4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline

Biodegradation in soil

Administrative data

Endpoint:

biodegradation in soil: simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Dec 2020 – Oct 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 307 (Aerobic and Anaerobic Transformation in Soil)

Version / remarks:

OECD (2002). Guidelines for the Testing of Chemicals. TG 307: Aerobic and Anaerobic Transformation in Soil. OECD, Paris.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

laboratory

Test material

Test material information

Constituent 1

Reference substance name:

4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline

EC Number:

233-215-5

EC Name:

4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline

Cas Number:

10081-67-1

Molecular formula:

C30H31N

IUPAC Name:

4-(2-phenylpropan-2-yl)-N-[4-(2-phenylpropan-2-yl)phenyl]aniline

Test material form:

solid

Details on test material:

Test substance name: 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline
Scymaris reference number: 1108TS001
CAS number: 10081-67-1
Batch/Lot number: EL0D14H276
Purity: 97%
Physical state and appearance: White to off-white powder
Expiry/re-test date: 14 April 2023
Storage conditions: Room Temperature

Specific details on test material used for the study:

2.3 [14C]Test Substance
Chemical name: [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl) phenyl]aniline
Scymaris reference number: 1108TS002
Alternative name/s: [phenyl-U-14C]4,4’-Bis(α,α-dimethylbenzyl)diphenylamine
Molecular weight: 407.44g/mol
Batch/Lot number: 11468YXC011-4
Supplier: Selcia
Purity: 99.0%
Specific activity: 5297 Bq/µg
Physical state and appearance: White solid
Storage conditions: Freezer

Radiolabelling:

yes

Study design

Oxygen conditions:

aerobic

Soil classification:

USDA (US Department of Agriculture)

Year:

2021

Soil properties

Soil properties 1

Soil no.:

#1

Soil type:

sandy loam

% Clay:

8

% Silt:

20

% Sand:

72

% Org. C:

0.9

pH:

6.22

CEC:

185.6

Soil properties 2

Soil no.:

#2

Soil type:

silt loam

% Clay:

19

% Silt:

68

% Sand:

13

% Org. C:

1

pH:

7.15

CEC:

100

Soil properties 3

Soil no.:

#3

Soil type:

silt loam

% Clay:

25

% Silt:

50

% Sand:

25

% Org. C:

4.83

pH:

5.96

CEC:

221.6

Soil properties 4

Soil no.:

#4

Soil type:

loamy sand

% Clay:

5

% Silt:

10

% Sand:

85

% Org. C:

3.17

pH:

5.98

CEC:

145.6

Details on soil characteristics:

Collection and characterisation
The four field fresh soils were collected from different sites in Germany and differed in carbon content and texture. Soil I had a low organic carbon content of 0.9% and a sandy loam texture. Soil II had a low organic carbon content of 1.0% and a silt loam texture. Soil III had a high organic carbon content of 4.83% and a silt loam texture. Soil IV had a medium organic carbon content of 3.17% and a loamy sand texture.
The soils were freshly collected from the field from the top layer (20 cm). Several subsamples were collected with a spade and the subsamples mixed to prepare a unified sample. Sampling and handling of the soils were performed with reference to ISO 10381-6 by a third party, Fraunhofer-Institut, Schmallenberg, Germany. Soils were sieved to 2 mm before shipment to Scymaris.
Particle size distribution (% sand, silt and clay) of the soils, cation exchange capacity, bulk density and water retention characteristics were determined by Smithers, Harrogate, UK as a separate GLP study. Organic carbon content, pH in water and calcium chloride, moisture content and microbial biomass measurement using the chloroform fumigation method of the soils were measured at Scymaris. All data obtained pertaining to the soil prior to start of the test is retained as non-study specific data.

Preparation
Soils were stored in loosely tied plastic bags at 4 ± 2°C in the dark. The storage period was 78 days prior to use in the study.
Aliquots of fresh soil equivalent to approximately 50 g oven dry equivalent (ODE) were added to the test vessels; the nominal wet weights of each soil were: 54.3 g of soil I; 54.5 g of soil II; 63.8 g of soil III and 54.9 g of soil IV. Additional RO water was added, when necessary, to reach the required moisture content of between pF 2.0 and 2.5 (equivalent to the water holding capacity at 0.1 and 0.33 bar respectively); vessels 101 – 117 and 401 – 417 did not require any moisture adjustment, vessels 201 – 217 had an additional 8 mL RO added and vessels 301 – 317 required the addition of 6.15 mL RO water. The soil then underwent a 16 day acclimation period under aerobic conditions in 250 mL glass vessels in the dark at 12 ± 2°C.

Measurement of soil microbial activity
Soil microbial activity was measured as microbial biomass. This was measured on blank soil on day 60 and 120 of the exposure phase. The microbial activity of soil in the solvent control vessels was measured at the end of the exposure phase to assess the effects of the dosing solvent on the microbiota.
The soil microbial biomass was determined using the chloroform fumigation-extraction method.

Duration of test (contact time)

Duration of test (contact time) 1

Soil No.:

#1

Duration:

120 d

Duration of test (contact time) 2

Soil No.:

#2

Duration:

120 d

Duration of test (contact time) 3

Soil No.:

#3

Duration:

120 d

Duration of test (contact time) 4

Soil No.:

#4

Duration:

120 d

Initial test substance concentration

Initial test substance concentration 1

Soil No.:

#1

Initial conc.:

1.02 mg/kg soil d.w.

Based on:

test mat.

Remarks:

The total amount of radioactivity dosed to the soils was 269783 Bq, which gave a concentration of 1.02 mg/kg in the vessels. These values were used for the calculating the mass balance.

Initial test substance concentration 2

Soil No.:

#2

Initial conc.:

1.02 mg/kg soil d.w.

Based on:

test mat.

Remarks:

The total amount of radioactivity dosed to the soils was 269783 Bq, which gave a concentration of 1.02 mg/kg in the vessels. These values were used for the calculating the mass balance.

Initial test substance concentration 3

Soil No.:

#3

Initial conc.:

1.02 mg/kg soil d.w.

Based on:

test mat.

Remarks:

The total amount of radioactivity dosed to the soils was 269783 Bq, which gave a concentration of 1.02 mg/kg in the vessels. These values were used for the calculating the mass balance.

Initial test substance concentration 4

Soil No.:

#4

Initial conc.:

1.02 mg/kg soil d.w.

Based on:

test mat.

Remarks:

The total amount of radioactivity dosed to the soils was 269783 Bq, which gave a concentration of 1.02 mg/kg in the vessels. These values were used for the calculating the mass balance.

Parameter followed for biodegradation estimation:

radiochem. meas.

Experimental conditions

Experimental conditions 1

Soil No.:

#4

Temp.:

12 ± 2 °C

Humidity:

Not specified

Microbial biomass:

172.62 µg/g

Experimental conditions 2

Soil No.:

#3

Temp.:

12 ± 2 °C

Humidity:

Not specified

Microbial biomass:

594.70 µg/g

Experimental conditions 3

Soil No.:

#2

Temp.:

12 ± 2 °C

Humidity:

Not specified

Microbial biomass:

182.42 µg/g

Experimental conditions 4

Soil No.:

#1

Temp.:

12 ± 2 °C

Humidity:

Not specified

Microbial biomass:

116.05 µg/g

Details on experimental conditions:

TEST METHOD AND CONDITIONS

Test system
The test vessels were glass bottles connected to a series of traps, labelled with the appropriate study number and test vessel content information. The glass vessels used for each soil were 250 mL glass bottles with screw thread necks. Humidified ambient air was drawn through each test vessel using negative pressure provided by a vacuum system (Figure 1). Each vessel was continuously ventilated with humidified air under negative pressure. With the exception of the day 0 and biomass vessels, the exhaust air of each metabolism vessel was passed through individual trapping systems by two types of sorbent material in the form of ORBO tubes (ORBO-32 followed by ORBO 91) to trap organic volatiles and then two liquid traps containing 2M sodium hydroxide (NaOH) solution to capture [14C]carbon dioxide (14CO2).
The test design is summarised in Table 1, and comprised of the following test vessels:
• Transformation vessels: Fourteen vessels of each soil type were dosed to give an exposure concentration of approximately 1 mg a.i./kg dry soil [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline, and duplicate vessels were sacrificed at each of the 6 pre-determined timepoints, with duplicate spare vessels to provide an additional point of analysis, if required.
• Characterisation vessels (microbial biomass): Two additional vessels of each soil type were prepared for measurement of microbial biomass. One of these vessels was sampled during the study (nominally day 60 ± 3), and one was sampled on Day 120 ± 3. There were no volatile or CO2 traps associated with these vessels.
• Characterisation vessels (microbial biomass in presence of carrier solvent): One vessel of each soil type was prepared and dosed with the same volume of solvent used to dose the transformation vessels, which was sampled on Day 120 ± 3. There were no volatile or CO2 traps associated with these vessels.
The spare 1 mg a.i./kg vessels were not analysed during this study, and therefore are not included in the reporting.

EXPERIMENTAL PROCEDURE

Acclimation period
All test and control vessels were acclimated prior to the addition of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline for a 16 day period at the test temperature of 12 ± 2°C. The soil moisture content of the soil was maintained between pF 2.0 and 2.5 during this period.

Stock solution preparation
1.144 mL aliquot containing 17.46 MBq of [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline from a bulk stock in acetone was made up to 16 mL volume with acetone, to give a final concentration of 206.0 mg [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline /L (Stock H1).
0.00107 g of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline was weighed into a 1 mL glass volumetric and made up to volume with acetonitrile, to give a final concentration of 1 mg 4 (1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline /mL (Stock C1).
100 µL was taken from Stock H1 and added to 900 µL of 1:1 ROW:ACN prior to dosing to determine the radiochemical purity of [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline in the dosing solution using high-performance liquid chromatography paired with radiochemical and ultraviolet detection (HPLC-RAD/UV).

Test substance application
Stock H1 was applied to the soil surface of the test vessels, in a single 243 µL aliquot, to give a test concentration of 1 mg/kg [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline.
The actual concentration of [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline in the dosing solution, at the time of application to the test vessels, was measured by LSC of triplicate aliquots taken pre and post dosing the test vessels and calculated using the specific activity.
The test solution was applied to the test vessels onto the soil surface using a pipette of an appropriate volume. Vessels being sacrificed on Day 0 were dosed last to minimise time between dosing and solvent extraction of the soil.

Incubation of test vessels
All test vessels were incubated in darkness at 12  2C. The temperature in the room was monitored continuously throughout the acclimation and exposure phase using a digital max-min thermometer. Incubation was for 120 days after dosing of the test substance.
The water content of the soil samples was maintained at a moisture content of between pF 2.0 and 2.5. Test vessels were weighed fortnightly and RO water added, if necessary, to return the water content of the soil to the specified level. The weight of each test vessel was recorded before and after this procedure.

Sampling intervals
Duplicate vessels treated with 1 mg/kg [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline were sacrificed on sampling days 0, 7, 14, 28, 60 and 120.

Measurement of soil microbial activity
Soil microbial activity was measured as microbial biomass. This was measured on blank soil on day 60 and 120 of the exposure phase. The microbial activity of soil in the solvent control vessels was measured at the end of the exposure phase to assess the effects of the dosing solvent on the microbiota.
The soil microbial biomass was determined using the chloroform fumigation-extraction method
SAMPLING PROCEDURE
Soil extraction
At each sampling interval, 0, 7, 14, 28, 60, and 120 days after application, duplicate vessels of each soil were taken for analysis. The soil was transferred from the test vessel to a pre-weighed high density polyethylene (HDPE) bottle and the test vessels was rinsed using 100 mL acetonitrile and added to the soil. The bottle was mechanically rolled for approximately 30 minutes. The soil and extract solution slurry was then centrifuged at 5000 g at 4°C for 5 minutes, and the supernatant decanted into pre-weighed glass bottles and sampled in triplicate for liquid scintillation counting (LSC). A further 100 mL acetonitrile was added to the soil, and the extraction repeated so that a total of three extractions were carried out. On Day 14 and Day 60 only, the second extract was collected in the same bottle as the first extraction, and then triplicate LSC aliquots taken of the two combined extracts. On all other occasions, each individual extract had triplicate aliquots taken for LSC.
For Day 28 Soil III vessels, Day 60 Soil II vessels, and Day 120 Soil II and III vessels, approximately 20 g w/w of soil was mixed with 10 g diatomaceous earth and the mixture homogenised by hand. An aliquot of this was weighed into a 33 mL cell. This was then extracted with acetonitrile under high temperature and pressure using accelerated solvent extraction (ASE) with the Dionex ASE200 (conditions below) into a pre-weighed 60 mL vial. The vial was reweighed, and triplicate 1 mL aliquots were weighed for LSC.
The parameters for the ASE extraction were:
Heat 6 min
Flush 50% vol
Purge 30 sec
Cycles 2
Pressure 1500 psi
Temperature 125oC
Static 2 min
The ambient temperature extracts for each sampling point were combined in a glass bottle, and triplicate aliquots taken for LSC.
For Day 0, 150 mL of the combined extracts for each vessel were concentrated by rotary evaporation at 40°C to approx. 2-5 mL, with the round bottom flask used rinsed with 2 mL reverse osmosis water (ROW)/acetonitrile (ACN) (1:1, v/v) into the same vial as the concentrate. Triplicate aliquots were taken for LSC analysis. V302 and V402 required a further 2 mL ACN rinse of the flask to aid in recovery, and further triplicate aliquots taken for LSC.
For Day 7, the same procedure as Day 0 was followed, but with the flask rinse carried out with 4 mL ACN, except for Vessel 203 which was concentrated to dryness and was reconstituted with a 6 mL ACN rinse. Triplicate aliquots were taken for LSC.
For Days 14, 28, and 60, the same procedure as Day 0 was followed, but with the flask rinse carried out with 2 mL ACN, except for Vessels 108 and 207 which were concentrated to dryness and reconstituted in with a 4 mL ACN rinse. Triplicate aliquots were taken for LSC.
For Day 120, 150 mL of the combined extracts were concentrated to approx. 2-5 mL on a Genevac Rocket Evaporator at 40°C in Sample Genie sample holders directly into the collection vial. Triplicate aliquots were taken for LSC.
HPLC-RAD/UV analysis was carried out on the ambient soil extract concentrates. No further analysis was carried out on the ASE extracts.

Quantification of bound radioactivity
The bound radioactivity was quantified through combustion of dried homogenised soil residue aliquots in triplicate, using a Packard 307 sample oxidiser or Harvery biological oxidiser (only Day 120 Vessel 112) followed by LSC. Recovery checks of a known amount of radioactivity were run on each oxidiser prior to samples each day, and samples were only combusted if recovered radioactivity on the Packard was between 95 and 105%, and above 70% for the Harvey. Data was not corrected for recovery due to all recoveries for both oxidisers being within 95-105%.

Quantification of volatile radioactivity
The 14CO2 traps were sampled by decanting the contents of each trap into a pre-weighed HDPE bottle, reweighing and triplicate aliquots taken for LSC.
At each sampling occasion, the ORBOTM tubes of the sacrificed transformation vessels were removed. Volatile organic radioactivity trapped in the ORBOTM tubes was extracted with 10 mL of methanol and analysed by LSC.

Mass balance
A mass balance was obtained by the summation of radioactivity measured in the soil extracts, volatile traps and unextractable radioactivity.

Storage of samples
Storage of samples prior to analysis was avoided if possible, however, if storage was required for the bulk samples of soil and extracts, they were stored refrigerated. Concentrated extracts were stored frozen. Trapping solutions were stored at room temperature.

DATA ACQUISTION AND PROCESSING

Liquid scintillation counting
Radioactivity in the prepared sample/scintillant mixtures was quantified using a Tri-Carb liquid scintillation counter (PerkinElmer) with automatic quench correction using an external standard. The analyser has the optimal channel settings and quench correction curves for the relevant isotope/scintillant combinations.
Samples were counted for 10 min or to a 2-sigma counting error of 0.5% (whichever was reached soonest). Disintegration rates per second (Bq) of appropriate blank sample vials were subtracted from each sample measurement to give a net Bq value for each sample. A radioactivity measurement of less than twice the mean background count was considered below the limit of accurate quantification.

HPLC-RAD/UV Analysis
Radioactivity in samples analysed by HPLC was quantified by either on-line radiodetection with peak evaluation using Laura v 6.1.1.20 software, or by fraction collection with TopCount and subsequent evaluation in Laura v 6.1.1.20.
The HPLC conditions used were:
HPLC system: Agilent 1100/1200
Column: Gemini-NX C18 150 x 4.6 mm, 5 µm
Eluent flow rate: 1.0 mL/min (if required)
Mobile phase A: LC-MS Water & 0.1% phosphoric acid
Mobile phase B: LC-MS Acetonitrile & 0.1% phosphoric acid
Column Temperature: 30°C
UV wavelength 290 nm
Regions of background and regions of interest (ROI) were manually added to chromatograms. Percentage of sample radioactivity per ROI was calculated. The proportions of sample radioactivity in each ROI was converted to % of applied radioactivity.
Day 120 V111 ambient soil extract concentrate was analysed in duplicate by HPLC-RAD to show the repeatability of the analytical method.

Identification of transformation products
The confirmation/identification of transformation product(s) detected was attempted by the analysis of Day 28 Vessel 107 and Day 120 Vessel 212 concentrated extracts by suitable LC-MS/MS techniques. Identification of transformation products was attempted when observed:
• At greater than 10% applied radioactivity at any single sampling interval
• At greater than 5% applied radioactivity at any two consecutive sampling intervals
• At greater than 5% applied radioactivity and steadily rising at the end of the study.
The selected sample was analysed by LC-RAD-MS/MS, including accurate mass analysis to attempt to identify the chromatographically separated radiolabelled components. Structural information on the observed radiochemical components in the samples was based on molecular ion and product ion information.
Using a Dionex U3000 liquid chromatography system and Thermo Scientific LTQ Orbitrap mass spectrometer along with a Raytest Mirastar radio detector, the LC-MS conditions used for the qualitative analysis of [14C] 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl) phenyl]aniline and any [14C]metabolites were utilised.
Online LC-MS radioactivity data in chromatogram format, with associated mass spectrometry data, was captured directly by Xcalibur v 2.0.7 software. Any structural elucidation work required was carried out using Compound Discoverer v 3.1.

Limit of detection and limit of quantification
Values of limit of detection (LOD) and limit of quantification (LOQ) for both LSC and HPLC-RAD were calculated for samples generated at Day 0 (as Vessel 101 had the smallest % AR equivalent to a peak in the HPLC-RAD chromatogram); this was considered representative of all study sample data.

Calculations
Analytical results were calculated using Microsoft Excel. The values presented in the tables and appendices of the report were rounded. Manual calculations using the rounded values may produce slightly different results.
For each sample by LSC, the mean Bq/mL or Bq/g was calculated from the measured radioactivity in each aliquot. The sample mean was multiplied by the aliquot size (mL or g) to determine the total radioactivity in each sample. The total radioactivity was divided by the amount of radioactivity applied to determine the % AR in each sample. The total radioactivity in the soil extracts, soil residue, plus the cumulative amounts in the traps were used to determine the total radioactivity recovered from each test chamber. The total radioactivity recovered was divided by the applied radioactivity to determine the mass balance for each test vessel.
The total amount of radioactivity in the soil was calculated as the sum of the total radioactivity in the ambient extracts, ASE extracts, and the solids after extraction.
For each sample analysed by HPLC-RAD/UV, the percentage of radioactivity in each ROI was multiplied by the % AR in each sample to determine the relative distribution of radioactivity within each sample.

Kinetics calculation
The behaviour of [14C] 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline in the total system was evaluated to determine the dissipation/transformation rate. The DT50 and DT90 (i.e. time taken for 50 or 90% of parent [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline to disappear by dissipation and transformation processes) in the test system was calculated using the % AR attributable to parent [14C] 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline. The methods to determine transformation rate followed FOCUS recommendations. CAKE v 3.4 was used to fit the experimental data to different kinetic models – simple first order (SFO), double first-order in parallel (DFOP), hockey stick (HS) and first-order multiple component (FOMC). The r2 and χ2 values from each model was compared and the graphs visually assessed to determine the best fit. Acceptable values are typically r2 > 0.7 and χ2 < 15%. The best fit model was used to derive the DT50 and DT90 values.

Results and discussion

Half-life / dissipation time of parent compound

Half-life / dissipation time of parent compound 1

Key result

Soil No.:

#4

DT50:

88.4 d

Type:

not specified

Temp.:

12.2 °C

Half-life / dissipation time of parent compound 2

Key result

Soil No.:

#3

DT50:

102 d

Type:

not specified

Temp.:

12.2 °C

Half-life / dissipation time of parent compound 3

Key result

Soil No.:

#2

DT50:

79.3 d

Type:

not specified

Temp.:

12.2 °C

Half-life / dissipation time of parent compound 4

Key result

Soil No.:

#1

DT50:

26.6 d

Type:

not specified

Temp.:

12.2 °C

Transformation products:

yes

Identity of transformation products

Identity of transformation products 1

No.:

#1

Reference

Reference substance name:

Unnamed

IUPAC name:

N,N-bis(4-(2-phenylpropan-2-yl)phenyl)nitrous amide

Molecular formula:

C30H30N2O

Molecular weight:

434.236

SMILES notation:

C1=CC=C(C=C1)C(C)(C(=CC=C1N(N=O)C(C=CC2C(C)(C)C(C=CC3)=CC=3)=CC=2)C=C1)C

Identity of transformation products 2

No.:

#2

Reference

Reference substance name:

Unnamed

IUPAC name:

2-nitro-4-(2-phenylpropan-2-yl)-N-(4-(2-phenylpropan-2-yl)phenyl)aniline

Molecular formula:

C30H30N2O2

Molecular weight:

450.231

SMILES notation:

C1=CC(NC(=CC=C2C(C)(C)C(C=CC3)=CC=3)C=C2)=C(C=C1C(C)(C)C(C=CC1)=CC=1)[N+](=O)[O-]

Identity of transformation products 3

No.:

#3

Reference

Reference substance name:

Unnamed

IUPAC name:

1,1,2,2-tetrakis(4-(2-phenylpropan-2-yl)phenyl)hydrazine

Molecular formula:

C60H60N2

Molecular weight:

808.476

SMILES notation:

C1=CC=C(C=C1)C(C)(C(C=CC1N(N(C(=CC=C2C(C)(C)C(C=CC3)=CC=3)C=C2)C(C=CC2C(C)(C)C(=CC=C3)C=C3)=CC=2)C(C=CC2C(C)(C)C(=CC=C3)C=C3)=CC=2)=CC=1)C

Details on transformation products:

Two transformation products were determined to be > 10% of the applied radioactivity at any single sampling interval by HPLC-RAD/UV; a third was found to be > 5% and steadily rising at the end of the study. All three transformation products were determined at appreciable levels in Vessel 107 Day 28 and Vessel 212 Day 120 soil extracts and therefore these samples were selected for identity analysis. These transformation products are nominally named with the approximate relative retention times (RRT) determined by the quantitative HPLC-RAD/UV method: RRT 0.98, RRT 1.07 and RRT 1.45. These nominal RRTs are used throught the report to distuguish between the transformation products.
Liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-multistage mass spectrometry (LC-MSn) accurate mass measurements were used to identify [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl) phenyl]aniline ([14C]TS) and proposed transformation products. The following structures and transformation pathways are suggested for the transformation products, for clarity structures and formulae are based on neutral unlabelled molecules.

Evaporation of parent compound:

no

Remarks:

The mean % AR captured in the volatile traps was < 2% for all soil types on all sampling occasions. The mean % AR in Orbo™ tubes remained below 0.1% on all sampling occasions.

Volatile metabolites:

no

Residues:

no

Remarks:

Soil extracts carried out on the ASE200 for Day 28 Soil III, Day 60 Soil II and Day 120 Soils II and III all contained < 2% AR

Details on results:

Chemical and physical analysis of soil
The characteristics of each soil are given below under “any other information on results incl. tables”

Soil microbial activity
Microbial biomass measured in the four soils prior to the start of the study was 116.05, 182.45, 594.70 and 172.62 mg C/kg (dry weight) in soils I, II, III and IV respectively. On Day 60, the microbial biomass in the designated blank characterisation vessels was 97.54, 359.90, 839.86 and 139.37 mg C/kg (dry weight) for soils I, II, III and IV respectively. By Day 120, the microbial biomass in the designated blank characterisation vessels was 60.15, 213.93, 630.41 and 58.58 mg C/kg (dry weight) in soils I, II, III and IV respectively.
Microbial biomass in the designated solvent control vessels was 96.73, 163.64, 593.80 and 94.11 mg C/kg by Day 120 for soils I, II, III and IV respectively. All data is displayed below under “any other information on results incl. tables”
The microbial biomass did not decrease significantly over time, and the addition of solvent did not have a negative impact on the microbial biota.

Temperature monitoring
The room temperature, monitored with a min/max thermometer, remained within 12 ± 2°C throughout the test.

Analysis of dosing solutions
The dosing solution was sub-sampled and analysed before and after dispensing to the test vessels. The radioactivity measured pre and post dosing varied by 5%.
The total amount of radioactivity applied to each vessel was calculated from the mean of the six replicates, and then adjusted to the volumes dosed to the test vessels (0.243 mL to all dosed vessels).
The total amount of radioactivity dosed to the soils was 269783 Bq, which gave a concentration of 1.02 mg/kg in the vessels. These values were used for the calculating the mass balance.
HPLC-RAD/UV on Stock H1 showed 97.2% AR was present as [14C] 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline.

Soil extraction
On Days 0-28, the ambient soil extracts contained > 90% AR in all soils.
On Day 60, the mean AR extracted from Soil II with ambient soil extracts was 85%, and Soils I, III, and IV all had a mean extraction > 90% AR.
On Day 120, the mean AR extracted from Soils II and III with ambient soil extracts were 81% and 88% respectively. Ambient extracts of Soils I and IV both contained > 90% AR.
Soil extracts carried out on the ASE200 for Day 28 Soil III, Day 60 Soil II and Day 120 Soils II and III all contained < 2% AR .
Total radioactivity extracted from Soils ! and IV was > 90% AR on all occasions. Soil II decreased to 82% AR by Day 60 and 86% AR by Day 120. Soil III decreased to 89% AR on Day 120.
HPLC-RAD was carried out on all ambient soil extracts as they all contained > 5% AR%.
On Day 0 the mean %AR attributable to [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline for Soils I, II, III, and IV was 87.0%, 93.7%, 84.8%, and 96.7% AR respectively.
In Soil I, this had decreased to 79.0% AR by Day 28 and to 67.8% AR by Day 120. In Soil II, parent had decreased to 85.9% by Day 120. In Soil III, parent had decreased to 83.7% AR by Day 28, and decreased even more to 41.7% AR by Day 120. In Soil IV, parent had decreased to 76.5% AR by Day 120.
Three transformation products were measured in the soil extracts at > 10% AR or > 5% AR and steadily rising. One, at a RRT of 0.97-0.98, first appeared on Day 0 at a maximum mean of 2.0% AR in Soil III. This had increased by Day 120 to 29.0% in Soil III, but was present at a maximum of 58.3% AR in Soil IV. The second transformation product at a RRT of 1.07-1.08 was present on Day 0 in Soils I, II and III at a maximum of 2.1% AR in Soil II. This had increased by Day 120 to a maximum of 26.8% AR in Soil I, and was present in all four soils. The third transformation product at a RRT of 1.43-1.46 was present on Day 0 in Soils I, III and IV, at a maximum of 1.8 % AR in Soil III. This had increased to 3.1% AR in Soil III by Day 14, and by Day 120 was present in all four soils, at a maximum of 6.9% AR in Soil II.

Bound radioactivity
The amount of bound or non-extractable residues (NER) in the soils was ≤ 10% for all soils on all sampling occasions up to Day 28. On Day 60, Soils I, II and IV remained below 10%, while Soil III had a mean AR% of 11%. By Day 120, the mean bound radioactivity in soil IV remained below 5% AR while the values for soils I, II, and III were between 10-14%.

Quantification of volatile radioactivity
The mean % AR captured in the volatile traps was < 2% for all soil types on all sampling occasions
The mean % AR in Orbo™ tubes remained below 0.1% on all sampling occasions.

HPLC repeatability
Day 120 V111 duplicate analysis by HPLC-RAD showed that the repeatability of the analytical method was acceptable, as the areas of both regions of interest in each chromatogram were within 20% of each other.

Mass balance
A mean mass balance of 90-110% was achieved for all soils at all sampling occasions.

Identification of transformation products
In summary, three transformation products were observed and structures based on LC-MS/MS were proposed with molecular weights of 434.24, 450.23 and 808.48 g/mol.

Limit of detection and limit of quantification
The limit of detection (LOD) and limit of quantification (LOQ) were deemed to be equivalent. The LOQ was below 1% of applied radioactivity for both LSC and HPLC-RAD.
An example LOQ for LSC analysis was calculated for each sample matrix using samples from Day 0 and are displayed in the table below. As a Bq value is not determined for the blank vial, the CPM value is converted to Bq using the lowest efficiency (therefore the worst-case scenario) for that set of samples. The LOQ equivalent % AR for each sediment type was then determined using the following calculation:
LOQ in %AR=(Blank vial cpm ×10000)/(efficiency × 60 ×Total Bq dosed to vessel)

For the HPLC-RAD analyses, the smallest applied radioactivity equivalent peak quantified was for Day 0 Soil I soil extract (Vessel 101), which was equivalent to 0.2% AR.

Kinetics
The data and regression statistics used to derive the DT50 and DT90 of [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline in four soils at an experimental temperature of 12°C are displayed below.
Since all ASE extractions carried out were less than 2% AR, the bound residues were considered to be irreversibly bound and subsequently not bioavailable. Irreversible binding was considered a path of disappearance of parent [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline from the soil systems. In this case, DT50 and DT90 values are disappearance kinetics, therefore any bound residues were not included as parent. The purity value of parent [14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline of 97.2% was used as Day 0 in all kinetics, as this was deemed to be a more accurate way to measure the degradation, due to some Day 0 values in the soils showing < 90% of AR as parent.
The soil extract data for all four soils were fit to simple first order (SFO), first-order multiple component (FOMC), double first-order in parallel (DFOP), and hockey stick (HS) models. For Soils I and II, the SFO model was chosen, as all fits were similar and this was the simplest model. For Soils III and IV, the HS model was chosen as this demonstrated the best χ2 and r2 values.

Any other information on results incl. tables

Analysis of soil

Name	Soil I	Soil II	Soil III	Soil IV
	SOIL/2020/06	SOIL/2020/07	SOIL/2020/08	SOIL/2020/09
Sampling location	Refesol 02-G (Germany)	Refesol 03-G (Germany)	Refesol 03-G (Germany)	Refesol 03-G (Germany)
Date sampled	22 September 2020	22 September 2020	22 September 2020	22 September 2020
Particle size (% w/w):
Clay (< 2 µm)^a	8	19	25	5
Silt (50-2 µm)^a	20	68	50	10
Sand (2000-50 µm)^a	72	13	25	85
Texture (USDA)^a	Sandy loam	Silt Loam	Silt Loam	Loamy sand
pH (water)^b	6.22	7.15	5.96	5.98
pH (0.01 M CaCl2)^b	5.50	6.51	5.46	5.13
Organic carbon (%)^b	0.9	1.0	4.83	3.17
CEC (mmol/kg)^a	185.6	100.0	221.6	145.6
Biomass mg org C/kg soil:
Before study start	116.05	182.45	594.70	172.62
During study (control)	97.54	359.90	839.86	139.37
End of exposure (control)	60.15	213.93	630.41	58.58
(solvent control)	96.73	163.64	593.80	94.11

All soils were sourced from Fraunhofer-Institut, Auf dem Aberg 1, 57392 Schmallenberg, Germany. 

^a        Data collected by Smithers as part of a non-study specific study.

^b        Data collected by Scymaris Ltd

Measured radioactivity in individual soil extractions displayed as % applied radioactivity (AR)

Day No	Soil	Extract 1		Extract 2		Extract 3		Extract 4 (ASE)		Total Extracted
		Bq	%	Bq	%	Bq		Bq	%	Bq	%
0	I	220553	82	27996	10	4021	1	-	-	252570	94
	II	223851	83	38178	14	5777	2	-	-	267805	99
	III	198624	74	49482	18	8011	3	-	-	256117	95
	IV	223968	83	36162	13	4826	2	-	-	264955	98
7	I	224136	83	32038	12	4558	2	-	-	260732	97
	II	215291	80	37771	14	6313	2	-	-	259374	96
	III	197925	73	52205	19	11505	4	-	-	261635	97
	IV	217417	81	34304	13	5941	2	-	-	257662	96
14	I	259834	96	-	-	4903	2	-	-	264737	98
	II	248009	92	-	-	5722	2	-	-	253731	94
	III	244586	91	-	-	10806	4	-	-	255391	95
	IV	252561	94	-	-	6628	2	-	-	259189	96
28	I	213455	79	32055	12	4751	2	-	-	250262	93
	II	214221	79	34502	13	5478	2	-	-	254201	94
	III	186884	69	49638	18	11190	4	3133	1	250844	93
	IV	222162	82	36120	13	6091	2	-	-	264373	98
60	I	248865	92	-	-	5696	2	-	-	254561	94
	II	224098	83	-	-	5282	2	2834	1	232214	86
	III	239184	89	-	-	12121	4	-	-	251305	93
	IV	247952	92	-	-	6990	3	-	-	254942	94
120	I	213044	79	28306	10	4046	1	-	-	245396	91
	II	186550	69	28436	11	4822	2	1667	0.6	221476	82
	III	180208	67	47414	18	11059	4	1260	0.5	239941	89
	IV	209014	77	36475	14	5012	2	-	-	250502	93

All extractions were acetonitrile

On Days 14 and 60, extracts 1 & 2 were pooled and analysed as extract 1

 

Mass balance by day (recovered radioactivity shown as measured value (Bq) and percentage of applied)

Day No	Soil	Soil extracts		Soil Residue		Mineralisation		Volatiles		Total
		Bq	%	Bq	%	Bq		Bq	%	Bq	%
0	I	252570	94	17971	7		-		-	270540	100
	II	267805	99	3316	1		-		-	271121	100
	III	256117	95	20335	8		-		-	276453	102
	IV	264955	98	1315	0		-		-	266270	99
7	I	260732	97	8911	3	127	<0.1	12	<0.1	269782	100
	II	259374	96	9304	3	238	<0.1	4	<0.1	268920	100
	III	261635	97	17817	7	259	<0.1	3	<0.1	279714	104
	IV	257662	96	7629	3	206	<0.1	27	<0.1	265523	98
14	I	264737	98	7854	3	158	<0.1	4	<0.1	272753	101
	II	253731	94	14171	5	802	0.3	0	<0.1	268703	100
	III	255391	95	23561	9	593	0.2	6	<0.1	279552	104
	IV	259189	96	5597	2	119	<0.1	0	<0.1	264906	98
28	I	250262	93	19003	7	851	0.3	15	<0.1	270130	100
	II	254201	94	15035	6	685	0.3	0	<0.1	269920	100
	III	250844	93	22768	8	873	0.3	8	<0.1	274493	102
	IV	264373	98	6594	2	193	<0.1	4	<0.1	271163	101
60	I	254561	94	21158	8	782	0.3	47	<0.1	276548	103
	II	232214	86	25008	9	3086	1	16	<0.1	260325	96
	III	251305	93	30451	11	792	0.3	5	<0.1	282553	105
	IV	254942	94	8059	3	518	0.2	6	<0.1	263525	98
120	I	245396	91	27135	10	697	0.3	17	<0.1	273244	101
	II	221476	82	32521	12	2133	0.8	22	<0.1	256152	95
	III	239941	89	36830	14	1121	0.4	18	<0.1	277911	103
	IV	250502	93	11233	4	660	0.2	8	<0.1	262402	97

Percentages above 1% were rounded to whole integers

Extract 4 was carried out using the Dionex ASE200

Percentages above 1% were rounded to whole integers

 

Mass balance by soil (recovered radioactivity shown as measured value (Bq) and percentage of applied)

Soil	Day No	Total soil extracts		Soil Residue		Mineralisation		Volatiles		Total
		Bq	%	Bq	%	Bq	%	Bq	%	Bq	%
Soil I	0	252570	94	17971	7	0	-	0	-	270540	100
	7	260732	97	8911	3	127	<0.1	12	<0.1	269782	100
	14	264737	98	7854	3	158	<0.1	4	<0.1	272753	101
	28	250262	93	19003	7	851	0.3	15	<0.1	270130	100
	60	254561	94	21158	8	782	0.3	47	<0.1	276548	103
	120	245396	91	27135	10	697	0.3	17	<0.1	273244	101
Soil II	0	267805	99	3316	1	0	-	0	-	271121	100
	7	259374	96	9304	3	238	<0.1	4	<0.1	268920	100
	14	253731	94	14171	5	802	0.3	0	<0.1	268703	100
	28	254201	94	15035	6	685	0.3	0	<0.1	269920	100
	60	232214	86	25008	9	3086	1	16	<0.1	260325	96
	120	221476	82	32521	12	2133	0.8	22	<0.1	256152	95
Soil III	0	256117	95	20335	8	0	-	0	-	276453	102
	7	261635	97	17817	7	259	<0.1	3	<0.1	279714	104
	14	255391	95	23561	9	593	0.2	6	<0.1	279552	104
	28	250844	93	22768	8	873	0.3	8	<0.1	274493	102
	60	251305	93	30451	11	792	0.3	5	<0.1	282553	105
	120	239941	89	36830	14	1121	0.4	18	<0.1	277911	103
Soil IV	0	264955	98	1315	0	0	-	0	-	266270	99
	7	257662	96	7629	3	206	<0.1	27	<0.1	265523	98
	14	259189	96	5597	2	119	<0.1	0	<0.1	264906	98
	28	264373	98	6594	2	193	<0.1	4	<0.1	271163	101
	60	254942	94	8059	3	518	0.2	6	<0.1	263525	98
	120	250502	93	11233	4	660	0.2	8	<0.1	262402	97

Percentages above 1% were rounded to whole integers

 

HPLC profiling of ambient sediment extracts expressed in mean % applied radioactivity (AR)

Sample Day	Soil	Mean %AR in extracts analysed	Relative Retention Time (RRT)
			0.25	0.43-0.44	0.51	0.58	0.63	0.64-0.65	0.72	0.76-0.78	0.86-0.87	0.88-0.90	0.91-0.92	0.94-0.95	0.97-0.98	1.0	1.07-1.08	1.11	1.43-1.46	1.48-1.52
0	I	93.6	0.5	2.5						0.7	0.3	0.2	0.8		0.6	87.0	0.8	0.3	1.0
	II	99.3		2.3						0.5					0.7	93.7	2.1
	III	94.9		4.3						1.2	1.0			0.7	2.0	84.8	1.0		1.8
	IV	98.2		0.8											0.4	96.7			0.9
7	I	96.6		0.7								0.9			9.7	78.6	6.0		0.7	0.9
	II	96.1		0.6							0.7		0.8		4.7	82.3	2.4		1.6	3.7
	III	97.0		4.0											8.5	79.0	2.7		1.6	1.2
	IV	95.5													4.0	88.5	2.4		0.6	0.7
14	I	98.1		0.6											7.9	83.7	4.6		0.9	1.2
	II	94.1		0.4		0.5	0.5	0.5		1.7	0.8	0.7	0.4		6.9	71.1	6.0	0.5	2.4	3.8
	III	94.7		3.0	1.1										12.8	67.8	5.5		3.1	1.4
	IV	96.1		0.4											7.0	85.9	1.9		0.6	0.4
28	I	92.8	0.6	0.9	0.8			0.6	1.2	1.0	1.3	2.9	0.6		22.2	41.7	19.5		1.9	1.3
	II	94.2													5.6	76.5	7.6		1.4	3.1
	III	91.8		1.3	0.6			0.8					0.8		13.7	63.7	7.7		2.8	1.2
	IV	98.0													6.4	88.0	3.6
60	I	94.4									1.2		5.7		35.2	13.2	39.0		1.4
	II	85.0													4.5	63.5	12.6		1.4	3.1
	III	93.2		3.7	1.0								1.2		13.9	64.8	6.6		1.9	1.1
	IV	94.5													7.4	80.0	6.3			0.8
120	I	91.0										1.4	4.1	1.6	37.4	16.8	26.8		4.3
	II	81.5											1.7		26.5	25.3	19.0		6.9	2.9
	III	88.5		1.0											29.0	41.6	11.1		4.4	1.8
	IV	92.9													58.3	22.4	9.9		1.9	0.7

Relative retention time of 1.0 is [¹⁴C] 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]

Applicant's summary and conclusion

Conclusions:

[14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl)phenyl]aniline disappeared over time in all soils, with DT50 ¬values of 26.6, 79.3, 102 and 88.4 days in Soils I, II, III and IV respectively.
Almost no mineralisation occurred, with < 2% measured in all soils over 120 days.
10%, 12%, 14% and 4% of applied radioactivity remained in the soil residue for Soils I, II, III and IV respectively at Day 120 after all extractions had been carried out. ‘Harsh’ ASE extractions removed less than 2% of applied radioactivity. Therefore, these residues are not considered to be bioavailable and binding to soils was considered a pathway for disappearance.

Executive summary:

STUDY SUMMARY
Materials & Methods
Test Substance	Name:	[^14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl) phenyl]aniline
	Lot Number:	ELOD14GH276
	Specific activity:	5297 Bq/µg
	Radiochemical purity:	99.0%
Test Systems	Soil ID	Soil I (SOIL/2020/06)	Soil II (SOIL/2020/07)	Soil III (SOIL/2020/08)		Soil IV (SOIL/2020/09)
	Sand/Silt/Clay (%):	72/20/8	13/68/19	25/50/25		85/10/5
	Organic Carbon (%):	0.9	1.0	4.8		3.2
	pH (water):	6.22	7.15	5.96		5.98
	CEC (mmol/kg)	185.6	100.0	221.6		145.6
	Microbial Biomass (µg/g):	116.05	182.42	594.70		172.62
Incubation Conditions	Duration:	120 ± 2 days
	Dates of Incubation:	7 January 2021 to 7 May 2021
	Temperature/ Light:	12 ± 2 °C in dark
	Concentration:	0.1 mg [^14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl) phenyl]aniline /L
	Replication:	2 vessels per soil per sampling interval
	Aeration:	Flow-through air
Analysis	Sampling Intervals:	0, 7, 14, 28, 60 & 120 days
	Processing:	Extract soils sequentially with acetonitrile at room temperature until <5% extracted, followed by accelerated solvent extraction using acetonitrile
	Methods:	Total radioactivity in soil extracts, combusted post-extraction soils, and volatile solutions by LSC
		Radioactivity profile of soil extracts by HPLC-RAD/UV
Results
Soil		I	II	III			IV
Mean Mass Balance (%)		101	99	103			98
[^14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl) phenyl]aniline by HPLC-RAD in total test system at Day 0 (%)		87.0	93.7	84.8			96.7
[^14C]4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl) phenyl]aniline by HPLC-RAD in total test system at Day 120 (%)		16.8	25.3	41.6			22.4
Kinetic Analyses		Days at 12ºC
DT50 of 4-(1-methyl-1-phenylethyl)-N-[4-(1-methyl-1-phenylethyl) phenyl]aniline in soil		26.6	79.3	102			88.4
Transformation products
Compound Name		RRT 0.98	RRT 1.07		RRT 1.45
Structure
Molecular Formula		C30H30N2O	C30H30N2O2		C60H60N2
Molecular Weight (g/mol)		434.2358	450.2307		808.4756
Chemical Name		N,N-bis(4-(2-phenylpropan-2-yl)phenyl)nitrous amide	2-nitro-4-(2-phenylpropan-2-yl)-N-(4-(2-phenylpropan-2-yl)phenyl)aniline		1,1,2,2-tetrakis(4-(2-phenylpropan-2-yl)phenyl)hydrazine
Compositional Change		-H, +NO	-H, +NO2		+C30H30N
Transformation pathway		Nitrosation	Nitration		Dimerisation