N,N'-bis(1,3-dimethylbutylidene)-2,2'-iminobis(ethylamine)

Hydrolysis

Administrative data

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

30 May 2002 to 25 July 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-bis(1,3-dimethylbutylidene)-2,2'-iminobis(ethylamine)

EC Number:

234-205-3

EC Name:

N,N'-bis(1,3-dimethylbutylidene)-2,2'-iminobis(ethylamine)

Cas Number:

10595-60-5

Molecular formula:

C16H33N3

IUPAC Name:

N-(1,3-dimethylbutylidene)-N'-{2-[(1,3-dimethylbutylidene)amino]ethyl}ethane-1,2-diamine

Test material form:

not specified

Details on test material:

- Storage conditions: room temperature in the original container in a dark ventilated cabinet

Radiolabelling:

no

Study design

Analytical monitoring:

yes

Details on sampling:

Samples were collected at four to six intervals, depending on pH, to monitor a fast hydrolysis rate. Samples for pH 1.2 were collected at time 0, 15 minutes, 1, 2, 4 and 18 hours. Samples at pH 4 were collected at time 0, 15 minutes, and 1, 2, 4 and 6 hours. Samples at pH 7 were collected at time 0, 5 and 15 minutes, and 1 and 3 hours. Samples at pH 9 were collected at time 0, 1, 3 and 15 minutes. At each sampling interval, the concentration of the test material and the presence of the degradate DETA (diethylenetriamine) in solution was determined by liquid chromatography/mass spectrometry (LC/MS).

Buffers:

Buffer solutions were prepared in the following manner:

-pH 1.2
50 mL of acetic acid was diluted to a volume of 100 mL with purified reagent water, and the pH adjusted with 10.0 mL of 0.1 N hydrochloric acid and 0.40 mL of concentrated hydrochloric acid. The pH of the resultant solution was 1.22.

-pH 4
50 mL of acetic acid was diluted to a volume of 100 mL with purified reagent water and the pH adjusted with 9.9 mL of concentrated ammonium hydroxide. The pH of the resultant solution was 4.00.

-pH 7
25 mL of acetic acid was diluted to a volume of 100 mL with purified reagent water and the pH adjusted with 31.5 mL of concentrated ammonium hydroxide. The pH of the resultant solution was 7.08.

-pH 9
25 mL of acetic acid was diluted to a volume of 100 mL with purified reagent water and the pH adjusted with 37.8 mL of concentrated ammonium hydroxide. The pH of the resultant solution was 9.00.

Buffers were filter-sterilised through a 0.45 µm Gelman filter disc prior to use. Prior to dosing, the buffer solutions were purged with nitrogen for approximately 5 minutes to exclude oxygen.

Details on test conditions:

TEST DESIGN
Experiments at pH 1.2, 4 and 7 were conducted twice, and the first experiments at these pHs were not reported. At pH 1.2, calibration standards were not analysed immediately, analytical instrument problems occurred during the first analysis with pH 4, and the test material was insoluble at the test concentration at pH 7.

The test apparatus consisted of 10 mL stoppered, glass volumetric flasks. Prior to testing, the volumetric flasks and pipettes for transfer were autoclaved at approximately 121 °C at 15 psi for 30 minutes, to minimise the potential for microbial degradation of the test material. The flasks, which were covered with aluminium foil to prevent photolytic degradation, were placed on a bench top in the laboratory at room temperature (approximately 20 °C).

TEST PROCEDURES
The number of test vessels at each pH varied based on the number of earlier sampling intervals that had to be added due to rapid degradation. At certain pH values, hydrolysis occurred in minutes but analyses could only be conducted approximately every 45 minutes in order to run blanks needed to prepare the analytical system for the next sample. Therefore, separate test solutions had to be made for more than one sampling interval within one hour. For instance, the number of test solutions that had to be made for pH 1.2, 4, 7, and 9 were two, two, three and four, respectively. A larger number of test solutions were prepared for the pH 9 experiment due to the required sampling intervals being at time 0, 15 minutes, 3 minutes and 1 minute. Sampling interval times were decreased as it was discovered that the half-life of the test material at pH 9 was only 1.3 minutes.

Individual 2500 mg/L test solutions were prepared for buffer solutions at pH 1.2 and 4 by weighing approximately 35 mg of the test material (25 mg as active ingredient) into separate 10 mL volumetric flasks and bringing each to volume with the appropriate buffer. This provided a molar concentration of 0.01 M. Both pH 1.2 and 4 solutions appeared clear and colourless. Each solution was shaken and sonicated for a few seconds. A 0.50 mL aliquot of each 0.01 M solution was diluted with 19.50 mL of acetonitrile. Individual solutions were mixed and an aliquot was analysed by LC/MS. Following these procedures, additional samples were prepared in the same manner and sampled for the remaining intervals as necessary.

Individual 270 mg/L test solutions were prepared for buffer solutions at pH 7 and 9 by weighing approximately 3.8 mg of the test material (2.7 mg as active ingredient) into separate 10 mL volumetric flasks and bringing each to a volume of 10 mL with the appropriate buffer. This provided a molar concentration of 0.001 M. Both pH 7 and 9 solutions appeared clear and colourless. Each solution was shaken and sonicated for a few seconds. A 1.0 mL aliquot of each 0.001 M solution was diluted with 3.0 mL of methanol. Individual solutions were mixed and an aliquot was analysed by LC/MS. Following these procedures, additional samples were prepared in the same manner and sampled for the remaining intervals as necessary.

Sample buffer matrix was also used to prepare the calibration standards, in order to match the matrix of the calibration standards with the samples.

All exposure solution samples were analysed for the test material (as active ingredient) and the degradate DETA, using LC/MS.

Due to the rapid degradation of the test material and speed that the compound needed to reach the detector on the chromatographic system, the r² of the calibration standard curves was used to evaluate the validity of the analytical method during each experiment.

TEST MONITORING
The pH of the test solutions was measured at test termination. The pH was determined to the nearest 0.01 pH unit using a Beckman 250 pH meter, except for the pH 1.2 solution, which was determined with pH paper at test termination. The temperature of the laboratory was measured throughout the test using a minimum/maximum calibrated thermometer.

Duration of test

Duration of test 1

Duration:

18 h

pH:

1.2

Temp.:

20 °C

Initial conc. measured:

2 500 mg/L

Duration of test 2

Duration:

6 h

pH:

4

Temp.:

20 °C

Initial conc. measured:

2 500 mg/L

Duration of test 3

Duration:

3 h

pH:

7

Temp.:

20 °C

Initial conc. measured:

270 mg/L

Duration of test 4

Duration:

15 min

pH:

9

Temp.:

20 °C

Initial conc. measured:

270 mg/L

Number of replicates:

Individual test solutions were prepared: two vessels for pH 1.2 and 4 buffer solutions, three vessels for pH 7 buffer solutions and four vessels for pH 9 buffer solutions.

Positive controls:

no

Negative controls:

no

Results and discussion

Transformation products:

yes

Identity of transformation products

No.:

#1

Reference

Reference substance name:

Unnamed

IUPAC name:

N-(2-aminoethyl)ethane-1,2-diamine

Inventory number:

InventoryMultipleMappingImpl [inventoryEntryValue=EC 203-865-4]

CAS number:

111-40-0

Identity:

1,2-Ethanediamine, N-(2-aminoethyl)-

Identity:

diethylene triamine

Molecular formula:

C4H13N3

Molecular weight:

103.166

SMILES notation:

NCCNCCN

InChl:

InChI=1/C4H13N3/c5-1-3-7-4-2-6/h7H,1-6H2

Details on results:

The temperature of the test solutions was maintained at approximately 20 °C during hydrolysis testing, and the pH of the test solutions was relatively unchanged when compared to the pH of the original buffers, indicating that the buffer capacity of the solutions was not exceeded.
Tables 1 to 4 present the results from the hydrolysis testing at four different pH values. Table 5 summarises the percent hydrolysis, hydrolytic rate constants and half lives of the test material experimentally determined during the study.

Based on these results, the test material is expected to hydrolyse rapidly in natural water bodies. In addition, the presence of the one degradate of the test material was confirmed during each of the tests.
Based on the r² for each analysis (ranging from 0.999 to 1.00), it was established that adequate precision was maintained during the analysis of the test solutions to determine half-lives based on the initial concentrations (0 hour).

Any other information on results incl. tables

Table 1 Concentrations of the Test Material Measured in pH 1.2 Replicates

Hours	Fortified Concentration (mg a.i./L)	Analytical Result (mg a.i./L)	Percent Hydrolysis
0	2500	2211	-
0.25	2500	1321	40.2
1	2500	898	59.4
2	2500	<796*	100
4	2500	<796*	100

*Concentrations expressed as < were below the limit of detection; therefore a concentration of zero was used in calculating percent hydrolysis.

 

Table 2 Concentrations of the Test Material Measured in pH 4 Replicates

Hours	Fortified Concentration (mg a.i./L)	Analytical Result (mg a.i./L)	Percent Hydrolysis
0	2500	1804	-
0.25	2500	967	46.4
1	2500	485	73.1
2	2500	474	73.7
4	2500	436	75.8
6	2500	430	76.1

 

Table 3 Concentrations of the Test Material Measured in pH 7 Replicates

Hours	Fortified Concentration (mg a.i./L)	Analytical Result (mg a.i./L)	Percent Hydrolysis
0	270	266	-
0.0833	270	55.5	79.2
0.25	270	28.4	89.3
1	270	28.4	89.3
3	270	24.9	90.6

 

Table 4 Concentrations of the Test Material Measured in pH 9 Replicates

Minutes	Fortified Concentration (mg a.i./L)	Analytical Result (mg a.i./L)	Percent Hydrolysis
0	270	203	-
1	270	103	49.2
3	270	24.1	88.1
15	270	<18.3	100

 

Table 5 Calculated Rate Constant and Half Lives

pH	Percent Hydrolysis	Rate Constant (Kobs)	Calculated HalfLife (t1/2)
			Hours	Minutes
1.2	71.3	0.813	0.853	51.2
4	76.1	1.22	0.566	34.0
7	90.6	8.25	0.0840	5.04
9	92.4	0.713	0.0162	0.972

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the percent hydrolysis and calculated half life at each of the pH levels was as follows:
pH 1.2: 100 %: t1/2 = 51 mins
pH 4: 76.1 %: t1/2 = 34 mins
pH 7: 90.6 %: t1/2 = 5 mins
pH 9: 100 %: t1/2 = 0.9 mins

Based on these results, the test material is expected to hydrolyse rapidly in natural water bodies.

Executive summary:

The hydrolytic stability of the test material was investigated in accordance with the standardised guideline OECD 111.

Hydrolysis testing was performed at 20 °C at pH 1.2, 4, 7 and 9. All samples were analysed for the test material and the degradate diethylenetriamine (DETA) using flow injection liquid chromatography/mass spectrometry (LC/MS). DETA was determined qualitatively to be present in the hydrolysis samples.

Under the conditions of this study, the percent hydrolysis and calculated half life at each of the pH levels was as follows:

pH 1.2: 100 %: t1/2 = 51 mins

pH 4: 76.1 %: t1/2 = 34 mins

pH 7: 90.6 %: t1/2 = 5 mins

pH 9: 100 %: t1/2 = 0.9 mins

Based on these results, the test material is expected to hydrolyse rapidly in natural water bodies.