2,2'-azobis[2-methylpropionamidine] dihydrochloride

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:

06.08.-21.08.2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 111 (Hydrolysis as a Function of pH)

Version / remarks:

only tier 1 pre-test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

no

Analytical monitoring:

yes

Duration:

5 d

pH:

4

Temp.:

50 °C

Initial conc. measured:

540 mg/L

Duration:

5 d

pH:

7

Temp.:

50 °C

Initial conc. measured:

540 mg/L

Duration:

5 d

pH:

9

Temp.:

50 °C

Initial conc. measured:

540 mg/L

Number of replicates:

2

Positive controls:

no

Negative controls:

no

Preliminary study:

The substance is hydrolytically unstable under test conditions. Tier 2 study was not performed since other available infomation was considered sufficient for the assessment.

Transformation products:

not specified

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

- Formation and decline of each transformation product during test:
- Pathways for transformation: see supplement to Werber et al. (2011) study
- Other:

Key result

Remarks on result:

not measured/tested

Remarks:

available information on degradation were considered sufficient

By comparing the molecular weight of the findings with the observations of Werber et al. following metabolites were identified: R₁OH (pH 7), R₁H (pH 4, 7, 9), Ring 1 (pH 4, 7, 9), R₂NNR₁ (pH 4, 7, 9) and R₂NNR₂ (pH 7, 9). While R₁OH, R₁H and Ring 1 can be identified as products, respectively intermediates, of the thermal process, R₂NNR₁ and R₂NNR₂ are the two steps of the hydrolysis. Full reaction scheme is attached to Werber et al. template (w_Werber_2011).

Validity criteria fulfilled:

yes

Conclusions:

The substance is hydrolytically instable.

Executive summary:

A solution of the test item in demineralised water was mixed with buffer solutions (pH values: 4, 7, and 9). The resulting solutions were sterilised by filtration using sterile 0.2 µm filters and stored at 50 °C for a period of five days. Samples were taken at the beginning and after five days. The analysis of the samples (performed with HPLC/UVD) showed significant changes in the concentration of the test item within five days. The areas of the detected test item lay below 90 % of the start areas at pH 4, 7, and 9; therefore, the test item is hydrolytically instable.

Reference 2

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Degradation of 2,2'-azobis(2-methylpropionamidine)dihydrochloride was examined over a wide range of pH values at three different temperatures. Kinetic of the reaction was obtained by comparison of changes in the UV spectra and the polarograpic signal of the solutions. The metabolites of hydrolysis were analysed by IR spectroscopy and elementar analysis.

GLP compliance:

no

Radiolabelling:

not specified

Analytical monitoring:

yes

Positive controls:

no

Negative controls:

no

Transformation products:

yes

No.:

#1

Key result

Remarks on result:

other: Please check details in any other information on results incl. tables

 

pH	kdx 10^-5s^-1
0.90	3.85
1.81	3.47
2.90	3.15
4.30	3.03
6.00	2.95
7.05	2.80

Table 1. Rates of decomposition of AAP at 60 °C

 

pH	kd/kh	(kd+ kh)x 10^-5s^-1	kdx 10^-5s^-1	khx 10^-5s^-1
7.7	0.90	5.7 ± 0.2	2.7	3.0 ± 0.2
8.5	0.29	11 ± 1	2.5	5.8 ± 0.7
9.5	0.13	22 ± 2	2.5	19.5 ± 2
9.85	0.095	29 ± 3	2.5	26.5 ± 3
10.2	0.062	45 ± 5	2.5	42.5 ± 5

Table 2. Rates of decomposition and hydrolysis at 60 °C

 

pH	Temperature (°C)	khx 10^-5s^-1	DT50 (h)
8.00	60	4.3	4.48
9.03	60	12.2	1.58
10.05	60	41.0	0.47
10.10	40	21.0	0.92
10.10	50	30.3	0.64
10.10	60	45.0	0.43
10.60	60	78.0	0.25

Table 3. Rates of Hydrolysis of AAP

Validity criteria fulfilled:

not applicable

Conclusions:

Both pathways of decomposition of AAP were examined over a wide pH range at 40 - 60 °C. Kinetic data were obtained by measurements using UV spectroscopy and polarography. Hydrolysis product was identified by infrared spectroscopy and elementar analysis. Findings show a high rate of hydrolysis at elevated temperatures and pH values which can be extrapolated to more environmental conditions.

Reference 3

Endpoint:

hydrolysis

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

AAPH was degraded in aqueous solution at 40 °C over a wide pH range. The transformation products were analysed by LC-MS/MS.

GLP compliance:

not specified

Remarks:

per-reviewed publication from academia, no GLP suspected

Radiolabelling:

no

Analytical monitoring:

yes

Details on sampling:

- Sampling intervals for the parent/transformation products:
- Sampling method:
- Sampling methods for the volatile compounds, if any:
- Sampling intervals/times for pH measurements:
- Sampling intervals/times for sterility check:
- Sample storage conditions before analysis:
- Other observation, if any (e.g.: precipitation, color change etc.):

Buffers:

- pH: 3-8
- Composition of buffer: 20 mM 2-(4-(2-hydroxyethyl)piperazin-1-yl)ethanesulfonic acid (HEPES)

- pH: 8-9.7
- Composition of buffer: 50 mM boric acid/ 50 mM KCl

- pH: 9.8-11
- Composition of buffer: 20 mM triethylamine

Exact pH were adjusted using 1 M HCl and 1 M NaOH.

Details on test conditions:

Solutions were stressed thermally at 40 °C until double half-life had passed at least.

Positive controls:

no

Negative controls:

no

Transformation products:

yes

No.:

#1

No.:

#2

No.:

#3

No.:

#4

No.:

#5

No.:

#6

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

- Formation and decline of each transformation product during test: Only stable endproducts of the different pathways are listed above.
- Pathways for transformation: The substance can be transformed either hydrolytically or thermally. Please check attached illustration
- Other:

Key result

pH:

8

Temp.:

40 °C

Hydrolysis rate constant:

0 s-1

Type:

not specified

shortened name	retention time (min)	molecular weight (Da)	compound name	MS/MS Fragments (m/z)
R1OH	3.7	102	2-Hydroxy-2-methylpropanimidamide	45, 58, 59, 68, 85, 86, 103
R2OH	4.3	103	2-Hydroxy-2-methylpropanamide	41, 58, 69, 71, 86, 104
R1OOR1	4.5	202	2,2‘-Peroxybis(2-methylpropanimidamide)	86, 101, 159, 186, 203
R1‘	5.2	84	Methacrylimidamide	41, 43, 44, 52, 53, 58, 68, 69, 85
R1H	5.6	86	Isobutyrimidamide	41, 43, 44, 54, 71, 87
R2‘	10.3	85	Methacrylamide	41, 44, 58, 70, 86
R2H	11.1	87	Isobutyramide	41, 43, 44, 45, 55, 57, 72, 73, 88
AAPH	12.3	198	2,2-Azobis (2-amidinopropane) dihydrochloride	71, 85, 86, 87, 154, 199
Ring 1	14.0	153	3,3,4,4-Tetramethylpyrrolidine-2,5-diimine	58, 68, 70, 71, 85, 86, 124, 137, 139, 154
R2NNR1	18.5	199	ACAP; Azo-2-carbamoyl-2,2‘-amidinopropane hydrochloride	only MS1 performed
Ring 2	19.3	154	3,3,4,4-Tetramethyl-5-oxopyrrolidin-2-imine	58, 67, 71, 85, 95, 110, 125, 138, 140, 155
R2NNR2	26.0	200	ACP; 2,2‘-Azobis(2-carbamoylpropane)	only MS1 performed
Ring 3	32.0	155	3,3,4,4-Tetramethylpyrrolidine-2,5-dione	58, 85, 86, 95, 101, 110, 113, 156

Table 1. Retention times and identification of compounds by LC-MS/MS

pH	kh1(x 10^-5s^-1)	kh2(x 10^-5s^-1)	Ratio
8.0	1.3	0.13	10
8.8	7.1	1.2	5.9
9.8	62	12	5.2
10.1	78	22	3.5
10.9	98	41	2.4
11	85	40	2.1

Table 2. rates of hydrolysis for various pH conditions

The degradation rate for thermal decomposition was calculated by subtraction of hydrolysis rate k_h1 from the observed overall degradation rate. The thermal degradation rate at 40 °C was found to be in the range of 1.5 x 10^-6 and 2.4 x 10^-6 s^-1 showing no clear trend with pH variations. This observation is in good agreement with the findings of Dougherty from which a value of 2.3 x 10^-6 s^-1 for the thermal decomposition rate can be calculated for 40 °C.

Validity criteria fulfilled:

not applicable

Conclusions:

Observation in the presented study show clear indications on thermal and hydrolytically degradation of 2,2-Azobis(2-amidinopropane) dihydrochloride at 40 °C with varying pH value. A complex degradation scheme was identified using LC-MS/MS. Kinetic observations using LC-UV indicate that thermal decomposition is pH independent while the hydrolysis rates increases with gradient pH value.

Executive summary:

The test item was degraded in aqueous solutions at 40 °C over a wide range of pH. Degradation rates were observed via LC/UVD and formation of metabolites as well as pathways were analysed by LC/MS-MS. Two major degradation processes were identified. On the one hand a pH independent thermal decomposition of the substance and on the other hand the hydrolysis with exponentially increasing degradation rate with pH.

Description of key information

The key value was estimated from the extrapolations of the findings from Werber and Ito. Additionally, the half-life under environmental conditions was doubled to accommodate the high uncertainties due to the extrapolation.

Key value for chemical safety assessment

Half-life for hydrolysis:

10 d

at the temperature of:

20 °C

Additional information

The hydrolysis of 2, 2’-azobis(2-amidinopropane) dihydrochloride has been in the focus of research groups for a long time. Findings show that generally the substance decomposes by two different competing multi-step mechanisms in aqueous solutions. On the one hand a pH value independent thermal decomposition and on the other hand a hydrolytically decomposition whose rate exponentially increases with pH is known.

Observations of Dougherty and Hammond et al. on thermal decomposition indicate that tertiary radicals are formed by loss of the labile azo group, which are stabilized by a solvent cage [Hammond, 1963]. In further analysis on the formation of metabolites under acidic conditions by elemental analysis, m.p. determination and infrared spectroscopy for a period of several month tetramethylsuccinimide and tetramethyl-5-imino-2-pyrrolidone were identified [Dougherty, 1963]. These findings are supported by observations and LC/MS analysis of Werber et al.. In addition, several other, non-cyclic, metabolites were identified showing a complex radical degradation pattern of AAPH [Werber, 2011]. 

Hydrolysis of AAPH is a two-step process. While consuming hydroxyl ions two NH₃ molecules are separated from the AAPH molecule forming 2, 2’-azobis(2-carbamylpropane (ACP)) [Werber, 2011; Ito, 1973]. As the first step still undergoes thermal decomposition processes the final product is considered to be stable to thermal decomposition [Ito, 1973].

Findings of Werber et al. (Werber, 2011) show clear indications that at 40 °C in acidic and neutral environments thermal and in basic environments hydrolytically decomposition predominates the degradation process of AAPH. The kinetic rate constant for thermal decomposition was found to be relatively constant over the examined pH range showing an average of 2.1x10^-6 s^-1while the rate constant for hydrolysis increased with pH from 1.3x10^-5 s^-1at pH 8.0 to 85x10^-5 s^-1at pH 11.0. Several metabolites of both pathways were identified by LC/MS. Ito found hydrolysis rates about two orders of magnitude lower than rates for thermal decomposition under acidic conditions. 

In a recent Tier 1 study according OECD guideline 111 (Brinkmann, 2013) several metabolites were found after five days. By comparing the molecular weight of the findings with the observations of Werber following metabolites were identified: R₁OH (pH 7), R₁H (pH 4, 7, 9), Ring 1 (pH 4, 7, 9), R₂NNR₁ (pH 4, 7, 9) and R₂NNR₂ (pH 7, 9). While R₁OH, R₁H and Ring 1 can be identified as products, respectively intermediates, of the thermal process, R₂NNR₁ and R₂NNR₂ are the two steps of the hydrolysis.

In absence of experimental data for the transformation products QSAR estimations of biodegradability were performed indicating a low rate of biodegradation, in general. These calculations are supported by the available study on readily biodegradation of 2, 2’-azobis(2-amidinopropane) dihydrochloride showing almost no degradation after 28 days.

Summarizing all available information on hydrolysis of AAPH two competing process can be identified. While decomposition of AAPH at acidic and neutral conditions is predominated by thermal decomposition the rate of hydrolysis increases with elevating pH value. However, both steps are relatively fast and full decomposition of AAPH after one week under environmental conditions can be assumed. From the observations of Ito a half-life of 115 hours can be calculated for pH 7.05 and 20 °C using equation R.16 -9 (TGD on information requirements and chemical safety assessment (Version 2, May 2010)). An identical half-life of 115 hours at pH 7 and 20 °C can be estimated by exponential fitting of the results of Werber et al. and subsequent extrapolation using equation R.16 -9. For the assessment a half-life DT50 of 10 days is assumed keeping in mind the uncertainties from extrapolating data.

Thus, the available information are considered to be adequate for assessing the abiotic degradation of AAPH and no further gain of knowledge is expected from conducting additional studies.

Brinkmann A (2013) Determination of pH-dependent Hydrolysis in Water of 2, 2'-Azobis(2 -amidinopropane)-dihydrochloride (=2,2'-azobis[2 -methylpropionamidine]dihydrochloride) according to OECD Guideline 111, Study No. 12010301G916, LAUS GmbH.

Dougherty TJ (1961) Chemistry of 2, 2'-Azobisisobutyramidine Hydrochloride in Aqueous Solution: A Water-Soluble Azo Initiator, J. Am. Chem. Soc., 83 (23), 4849 -4853.

Hammond GS, Neuman RC (1963) The Mechanism of Decomposition of Azo Compounds. III. Cage Effects with Positively Charged Geminate Radical Pairs, J. Am. Chem. Soc., 85 (10), 1501 -1508.

Ito K (1973) Competition between Thermal Decomposition and Hydrolysis of 2, 2'-Azobis(2 -amidinopropane) in Aqueous Solution, J. Polymer Sci., 11, 1673 -1681.

Werber J et al. (2011) Analysis of 2, 2'-Azobis (2 -Amidinopropane) Dihydrochloride Degradation and Hydrolysis in Aqueous Solutions, J. Pharm. Sci., 100 (8), 3307 -3315.