N-[3-({[2,2-dimethyl-3-(morpholin-4-yl)propylidene]amino}methyl)-3,5,5-trimethylcyclohexyl]-2,2-dimethyl-3-(morpholin-4-yl)propan-1-imine

Hydrolysis

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2014-06-13 to 2014-07-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study is regarded as reliable without restrictions. The study was conducted under GLP equivalent conditions. The study was conducted under supervision of a quality assurance unit which had ensured e.g instrumentation validation, reagent/materials certification, analyst certification and providing standard operating procedures. The study was conducted according to OECD guideline but have deviation to the guideline requirements. Deviations: The study was conducted only at the temperature of 10 °C. It was assumed that the slowest rate of hydrolysis will be observed at 10 °C. Thus, this deviation was not regarded to have an influence of the outcome of the study.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Deviations:

yes

Remarks:

Deviations: The study was conducted only at the temperature of 10 °C. It was assumed that the slowest rate of hydrolysis will be observed at 10 °C. Thus, this deviation was not regarded to have an influence of the outcome of the study.

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)

Deviations:

yes

Remarks:

Deviations: The study was conducted only at the temperature of 10 °C. It was assumed that the slowest rate of hydrolysis will be observed at 10 °C. Thus, this deviation was not regarded to have an influence of the outcome of the study.

GLP compliance:

yes

Remarks:

The study was conducted under supervision of a quality assurance unit which had ensured e.g instrumentation validation, reagent/materials certification, analyst certification and providing standard operating procedures.

Specific details on test material used for the study:

PHYSICO-CHEMICAL PROPERTIES
- Vapour pressure: 0.000000044 Pa
- Water solubility: The substance hydrolysis fast in contact with water, thus it was technically not feasible to determine the water solubility.
- log Pow: The log Pow of the hydrolysis Product was determined to be 5.71
- pKa: not determined
- Base or acid catalysis of test material: The susbance has no basic or acid properties.
- UV absorption: above 220 nm
- Stability of test material at room temperature: The substance is stable at room temperature under dry conditions.

Radiolabelling:

no

Analytical monitoring:

yes

Details on sampling:

- Sampling intervals for the parent/transformation products: The stability measurement of the test item was performed initially after sample preparation and again after one and eight days.
- Sampling method: A microliter pipette was used.
- Sampling intervals/times for pH measurements: The pH value was once measured immediately after sample preparation and again after one and eight days.
- Sample storage conditions before analysis: The samples are kept at 10 °C and at room temperature.
- Other observation: No alteration of the test solution was observed.

The substance was dissolved in dry acetonitrile (approx. 100 mg in 5 mL ACN). The organic solutions (26 - 27 μL) were added to 5 mL of the tempered buffer solutions (10 °C). Immediately after vortexing, the sample solution (100 μg test item/mL water) was injected by a 500 μL syringe at a constant flow rate (10 μL/min) into the mass spectrometer.

Buffers:

- pH 4: 5.7 g ammonium formate and 1.885 mL (0.1 M) of formic acid were dissolved and diluted to 500 mL with ultra-pure water. The pH value was adjusted with formic acid to pH 4.03 (degassed with argon).
- pH 7: ultra-pure water, measured after degassing with argon pH: 6.8.
- pH 9: 3.2 g ammonium formate (0.1 M) and 2.0 mL ammonia solution (25 %) were dissolved and diluted to 500 mL with ultra-pure water. The pH value was adjusted with formic acid to pH 9.04 (degassed with argon).
The pH of each buffer solution was checked with a calibrated pH meter.

Details on test conditions:

TEST SYSTEM
- Type, material and volume of test flasks, other equipment used: 5 mL volumetric flask.
- Measures taken to avoid photolytic effects: The samples were stored in the dark.
- Measures to exclude oxygen: The solvents used were degassed with argon and the samples were stored under argon.
- If no traps were used, is the closed / open. A closed test system was used.
- Is there any indication of the test material adsorbing to the walls of the test apparatus? There was no evidence that the test material had an affinity for adsorption on the walls of the test apparatus.
TEST MEDIUM
- Volume used/treatment: 5 mL Buffer solution containing not more than 1 % (v/v) of acetonitrile
- Kind and purity of water: degassed ultra-pure water

Number of replicates:

3 replicates

Positive controls:

no

Negative controls:

no

Preliminary study:

The substance was known to be hydrolytically unstable at environmentally relevant temperature. Thus, no preliminary test was conducted.

Transformation products:

yes

No.:

#1

No.:

#2

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

The substance contains two imine groups which are known to be hydrolytically unstable. After getting into contact with the water (buffer solution) the hydrolysis reaction forms the respective aldehyde and amine compound.

#1 degradation product:
- CAS no.: 23588-51-4
- CAS name: 4-Morpholinepropanal, a,a-dimethyl-
- IUPAC name: 2,2-dimethyl-3-(morpholin-4-yl)propanal
- Common name: Aldehyde M

#2 degradation product:
- CAS: 2855-13-2
- IUPAC name: 3-aminomethyl-3,5,5-trimethylcyclohexylamine
-Common name: IPDA

% Recovery:

0

pH:

4.03

Temp.:

10 °C

Duration:

1.25 min

% Recovery:

0

pH:

6.8

Temp.:

10 °C

Duration:

1.25 min

% Recovery:

0

pH:

9.04

Temp.:

10 °C

Duration:

1.25 min

Key result

pH:

4

Temp.:

25 °C

DT50:

< 1 min

Type:

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

Key result

pH:

7

Temp.:

25 °C

DT50:

< 1 min

Type:

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

Key result

pH:

9

Temp.:

25 °C

DT50:

< 1 min

Type:

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

Other kinetic parameters:

Due to the fast hydrolysis it was technically not possible to determine the kinetic parameters of the hydrolysis. The first measurement was immediately after mixing of the stock solution of the substance with the respective buffer solution. This procedure took 1 min 15 sec. No parent compound could be detected in the test solution. Thus, it was concluded that the substance was fully hydrolysed after 1.25 min at 10 °C. Thus, further test at different temperature were omitted as it was predicted that at higher temperature the same results would be achieved.

Details on results:

The test item was completely hydrolysed after 1 min 15 sec. Neither the substance nor its partly hydrolysed form was observed, independently of the pH value (pH 4.03, 6.8 and 9.04). The first measurement was taken 1 min 15 sec after mixing and a stable MS signal was observed. The second and the third measurements after 4 and 7 min do not indicate a significant increase of the signal intensities. Consequently the substance was dissolved in water due to the hydrolysis reaction after 1 min 15 sec.
It was technically not possible to achieve six spaced data points showing hydrolysis of the test substance between 10 % and 90 % hydrolysis as requested by the guideline. The first data point was taken after 1 min 15 sec which was the time from mixing the stock solution of the test item with the respective buffer solution. This was confirmed in all replicate measurements. Thus, it was not possible to prepare a log-transformed presentation of the date nor to calculate the DT50 values.
As the substance was fully hydrolysed the respective degradation products were identified. The degradation products were identified at any time point and pH values.

Validity criteria fulfilled:

not applicable

Conclusions:

The hydrolysis was investigated using a flow-injection mass spectrometer. The test item was fully hydrolysed after 1 min and 15 sec at pH 4, 7 and 9 at temperature of 10 °C. It was concluded that the substance will undergoes fast hydrolysis when getting into contact with water under all conditions. Measurements revealed a very fast hydrolysis of SIKA Hardener MI in water and water-based media (t1/2 < 0.5 minutes in water). The degradation products of the hydrolysis of SIKA Hardener MI are 2,2-dimethyl-3-(morpholin-4-yl)propanal (Aldehyde M) and 3-Aminomethyl-3,5,5-trimethylcyclohexylamine.

Executive summary:

A study was conducted in accordance with OECD TG 111 to determine the kinetics of the hydrolysis at different pH values (4, 7 and 9). The substance consists of two imine groups which are known to be hydrolytically unstable. It was assumed that when getting into contact with water the substance will undergo hydrolysis forming the respective aldehyde and amine compounds. Thus no preliminary test was conducted. A stock solution of the test item in acetonitrile was prepared. 26 - 27 µL of the stock solution was mixed with 5 mL of the tempered buffer solution. Thus, the test solution did not contain more than 1 % organic solvent. The test solution as measured immediately after mixing. This took 1 min 15 sec. Neither the parent compound nor its partly hydrolysed form was detected independently of the pH values (4, 7 and 9) in the first measurements after 1.25 min. This was confirmed in all replicate measurements. It was therefore technically not possible to provide a minimum of six spaced data points showing hydrolysis between 10 and 90 %. Furthermore it was not possible to calculate the accurate DT50 values of the test item. It was concluded that the DT50 (pseudo first order) will be less than 1 min. As no parent compound was observed the two hydrolysis products were identified. The initial assumption was confirmed. The respective aldehyde 2,2-dimethyl-3-(morpholin-4-yl)propanal (Aldehyde M) and amine amine 3-aminomethyl-3,5,5-trimethylcyclohexylamine (Isophorondiamine; IPDA) were detected using the APCI-MS method and identified using the mass spectra in combination with the exact mass. In deviation to the guideline requirements the test was only conducted at 10 °C. This temperature was the lowest value requested by the guideline. As the hydrolysis depends on the temperature it was assumed that the slowest hydrolysis rate will be at 10 °C. As the parent compound could not be detected at all three pH values immediately after mixing of the test solution, it was concluded that the same results will be achieved at higher temperatures. Thus, test at other temperatures were omitted. In conclusion, it was stated that the substance will undergo fast hydrolysis when getting into contact with water under all conditions.

Reference 2

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2011-05-26

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.7 (Degradation: Abiotic Degradation: Hydrolysis as a Function of pH)

Principles of method if other than guideline:

In this study the stability of SIKA Hardener MI in water and in organic solvents (acetonitrile and methanol) was investigated. In order to assess the hydrolysis sampling was performed at representative and relevant points in time. The degradation products of SIKA Hardener MI were identified and the degradation product's stability was investigated in media used for ecotoxicological studies (OECD and ISO medium).

GLP compliance:

yes

Remarks:

equivalent to GLP

Radiolabelling:

no

Analytical monitoring:

yes

Details on sampling:

Stock solutions:
- 25 mg/mL SIKA Hardener MI in acetonitrile (for stability test in acetonitrile)
- 25 mg/mL SIKA Hardener MI in methanol (for stability test in methanol)
- 10 mg/mL SIKA Hardener MI in acetonitrile (for stability test in water)
- 1 mg/mL degradation product of SIKA Hardener MI (Aldehyde M) in acetonitrile (for stability test in OECD and ISO medium)

Preparation of working solutions for investigating the stability/hydrolysis via LC/MS and LC/MS/MS, respectively:
- For stability test in organic solvents:
The stock solutions were diluted with methanol or acetonitrile in three steps (25x, 10x, 10x) from 25 mg/mL to 10 µg/mL. The stock solution prepared on methanol was diluted with methanol containing 1mM ammonium formate.
- For stability test in water:
The stock solution (10 mg/mL SIKA Hardener MI in acetonitrile) was quickly diluted with ultra-pure water in one step from 10 mg/mL to 10 µg/mL and 100 µg/mL, respectively.
- For stability test in OECD and ISO medium:
The stock solutions were diluted to 2.5, 5, 10, 25, 50, 75, 100, 150 ng/mL concentrations with water, ISO or OECD medium.
The protriptyline hydrochloride concentration was 125 ng/mL in each working solution. Protriptyline hydrochloride was used as internal standard.

Buffers:

- OECD medium: aqueous buffer solution prepared in accordance with OECD guideline no. 201 (Algae), pH = 8.1
- ISO medium: aqueous buffer solution prepared in accordance with OECD guideline no. 202 (Daphnia sp.), pH = 6 - 9

Details on test conditions:

- Solvent: ultra-pure water, acetonitrile and methanol (for SIKA Hardener MI), ultra-pure water, OECD and ISO medium (for Aldehyde M)
- Sampling: immediately after dilution, 0.5 and 1 minutes later (in water); immediately after dilution, 2 and 4 hours later (in acetonitrile); immediately after dilution (in methanol); immediately after dilution, 13 and 26 hours later (in ISO and OECD medium)
- Other:
In order to assess the adsorption of the test item to the vial's surface the stock solution (10 mg/mL SIKA Hardener MI in acetonitrile) was quickly diluted to 10 µg/mL or 100 µg/mL in one step with ultra-pure water. After homogenization the solution was transferred out into another vial. To recover the adsorbed test item the original vial was washed with equal volume of acetonitrile. Then the rinsing acetonitrile was injected directly into the LC/MS system.

Number of replicates:

3 replicates (in OECD and ISO medium for SIKA Hardener MI's degradation product)

Positive controls:

no

Negative controls:

no

Transformation products:

yes

No.:

#1

No.:

#2

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

#1 degradation product:
- CAS no.: 23588-51-4
- CAS name: 4-Morpholinepropanal, a,a-dimethyl-
- IUPAC name: 2,2-dimethyl-3-(morpholin-4-yl)propanal
- Common name: Aldehyde M

#2 degradation product:
- CAS: 2855-13-2
- CAS name: 3-Aminomethyl-3,5,5-trimethylcyclohexylamine

% Recovery:

0

Temp.:

25 °C

Duration:

ca. 0.5 min

Details on results:

Calibration:
Calibration series were prepared from separate stock solutions for Aldehyde M (as reference compound) and for Aldehyde M originated as degradation product from the hydrolysis of SIKA Hardener MI in water, OECD and ISO medium immediately after dilution, 13 and 26 hours later. The concentration of Aldehyde M in these different media showed good linearity in the 2.5-150 ng/mL concentration range. The calibration curves were constructed applying linear regression.

Investigation of the hydrolysis of SIKA Hardener MI and identification of its degradation products:

In acetonitrile no degradation products of SIKA Hardener MI (Aldehyde M, m/z: 172.1 amu) could be detected, even after a storage for 24 hours (see Figures 1-3). In contrast after mixing SIKA Hardener MI with the protic solvent methanol an intense signal of the degradation product Aldehyde M (m/z: 172.1 amu) and its methanol-adduct (m/z: 204.0) could be observed (see Figure 4). MS spectra of SIKA Hardener MI in ultrapure water at a low concentration level (10 µg/mL) (see Figure 5) showed immediately after dilution an almost complete hydrolysis. No signals of SIKA Hardener MI (m/z: 477.2 amu for [MI+H+]) could be detected. At the high concentration level (100 µg/mL) (see Figure 6) weak MS signals of SIKA Hardener MI (~ 8 % relative abundance) and intense signals of the degradation products Aldehyde M (m/z: 172.1 amu), its methanol-adduct (m/z: 204.0) and the corresponding amine (m/z: 171.1 amu) could be detected immediately after preparation. The weak signal of the parent compound practically disappeared within approximately 1 minute (see Figure 7, 8).

Furthermore via the analysis of the solvent that was used for washing the vial containing test item solution, it could be shown that the very fast and intense degradation of SIKA Hardener MI is the predominant process resulting in prompt disappearance of the test items in water. Thus the adsorption of the test item on the vial's surface can be excluded.

The degradation products (Aldehyde M and 3-Aminomethyl-3,5,5-trimethylcyclohexylamine) were identified via their MS signals (m/z: 172.1 amu and m/z: 171.1 amu, respectively) (see Figure 5 -8).

The storage stability of the degradation product of SIKA Hardener MI (Aldehyde M) was evaluated in aqueous solutions (ultra-pure water, OECD and ISO medium). In ultrapure water (see Figure 9-10) Aldehyde M (reference item as well as item originated from the hydrolysis of SIKA Hardener MI) is stable for at least 26 hours. In aqueous buffer solutions (OECD and ISO medium) a rapidly decreasing signal of Aldehyde M (during 26 h storage) was observed (see Figure 11-14). In OECD medium approximately 5 times and in ISO medium approximately 1.5 times lower Aldehyde M concentration were measured after 26 hours.

Validity criteria fulfilled:

not specified

Conclusions:

The hydrolysis of SIKA Hardener MI in different media (ultra-pure water, aprotic and protic organic solvents like acetonitrile and methanol) and the stability of SIKA Hardener MI's degradation product (Aldehyde M) in water and aqueous buffer solutions (OECD and ISO medium) at room temperature were investigated using LC/MS and LC/MS/MS, respectively.
Measurements revealed a very fast hydrolysis of SIKA Hardener MH in water and water-based media (t1/2 < 0.6 minutes in water). The degradation products of the hydrolysis of SIKA Hardener MH are 2,2-dimethyl-3-(morpholin-4-yl)propanal (Aldehyde M) and hexanediamine.

Executive summary:

The hydrolysis of SIKA Hardener MI and the identification of the decomposition products was assessed in a way similar to the methods described in OECD guideline no. 111 and EU method C.7.

The hydrolysis of SIKA Hardener MI in different media (water, aprotic and protic organic solvents like acetonitrile and methanol) and the stability of the degradation product (2,2-dimethyl-3-(morpholin-4-yl)propanal (Aldehyde M)) in water and aqueous buffer solutions (OECD and ISO medium) at room temperature was investigated using LC/MS/MS.

Measurements revealed a very fast hydrolysis of SIKA Hardener MI in water and water-based media (t1/2 < 0.5 minutes in water). The degradation products of the hydrolysis of SIKA Hardener MI 2,2-dimethyl-3-(morpholin-4-yl)propanal (Aldehyde M) and 3-Aminomethyl-3,5,5-trimethylcyclohexylamine were successfully identified via their MS signals. In the aprotic organic solvent acetonitrile SIKA Hardener MI is stable for at least 26 hours storage. In contrast in the protic solvent methanol SIKA Hardener MI is not stable.

The storage stability of the degradation product of SIKA Hardener MI (Aldehyde M) was evaluated in aqueous solutions (ultra-pure water, OECD and ISO medium). In ultra-pure water Aldehyde M is stable for at least 26 hours. In aqueous buffer solutions (OECD and ISO medium) a rapidly decreasing signal of Aldehyde M was observed. In OECD medium approximately 5 times and in ISO medium approximately 1.5 times lower Aldehyde M concentration were measured after 26 hours. This observation indicates that the measured concentration of Aldehyde M in these ecotoxicological media does not reflect its real concentration.

Description of key information

The hydrolysis of Sika Hardener MI in different media (ultra-pure water, aprotic and protic organic solvents like acetonitrile and methanol)  and the stability of Sika Hardener MI's degradation product (Aldehyde M) in water and aqueous buffer solutions (OECD and ISO medium) at room temperature were investigated using LC/MS and LC/MS/MS, respectively. Measurements revealed a very fast hydrolysis of Sika Hardener MI in water and water-based media (t1/2 < 0.5 minutes in water).  The degradation products of the hydrolysis of Sika Hardener MI are 2,2-dimethyl-3-(morpholin-4-yl)propanal (Aldehyde M) and 3-Aminomethyl-3,5,5-trimethylcyclohexylamine.

Key value for chemical safety assessment

Half-life for hydrolysis:

0.5 min

at the temperature of:

25 °C

Additional information

A study was conducted in accordance with OECD TG 111 to determine the kinetics of the hydrolysis at different pH values (4, 7 and 9). The substance consists of two imine groups which are known to be hydrolytically unstable. It was assumed that when getting into contact with water the substance will undergo hydrolysis forming the respective aldehyde and amine compounds. Thus no preliminary test was conducted. A stock solution of the test item in acetonitrile was prepared. 26 - 27 µL of the stock solution was mixed with 5 mL of the tempered buffer solution. Thus, the test solution did not contain more than 1 % organic solvent. The test solution as measured immediately after mixing. This took 1 min 15 sec. Neither the parent compound nor its partly hydrolysed form was detected independently of the pH values (4, 7 and 9) in the first measurements after 1.25 min. This was confirmed in all replicate measurements. It was therefore technically not possible to provide a minimum of six spaced data points showing hydrolysis between 10 and 90 %. Furthermore it was not possible to calculate the accurate DT50 values of the test item. It was concluded that the DT50 (pseudo first order) will be less than 1 min. As no parent compound was observed the two hydrolysis products were identified. The initial assumption was confirmed. The respective aldehyde 2,2-dimethyl-3-(morpholin-4-yl)propanal (Aldehyde M) and amine amine 3-aminomethyl-3,5,5-trimethylcyclohexylamine (Isophorondiamine; IPDA) were detected using the APCI-MS method and identified using the mass spectra in combination with the exact mass. In deviation to the guideline requirements the test was only conducted at 10 °C. This temperature was the lowest value requested by the guideline. As the hydrolysis depends on the temperature it was assumed that the slowest hydrolysis rate will be at 10 °C. As the parent compound could not be detected at all three pH values immediately after mixing of the test solution, it was concluded that the same results will be achieved at higher temperatures. Thus, test at other temperatures were omitted. In conclusion, it was stated that the substance will undergo fast hydrolysis when getting into contact with water under all conditions.

 

In a supporting study the hydrolysis of Sika Hardener MI and the identification of the decomposition products was assessed in a way similar to the methods described in OECD guideline no. 111 and EU method C.7. The hydrolysis of Sika Hardener MI in different media (water, aprotic and protic organic solvents like acetonitrile and methanol) and the stability of the degradation product (2,2-dimethyl-3-(morpholin-4-yl)propanal (Aldehyde M)) in water and aqueous buffer solutions (OECD and ISO medium) at room temperature was investigated using LC/MS/MS.

Measurements revealed a very fast hydrolysis of Sika Hardener MI in water and water-based media (t1/2 < 0.5 minutes in water). The degradation products of the hydrolysis of SIKA Hardener MI 2,2-dimethyl-3-(morpholin-4-yl)propanal (Aldehyde M) and 3-Aminomethyl-3,5,5-trimethylcyclohexylamine were successfully identified via their MS signals. In the aprotic organic solvent acetonitrile Sika Hardener MI is stable for at least 26 hours storage. In contrast in the protic solvent methanol Sika Hardener MI is not stable.

The storage stability of the degradation product of Sika Hardener MI (Aldehyde M) was evaluated in aqueous solutions (ultra-pure water, OECD and ISO medium). In ultra-pure water Aldehyde M is stable for at least 26 hours. In aqueous buffer solutions (OECD and ISO medium) a rapidly decreasing signal of Aldehyde M was observed. In OECD medium approximately 5 times and in ISO medium approximately 1.5 times lower Aldehyde M concentration were measured after 26 hours. This observation indicates that the measured concentration of Aldehyde M in these ecotoxicological media does not reflect its real concentration.