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Phototransformation in water

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Reference
Endpoint:
phototransformation in water
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 October 2018 - 24 September 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Study type:
direct photolysis
Qualifier:
according to
Guideline:
OECD Guideline 316 (Phototransformation of Chemicals in Water - Direct Photolysis)
Version / remarks:
adopted 03 October 2008
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
RADIOLABELLED TEST MATERIAL

Identification: [carbonyl-14C] 2-(dimethylamino)-2-[(4-methylphenyl)methyl]-1-[4-(morpholin-4-yl)phenyl]butan-1-one
Preparation of test material: Spike solutions were prepared by dissolving 14C-labelled Omnirad 379 in acetonitrile
Physical Description: Pale yellow solution in acetonitrile
Radiochemical purity: 98.9% (at use); 99.3% (as received)
Chemical purity: 97.5% (at use); 98.6% (as received)
Specific activity: 3.45 MBq/mg (1317 MBq/mmol)
Molecular formula: C24H32N2O2
Molecular weight: 381.66 g/mol (at this specific activity)

Test item storage: In freezer (≤ -15°C) protected from light
Test item handling: Use amber glassware or wrap container in aluminium foil
Supplier: Selcia Limited, Fyfield Business & Research park, Fyfield Road, Ongar, Essex, CM5 OGS, UK
Justification for Type of Labelling: Carbon-14 is the isotope of choice in environmental fate studies
(characterization in appendix 1 photolysis report)


UNLABELLED TEST ITEM

Identification: 2-(dimethylamino)-2-[(4-methylphenyl)methyl]-1-[4-(morpholin-4-yl)phenyl]butan-1-one
Physical Description: Slightly yellow powder
Chemical purity: 96.416%
Test item storage: At room temperature protected from light
Purity/Composition correction factor: No correction factor required
Test item handling: Use amber glassware or wrap container in aluminium-foil
Chemical name (IUPAC): 2-dimethylamino-2-(4-methyl-benzyl)-1-(4-morpholin-4-yl-phenyl)-butan-1-one
CAS number: 119344-86-4
EC number: 438-340-0
Solubility in water: 0.0025 g/L at 20˚C
(characterization in appendix 2 photolysis report)

TEST ITEM CHARACTERIZATION
Documentation of the identity, purity, composition, and stability for the test item and radiolabeled test item was provided to the Test Facility by the Sponsor and synthesis lab, respectively. The characterization was conducted in a Sponsor or Sponsor subcontractor quality environment. Certificates of Analysis or equivalent documents were provided to the Test Facility and are presented in Appendix 1 (photolysis report) and Appendix 2 (photolysis report).

Radiolabelling:
yes
Remarks:
[carbonyl-14C] 2-(dimethylamino)-2-[(4-methylphenyl)methyl]-1-[4-(morpholin-4-yl)phenyl]butan-1-one
Analytical method:
high-performance liquid chromatography
Details on sampling:
Sampling intervals for the parent/transformation products
Prior to performance of the main study, a preliminary test was performed. Time points in the final experiment were based on the results of this preliminary test.
Samples were taken at twelve time intervals, including t=0:
0, 0.0035, 0.0069, 0.010, 0.021, 0.042, 0.13, 0.25, 1.00, 2.00, 6.95, 13.92

Sampling Method
3 weighed 1 mL subsamples were taken at each timepoint.

Sampling methods for the volatile compounds
Polyurethane foam (PUF) traps were used for CO2
The weight of the test solution was determined to account for loss of volume due to evaporation


Sampling intervals/times for pH measurements
pH of the test solutions was determined at each sampling point - The pH was measured in ~4 mL aliquots taken from the test solutions (irradiated solution and dark control)
The pH remained stable during the incubation period and ranged between 3.9 and 4.1 at pH 4, and between 6.9 and 7.2 at pH 7.

Sampling intervals/times for sterility check
Sterility of the test solutions was determined at each sampling point.
For the sterility test, 0.1 mL of test solution was added to approximately 3 mL of sterile BHI medium and the mixture was incubated for at least two days at 37°C. Absence of turbidity (observed visually) was considered as proof for sterile conditions.
No turbidity was observed in the BHI medium at all sampling points for irradiated solutions and dark controls. These negative readings indicate all vessels were sterile at the time of sampling


Samples were analysed by HPLC immediately after sampling
Buffers:
Acetate buffer pH 4, 0.01 M Solution of 0.2460 gram sodium acetate in 300 mL water and 0.9052 gram acetic acid in 1500 mL water combined
Phosphate buffer pH 7, 0.01 M Solution of 2.7304 gram dihydrogen phosphate in 1000 mL water, adjusted to pH 7 using 1 M sodium hydroxide and made up to 2000 mL with water
Light source:
Xenon lamp
Remarks:
Suntest CPS+ system (Atlas GmbH, Linsengericht, Germany), equipped with a xenon light source providing a maximum output of approximately 1.5 kW
Light spectrum: wavelength in nm:
> 250 - < 765
Relative light intensity:
2.22
Details on light source:
Suntest CPS+ system (Atlas GmbH, Linsengericht, Germany), equipped with a xenon light source providing a maximum output of approximately 1.5 kW.
The xenon light source is equipped with a quarts glass filter with IR reflective coating for heat reflection and a UV-filter for cut-off of wavelengths below 290 nm.
The spectrum of the xenon light source simulates the spectrum of natural sunlight - see figure 2 in appendix 3 (photolysis report).
Irradiance could be controlled between approximately 250 and 765 W/m2 in the 300-800 nm band.
The mean ratio xenon/sun is 2.22

Determination of irradiance
The intensity of the xenon light source at the position of the exposed test solutions was determined before the start and at the end of the photolytic degradation test, using a XenoCal irradiance sensor (Atlas GmbH). Light intensity was measured in the 300-400 nm band.

Comparison with Natural Sunlight
According to the OECD guideline for Phototransformation of Chemicals on Soil Surfaces, the maximum intensity of the solar irradiance is 68.98 W/m2 (summer, 300-400nm, 40°N).
The average daily sun radiance intensity (300-400 nm) over a 12 h period is approximately 75% of the maximum intensity due to diurnal variations between sunrise and sunset. After application of a correction factor of 2 (day-night) as well as the correction factor of 0.75 for diurnal variations to the maximum intensity of 68.98 W/m2, this leads to an overall value for sunlight intensity (300-400 nm) of 25.87 W/m2 (summer, 40°N).

Light intensity and Comparison with Natural Sunlight data is in appendix 5 (photolysis report)

The surface area of test solution exposed to the light source was 10.3 cm2. Distance of test solution surface to the light source was approximately 19 cm.

See 'Any other information on materials and methods incl. tables' for calculations.
Details on test conditions:
TEST SYSTEM
- Type, material and volume of test apparatus/vessels (see Figure 1, identification report):
Vessels were closed with a quartz glass lid using vacuum grease.
Vessels were placed on a cooling table connected to a flow-through cooling water system to allow adequate temperature control.
The vessels were placed in a thin layer of glycerol to ensure good contact with the cooling plate.
Solutions of Omnirad 379 in 0.01 M buffer were irradiated in a Suntest CPS+ system (Atlas GmbH, Linsengericht, Germany)
Vessels were placed in the Suntest system at approximately 25°C. One additional vessel containing 18 mL buffer but without test item was placed in the Suntest chamber to monitor the temperature.
Test item solutions, glassware, and trapping systems for the dark controls were identical to those for irradiated samples. Dark controls were placed in a water bath adjusted to a temperature of 25°C. One additional vessel containing 18 mL buffer without test item was used for temperature monitoring by means of a thermocouple inserted in the solution.

- Sterilisation method:
Sterility and pH of the test solutions were determined at each sampling point. For the sterility test, 0.1 mL of test solution was added to approximately 3 mL of sterile BHI medium and the mixture was incubated for at least two days at 37°C. Absence of turbidity (observed visually) was considered as proof for sterile conditions.
All glassware was sterilised prior to use at 160°C for 3 hours. Buffer solutions were sterilised at 121°C for 20 min prior to use.

- Measures to saturate with oxygen: not reported

- Details on test procedure for unstable compounds: see dark controls

- Details of traps for volatile, if any:
Test solutions were irradiated in small irradiation vessels equipped with two traps; polyurethane foam for volatile organics, and soda lime for CO2 (solid traps).
If radioactivity recovered from test solutions was less than 95% of applied, radioactivity in the traps was analysed.
Polyurethane foam (PUF) traps were extracted by vortexing for one minute with a weighed 10 mL acetonitrile aliquot.
Total radioactivity was determined by LSC of a 1 mL subsample. Soda lime granules were transferred to a 3-neck round-bottom flask, connected to traps containing approximately 100 mL 2M NaOH. Thereafter, 2M HCl was added to the soda lime. Humidified air was passed over the continuously stirred soda lime mixture and was led through the traps until all soda lime had dissolved. Radioactivity in the weighed traps and acidified soda lime mixture was determined by LSC of a weighed 1 mL subsample.
In case of an incomplete mass balance, test vessels of irradiated buffer solutions were rinsed with a two weighed aliquots (10 mL in total) of acetonitrile. Total radioactivity in the pooled rinsate was determined by LSC of a weighed 1 mL subsample.

- Indication of test material adsorbing to the walls of test apparatus: at pH 4 and pH 7 the mass balances up to 1 day of incubation were >80%


TEST MEDIUM
- Volume used/treatment: 18 mL
- Kind and purity of water: tap water purified by a Milli-Q water purification system (Millipore, Bedford, MA, USA).
- Preparation of test medium: see preparation of buffers
- Renewal of test solution: not applicable for this test system
- Identity and concentration of co-solvent: acetonitrile
- Concentration of co-solvent (pH 4): 0.87% v/v
- Concentration of co-solvent (pH 7): 0.22% v/v
- Concentration of solubilising agent: not reported

REPLICATION
- No. of replicates (dark): 1 at each pH
- No. of replicates (irradiated): 2 at each pH
Duration:
14 d
Temp.:
21.4 °C
Initial conc. measured:
4.64 other: mg/ L at pH 4
Duration:
14 d
Temp.:
24.2 °C
Initial conc. measured:
1.16 other: mg/ L at pH 7
Reference substance:
no
Dark controls:
yes
Computational methods:
For calculations see "Any other information on materials and methods incl. tables"

Critical computerized systems used in the study are listed below. All computerized systems used in the conduct of this study have been validated; when a particular system has not satisfied all requirements, appropriate administrative and procedural controls were implemented to assure the quality and integrity of data.

COMPUTERIZED SYSTEMS (photolysis report)

System name - Version No. - Description of Data Collected and/or Analyzed
REES Centron - SQL 2.0 - Temperature, relative humidity and/or atmospheric pressure monitoring
QuantaSmart - 2.03 - System control and data acquisition
Laura - 4.2.11.129 - System control, data acquisition and processing
Cary WinUV - 5.1.0.1016 - System control and data acquisition
XenoSoft - 2.85 - System control and data acquisition
Deviation Information Library - 2.1.29 - Deviations


COMPUTERIZED SYSTEMS (identification report)

System name - Version No.- Description of Data Collected and/or Analyzed
Deviation Information Library - 2.1.61 - Deviations
Flo-one - 3.65 - System control, data acquisition and integration
QuantaSmart - 4.00 and 4.02 - System control and data acquisition
REES Centron - SQL 2.0 - Temperature, relative humidity and/or atmospheric pressure monitoring
Xcalibur - 2.1 - System control, data acquisition and integration
Preliminary study:
The UV/VIS spectral data for Omnirad 379 are shown in Table 12 and Table 13, appendix 4 (photolysis report).
Molar extinction coefficients were calculated and are included in the tables. As these were above the trigger value of 10 L mol-1 cm-1, further experimental work was performed
Parameter:
max epsilon
Value:
18 400 1/cm dm³ 1/mol
Remarks:
At 352.5 nm for pH 4 using 0.066mM
Parameter:
max epsilon
Value:
15 000 1/cm dm³ 1/mol
Remarks:
At 357.5 nm pH7 0.066mM
Key result
% Degr.:
100
Sampling time:
3 h
Test condition:
pH4
Key result
% Degr.:
100
Sampling time:
1 h
Test condition:
pH7
Quantum yield (for direct photolysis):
0.002
Key result
DT50:
0.019 d
Test condition:
pH4
Key result
DT50:
0.009 d
Test condition:
pH7
Predicted environmental photolytic half-life:
Upon incubation under simulated sunlight, Omnirad 379 degraded to 0% (pH 4 and pH 7) after an incubation time of 3 hours and 1 hour, respectively, corresponding to approximately 6.5 and 2.2 hours of natural sunlight
Transformation products:
yes
No.:
#3
Details on results:
HALF-LIFE

Test conditions (pH, sterility, temperature, and other experimental conditions) maintained throughout the study: Yes
Details:

- Radiochemical purtiy of Test Item in stock solution
Based on HPLC analysis, radiochemical purity of stock solution used to spike pH 4 and pH 7 buffer solutions was 98.9% on the first day of spiking and 96.2% on the last day of spiking. HPLC chromatograms of the spike solution before the first and after the last spiking occasion are shown in Appendix 7 (photolysis report).

- Light Intensity During the Exposure Phase
Test vessels were incubated in two different Suntest systems. Light intensity in a range of 300-400 nm, measured before and after the incubation period, varied between 54.7 and 56.2 W/m2. All calculations were performed using average light intensity for each respective Suntest system.
After correction for passage through a quartz plate (Cquartz) and position of the samples (Fposition X), light intensity at the surface of each sample was calculated. Days of average light under test conditions for each sample were calculated as described in Equation 3 ('any other information on materials and methods incl. tables'). Results of these calculations are shown in Table 14 and Table 15 (appendix 5, photolysis report). Furthermore, the light intensity was converted to units of photons second-1 m-2 as described in Equation 4 and Equation 5 ('any other information on materials and methods incl. tables').

- Temperature
Average temperatures and temperature range of irradiated solutions and dark controls are shown in Table 1 of the photolysis report 'Average Temperature of Test Solutions' (in 'any other information on results incl. tables'). Average temperature of dark control samples was 23.5°C at pH 4 and pH 7, and ranged between 19.8 and 24.8°C. Average temperature of irradiated solutions was between 21.4 and 24.2°C, with a full range between 14.8 and 26.3°C. See also deviation in Appendix 8 (photolysis report).

- Sterility and pH of Test Solutions
No turbidity was observed in the BHI medium at all sampling points for irradiated solutions and dark controls. These negative readings indicate all vessels were sterile at the time of sampling.
The pH remained stable during the incubation period and ranged between 3.9 and 4.1 at pH 4, and between 6.9 and 7.2 at pH 7.

Results:

- Kinetics as described in ('any other information on materials and methods incl. tables') were fitted to the experimental data for the test item obtained by LC-RAD. Goodness of fit assessment is presented in Table 2 'Goodness of Fit Assesment (Parent)'.
The resulting degradation curves of the irradiated samples are in Figure 10 and Figure 11 (appendix 6, photolysis report) at pH 4 and pH 7, respectively.
The resulting degradation curves of the dark samples at pH 4 and pH 7 are appended in 'SFO Fit for Dark Control solutions' in the attached background materials.
- Kinetic rate constants (kdark and kphot), DT50 and DT90 for degradation of Omnirad 379 in dark controls and in natural sunlight are shown in Table 4 'Photolytic Degradation Rate of [Test item]'; parameters obtained during modelling are listed in Table 3 'Parameter estimates (Parent)'. As there were several orders of magnitude difference between kdark and kphot, kphot was not corrected for degradation in the dark controls.

Tables 1-4 are appended in the attached background material

DISTRIBUTION OF RADIOACTIVITY
The recovery of radioactivity in solution for irradiated samples and dark controls are given in Table 5 'Distribution of Radioactivity at pH 4 (% of Applied Radioactivity)' and Table 6 'Distribution of Radioactivity at pH 7 (% of Applied Radioactivity)'. The recovery of radioactivity in traps and rinsates, including mass balances, of the irradiated solutions are shown in Table 7 'Distribution of Radioactivity in Traps and Rinsates at pH 4 and Mass Balance (% of Applied Radioactivity)' and Table 8 'Distribution of Radioactivity in Traps and Rinsates at pH 7 and Mass Balance (% of Applied Radioactivity)'. Detailed chromatographic results are reported in Appendix 7 (photolysis report). Summarised LC results for parent and degradation products are shown in Table 16 and Table 17 (appendix 7, photolysis report).
At the end of the experiment 27.6-38.8% (pH 4) and 44.5-62.6% (pH 7) of applied radioactivity was recovered. Variable amounts of radioactivity were recovered from the vessel rinsates (see Table 7 and Table 8). A maximum of 0.3% of applied radioactivity was recovered from the PUF extracts. Radioactivity recovered as CO2 in the NaOH traps after acidifying of soda lime increased to a maximum value of 27.4% and 20.6% of applied, for pH 4 and pH 7, respectively. Up to 1.4% of applied radioactivity remained in soda lime after acidifying.
Mass balances for the dark controls were within the guideline criterion of 90-110 % of applied radioactivity with no exceptions. At pH 4 and pH 7 the mass balances up to 1 day of incubation were >80%. The cause of the low mass balance was not known, but it might be related to the high reactivity of the molecule and incomplete trapping of formed CO2, as supported by the inconsistency between replicates and apparent decrease of CO2 for successive time points. To allow for a worst case assessment, calculations based on chromatograms were performed under the assumption that radioactivity missing from the mass balance should be attributed to the LC samples. After applying this worst case correction factor, all data were accepted and used for DT50/DT90 calculations.
Activity recovered in the dark controls was 94.6-96.9% of applied at the end of the incubation period.

Tables 5-8 are appended in the attached background material


PHOTODEGRADATION
Samples from irradiated vessels and dark controls were analysed by LC-RAD. Amount of parent compound (as % of applied radioactivity) is reported in Table 5 'Distribution of Radioactivity at pH 4 (% of Applied Radioactivity)' and Table 6 'Distribution of Radioactivity at pH 7 (% of Applied Radioactivity)'. Vessel rinsates were not analysed by LC-RAD for all samples; in such cases, it was assumed that rinsates contained the same percentage of parent as the vessel sample. Detailed results and selected chromatograms are shown in Appendix 7 (photolysis report). Minor degradation was also observed in the dark control samples. However, no major degradation products were formed in the dark controls.
In irradiated buffer solution at pH 4, Omnirad 379 degraded to undetectable levels within 3 hours of irradiation. At pH 7, the test item degraded to undetectable levels within 1 hour of irradiation.
Seven major photolytic degradation products were detected, which exceeded 10% of applied radioactivity. Identification of major degradation products will be carried out in a separate project.

MAJOR PHOTODEGRADATION PRODUCTS at pH 4
Degradation product M-1 increased to a maximum of 24.5% of applied radioactivity after an incubation time of 2 days, after which concentrations decreased to 3.4% of applied radioactivity after an incubation time of 14 days.
Degradation product M-2 increased to a maximum of 12.7% of applied radioactivity after an incubation time of 1 day, after which concentrations decreased to 0% of applied radioactivity after an incubation time of 7 days.
Degradation product M-5 increased to a maximum of 17.8% of applied radioactivity after an incubation time of 1 hour, after which concentrations decreased to 0% of applied radioactivity after an incubation time of 2 days.
Degradation product M-6 increased to a maximum of 59.8% of applied radioactivity after an incubation time of 30 minutes, after which concentrations decreased to 0% of applied radioactivity after an incubation time of 2 days.
Degradation product M-7 increased to a maximum of 14.1% of applied radioactivity after an incubation time of 1 hour, after which concentrations decreased to 0% of applied radioactivity after an incubation time of 1 day.


MAJOR PHOTODEGRADATION PRODUCTS at pH 7
Degradation product M-1 increased to a maximum of 12.9% of applied radioactivity after an incubation time of 2 days, after which concentrations decreased to 3.6% of applied radioactivity after an incubation time of 14 days.
Degradation product M-2 increased to a maximum of 18.7% of applied radioactivity after an incubation time of 1 hour, after which concentrations decreased to 0% of applied radioactivity after an incubation time of 2 days.
Degradation product M-3 increased to a maximum of 10.0% of applied radioactivity after an incubation time of 15 minutes, after which concentrations decreased to 0% of applied radioactivity after an incubation time of 1 day.
Degradation product M-5 increased to a maximum of 16.4% of applied radioactivity after an incubation time of 1 hour, after which concentrations decreased to 0% of applied radioactivity after an incubation time of 1 day.
Degradation product M-6 increased to a maximum of 35.3% of applied radioactivity after an incubation time of 30 minutes, after which concentrations decreased to 0% of applied radioactivity after an incubation time of 2 days.
Degradation product M-7 increased to a maximum of 12.0% of applied radioactivity after an incubation time of 1 hour, after which concentrations decreased to 0% of applied radioactivity after an incubation time of 2 days.
Degradation product M-9 increased to a maximum of 20.1% of applied radioactivity after an incubation time of 7 days, after which concentrations decreased to 13.0% of applied radioactivity after an incubation time of 14 days.
Degradation product M-3 was identified to be 4-(4-morpholinyl)benzaldehyde by co- chromatography against reference standard AS1842, see also Figure 42, Figure 43, and Figure 44 (appendix 7, photolysis report).


PHOTODEGRADATION PATHWAYS
Photodegradation pathways are appended in the attached background material 'pH 4 photodegradation pathway' and 'pH 7 photodegradation pathway'

FORMATION AND PHOTOLYTIC DEGRADATION RATE OF DEGRADATION PRODUCTS
The DT50 and DT90 calculations for photolytic degradation of Omnirad 379, and formation and photolytic degradation of major degradation products are based on individual LC results as shown in Table 16 and Table 17 (appendix 7, photolysis report). The goodness of fit assessment is presented in Table 9 'Goodness of Fit Assesment (Parent and Degradation Products)', estimated parameters in Table 10 'Parameter Estimates (Parent and Degradation Product Fitted Simultaneously)', and graphs in Figure 12 - Figure 23 (appendix 6, photolysis report).
The fits obtained by the SFO model for simultaneously fitting parent and degradation products was generally good for parent, except at pH 7 for degradation products M-3, M-5, M-6, and M-7, where the DFOP model was used for parent. The relatively high χ2 for some of the degradation products was expected to be due to the low concentrations and differences between duplicate vessels. However, calculated DT50 and DT90 values are deemed reliable.

Tables 9-10 are appended in the attached background material

COMPARISON WITH NATURAL SUNLIGHT
Based on a sunlight intensity of 25.87 W/m2 and the average light intensity of the xenon lamp, the ratio xenon lamp / natural sunlight was 2.11-2.29 at pH 4 and 2.11-2.34 at pH 7 (see Table 14 and Table 15, appendix 5, photolysis report). The photolytic half-life of test item in natural sunlight was equivalent to 26.6 minutes and 12.8 minutes at pH 4 and pH 7, respectively (see also Table 4 'Photolytic Degradation Rate of [Test Item]).

QUANTUM YIELD
According to the OECD 316 (point 19), the determination of the quantum yield and its use to estimate direct photolysis rate constants is optional.
Using the information reported, the quantum yield was calculated to be:
- pH 4: 9.543 x 10-4
- pH 7: 2.037 x 10-3
These calculations have not been through quality control. The equations (equation 4 & 5) are detailed in the 'any other information on materials and methods incl. tables'
Estimated direct photolysis rate constants for the test chemical in natural water that are estimated from the test chemical quantum yield for seasons, latitudes, and water body types of interest have not been calculated as they are optional in the OECD 316.


LC-PDA-(RAD)MS DATA OF [14C]-TEST ITEM
A [14C]-Test Item standard solution was analyzed to determine the retention time, PDA, RAD, MS and MSn data in the positive ion mode. The retention time of [14C]-Test Item obtained with the described method (Table 1, Appendix 4, identification report) was 13.85 min on the PDA detector (Figure 1A, appendix 1, identification report), 14.10 min on the radioactivity detector (Figure 2, appendix 1, identification report) and 13.90 min on the MS detector (Figure 3, appendix 1, identification report). The slight differences in retention time are due to serial connection of the different detectors. Figure 1A, appendix 1, identification report shows the light absorbance chromatogram at a wavelength of 355 nm (specific absorbance, Figure 1B, appendix 1, identification report).
The mono-isotopic molecular mass of the test item and [14C]-Test Item as well as the m/z of possibly relevant ions in the positive ion mode were calculated and are depicted below.

Calculation of the Mono-isotopic Molecular Mass and m/z of Mono-isotopic [M+H]+ of Test Item and [14C]-Test Item
Element Number of atoms Mono isotopic atomic mass (a) Subtotal mono isotopic molecular mass (Da)
C 24 12.0000 288.0000
H 32 1.0078 32.2504
N 2 14.0031 28.0061
O 2 15.9949 31.9898

Total monoisotopic molecular mass of Omnirad 379 380.2464
Total monoisotopic molecular mass of [14C]-Omnirad 379 (b) 382.2496
m/z of monoisotopic of Omnirad 379 [M+H]+ 381.2537
m/z of monoisotopic of [14C]-Omnirad 379 [M+H]+ (b) 383.2569

(a) From: Handbook of chemistry and physics, 67th edition, CRC Press Inc. (Boca Raton, Florida, USA)
(b) Representing 23 C-atoms and one 14C atom (=one 12C atom (12.0000 Da) is replaced by one 14C atom (14.0032 Da))

The reconstructed ion chromatogram of m/z 383.257 (the expected m/z of the [M+H]+ ion of [14C]-Test Item), the MS, MS2 and MS3 spectrum are given in Figure 3, appendix 1, identification report. [14C]-Test Item was observed in the MS spectrum as [M+H]+ at m/z 383.256 and m/z 381.253 corresponding to [14C]-Test Item and the non-labeled Test item, respectively. The accurate mass measurement of [14C]-Test Item showed an accuracy of ≤1 mDa relative to the theoretical mass of [14C]-Test Item.
Identification will be based on the protonated molecule of [14C]-Test Item. The molecular structure of [14C]-Test Item is given in Figure 4, appendix 1, identification report. For convenient description of the fragmentation, the rings in the structure are marked A through C.

In the MS2 spectrum of the protonated molecule of [14C]-Omnirad 379 (Figure 3, appendix 1, identification report), i.e. m/z 383.256, a major fragment ion was observed with m/z 190.159 (due to the loss of 193.097 Da, C1014CH13NO2 (AB-ring)). Additional fragment ions with m/z 338.200 (due to the loss of 45.056 Da, C2H7N) indicative for the loss of NH-dimethyl, m/z 308.202 (due to the loss of 75.054 Da, 338-14C=O), m/z 192.090 (due to the loss of 191.166 Da, C13H20N) indicative for C-ring part, m/z 176.108 (due to the loss of 207.148 Da, 308-C10H12) and m/z 161.120 (due to the loss of 222.136 Da, 190-•C2H5) were observed. In the MS3 spectrum of the major MS2 fragment ion with m/z 190.159, fragment ions with m/z 175.136 (due to the loss of 15.023, •CH3), m/z 161.120 (due to the loss 29.040 Da, •C2H5), m/z 145.101 (due to the loss of 45.058 Da, C2H7N) and m/z 105.070 (due to the loss of 85.089 Da, C5H11N). The proposed structures of the MS2 and MS3 fragment ions are presented in Figure 5, appendix 1, identification report.


LC-MS DATA OF REFERENCE ITEM
A 4-(4-Morpholinyl)benzaldehyde standard solution was analyzed to determine the retention time, MS and MSn data in the positive ion mode. The retention time of Reference Item obtained with the described method (Table 1 in Appendix 4, identification report) was 14.85 min on the MS detector (Figure 6, appendix 1, identification report).
The mono-isotopic molecular mass of 4-(4-Morpholinyl)benzaldehyde as well as the m/z of possibly relevant ion in the positive ion mode were calculated and are depicted below.

Calculation of the Mono-isotopic Molecular Mass and m/z of Mono-isotopic [M+H]+ of 4-(4-Morpholinyl)benzaldehyde
Element Number of atoms Mono isotopic atomic mass (a) Subtotal mono isotopic molecular mass (Da)
C 11 12.0000 132.0000
H 13 1.0078 13.1017
N 1 14.0031 14.0031
O 2 15.9949 31.9898

Total monoisotopic molecular mass of 4-(4-Morpholinyl)benzaldehyde 191.0946
m/z of monoisotopic of 4-(4-Morpholinyl)benzaldehyde [M+H]+ 192.1019
(a) From: Handbook of chemistry and physics, 67th edition, CRC Press Inc. (Boca Raton, Florida, USA)

The reconstructed ion chromatogram of m/z 192.102 (the expected m/z of the [M+H]+ ion of 4-(4-Morpholinyl)benzaldehyde, the MS, MS2 and MS3 spectrum are given in Figure 6, appendix 1, identification report. 4-(4-Morpholinyl)benzaldehyde was observed in the MS spectrum as [M+H]+ at m/z 192.102. The accurate mass measurement of 4-(4-Morpholinyl)benzaldehyde showed an accuracy of ≤1 mDa relative to the theoretical mass of 4-(4-Morpholinyl)benzaldehyde. The molecular structure of 4-(4-Morpholinyl)benzaldehyde is given in Figure 7, appendix 1, identification report. For convenient description of the fragmentation, the rings in the structure are marked A and B. In the MS2 spectrum of the protonated molecule of 4-(4-Morpholinyl)benzaldehyde (Figure 6, appendix 1, identification report), i.e. m/z 192.102, a major fragment ion was observed with m/z 164.107 (due to the loss of 27.995 Da, C=O). In the MS3 spectrum of the major MS2 fragment ion with m/z 164.107, fragment ions with m/z 146.096 (due to the loss of 18.011, H2O) and m/z 120.081 (due to the loss 44.027 Da, 164-C2H4O). The proposed structures of the MS2 and MS3 fragment ions are presented in Figure 8, appendix 1, identification report.


LC-PDA-RAD ANALYSES OF SAMPLES
The amount of radioactivity in the selected samples was determined at the start of this study. For several samples the amount of radioactivity in the samples was higher than the amount of radioactivity in the same sample resulting in recoveries of 158-265%, individual results are not reported, these data are stored in the study file only. This high recovery values might be due to evaporation of the solvent during storage and thawing procedures. However, as the radioactivity profiles analysed within this study were comparable to the radioactivity profiles obtained in the photolysis report, the samples were acceptable for identification purposes.
The radioactivity chromatogram obtained from the concentrated (freeze-dried) water sample 7A pH 7 Day 1 is presented in Figure 9. The amount of radioactivity in this samples was too low to obtain a reliable radioactivity chromatogram from this sample. Based on this result, identification of degradation product M-9 was not possible.
The radioactivity chromatogram obtained from the concentrated (i.e., water phase extracted in ethyl acetate, evaporated to dryness and dissolved in ACN) sample 20 ACN pH 7 180 min (i.e., water phase extracted in ethyl acetate, evaporated to dryness and dissolved in ACN) samples 1 ACN pH 4 180 min, 1b ACN pH 4 60 min and 6b ACN pH 4 Day 1 are presented in Figure 11, Figure 12 and Figure 13 (appendix 1, identification report), respectively.
The radioactivity chromatograms were comparable to the earlier obtained radioactivity chromatograms. The peak eluting at 11.9 minutes corresponds to peak M-1, the peak eluting at 13.9-14.0 minutes corresponds to M-2, the peak eluting at 15.4 minutes corresponds to M-3, the peak eluting at 21.8 minutes corresponds to M-4, the peak eluting at 22.5-22.6 minutes corresponds to M-5, the peak eluting at 23.6 minutes corresponds to M-6 and the peak eluting at 24.6-24.8 minutes corresponds to M-7.


LC-PDA-MSn ANALYSES OF SAMPLES
Based on the radioactivity chromatograms, peaks M-1, M-2, M-3, M-4, M-5, M-6 and M-7 were selected for screening of m/z values present in the accurate-mass spectral data. In case ions with m/z values were present in these samples that were not present in the corresponding blank samples, MS2 mass spectra were acquired. The obtained MS data and the possible m/z values representing degradation products M-1, M-2, M-3, M-4, M-5, M-6 and M-7 are discussed in the following sections.


IDENTIFICATION OF MAJOR DEGRADATION PRODUCTS
Samples to be used for identification of degradation products were selected based on total activity (LSC) and relative abundance of each major degradation product (LC). Stability of selected extracts was checked by LC on 02 Apr 2019 and 04 Apr 2019. After confirming stability extracts were freeze-dried to dryness and dissolved in Milli-Q. Further identification of degradation products was carried out in and reported in th identification report.
Degradation product M-9 could not be identified, as activity remaining after freeze-drying was not sufficient to allow proper identification. However, labelling M-9 as a major degradation product was based on extensive extrapolation due to the poor mass balance.
Therefore, the chance M-9 is actually a major degradation product is relatively slim.


Results with reference substance:
One degradation product (M-3) could be identified as 4-(4-morpholinyl)benzaldehyde by co- chromatography against the reference standard.

CHARACTERIZATION/IDENTIFICATION OF DEGRADATION PRODUCT M-1

Based on the accurate mass m/z 240.074 is most likely the loss of N,N-dimethyl-1-(p-tolyl)butan-2-amine in combination with a desaturation (+H2) and 3 oxidations (+3 O-atoms). Additional MSn analyses were performed using the concentrated sample 6b ACN pH 4 Day 1. The MS data of m/z 240.074 are presented in Figure 14, appendix 1, identification report. In the MS spectrum, the ion with m/z 240.074 is clearly visible. The 14C-isotope pattern of this ion was comparable to the isotope pattern observed for the parent compound. The mass shift of this degradation product is -143.182 Da relative to the protonated molecule of the parent compound. Consistently, from the accurate mass measurements the most likely molecular formula for this protonated degradation product is C1014CH12NO5+ (error < 1 mDa, RDB= 6.5).

The MS2 spectrum of m/z 240.074 showed a main fragment ion with m/z 194.069 (due to the loss of 46.005 Da, HCOOH) indicative for the loss of most likely the loss of a carboxyl and a minor fragment ion with m/z 222.065 (due to the loss of 18.009 Da, H2O). In the MS3 spectrum of the major MS2 fragment ion with m/z 194.069 fragment ions with m/z 166.074 (due to the loss of 27.995, C=O), m/z 148.063 (due to the loss 46.007 Da, 166-H2O), m/z 120.081 (due to the loss of 73.988 Da, 166-14CO2) indicative that hydrolysis at the 14C-atom and m/z 118.065 (due to the loss of 76.004 Da, 166-H14COOH) were observed.

Based on the molecular formula, the loss of 14CO2 and the loss of CH2O2 in the MS2 fragmentation, the molecule is most likely hydrolysed at the 14C-atom, and desaturated and oxidized twice at the A-ring of the molecule. The proposed structures of MS2 and MS3 fragment ions are presented in Figure 15, appendix 1, identification report. A structure for the degradation product M-1 is proposed in 'Proposed Structure of the Possible Photolytic Degradation Products' appended in the attached background material.

The reconstructed ion chromatograms of m/z 240.074 in the selected concentrated samples (pH 4 and pH 7) are presented in Figure 16, appendix 1, identification report.

CHARACTERIZATION/IDENTIFICATION OF DEGRADATION PRODUCT M-2

In the MS screening at the retention of degradation product M-2, a major ion with m/z 210.100 (coded M-2a), a medium ion with m/z 383.256 (coded M-2b) and a minor ion with m/z 399.252 (coded M-2c) were observed. Additional MSn analysis was performed for these three peaks.

Peak M-2a

Based on the accurate mass m/z 210.100 is most likely the result of hydrolysis of the 14C-atom, and thus the loss of N,N-dimethyl-1-(p-tolyl)butan-2-amine. Additional MSn analyses were performed using the concentrated sample 6b ACN pH 4 Day 1. The MS data of m/z 210.100 are presented in Figure 17, appendix 1, identification report. In the MS spectrum, the ion with m/z 210.100 is clearly visible. The 14C-isotope pattern of this ion was comparable to the isotope pattern observed for the parent compound. The mass shift of this degradation product is -173.156 Da relative to the protonated molecule of the parent compound. Consistently, from the accurate mass measurements the most likely molecular formula for this protonated degradation product is C1014CH14NO3+ (error < 1 mDa, RDB= 5.5).

The MS2 spectrum of m/z 210.100 showed a main ion with m/z 210.100 indicating a relatively low fragmentation. Therefore, the results of the MS3 spectrum of the MS2 fragment ion with m/z 210.100 is mainly used for the identification of M-2a. In the MS3 spectrum of the major MS2 fragment ion with m/z 210.100 a main fragment ion with m/z 164.107 (due to the loss of 45.993 Da, 14CO2) indicative for hydrolysis at the 14C-atom. Additionally, fragment ions with m/z 192.089 (due to the loss 18.011 Da, H2O), m/z 166.074 (due to the loss of 44.026 Da, C2H4O), m/z 146.096 (due to the loss of 64.004 Da, 164-H2O), m/z 131.073 (due to the loss of 79.027 Da, 146-•CH3), m/z 120.081 (due to the loss of 90.019 Da, 166-14CO2) and m/z 118.065 (due to the loss of 92.035 Da, 166-H14COOH) were observed. The proposed structures of the MS3 fragment ions are presented in Figure 18, appendix 1, identification report. Based on the molecular formula and the loss of 14CO2, the molecule is most likely hydrolysed at the 14C-atom. A structure for the degradation product M-2a is proposed in 'Proposed Structure of the Possible Photolytic Degradation Products' appended in the attached background material.

The reconstructed ion chromatograms of m/z 210.100 (coded M-2a) in the selected concentrated samples (pH 4 and pH 7) are presented in Figure 19, appendix 1, identification report.

Peak M-2b

Based on the accurate mass m/z 383.256 this molecule is comparable to the parent compound. However, as the retention time of M-2b is slightly different than the retention time of the parent compound, this degradation product is further investigated. Additional MSn analyses were performed using the concentrated sample 1 ACN pH 4 180 min. The MS data of m/z 383.256 eluting at 13.7 minutes are presented in Figure 20, appendix 1, identification report. In the MS spectrum, the ion with m/z 383.256 is not the main ion, however clearly visible. The 14C-isotope pattern of this ion was comparable to the isotope pattern observed for the parent compound. As the mass is comparable to the parent compound the most likely molecular formula for this protonated degradation product is C2314CH33N2O2+ (error < 1 mDa, RDB= 9.5).

The MS2 spectrum of m/z 383.256 showed a main ion with m/z 383.256 indicating a relatively low fragmentation. As the results of the MS3 spectrum of the MS2 fragment ion with m/z 383.256 did not result in reliable results due to a too low signal, the MS2

fragmentation is used for identification. In the MS2 spectrum of m/z 383.256, fragment ions with m/z 340.214 (due to the loss of 43.042 Da, C2H5N) indicating the loss of aziridine (=desaturated N-dimethyl-part), m/z 190.159 (due to the loss of 193.097 Da, C1014CH13NO2) indicating that the AB-ring including the 14C=O part was unchanged, m/z 162.128 (due to the loss of 221.128 Da, 190-C2H4). The proposed structures of the MS2 fragment ions are presented in Figure 21, appendix 1, identification report.

Based on the molecular formula and the loss of C2H5N, the molecule is most likely desaturated at the dimethylamine and reduced at the C-moiety. A structure for the degradation product M-2b is proposed in 'Proposed Structure of the Possible Photolytic Degradation Products' appended in the attached background material.

The reconstructed ion chromatograms of m/z 383.256 (coded M-2b) in the selected concentrated samples (pH 4 and pH 7) are presented in Figure 22, appendix 1, identification report.

Peak M-2c

Based on the accurate mass m/z 399.252 is most likely the result of an oxidation of the parent compound. Additional MSn analyses were performed using the concentrated sample 1 ACN pH 4 180 min. The MS data of m/z 399.252 eluting at 13.7 minutes are presented in Figure 23, appendix 1, identification report. In the MS spectrum, the ion with m/z 399.252 is only a minor ion. The 14C-isotope pattern of this ion was comparable to the isotope pattern observed for the parent compound. The mass shift of this degradation product is +15.996 Da relative to the protonated molecule of the parent compound. Consistently, from the accurate mass measurements the most likely molecular formula for this protonated degradation product is C2314CH33N2O3+ (error < 1 mDa, RDB= 9.5).

As the MS2 spectrum quality of m/z 399.252 is very poor it is not possible to identify this degradation product.

The reconstructed ion chromatograms of m/z 399.252 (coded M-2c) in the selected concentrated samples (pH 4 and pH 7) are presented in Figure 24, appendix 1, identification report.

CHARACTERIZATION/IDENTIFICATION OF DEGRADATION PRODUCT M-3

In the MS screening at the retention of degradation product M-3, a major ion with m/z 194.105 (coded M-3a) and a medium ion with m/z 354.193 (coded M-3b) were observed. Additional MSn analysis was performed for both ions.

Peak M-3a

Based on the accurate mass m/z 194.105 is most likely the result of cleavage of the moiety containing the AB-ring and the moiety containing the C-ring. Additional MSn analyses were performed using the concentrated sample 1 ACN pH 4 180 min. The MS data of m/z 194.105 are presented in Figure 25, appendix 1, identification report. In the MS spectrum, the ion with m/z 194.105 is clearly visible. The 14C-isotope pattern of this ion was comparable to the isotope pattern observed for the parent compound. The mass shift of this degradation product is -189.151 Da relative to the protonated molecule of the parent compound. Consistently, from the accurate mass measurements the most likely molecular formula for this protonated degradation product is C1014CH14NO2+ (error < 1 mDa, RDB= 5.5).

The MS2 spectrum of m/z 194.105 showed a main ion with m/z 194.105 indicating a relatively low fragmentation. Therefore, the results of the MS3 spectrum of the MS2 fragment ion with m/z 194.105 is mainly used for the identification of M-3a. In the MS3 spectrum of the major MS2 fragment ion with m/z 194.105 a main fragment ion with m/z 164.107 (due to the loss of 29.998 Da, 14C=O). Additionally, fragment ions with m/z 150.079 (due to the loss 44.026 Da, C2H4O), m/z 146.096 (due to the loss of 48.009 Da, 164-H2O), m/z 120.081 (due to the loss of 74.024 Da, 164-C2H4O), m/z 119.073 (due to the loss of 75.032 Da, 126-•C2H3) and m/z 118.065 (due to the loss of 76.040 Da, 164-C2H6O) were observed. The fragmentation of this degradation product is comparable to the fragmentation of the reference item. The proposed structures of the MS3 fragment ions are presented in Figure 26, appendix 1, identification report.

Based on the molecular formula, the comparable retention time and fragmentation of M-3a and reference item AS1842, this molecule is most likely 4-(4-Morpholinyl)benzaldehyde.

The reconstructed ion chromatograms of m/z 194.105 (coded M-3a) in the selected concentrated samples (pH 4 and pH 7) are presented in Figure 27, appendix 1, identification report.

Peak M-3b

Based on the accurate mass m/z 354.193 is most likely the result of the loss of the dimethylamine (C2H7N) moiety resulting in a desaturation in combination with an oxidation. Additional MSn analyses were performed using the concentrated sample 1 ACN pH 4

180 min. The MS data of m/z 354.193 are presented in Figure 28, appendix 1, identification report. In the MS spectrum, the ion with m/z 354.193 is clearly visible. The 14C-isotope pattern of this ion was comparable to the isotope pattern observed for the parent compound. The mass shift of this degradation product is -29.063 Da relative to the protonated molecule of the parent compound. Consistently, from the accurate mass measurements the most likely molecular formula for this protonated degradation product is C2114CH26NO3+ (error < 1 mDa, RDB= 10.5).

The MS2 spectrum of m/z 354.193 showed a main ion with m/z 337.191 (due to the loss of 17.003 Da, •OH) indicating an N-oxidation. In the MS3 spectrum of the major MS2 fragment ion with m/z 337.191 main fragment ions with m/z 322.168 (due to the loss of 15.023 Da, •CH3), m/z 308.153 (due to the loss of 29.038 Da, •C2H5), m/z 251.131 (due to the loss 86.060 Da, C4H8NO) indicating the loss of the A-ring and m/z 232.121 (due to the loss of 105.070 Da, C8H9) indicating the loss of the moiety containing the C-ring were observed.

Based on the molecular formula, the loss of dimethylamine (C2H7N) resulting in a desaturation and the loss of an •OH, M3b is most likely oxidized at the N-atom in the A-ring. The proposed structures of MS2 and MS3 fragment ions are presented in Figure 29, appendix 1, identification report. A structure for the degradation product M-3b is proposed in 'Proposed Structure of the Possible Photolytic Degradation Products' appended in the attached background material.

The reconstructed ion chromatograms of m/z 354.193 (coded M-3b) in the selected concentrated samples (pH 4 and pH 7) are presented in Figure 30, appendix 1, identification report.

CHARACTERIZATION/IDENTIFICATION OF DEGRADATION PRODUCT

M-4

In the MS screening at the retention of degradation product M-4, three major ions with m/z 354.193 (coded M-4a), m/z 352.178 (coded M-4b) and m/z 368.173 (coded M-4c) were observed. Additionally, a medium ion with m/z 368.173 (coded M-4d) and three minor ions with m/z 399.251 (coded M-4e), m/z 413.231 (coded M-4f) and m/z 397.236 (coded M-4g) were observed. Additional MSn analysis was performed for all ions.

Peak M-4a

Based on the accurate mass m/z 354.193 is most likely the result of the result of the loss of the NH-dimethyl moiety in combination with an oxidation. Additional MSn analyses was performed using the concentrated sample 1 ACN pH 4 180 min. The MS data of m/z 354.193 are presented in Figure 31, appendix 1, identification report. In the MS spectrum, the ion with m/z 354.193 is clearly visible. The 14C-isotope pattern of this ion was comparable to the isotope pattern observed for the parent compound. The mass shift of this degradation product is -29.063 Da relative to the protonated molecule of the parent compound. Consistently, from the accurate mass measurements the most likely molecular formula for this protonated degradation product is C2114CH26NO3+ (error < 1 mDa, RDB= 10.5).

The MS2 spectrum of m/z 354.193 showed a main ion with m/z 192.090 which is C1014CH12NO2+ (AB–14CO part of the molecule), (due to the loss of 162.104 Da, C11H14O) indicating that the AB-moiety and the attached 14C=O is unchanged and that the oxidation occurred at the moiety containing the C-ring and a minor ion with m/z 190.086 (due to the loss of 164.107, •C10H14NO) confirming the loss of the AB-moiety. In the MS3 spectrum of the major MS2 fragment ion with m/z 192.090, a main fragment ion with m/z 162.091 (due to the loss of 29.999 Da, 14C=O). Additionally, fragment ions with m/z 164.094 (due to the loss 27.995 Da, C=O), m/z 144.080 (due to the loss of 48.009 Da, 162-H2O), m/z 134.096 (due to the loss of 57.994 Da, 162-C=O), m/z 132.081 (due to the loss of 60.009 Da, 164 -C14CH2O2), m/z 120.081 (due to the loss of 72.009 Da, 162 –C2H2O), m/z 118.065 (due to the loss of 74.025 Da, 162 - C2H4O) and m/z 91.054 (due to the loss of 101.036 Da, 118-CHN) were observed.

Based on the molecular formula, the loss of dimethylamine (C2H7N) resulting in a desaturation, the intact AB-moiety M4a is most likely oxidized in the moiety containing the C-ring. The proposed structures of MS2 and MS3 fragment ions are presented in Figure 32, appendix 1, identification report.

A structure for the degradation product M-4a is proposed in 'Proposed Structure of the Possible Photolytic Degradation Products' appended in the attached background material.

The reconstructed ion chromatograms of m/z 354.193 (coded M-4a) in the selected concentrated samples (pH 4 and pH 7) are presented in Figure 30, appendix 1, identification report.

Peak M-4b

Based on the accurate mass m/z 352.178 is most likely the result of the loss of dimethylamine (C2H7N) moiety resulting in a desaturation in combination with a desaturation and oxidation. Additional MSn analyses was performed using the concentrated sample 6b ACN pH 4 Day 1. The MS data of m/z 352.178 are presented in Figure 33, appendix 1, identification report. In the MS spectrum, the ion with m/z 352.178 is clearly visible. The 14C-isotope pattern of this ion was comparable to the isotope pattern observed for the parent compound. The mass shift of this degradation product is -31.078. Da relative to the protonated molecule of the parent compound. Consistently, from the accurate mass measurements the most likely molecular formula for this protonated degradation product is C2114CH24NO3+ (error < 1 mDa, RDB= 11.5).

The MS2 spectrum of m/z 352.178 showed a main ion with m/z 189.078 (due to the loss of 163.099 Da, C10H13NO) indicating that the AB-moiety is unchanged and thus the desaturations and oxidation occurred in the moiety containing the C-ring. Additionally, fragment ions with m/z 310.168 (due to the loss 42.010 Da, C2H2O) indicating a desaturation and oxidation at the ethyl chain, m/z 260.116 (due to the loss of 92.062 Da, C7H8) indicating that the C-ring is unchanged, m/z 147.068 (due to the loss of 205.110 Da, 189-C2H2O) and m/z 117.070 (due to the loss of 235.108 Da, 147-14C=O) were observed.

Based on the molecular formula, the loss of dimethylamine (C2H7N) resulting in a desaturation, the unchanged AB-moiety and unchanged C-ring M4b is most likely desaturated and oxidized in the part between the AB-moiety and C-ring. The proposed structures of MS2 ions are presented in Figure 34, appendix 1, identification report. A structure for the degradation product M-4a is proposed in 'Proposed Structure of the Possible Photolytic Degradation Products' appended in the attached background material.

The reconstructed ion chromatograms of m/z 352.178 (coded M-4b) in the selected concentrated samples (pH 4 and pH 7) are presented in Figure 35, appendix 1, identification report.

Peak M-4c

Based on the accurate mass m/z 368.173 is most likely the result of the loss of dimethylamine (C2H7N) moiety resulting in a desaturation in combination with a carboxylation. Additional MSn analyses was performed using the concentrated sample 6b ACN pH 4 Day 1. The MS data of m/z 368.173 are presented in Figure 36, appendix 1, identification report. In the MS spectrum, the ion with m/z 368.173 is clearly visible. The 14C-isotope pattern of this ion was comparable to the isotope pattern observed for the parent compound. The mass shift of this degradation product is -15.083. Da relative to the protonated molecule of the parent compound. Consistently, from the accurate mass measurements the most likely molecular formula for this protonated degradation product is C2114CH24NO4+ (error < 1 mDa, RDB= 11.5).

The MS2 spectrum of m/z 368.173 showed a main ion with m/z 322.167 (due to the loss of 46.006 Da, HCOOH) indicating a carboxylation. Additionally, fragment ions with m/z 350.162 (due to the loss 18.011 Da, H2O), m/z 340.178 (due to the loss of 27.994 Da, C=O), m/z 276.111 (due to the loss of 92.062 Da, C7H8) indicating an unchanged C-ring and m/z 230.105 (due to the loss of 138.068 Da, 276-HCOOH) were observed. In the MS3 spectrum of the major MS2 fragment ion with m/z 322.167, a main fragment ion with m/z 230.105 (due to the loss of 92.062 Da, C7H8) was observed. Additionally, fragment ions with m/z 293.128 (due to the loss of 29.039 Da, •C2H5), m/z 202.110 (due to the loss of 120.057 Da, 230-C=O), m/z 176.058 (due to the loss of 146.109 Da, C11H14), m/z 148.063 (due to the loss of 174.104 Da, 176-C=O), m/z 145.101 (due to the loss of 177.066 Da, C914CH9NO2) and m/z 105.070 (due to the loss of 217.097 Da, C1214CH13NO2) were observed.

Based on the molecular formula, the loss of dimethylamine (C2H7N) resulting in a desaturation, the loss of HCOOH indicating carboxylation and the unchanged C-ring M4c is most likely carboxylated at the ethyl chain. The proposed structures of the most relevant MS2 and MS3 ions are presented in Figure 37, appendix 1, identification report. A structure for the degradation product M-4c is proposed in 'Proposed Structure of the Possible Photolytic Degradation Products' appended in the attached background material.

The reconstructed ion chromatograms of m/z 368.173 (coded M-4c) in the selected concentrated samples (pH 4 and pH 7) are presented in Figure 38, appendix 1, identification report.

Peak M-4d

Based on the accurate mass m/z 368.173 is most likely the result of the loss of dimethylamine (C2H7N) moiety resulting in a desaturation in combination with desaturation and 2 times oxidation. Additional MSn analyses was performed using the concentrated sample 1 ACN pH 4 180 min. The MS data of m/z 368.173 are presented in Figure 39, appendix 1, identification report. In the MS spectrum, the ion with m/z 368.173 is clearly visible. The 14C-isotope pattern of this ion was comparable to the isotope pattern observed for the parent compound. The mass shift of this degradation product is -15.083. Da relative to the protonated molecule of the parent compound.

Consistently, from the accurate mass measurements the most likely molecular formula for this protonated degradation product is C2114CH24NO4+ (error < 1 mDa, RDB= 11.5). The MS2 spectrum of m/z 368.173 showed a main ion with m/z 322.167 (due to the loss of 46.006 Da, HCOOH) indicating a carboxylation and a minor fragment ion with m/z 340.178 (due to the loss 27.994 Da, C=O). In the MS3 spectrum of the major MS2 fragment ion with m/z 322.167 a major fragment ion with m/z 217.097 (due to the loss of 105.070 Da, •C8H9) indicating the loss of the C-ring was observed. Additionally, fragment ions with m/z 294.172 (due to the loss 27.994 Da, C=O), m/z 202.110 (due to the loss of 120.057 Da, 294-C7H8), m/z 176.058 (due to the loss of 146.109 Da, C11H14), m/z 145.101 (due to the loss of 177.066 Da, C914CH9NO2) and m/z 105.070 (due to the loss of 217.097 Da, C1214CH13NO2) were observed. This degradation product shows similar fragmentation as M-4c, especially in the key fragments, i.e., the ions with m/z 176 and m/z 145. Therefore M-4d is most likely an isomer of degradation product M-4c. The difference is most likely the position of the double bond in the C-part.

Based on the molecular formula, the loss of dimethylamine (C2H7N) resulting in a desaturation, the loss of HCOOH indicating carboxylation and the unchanged C-ring M-4d is most likely desaturated and 2 times oxidized at the A-ring. The proposed structures of the most relevant MS2 and MS3 ions are presented in Figure 40, appendix 1, identification report. A structure for the degradation product M-4d is proposed in 'Proposed Structure of the Possible Photolytic Degradation Products' appended in the attached background material.

The reconstructed ion chromatograms of m/z 368.173 (coded M-4d) in the selected concentrated samples (pH 4 and pH 7) are presented in Figure 38, appendix 1, identification report.

M-4e, M-4f and M-4g

For the three minor ions with m/z 399.251 (coded M-4e), m/z 413.231 (coded M-4f) and m/z 397.236 (coded M-4g) the MS2 and MS3 data were very limited due to the relatively low signals. For this reason, only the m/z value and proposed molecular formula and most likely change are given in the table below. The reconstructed ion chromatograms of m/z 399.251 (coded M-4e), m/z 413.231 (coded M-4f) and m/z 397.236 (coded M-4g) in the selected concentrated samples (pH 4 and pH 7) are presented in Figure 24, Figure 43 and Figure 45 (appendix 1, identification report) respectively.

Information on Degradation Products M-4e, M-4f and M-4g

Degradation product m/z value Proposed molecular formula Most likely change
M-4e  399.251 C2314CH33N2O3+  –CH2OH instead of –CH3 
M-4f  413.231 C2314CH31N2O4+ –COOH instead of a CH3 
M-4g  397.236 C2314CH31N2O3+  –CHO instead of a –CH3 

CHARACTERIZATION/IDENTIFICATION OF DEGRADATION PRODUCT M-5

In the MS screening at the retention of degradation product M-5, a major ion with m/z 356.209 (coded M-5a) and a minor ion with m/z 401.267 (coded M-5b) were observed. Additional MSn analysis was performed for both ions.

Peak M-5a

Based on the accurate mass m/z 356.209 is most likely the result of loss of NH-dimethyl moiety in combination with an oxidation. Additional MSn analyses were performed using the concentrated sample 1 ACN pH 4 180 min. The MS data of m/z 356.209 are presented in Figure 46, appendix 1, identification report. In the MS spectrum, the ion with m/z 356.209 is clearly visible. The 14C-isotope pattern of this ion was comparable to the isotope pattern observed for the parent compound.

The mass shift of this degradation product is -27.047 Da relative to the protonated molecule of the parent compound. Consistently, from the accurate mass measurements the most likely molecular formula for this protonated degradation product is C2114CH28NO3+ (error < 1 mDa, RDB= 9.5).

The MS2 spectrum of m/z 356.209 showed a main ion with m/z 356.209 indicating a relatively low fragmentation. Therefore, the results of the MS3 spectrum of the MS2 fragment ion with m/z 356.209 is mainly used for the identification of M-5a. In the MS3 spectrum of the major MS2 fragment ion with m/z 356.209 a main fragment ion with m/z 192.089 (due to the loss of 164.120 Da, C11H16O) indicating that the AB- moiety including the 14C=O are unchanged and that the oxidation occurred at the moiety containing the C-ring. Additionally, fragment ions with m/z 338.198 (due to the loss 18.011 Da, H2O), m/z 308.200 (due to the loss of 48.009 Da, 338-14C=O), m/z 282.172 (C1814CH22NO+, due to the loss of H2O and C3H4O from the morpholine ring), m/z 251.138 (due to the loss of 105.071 Da, •C8H9) indicating that the hydroxy-group is not in the –CH2–Phenyl–CH3 part (C-ring) of the structure, m/z 192.089 (C1014CH12NO2+, due to the loss of 164.120 Da, C1114CH14O) indicating the AB-moiety including the 14C=O was unchanged, m/z 164.107 (due to the loss of 192.102 Da, of C1114CH14O2) and m/z 163.111 (due to the loss of C1014CH15NO3) confirming oxidation occurred at the part between the 14C=O and C-ring were observed.

Based on the loss of the unchanged AB-moiety and the unchanged C-ring the oxidation occurred most likely at the ethyl chain. The proposed structures of MS2 and MS3 fragment ions are presented in Figure 47, appendix 1, identification report. A structure for the degradation product M-5a is proposed in 'Proposed Structure of the Possible Photolytic Degradation Products' appended in the attached background material.

The reconstructed ion chromatograms of m/z 356.209 (coded M-5a) in the selected concentrated samples (pH 4 and pH 7) are presented in Figure 48, appendix 1, identification report.

Peak M-5b

Based on the accurate mass m/z 401.267 is most likely the result of an oxidation in combination with a desaturation of a double bond of the parent compound. Additional MSn analyses were performed using the concentrated sample 1 ACN pH 4 180 min. The MS data of m/z 401.267 eluting at 22.3 minutes are presented in Figure 49, appendix 1, identification report. In the MS spectrum, the ion with m/z 401.267 was a minor ion. The 14C-isotope pattern of this ion was comparable to the isotope pattern observed for the parent compound. The mass shift of this degradation product is +18.011 Da relative to the protonated molecule of the parent compound.

Consistently, from the accurate mass measurements the most likely molecular formula for this protonated degradation product is C2314CH35N2O3+ (error < 1 mDa, RDB= 8.5). The MS2 spectrum of m/z 401.267 did not result in reliable results due to a too low signals. Due to the very limited MS data it is not possible to identify this degradation product. The reconstructed ion chromatograms of m/z 401.267 (coded M-5b) in the selected concentrated samples (pH 4 and pH 7) are presented in Figure 50, appendix 1, identification report.

CHARACTERIZATION/IDENTIFICATION OF DEGRADATION PRODUCT M-6

Based on the accurate mass m/z 338.198 is most likely the loss of dimethylamine (C2H7N) resulting in the formation of a desaturation. Additional MSn analyses were performed using the concentrated sample 1 ACN pH 4 180 min. The MS data of m/z 338.198 are presented in Figure 51, appendix 1, identification report. In the MS spectrum, the ion with m/z 338.198 is clearly visible. The 14C-isotope pattern of this ion was comparable to the isotope pattern observed for the parent compound. The mass shift of this degradation product is -45.058 Da relative to the protonated molecule of the parent compound. Consistently, from the accurate mass measurements the most likely molecular formula for this protonated degradation product is C2114CH26NO2+ (error < 1 mDa, RDB= 10.5).

The MS2 spectrum of m/z 338.198 showed a main ion with m/z 338.198 indicating a relatively low fragmentation. Therefore, the results of the MS3 spectrum of the MS2 fragment ion with m/z 338.198 is mainly used for the identification of M-6. In the MS3 spectrum of the major MS2 ion with m/z 338.198 main fragment ions with m/z 320.188 (due to the loss of 18.010, H2O), m/z 233.128 (due to the loss 105.070 Da, •C8H9), m/z 192.089 (due to the loss of 146.109 Da, C11H14), m/z 175.099 (due to the loss of 163.099 Da, C10H13NO) and m/z 145.101 (due to the loss of 193.097 Da, C1014CH13NO2) were observed. The fragment ion with m/z 233.128 corresponds most likely to the AB- moiety including the 14C=O and a mono-desaturated propyl group. The fragment ion with m/z 192.089 confirms that the AB- moiety including the 14C=O is unchanged. The complementary fragment ion with m/z 145.101 and the fragment ion with m/z 175.099 confirms that the desaturation is localized on the moiety containing the C-ring.

Based on the molecular formula and the fragmentation of this molecule, the desaturation occurred most likely at the CH2-CH3 chain. The proposed structures of MS2 and MS3 fragment ions are presented in Figure 52, appendix 1, identification report. A structure for the degradation product M-6 is proposed in 'Proposed Structure of the Possible Photolytic Degradation Products' appended in the attached background material.

The reconstructed ion chromatograms of m/z 338.198 in the selected concentrated samples (pH 4 and pH 7) are presented in Figure 53, appendix 1, identification report.

CHARACTERIZATION/IDENTIFICATION OF DEGRADATION PRODUCT M-7

Based on the accurate mass m/z 338.198 is most likely the loss of dimethylamine (C2H7N) resulting in the formation of a desaturation. The difference of retention time between M-7 and M-6 indicates that both degradation product are isomers. Additional MSn analyses were performed using the concentrated sample 1 ACN pH 4 180 min. The MS data of m/z 338.198 are presented in Figure 54, appendix 1, identification report. In the MS spectrum, the ion with m/z 338.198 is clearly visible. The 14C-isotope pattern of this ion was comparable to the isotope pattern observed for the parent compound. The mass shift of this degradation product is -45.058 Da relative to the protonated molecule of the parent compound. Consistently, from the accurate mass measurements the most likely molecular formula for this protonated degradation product is C2114CH26NO2+ (error < 1 mDa, RDB= 10.5).

The MS2 spectrum of m/z 338.198 showed a main ion with m/z 338.198 indicating a relatively low fragmentation. Therefore, the results of the MS3 spectrum of the MS2 fragment ion with m/z 338.198 is mainly used for the identification of M-7. In the MS3 spectrum of the major MS2 ion with m/z 338.198 fragment ions with m/z 320.188 (due to the loss of 18.010, H2O), m/z 309.160 (due to the loss of 29.038, •C2H5), m/z 294.172 (due to the loss 44.026 Da, C2H4O), m/z 246.136 (due to the loss 92.062 Da, C7H8), m/z 192.089 (due to the loss of 146.109 Da, C11H14), m/z 175.099 (due to the loss of 163.100 Da, C10H13NO) and m/z 145.101 (due to the loss of 193.097 Da, C1014CH13NO2) were observed. The fragment ion with m/z 246.136 corresponds most likely to the AB- moiety including the 14C=O and a mono-desaturated sec-butyl group. The fragment ion with m/z 192.089 confirms that the AB-moiety including the 14C=O is unchanged. The complementary fragment ion with m/z 145.101 and the fragment ion with m/z 175.099 confirms that the desaturation is localized between the 14C and the C-ring. Based on the difference in fragmentation, in M-7, the loss of C7H8 and of •C2H5, and in M-6, the loss of •C8H9 and the absence of the ethyl radical loss, the desaturation in degradation product M-6 most likely occurred at the ethyl substituent and in M-7, it is most likely desaturated in the position shown below.

Based on the molecular formula, the loss of an ethyl radical and the main fragmentation of this molecule, the desaturation occurred most likely between the two carbons linking 14C=O and the C-ring. The proposed structures of MS2 and MS3 fragment ions are presented in Figure 55, appendix 1, identification report. A structure for the degradation product M-7 is proposed in 'Proposed Structure of the Possible Photolytic Degradation Products' appended in the attached background material.

The reconstructed ion chromatograms of m/z 338.198 in the selected concentrated samples (pH 4 and pH 7) are presented in Figure 53, appendix 1, identification report.

Validity criteria fulfilled:
yes
Conclusions:
Upon incubation under simulated sunlight, the test item degraded to 0% (pH 4 and pH 7) after an incubation time of 3 hours and 1 hour, respectively, corresponding to approximately 6.5 and 2.1 hours of natural sunlight. Seven major photolytic degradation products were detected, which exceeded 10% of applied radioactivity. Identification of major degradation products will be carried out in a separate project. From the seven major photolytic degradation products (M-1, M-2, M-3, M-5, M-6, M-7 and M-9) detected during photochemical degradation of the test item in water, it was observed using a LC-PDA-(RAD)MSn method that several degradation products were co-eluting at the retention time of M-2 (coded M-2a/b/c), M-3 (coded M-3a/b), M-4 (coded M-4a/b/c/d/e/f/g) and M-5 (coded M-5a/b) whereas one degradation product was observed at the retention time of M-1, M-6 and M-7.
The identification of degradation product M-9 was not possible due to a too low amount of radioactivity. Due to no or very limited MS data it was not possible to propose structures for five degradation products with a low MS intensity (i.e., M-2c, M-4e, M-4f, M-4g and M-5b).
Based on accurate masses and MSn fragmentations, molecular structures were proposed for twelve degradation products with a medium or high MS intensity (i.e., M-1, M-2a, M-2b, M-3a, M-3b, M-4a, M-4b, M-4c, M-4d, M-5a, M-6 and M-7).


At pH 4, degradation product M-1 increased to a maximum of 24.5% of applied radioactivity after an incubation time of 2 days, after which concentrations decreased to 3.4% of applied radioactivity after an incubation time of 14 days. Degradation product M-2, M- 5, M-6, and M-7 all increased to their maximum concentration within 24 hours of incubation, and decreased to 0% of applied radioactivity by the end of the incubation period.

At pH 7, degradation product M-1 increased to a maximum of 12.9% of applied radioactivity after an incubation time of 2 days, after which concentrations decreased to 3.6% of applied radioactivity after an incubation time of 14 days. Degradation product M-9 increased to a maximum of 20.1% of applied radioactivity after an incubation time of 7 days, after which concentrations decreased to 13.0% of applied radioactivity after an incubation time of 14 days. Degradation product M-2, M-3, M-5, M-6, and M-7 all increased to their maximum concentration within 24 hours of incubation, and decreased to 0% of applied radioactivity by the end of the incubation period. One degradation product (M-3) could be identified as 4- (4- morpholinyl)benzaldehyde by co- chromatography against reference standard AS1842.

Significant amounts of CO2 were formed in a few samples (up to 27.4% and 20.6% of applied radioactivity at pH 4 and pH 7, respectively). Negligible amounts of radioactivity (<0.3% of applied) were detected in the PUF traps. Recovery from vessel rinsates at pH 4 increased to 6.7% of applied radioactivity after an incubation time of 3 hours, and then gradually declined to <0.1% after 14 days of incubation. At pH 7, recovery from vessel rinsates was highest at the start of incubation, with up to 29.7% of applied radioactivity. This gradually decreased with incubation time, to <0.1% after 14 days of incubation, except for the dark controls where recovery from rinsates was still approximately 20% (on average) after 14 days of incubation.

Degradation was also observed in the dark control samples. After an incubation time of 14 days, 90.8% and 88.7% was recovered as parent at pH 4 and pH 7, respectively, assuming equal concentration of parent. No major degradation products were formed in the dark controls. The degradation rates of The test item in the dark and in natural sunlight (photolytic degradation rate) are summarised in the table below. The photolytic half-life of the test item in natural sunlight was equivalent to 26.6 minutes and 12.8 minutes at pH 4 and pH 7, respectively.
Photolytic Degradation Rate of the test item

DT50, dark [days] DT50, phot [days] DT90, dark [days] DT90, phot [days]
pH 4 >10,000 0.019 >10,000 0.062
pH 7 229 0.0089 762 0.030


Executive summary:

The objective of this study was to determine the photolytic degradation rate of the test item in water, and to determine major photolytic degradation products. Radiolabelled test item was incubated in aqueous buffer solutions at pH 4 and pH 7, for a maximum period of 14 days. Sunlight was simulated by irradiating sample vessels containing the test solutions using two xenon arc lamps. Dark controls were included to distinguish between photolytic degradation and degradation by other processes (e.g. hydrolysis).

Temperature in test solutions was monitored continuously.

Upon incubation under simulated sunlight, the test item degraded to 0% at pH 4 and pH 7, after an incubation time of 3 hours and 1 hour, respectively, corresponding to approximately 6.5 and 2.1 hours of natural sunlight. Seven major photolytic degradation products were detected, which exceeded 10% of applied radioactivity. Identification of major degradation products will be carried out in a separate project.

At pH 4, degradation product M-1 increased to a maximum of 24.5% of applied radioactivity after an incubation time of 2 days, after which concentrations decreased to 3.4% of applied radioactivity after an incubation time of 14 days. Degradation product M-2, M- 5, M-6, and M-7 all increased to their maximum concentration within 24 hours of incubation and decreased to 0% of applied radioactivity by the end of the incubation period.

At pH 7, degradation product M-1 increased to a maximum of 12.9% of applied radioactivity after an incubation time of 2 days, after which concentrations decreased to 3.6% of applied radioactivity after an incubation time of 14 days. Degradation product M-7 increased to a maximum of 12.0% of applied radioactivity after an incubation time of 1 hour, after which concentrations decreased to 0% of applied radioactivity after an incubation time of 2 days.

Degradation product M-9 increased to a maximum of 20.1 % of applied radioactivity after an incubation time of 7 days, after which concentrations decreased to 13.0% of applied radioactivity after an incubation time of 14 days. Degradation product M-2, M-3, M-5, and M-6 all increased to their maximum concentration within 24 hours of incubation, and decreased to 0% of applied radioactivity by the end of the incubation period. One degradation product (M-3) could be identified as 4-(4-morpholinyl)benzaldehyde by co- chromatography against the reference standard.

Significant amounts of CO2 were formed in some samples (up to 27.4% and 20.6% of applied radioactivity at pH 4 and pH 7, respectively). Negligible amounts of radioactivity (<0.3% of applied) were detected in the PUF traps. Recovery from vessel rinsates at pH 4 increased to 6.7% of applied radioactivity after 3 hours, and then gradually declined to <0.1% after 14 days of incubation. At pH 7, recovery from vessel rinsates was highest at the start of incubation, with up to 29.7% of applied radioactivity. This gradually decreased with incubation time, to <0.1% after 14 days of incubation, except for the dark controls where recovery from rinsates was still approximately 20% (on average) after 14 days of incubation. Degradation was also observed in the dark control samples. After an incubation time of 14 days, 90.8% and 88.7% was recovered as parent at pH 4 and pH 7, respectively. No major degradation products were formed in the dark controls.

The photolytic half-life of the test item in natural sunlight was equivalent to 26.6 minutes and 12.8 minutes at pH 4 and pH 7, respectively.

For the purpose of identification of the degradation products, the following samples were analyzed:

- Sample 1_ACN_pH 4_180 min

- Sample 1b_ACN_pH 4_60 min

- Sample 6b_ACN_pH 4_1 day

- Sample 7a_pH 7_1 day

- Sample 20_ACN_pH 7_180 min

- Corresponding blanks (pH4_A_ACN and pH7_A_ACN)

All samples were analyzed using an LC-PDA-(RAD)MSn system. The retention time of [14C]-test item was 13.85 min on the PDA detector, 14.10 min on the radioactivity detector and 13.90 min on the MS detector. The PDA spectrum of the test item showed specific absorbance at 355 nm.

The amount of radioactivity in sample 7a pH 7 Day 1 was too low to obtain a reliable radioactivity chromatogram from this sample. Based on this result, identification of degradation product M-9 was not possible.

The radioactivity chromatograms obtained for samples 20 ACN pH 7 180 min, 1 ACN pH 4 180 min, 1b ACN pH 4 60 min and 6b ACN pH 4 Day 1 were comparable to the earlier obtained radioactivity chromatograms in Test Facility Study No. 20153931. The peak eluting at 11.9 minutes was corresponding to M-1, the peak eluting at 13.9-14.0 minutes was corresponding to M-2, the peak eluting at 15.4 minutes was corresponding to M-3, the peak eluting at 21.8 minutes was corresponding to M-4, the peak eluting at 22.5-22.6 minutes was corresponding to M-5, the peak eluting at 23.6 minutes was corresponding to M-6 and the peak eluting at 24.7-24.8 minutes was corresponding to M 7.

In the MS screening at the retention of degradation product M-1, M-6 and M-7, only one ion per degradation product was observed. In the MS screening at the retention of degradation product M-2, M-3, M-4 and M-5, multiple ions of various intensities per degradation product were observed indicating most likely that co-elution occurred. A summary of the possible photolytic degradation products detected in the samples from Test Facility Study No. 20153931 and their respective proposed structures are presented in the tables appended in in the attached background material 'Proposed Structure of the Possible Photolytic Degradation Products' & ‘Possible Photolytic Degradation Products Detected in the Samples’.

In conclusion, from the seven major photolytic degradation products (M-1, M-2, M-3, M-5, M-6, M-7 and M-9) detected during photochemical degradation of the test item in water (Test Facility Study No. 20153931), it was observed using a LC-PDA-(RAD)MSn method that several degradation products were co-eluting at the retention time of M-2 (coded M-2a/b/c), M-3 (coded M-3a/b), M-4 (coded M-4a/b/c/d/e/f/g) and M-5 (coded M-5a/b) whereas one degradation product was observed at the retention time of M-1, M-6 and M-7.

The identification of degradation product M-9 was not possible due to a too low amount of radioactivity. Due to no or very limited MS data it was not possible to propose structures for five degradation products with a low MS intensity (i.e., M-2c, M-4e, M-4f, M-4g and M-5b). Based on accurate masses and MSn fragmentations, molecular structures were proposed for twelve degradation products with a medium or high MS intensity (i.e., M-1, M-2a, M-2b, M-3a, M-3b, M-4a, M-4b, M-4c, M-4d, M-5a, M-6 and M-7).

M-1: Retention time 11.5 mins, C1014CH12NO5+

Based on the accurate mass m/z 240.074 is most likely the loss of N,N-dimethyl-1-(p-tolyl)butan-2-amine in combination with a desaturation (+H2) and 3 oxidations (+3 O atoms).  

M-2aRetention time 13.7 mins, C1014CH14NO3+

Based on the accurate mass m/z 210.100 is most likely the result of hydrolysis of the 14C atom, and thus the loss of N,N-dimethyl-1-(p-tolyl)butan-2-amine.

M-2bRetention time 13.7 mins, C2314CH33N2O2+

Based on the accurate mass m/z 383.257 this molecule is comparable to the parent compound.  However, as the retention time of M-2b is slightly different than the retention time of the parent compound, this degradation product was further investigated.   Based on the molecular formula and the loss of C2H5N, the molecule is most likely desaturated at the dimethylamine and reduced at the C-moiety

M-2cRetention time 13.7 mins, C2314CH33N2O3+

Based on the accurate mass m/z 399.252 is most likely the result of an oxidation of the parent compound

M-3aRetention time 14.8 mins, C1014CH14NO2+

Based on the accurate mass m/z 194.105 is most likely the result of cleavage of the moiety containing the AB-ring and the moiety containing the C-ring to give 4-(4-Morpholinyl)benzaldehyde (CAS 1204 -86 -0).

M-3bRetention time 15.0 mins, C2114CH26NO3+

Based on the accurate mass m/z 354.193 is most likely the result of the loss of the dimethylamine (C2H7N) moiety resulting in a desaturation in combination with an oxidation.  

M4aRetention time 21.4 mins, C2114CH26NO3+

Based on the accurate mass m/z 354.193 is most likely the result of the result of the loss of the NH dimethyl moiety in combination with an oxidation

M4b, Retention time 21.6 mins, C2114CH24NO3+

Based on the accurate mass m/z 352.178 is most likely the result of the loss of dimethylamine (C2H7N) moiety resulting in a desaturation in combination with a desaturation and oxidation.  

M4c,Retention time 21.6 mins, C2114CH24NO4+

Based on the accurate mass m/z 368.173 is most likely the result of the loss of dimethylamine (C2H7N) moiety resulting in a desaturation in combination with a carboxylation.

M4d, Retention time 21.6 mins, C2114CH24NO4+

Based on the accurate mass m/z 368.173 is most likely the result of the loss of dimethylamine (C2H7N) moiety resulting in a desaturation in combination with desaturation and 2 times oxidation.  

M5a, Retention time 22.3, C2114CH28NO3+

Based on the accurate mass m/z 356.209 is most likely the result of loss of NH dimethyl moiety in combination with an oxidation

M5b, Retention time 22.3 mins, C2314CH35N2O3+

Based on the accurate mass m/z 401.267 is most likely the result of an oxidation in combination with a desaturation of a double bond of the parent compound

M6, Retention time 23.4 mins, C2114CH26NO2+

Based on the accurate mass m/z 338.198 is most likely the loss of dimethylamine (C2H7N) resulting in the formation of a desaturation.  

M7, Retention time 24.4 mins, C2114CH26NO2+

Based on the accurate mass m/z 338.198 is most likely the loss of dimethylamine (C2H7N) resulting in the formation of a desaturation

Description of key information

Study conducted to internationally recognised testing guideline with GLP

Key value for chemical safety assessment

Half-life in water:
0.012 d

Additional information

Upon incubation under simulated sunlight, Omnirad 379 degraded to 0% (pH 4 and pH 7) after an incubation time of 3 hours and 1 hour, respectively, corresponding to approximately 6.5 and 2.2 hours of natural sunlight. Seven major photolytic degradation products were detected, which exceeded 10% of applied radioactivity.

Photolytic Degradation Rate of Omnirad 379

   

DT50, dark [days]

 

DT50, phot [days]

 

DT90, dark [days]

 

DT90, phot [days]

 pH4  >10,000  0.019  >10,000  0.062
 pH7  229  <0.012  762  0.036