Registration Dossier

Administrative data

Endpoint:
hydrolysis
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
11-2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 111 (Hydrolysis as a Function of pH)
Deviations:
yes
Remarks:
substance prepared in situ in acidic solution. At t0 the solution was neutralized to pH 7 and test was started
Principles of method if other than guideline:
The study was performed according to the principles described in the guideline, i.e. preliminary screening at 50°C and assessment of hydrolysis at 20°C and neutral pH. Unlike the OECD guideline, only pH 7 was tested. Moreover, due to the instability of the substance, the substance was made in situ by a diazotization reaction. The resulting acidic solution containing the newly formed diazonium salt was neutralized to pH 7 on t0 and the test was started.
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
IN SITU SYNTHESIS OF THE DIAZONIUM SALT
2-nitro-p-anisidine (CAS 96-96-8) was converted in a diazotisation reaction to its corresponding diazonium salt.

1) 2-nitro-p-anisidine (13.5 g) was suspended in mixture of 25 mL water and 21.3 mL 30 % HCl at 0°C.
2) The mixture was cooled in an ice bath to 0°C and stirred for 30 min
3) A pre-cooled 30 % aq. solution of NaNO2 (80 mmol) was added dropwise over 2h to induce the diazotisation reaction. This reaction is highly exothermic and needs to be carried out carefully. The internal temperature was continuously monitored and maintained below 5°C.
4) Afterwards, the reaction mixture was diluted with water (100 mL) and stirred for another 30 minutes and kept at 0°C.
5) The products were not isolated, but kept in this aqueous solution in an ice bath. The hydrolysis experiments were immediately started.
Radiolabelling:
no

Study design

Analytical monitoring:
yes
Remarks:
LC-UV-MS
Details on sampling:
Pre-test at 50°C:
- Sampling intervals: 5 days

Main test at 20°C:
- Sampling intervals: 0, 3, 6, 12, 24, 48, 72, 96, 120 h
- Sampling method: vials from which sample was taken were not used anymore after sampling
- Sampling intervals/times for pH measurements: pH measurement at test start
- Sampling intervals/times for sterility check: not performed
- Sample treatment: filtered in case of precipitation or diluted in 1:5 methanol
- Sample storage conditions before analysis: direct analysis
Buffers:
phosphate buffer 0.1M at pH 7
Details on test conditions:
TEST SYSTEM
- Type, material: glass test flasks and vials
- Sterilisation method: none
- Lighting: none
- Measures taken to avoid photolytic effects: incubation in the dark
- Temperature: pre-test at 50°C and main test at 20°C in thermostated oven


TEST MEDIUM
- Preparation of test medium: synthesis solution with concentration 60 mg/mL dissolved 1:30 in 0.1 M phosphate buffer of pH 7
- For main test at 20°C: synthesis solution divided over 20 separate vials
- Renewal of test solution: samples were not disturbed during hydrolysis period

OTHER TEST CONDITIONS
- Adjustment of pH: after dissolution, pH was checked
Duration of test
Duration:
120 h
pH:
7
Temp.:
20 °C

Results and discussion

Preliminary study:
After 5 days, significant amount of precipitation, which is removed by filtering. Dilution of 1:10 in MeOH dissolved precipitation.
Degradation after 5 days at 50°C was complete.
Transformation products:
yes
Identity of transformation products
No.:
#1
Reference
Reference substance name:
Unnamed
Dissipation DT50 of parent compoundopen allclose all
Key result
pH:
7
Temp.:
20 °C
DT50:
< 24 h
Remarks on result:
other: final test
pH:
7
Temp.:
50 °C
DT50:
< 5 d
Remarks on result:
other: preliminary test
Details on results:
see any other information on results incl.tables

Any other information on results incl. tables

The concentration of the diazonium salt was found to drop to ca. 22% of the starting concentration after 24 hours. After 48 hours, none of the parent diazonium salt is left in the reaction mixture. These findings demonstrate that the diazonium salt is hydrolytically unstable, and its DT50 is < 24 hours. Hence, the identification of any relevant hydrolysis products was attempted.

 

The main degradation product that is formed during the hydrolysis experiment is 4-methoxy- benzenediazonium chloride. Unexpectedly, this hydrolysis product is in itself again a diazonium salt, in which the nitro group present in the parent substance has been replaced by a hydrogen atom.

Furthermore, the analysis of the sample at t0 shows that the diazotization reaction does not proceed very cleanly, and many side products are already visible before the start of the hydrolysis experiment. The main side product formed during the generation of the diazonium salt is 2-nitroanisole, i.e. the diazonium salt from which the N2 group was lost. Its concentration drops over time during the hydrolysis experiment.

Furthermore, also the aniline used for the synthesis of the diazonium salt is present in the t0 sample. Its concentration increases slightly during the hydrolysis experiment, but does not reach more than 15% of the starting concentration of the parent diazonium salt after 5 days.

Many other degradation and/or transformation products are found in the hydrolysis solution. However, it was not possible to (fully) identify most of them, and none of them reaches a concentration >5% of the parent diazonium salt.

Applicant's summary and conclusion

Validity criteria fulfilled:
not applicable
Conclusions:
The 4-methoxy-2-nitrobenzenediazonium chloride was found to rapidly hydrolyse. The half-life at 20°C and pH 7 is < 24h. The main degradation product formed is another diazonium salt: 4-methoxybenzenediazonium chloride.
Executive summary:

The hydrolysis of the diazonium salt 4-methoxy-2-nitrobenzenediazonium chloride was investigated in a study based on the principles of OECD 111. Deviation compared to the guideline was needed since the diazonium salt had to be generated in situ due to it's reactivity and explosive properties when isolated.

First, 2-nitro-p-anisidine (CAS 96-96-8) was converted in a diazotisation reaction to its corresponding diazonium salt, 4-methoxy-2-nitrobenzenediazonium chloride. This acidic cooled solution was neutralized to pH 7 in a phosphate buffer. This was considered to be t0 of the experiment.

In the main study at 20°C, the hydrolysis was followed by sampling after 0, 3, 6, 12, 24, 48, 72, 96 and 120h. Samples were filtered or diluted in methanol (1:5) and directly measured by LC-UV-MS. This analysis allowed a semi-quantitative analysis of the degradation products.

The concentration of diazonium salt was found to drop rapidly to 25 % of the starting concentration after 24h. After 48h, complete hydrolysis is assumed as no parent diazonium salt is left in the reaction mixture. The main degradation product that is formed during the hydrolysis experiment is another diazonium salt 4-methoxy- benzenediazonium chloride. Many other degradation and/or transformation products are found in the hydrolysis solution. However, it was not possible to (fully) identify most of them, and none of them reaches a concentration >5% of the parent diazonium salt.