed as % test solution radioactivity)	Proportion of test substance in solution (expressed as mg/L (ppm))
0	NM	0.96(a)	94.3	4.84
	NM	0.96(a)
0.5	99.7	0.98	87.5	4.49
	98.5	0.88
1	99.8	0.91	80.0	4.11
	99.0	0.88
2	100.0	0.99	68.0	3.49
	98.6	0.91
3	99.1	0.91	5.5	2.96
	98.8	0.95
6	99.3	0.94	38.6	1.98
	98.4	0.90
24	99.4	0.95	22.2	1.11
	99.7	0.96
48	100.0	0.94	22.0	1.10
	99.6	0.97

a) pH value of the treated buffer solution

NM – Not measured

 

Table: Combined results of recovery, measured pH, mean proportions (in %AR and mg/L) at 50°C for pH 1

Time (hours)	Recovery (%)	Measured pH	Proportion of test substance in solution (expressed as % test solution radioactivity)	Proportion of test substance in solution (expressed as mg/L (ppm))
0	NM	NM	95.0	3.61
0.5	97.8	1.13	54.0	2.06
1	98.8	1.09	26.4	1.01
2	98.2	1.17	11.8	0.45
3	98.4	1.14	10.7	0.41
6	97.7	1.12	9.8	0.37
24	98.5	1.11	10.7	0.41

NM – Not measured

 

Table: Combined results of recovery, measured pH, mean proportions (in %AR and mg/L) at 70°C for pH 1

Time (hours)	Recovery (%)	Measured pH	Proportion of test substance in solution (expressed as % test solution radioactivity)	Proportion of test substance in solution (expressed as mg/L (ppm))
0	NM	NM	96.1	4.98
0.5	98.8	0.91	9.9	0.51
1	99.1	1.13	7.5	0.39
2	98.4	1.09	7.4	0.38
3	98.9	1.08	7.3	0.38
6	98.8	0.92	7.6	0.39
24	98.1	1.16	7.8	0.40

NM – Not measured

 

PH 5 RESULTS

Table: Combined results of recovery, measured pH, mean proportions (in %AR and mg/L) at 25°C for pH 5

Time (days)	Recovery (%)	Measured pH	Proportion of test substance in solution (expressed as % test solution radioactivity)	Proportion of test substance in solution (expressed as mg/L (ppm))
0	98.5	5.16	96.1	5.07
	99.7	5.15
5	100.8	5.10	78.3	4.13
	100.0	5.08
10	100.2	5.13	65.2	3.44
	100.4	5.06
15	99.2	5.07	55.1	2.91
	100.1	5.13
20	99.5	5.20	49.2	2.60
	98.9	5.18
25	99.2	5.06	44.5	2.35
	100.1	5.09
30	100.0	5.06	42.4	2.24
	98.9	5.06

           

 

Table: Combined results of recovery, measured pH, mean proportions (in %AR and mg/L) at 25°C for the additional vesselsto investigate the pattern of formation of degradation productspH 5

Time (days)	Recovery (%)	Measured pH	Proportion of test substance in solution (expressed as % test solution radioactivity)	Proportion of test substance in solution (expressed as mg/L (ppm))
0	100.3	4.05(a)	95.8	4.92
	100.8	4.05(a)
3	100.4	5.21	87.6	4.50
	100.6	5.17
6	101	5.23	79.1	4.07
	101.4	5.3
10	101.6	5.09	69.9	3.60
	100.9	5.13
15	101.5	5.47	61.4	3.16
	102.3	5.46
20	101.1	5.23	55.1	2.83
	101.2	5.14
25	102	5.31	48.9	2.51
	101.6	5.36
32	101.8	5.36	44.7	2.30
	101.1	5.41

a) pH value of the treated buffer solution

 

Table: Combined results of recovery, measured pH, mean proportions (in %AR and mg/L) at 50°C for pH 5

Time (days)	Recovery (%)	Measured pH	Proportion of test substance in solution (expressed as % test solution radioactivity)	Proportion of test substance in solution (expressed as mg/L (ppm))
0	99.9	5.14	96.4	5.02
0.25	99.1	5.15	90.7	4.73
1	99	5.13	74.2	3.87
2	100.9	5.22	59.2	3.08
4	100.5	5.17	36.0	1.88
7	98.7	5.16	22.1	1.15
10	100.1	5.22	17.1	0.89

 

Table: Combined results of recovery, measured pH, mean proportions (in %AR and mg/L) at 70°C for pH 7

Time (hours)	Recovery (%)	Measured pH	Proportion of test substance in solution (expressed as % test solution radioactivity)	Proportion of test substance in solution (expressed as mg/L (ppm))
0	NM	NM	96.4	5.02
0.5	99.1	4.88	93.5	4.87
1	99.1	5.03	90.3	4.70
3	98.6	4.91	78.5	4.09
6	99.3	4.97	63.8	3.32
24	98.7	4.95	19.7	1.03
48	98.4	4.96	8.8	0.46

NM – Not measured

 

PH7 RESULTS

Table: Combined results of recovery, measured pH, mean proportions (in %AR and mg/L) at 25°C for pH 7

Time (days)	Recovery (%)	Measured pH	Proportion of test substance in solution (expressed as % test solution radioactivity)	Proportion of test substance in solution (expressed as mg/L (ppm))
0	99.8	7.07	97.2	5.04
	98.6	7.07
5	101.1	7.09	96.6	5.01
	100.9	7.10
9	100.3	7.16	95.9	4.98
	100.5	7.14
15	100.5	7.11	95.6	4.96
	100.7	7.12
20	100.1	7.04	94.8	4.82
	100.1	7.09
25	100.4	7.11	94.5	4.90
	99.4	7.10
30	99.2	7.14	93.9	4.87
	100.3	7.14

 

Table: Combined results of recovery, measured pH, mean proportions (in %AR and mg/L) at 50°C for pH 7

Time (days)	Recovery (%)	Measured pH	Proportion of test substance in solution (expressed as % test solution radioactivity)	Proportion of test substance in solution (expressed as mg/L (ppm))
0	99	7.15	96.8	4.93
5	99.8	7.09	92.8	4.72
10	98.7	7.12	88.1	4.48
15	96.6	7.08	84.6	4.31
20	97.3	7.09	82.3	4.19
25	98.1	7.16	78.6	4.00
30	93.2	6.95	73.6	3.74

 

Table: Combined results of recovery, measured pH, mean proportions (in %AR and mg/L) at 70°C for pH 7

Time (days)	Recovery (%)	Measured pH	Proportion of test substance in solution (expressed as % test solution radioactivity)	Proportion of test substance in solution (expressed as mg/L (ppm))
0	100.4	7.12	96.3	5.22
5	101.7	7.06	78.4	4.50
10	101.8	7.07	64.0	3.49
15	98.3	6.99	52.2	2.03
20	101.4	6.85	41.6	1.38
25	400.3	6.36	30.6	1.02
30	100.4	6.52	26.1	0.54

 

PH9 RESULTS

Table: Combined results of recovery, measured pH, mean proportions (in %AR and mg/L) at 25°C for pH 9

Time (days)	Recovery (%)	Measured pH	Proportion of test substance in solution (expressed as % test solution radioactivity)	Proportion of test substance in solution (expressed as mg/L (ppm))
0	99.0	9.00	97.2	5.22
	100.1	9.00
5	100.5	9.12	96.7	5.19
	100.7	9.03
9	100.4	9.13	96.4	5.18
	100.4	9.11
15	101.2	9.11	96.2	5.17
	100.6	9.13
20	100.0	9.05	95.6	5.13
	99.4	9.10
25	100.2	9.14	95.9	5.15
	100.4	9.07
30	99.3	9.03	95.4	5.12
	100.8	9.15

  

Table: Combined results of recovery, measured pH, mean proportions (in %AR and mg/L) at 50°C for pH 9

Time (days)	Recovery (%)	Measured pH	Proportion of test substance in solution (expressed as % test solution radioactivity)	Proportion of test substance in solution (expressed as mg/L (ppm))
0	100.1	9.08	96.6	5.04
5	99.7	9.08	90.2	4.71
10	99.8	9.06	85.6	4.47
15	100.4	9.27	75.3	3.93
20	98.5	9.18	74.2	3.87
25	98.3	9.06	63.3	3.30
30	92.7	9.12	61.9	3.23

 

 

Table: Combined results of recovery, measured pH, mean proportions (in %AR and mg/L) at 70°C for pH 9

Time (days)	Recovery (%)	Measured pH	Proportion of test substance in solution (expressed as % test solution radioactivity)	Proportion of test substance in solution (expressed as mg/L (ppm))
0	100.3	9.14	96.7	5.22
1	100	8.86	83.3	4.50
3	101.2	8.9	64.6	3.49
7	100.7	8.97	37.6	2.03
10	101.5	8.97	25.6	1.38
15	99	8.87	18.9	1.02
20	103.5	8.67	10.0	0.54

 

DEGRADATION

Table: Mean proportions of radioactive components in pH1 test solutions stored at 25°C after addition of the radiolabelled test substance at a nominal concentration of 5 mg/L (Results are expressed as % test solution radioactivity)

Component	Time after application (hours)
	0	0.5	1	2	3	6	24	48
Unknown peak	1.9	2.0	3.6	6.7	9.2	13.3	12.2	14.4
M5		1.1	2.0	2.2	2.7	3.3	2.9	2.9
M2 + M11		3.9	7.2	14.0	19.2	30.6	47	46.6
Unknown zone		3.0	4.3	5.9	7.7	9.4	11.7	9.8
Test substance	94.3	87.5	80.0	68.0	57.5	38.6	22.2	22.0
Diffuse radioactivity (a)	4	2.6	3.0	3.4	3.8	4.9	4.2	4.4

a) Radioactivity not associated with any of the components listed in the table

 

Table: Mean proportions of radioactive components in pH5 test solutions stored at 25°C after addition of the radiolabelled test substance at a nominal concentration of 5 mg/L (Results are expressed as % test solution radioactivity)

Component	Time after application (days)
	0	3	6	10	15	20	25	32
Unknown 1	0.2	0.8	0.9	0.9	1.0	1.1	1.1	1.0
M2	0.7	8.7	16.4	24.2	31.5	38.7	42.8	47.7
M11	0.2	0.5	1.0	1.1	1.7	1.2	2.6	1.8
Unknown 2	b	0.2	b	0.2	0.8	0.8	0.4	0.8
Test substance	95.8	87.6	79.1	69.9	61.4	55.1	48.9	44.7
Unknown 3	0.3	0.2	0.2	0.3	0.4	0.4	0.3	0.4
Diffuse radioactivity (a)	2.9	2.4	2.5	3.5	3.3	2.8	4.0	3.8

a) Radioactivity not associated with any of the components listed in the table

b) Not present as a discrete component; included in 'Diffuse radioactivity'

 

Validity criteria fulfilled:

yes

Remarks:

Recoveries ranged from 93-100.6%; see 'Test performance'

Conclusions:

The test substance is hydrolysed in aqueous solutions at rates dependent upon solution pH and temperature. The rate of hydrolysis increased with temperature, while at each temperature (25°C, 50°C or 70°C) the rate increased in the order pH7 < pH9 < pH5 < pH 1. At 25°C at pH values of 7 and 9, the test substance was essentially stable, whereas in pH1 solution at this temperature the test substance was extensively hydrolysed, with a half-life of 2. 7 hours. Half-lives at higher temperatures were correspondingly shorter, ranging from 0.1 hours (pH1) to 16 days (pH7) for the test substance incubated at 70°C. At acid pHs, the test substance appeared to reach an equilibrium with its hydrolysis products. The position of this equilibrium was dependent on temperature. A number of degradation products were formed in incubated solutions. The major product formed in solution buffered to pH5 and incubated at 25°C was identified, chromatographically and spectroscopically, as M2. A minor component was similarly identified, as M11.

Executive summary:

The hydrolytic stability of the test substance was studied in aqueous solutions buffered to pH values of 1, 5, 7 or 9 and incubated at temperatures of 25°C, 50°C or 70°C in accordance with OECD TG 111 and in compliance with GLP-criteria. The test substance was labelled with carbon-14 in its triazine ring. The approximate concentration of the test substance in the hydrolysis media was 5 mg/L, which is below its water solubility of 270 mg/L. Test solutions were incubated under sterile conditions, in darkness. For each experiment (combination of pH and temperature), samples were analysed at zero time and at six further times, distributed over about two half-lives (estimated from a preliminary experiment), or over 30 days whichever was the shorter. Total recoveries of the applied radioactivity were in almost all cases in the range 97 - 104 % . Incubated solutions were analysed by high performance liquid chromatography to determine the relative proportions of the test substance and, at pH1 and pH5 at 25°C, its degradation products. The test substance was hydrolysed at various rates dependent upon pH and temperature. At each pH, the rate of hydrolysis increased with temperature. At each temperature, the test substance was hydrolysed most rapidly in the order pH7 < pH9 < pH5 < pH 1. At 25 °C, half-lives for the hydrolysis of the test substance ranged from 2.7 hours at pH1 to 9.7 days at pH5. (At pH5/25°C, 50% degradation of the test substance was not actually reached over this calculated half-life period. This was due to a fall in the rate of decline of the test substance, presumably due to an equilibrium between the test substance and its initial hydrolysis products.) the test substance was essentially stable at pH7 and pH9 at 25°C. At 50°C, half-lives ranged from 0.5 hours at pH1 to 79 days at pH7, while corresponding values at 70°C were 0.1 hours (pH1) and 16 days (pH7). At pH1, conditions under which the test substance was degraded to the greatest extent, the rate of hydrolysis declined to zero, presumably due to the equilibrium between the test substance and its initial hydrolysis products. The position of this equilibrium varied with temperature. Hydrolysis at different pHs yielded a number of products. In solution buffered to pH5 and incubated at 25°C, M2 was the major product with a triazine moiety, representing about 48 % of the initial radioactivity after 32 days. M11 reached a maximum proportion of 2.6% after 25 days. No other hydrolysis product, at pH5/25°C, represented more than 1.1 % of the initial radioactivity. Unchanged test substance in incubated solution was also confirmed by mass spectrometry. In solution buffered to pH1 and incubated at 25°C, one major component, representing about 47% of the initial radioactivity, was shown to comprise both of the reference substances M2 and M11. Both of these substances have been identified as hydrolysis products of the test substance. One minor component, representing up to 3 % of the initial radioactivity, corresponded to M5. A polar fraction was shown to comprise a number of components, none of which represented more than 4 % of the initial radioactivity.

Reference 3

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

10 Jan 1996 to 25 Mar 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA Guideline Subdivision N 161-1 (Hydrolysis)

Deviations:

no

GLP compliance:

yes

Radiolabelling:

yes

Remarks:

[14C]-labelled at position 6 of triazine ring

pH:

5

Temp.:

25 °C

Hydrolysis rate constant:

0.057 d-1

DT50:

12.3 d

Type:

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

Based on the radioassay analysis, the radiochemical balance (% of total dose) ranged from 96.51% to 105.19%. There were a total of 8 degradates. The primary degradate, accounting for up to 41.67%, was M2. M11 was also present and accounted for a maximum of 2.73%. The estimated half-life value is 12.1 days with a rate constant of 0.057 per day . This half-life value closely correlates with the extrapolated DT50 value of 12.3 days from the graph. The percent of total dose for parent declined to 37.26% by Day 35. The percent of total dose represented by parent and M2 both reached a plateau of approximately 40% by Day 35, apparently reaching an equilibrium between the cleavage and formation of the bridge between the two rings.

Conclusions:

The DT50 value was found to be 12.3 days, and the rate constant for degradation 0.057 per day. Degradation products included M2 (up to 41.67%) and M11 (up to 2.73%).

Executive summary:

The hydrolytic stability and degradation of the test substance was studied in aqueous solution at pH 5 and incubated at a temperature of ca 25°C in accordance withEPA Environmental Fate Requirement 40CFR Section 158 Subdivision N, Section 161-1and in compliance with GLP-criteria. The test substance was labelled with carbon-14 in its triazine ring. The test substance was incubated in the pH 5 buffered solution at 25 °C in the dark for a maximum of 35 days. Samples were harvested in replicate at Day 0, 3, 7, 9, 11, 14, 22, 30 and 35. Radiochemical balance was determined by radioassay. The HPLC, TLC and mass spectrometry analysis were performedfor the identification and quantification of the test substance and the hydrolysis products in the samples.

Based on the radioassay analysis, the radiochemical balance (% of total dose) ranged from 96.51% to 105.19%. There were a total of 8 degradates. The primary degradate, accounting for up to 41.67%, was M2. M11 was also present and accounted for a maximum of 2.73%. The estimated half-life value is 12.1 days with a rate constant of 0.057 per day. This half-life value closely correlates with the extrapolated DT50 value of 12.3 days from the graph. The percent of total dose for parent declined to 37.26% by Day 35. The percent of total dose represented by parent and M2 both reached a plateau of approximately 40% by Day 35, apparently reaching an equilibrium between the cleavage and formation of the bridge between the two rings.

Description of key information

The test substance was stable under neutral (pH 7) and alkaline (pH9) conditions. Under acidic conditions, an equilibrium of the test substance and its hydrolysis products was quickly reached, OECD TG 111, Kirkpatrick, 1995a/b.

DT50 values at 25 °C at pH 1, 5, 7, and 9 were 2.8 h, 5 d, 800 d and 510 d, respectively, for the pyridine-labelled test substance (Kirkpatrick 1995a).

DT50 values at 25 °C at pH 1, 5, 7, and 9 were 2.7 - 2.9 h, 9.7 - 13.2 d, 616 d and 1210 d, respectively, for the triazine-labelled test substance (Kirkpatrick 1995b).

The highest DT50 value measured at pH=7 is used as a key value for the chemical safety assessment.

All available data was assessed and the studies representing the worst-case effects were included as weight-of-evidence studies. The other studies are included as supporting information. The weight-of-evidence studies are considered to be worst-case and were selected for the CSA.

Key value for chemical safety assessment

Half-life for hydrolysis:

800 d

at the temperature of:

25 °C

Additional information

Table: Overview of available data on hydrolysis

Method			Guideline / GLP	Endpoint	Value (DT50) *	Remark	Reference
Temperature	pH	Duration
25 °C	1	48 h	OECD TG 111 / GLP	DT50	2.8 h	Hydrolysis of the pyridine-labelled test substance	Kirkpatrick, 1995a
	5	30 d		DT50	5 d
	7	30 d		DT50	800 d
	9	30 d		DT50	510 d
25 °C	1	48 h	OECD TG 111 / GLP	DT50	2.7 – 2.9 h	Hydrolysis of the triazine-labelled test substance	Kirkpatrick, 1995b
	5	30 d		DT50	9.7 - 13.2 d
	7	30 d		DT50	616 d
	9	30 d		DT50	1210 d
50 °C	1	24 h		DT50	0.5 h
	5	10 d		DT50	2.2 d
	7	30 d		DT50	79 d
	9	30 d		DT50	44 d
70 °C	1	24 h		DT50	0.1 h
	5	48 h		DT50	13 h
	7	30 d		DT50	16 d
	9	20 d		DT50	6.2 d

*: Values > 30 days to be regarded with caution since they were extrapolated.