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Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.
Objective of study:
absorption
distribution
excretion
metabolism
Qualifier:
according to guideline
Guideline:
OECD Guideline 417 (Toxicokinetics)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)
Deviations:
no
GLP compliance:
yes
Radiolabelling:
yes
Remarks:
14C
Species:
rat
Strain:
other: Wistar Hanover
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Wilmington, MA
- Age at study initiation: approx. 10 weeks
- Weight at study initiation: approx. 200-300 g
- Fasting period before study: Immediately prior to dosing, the rats were fasted for approximately 10 hours.
- Housing: Rats in the low dose experiments (LDE) were housed in individual Nalgene rodent metabolic cages (Havard Bioscience, South Natick, MA) which allowed separation and collection of urine, feces, and respired 14CO2 and volatile metabolites. Rats in the bile cannulation experiments (BCE) were housed in individual rodent restraint cages which allowed separation and collection of urine, feces, and bile.
- Individual metabolism cages: yes
- Diet: Rodient Diet (PMI Nutrition International, Inc., St. Louis, MO), ad libitum after dosing
- Water: ad libitum after dosing
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 70±5
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: canola oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Low Dose Experiments:
Individual dosing solutions of [Indane-3-14C] AE1170437 or [Triazine-2,4-14C] AE1170437 were prepared for each rat in 10-ml pear-shaped flasks. A total of five dosing solutions per experiment were prepared.

For the I-LDE-M ([Indane-3-14C] low dose) group, an aliquot (1.01 mL, 5.00 mg) of the [Indane-3-14C] AE 1170437 stock solution was placed in a 10-mL volumetric flask and an aliquot (16.10 mg) of nonlabelled AE1170437 (Vial No. K-1480) was added. The solution was dissolved in a total of 10.00 mL of acetonitrile (ACN) and vortex mixed for 2 min. Triplicate 10-μl aliquots of the diluted solution were radioassayed. A 2.00-mL aliquot of the ACN solution was placed in each of five, 10-mL pear-shaped flasks, and the flasks were individually labelled with the compound, study number, test group, dose number, and date prepared. The solvent was removed in vacuo on a rotary-evaporator at ambient temperature, and the flasks with residue were sealed and stored in laboratory freezer (< -20 °C) until used. Immediately prior to dosing, the individual flasks holding the dry residue were removed from the freezer and allowed to warm to ambient temperature. To each flask was added 1.50 mL of canola oil; each flask was vortexed for 2 min and sonicated (Branson Ultrasonics Corp., Model 8510, Danbury, CT) for 2 min to generate a homogeneous suspension.

Each rat was dosed by oral gavage, using a separate syringe and needle, with the contents of one of the flasks. Each individual syringe was labeled with the identification number of the rat dosed. Following dosing, the amount of residual material in the flask and syringe used to dose each rat was measured. Based on the recovered radioactivity and the initial amount of material in each flask, the theoretical dose administered to each rat was determined. The residue in the dose flask and dosing syringe for each rat was analyzed by HPLC.

For the T-LDE-M ([Triazine-2,4-14C] low dose) group, an aliquot (1.50 mL, 5.00 mg) of the [Triazine-2,4-14C] AE1170437 stock solution was placed in a 10-mL volumetric flask and an aliquot (16.10 mg) of nonlabelled AE1170437 (Vial No. K-1480) was added. The subsequent steps were identical to that for the I-LDE-M group.

Bile Cannulation Experiments:
For the I-BCE-M ([Indane-3-14C] Bile Cannulation) group, an aliquot (1.01 mL, 5.00 mg) of the [Indane-3-14C] AE1170437 stock solution was placed in a 10-mL volumetric flask and an aliquot (23.14 mg) of nonlabelled AE1170437 (Vial No. K-1480) was added. The subsequent steps were identical to that for the I-LDE-M group.

For the T-BCE-M ([Triazine-2,4-14C] Bile Cannulation) group, an aliquot (1.52 mL, 5.00 mg) of the [Triazine-2,4-14C] AE1170437 stock solution was placed in a 10-mL volumetric flask and an aliquot (23.14 mg) of nonlabelled AE1170437 (Vial No. K-1480) was added. The subsequent steps were identical to that for the I-LDE-M group.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Vegetable oils are commonly used vehicles for oral dosing by gavage
- Concentration in vehicle: 2.813 mg/mL and 3.752 mg/mL in the low dose and the bile cannulation experiments, respectively.
- Amount of vehicle (if gavage): 1.5 mL/animal
- Purity: Edible, for human nutrition; received from local grocery store.

HOMOGENEITY AND STABILITY OF TEST MATERIAL:
Dosing preparations were throroughly vortexed and sonicated prior to administration to ensure a homogeneous suspension.
Duration and frequency of treatment / exposure:
Single exposure by gavage
Dose / conc.:
4.22 other: mg/animal
Remarks:
in the low dose experiments (corresponding to an average dose of 11.50 mg/kg bw for the I-LDE and 14.98 mg/kg bw for the T-LDE)
Dose / conc.:
5.628 other: mg/animal
Remarks:
in the bile cannulation experiments (corresponding to an average dose of 14.00 mg/kg bw for the I-BCE and 13.35 mg/kg bw for the T-BCE)
No. of animals per sex per dose / concentration:
4 and 5 males, respectively (5 males in bile cannulation experiment with [Triazine-2,4-14C] AE1170437 only)
Control animals:
no
Positive control reference chemical:
not applicable
Details on dosing and sampling:
PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, feces, blood, bone, brain, fat, heart, kidney, liver, lung, muscle, skin, spleen, testes, thyroid, gastrointestinal tract, remaining carcass, cage washes, bile, expired CO2 and volatile metabolites.
- Time and frequency of sampling: Urine was collected at 6, 12, 24, and 48 hours posttreatment. During the I-LDE-M and T-LDE-M experiments, urine was also collected at 72 hours posttreatment. The weight of urine at each interval was determined, and triplicate aliquots (0.010 to 0.100 mL) were radioassayed.

Feces were collected in separate containers at 24 and 48 hours posttreatment intervals until sacrifice. As with urine, during the I-LDE-M and T-LDE-M experiments feces were also collected at 72 hours posttreatment. Each feces sample was weighed and homogenized manually using a mortar and pestle. Aliquots (0.0081 to 0.0425 g) of the fecal homogenates were oxidized and radioassayed.

In the I-BCE-M and T-BCE-M experiments bile was collected 1, 2, 3, 4, 6, 8, 12, 24, 30, and 48 hours posttreatment. The weight of bile at each interval was determined, and triplicate aliquots (0.010 mL) were radioassayed.

At the conclusion of each experiment , individual rats were anesthetized with isofluorane exsanguinated through cardiac puncture using a 10-ml disposable syringe pretreated with heparin. Triplicate blood samples were pipetted into combustion pads for oxidation and radioassay. In all of the experiments, bone, brain, fat, heart, kidney, liver, lung, muscle, skin, spleen, testes, and thyroid were collected and weighed; aliquots of each tissue were oxidized and radioassayed.

The gastrointestinal tract (GIT) of each rat was collected and weighed, and the entire sample was homogenized using liquid nitrogen and an Ultra-Turrax Tissumizer. The homogenized GIT samples were placed in open plastic bags in a walk-in freezer (<-20 °C), and the liquid nitrogen was allowed to evaporate. Triplicate aliquots from each GIT were oxidized and radioassayed.

The remaining carcass was dissolved in 3 N ethanolic potassium hydroxide (KOH) solution (75 g of KOH dissolved in 425 mL of ethanol and 25 mL of H2O). Aliquots (1.0 mL) of the KOH solution were radioassayed.

In an attempt to detect and collect respired 14CO2 and all other volatile compounds in the I-LDE-M and T-LDE-M experiments, the rats were housed in metabolism cages equipped with a flow-through system which allowed for the separation and collection of respired gases. Atmospheric air was drawn through each cage at approximately 2000 mL/min using a vacuum pump. The air leaving each cage was sequentially passed through ethylene glycol (200 mL) to trap organic volatile compounds and 1 N aqueous sodium hydroxide (NaOH) solution (200 mL) to trap carbon dioxide (14CO2). Respired gas collection was discontinued 48 hours posttreatment. Respired gases were not collected during the I-BCE-M and T-BCE-M experiments.

- Other:
Following completion of each experiment, each metabolism cage was washed with methanol/water (MeOH/H2O; 1:1), and the washes from each cage were separately radioassayed. The urine and feces collection containers used in the I-LDE-M and T-LDE-M experiments were also washed with MeOH/H2O (1:1), and the combined washes from the urine and feces containers for each rat were radioassayed.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, faeces, bile
- Time and frequency of sampling: Quantitation and identification/characterization of radioactive components in urine, feces and bile were made from composite urine samples. Composite urine, feces and bile samples were prepared for each time interval described above.
- From how many animals: Composite samples for each time interval were prepared by combining one-half of the urine, feces and bile, respectively, collected from each rat at a given time interval.
- Method type(s) for identification (HPLC, LC/ESI-MS, NMR): Aliquots (0.010 to 0.100 mL) of the composite urine samples were radioassayed and analysed by HPLC. Individual components were isolated by HPLC, and isolated metabolites were analysed by liquid chromatography/electrospray interface - mass spectrometry (LC/ESI-MS).
Aliquots (0.0160 to 0.0526 g) of the composite feces samples were oxidised and radioassayed. A portion of each composited samples was extracted with ACN/H2O (4:1 v/v) using a tissumizer and centrifuged. Washing with fresh solvent was repeated twice, and supernatants and filtered solids, respectively, were combined. The combined supernatants were radioassayed and analysed by HPLC; the filtered solids were radioassayed. Individual components were isolated by HPLC and isolated metabolites were analysed by LC/ESI MS.
The composite bile samples were radioassyed and analysed by HPLC. Individual components were isolated by HPLC, and isolated metabolites were analysed by LC/ESI-MS and nuclear magnetic resonance (NMR) spectroscopy.
- Limits of detection and quantification:
Minimum sensitivity (µg/g; ppm) liquid samples: I-LDE-M and T-LDE-M: 0.0009; I-BCE-M and T-BCE-M: 0.0011
Minimum sensitivity (µg/g; ppm) solid samples: I-LDE-M and T-LDE-M: 0.0027; I-BCE-M: 0.0036; T-BCE-M: 0.0037
Statistics:
All calculations were performed using Microsoft Excel 2002 (10.6841.6839; SP-3).
Preliminary studies:
Not performed.
Type:
absorption
Results:
approximately 90.6%
Type:
distribution
Results:
in total approximately 0.2-5.0% in tissues (highest in GI-tract, liver, skin and thyroid gland)
Type:
excretion
Results:
approx. 35-49% of dose in urine and approx. 62-70% in the feces (the latter including 38-48% contribution of the bile)
Details on absorption:
In the low dose experiments the majority of the dose was excreted in the feces (62.2%), while 38.1% was excreted in the urine. Approximately 10% of the administered dose was excreted as unmetabolised parent compound in the feces and was therefore considered not to be absorbed. In conclusion, about 90% of the administered dose have been absorbed via the gastrointestinal tract. Total radioactive recovery in the respective experiments ranged from 101.4 to 106.7%.
Similar results were obtained from the bile cannulation experiments; summing the renal (37.0-48.8%) and biliary (37.9-48.3%) excretion with the residual amount of the dose in tissues shows a minimum bioavailability of 90.1-90.7% of the administered dose. Total radioactive recovery in the respective experiments ranged from 100.8 to 106.7%.
Details on distribution in tissues:
Less than 1% of the administered dose remained in the carcass and tissues at sacrifice with the highest amounts in the gastrointestinal tract and the liver, followed by skin and thyroid gland. Slight differences in amount of radioactivity occurred depending on the location of the radioactive label in the test substance, especially in the bile cannulation experiments. In these experiments up to 4.9% of radioactivity was found in the tissues, in contrast to only 0.2% in the low dose experiments.
Details on excretion:
In the low dose experiments a total of 10 metabolites were detected in urine accounting for between <1% of the administered dose and approximately 23% of the administered dose. Five of the isolated metabolites accounted for >32% of the administered dose. A total of five metabolites were detected in composite feces extracts accounting for between 2% and approximately 40% of the administered dose. All five of the isolated metabolites accounted for approximately 58% of the administered dose. A total of approximately 90% of the administered dose was identified in the I-LDE-M experiment.

In the T-LDE-M experiment a total of six metabolites were detected in urine accounting for between <1% and approximately 22% of the administered dose. Individual metabolites were isolated from composite urine for identification. All six of the isolated metabolites accounted for >34% of the administered dose. A total of 10 metabolites were detected in feces extracts accounting for between <1% and approximately 44% of the administered dose. Individual metabolites were isolated from composite feces extract for identification. Five of the isolated metabolites, accounting for approximately 59% of the administered dose, were identified in the T-LDE-M feces extracts. A total of approximately 94% of the administered dose was identified in the T-LDE-M experiment.

In the bile cannulation experiments a total of 10 metabolites were detected in I-BCE-M urine accounting for between <1% and approximately 16% of the administered dose. Five of the isolated metabolites accounted for >29% of the administered dose. A total of nine metabolites were detected in feces extracts accounting for between <1% and approximately 12% of the administered dose. Six of the isolated metabolites accounted for approximately 18% of the administered dose. A total of sixteen metabolites were detected in bile extracts accounting for between <1% and approximately 19% of the administered dose. Five of the isolated metabolites accounted for approximately 38% of the administered dose. A total of approximately 85% of the administered dose was identified in the I-BCE-M experiment.

In the T-BCE-M experiment A total of 12 metabolites were detected in urine accounting for between <1% and approximately 25% of the administered dose. Six of the isolated metabolites accounted for >39% of the administered dose. A total of nine metabolites were detected in feces extracts accounting for between <1% and approximately 5% of the administered dose. Six of the isolated metabolites accounted for approximately 9% of the administered dose. A total of 13 metabolites were detected in bile accounting for between <1% and approximately 13% of the administered dose. Five of the isolated metabolites, accounted for >33% of the administered dose. A total of approximately 81% of the administered dose was identified in the T-BCE-M experiment.

The majority (84.1% in the low dose and 94.6% in the bile cannulation experiment) of the absorbed doses was excreted within 24 hours.
Metabolites identified:
yes
Details on metabolites:
unmetabolised AE 1170437 parent: 1-12% feces, 4% bile;
AE 1170437-carboxylic acid: 16-25% urine, 1-44% feces, 13-19% bile;
AE 1170437-dihydroxy: 6-10% urine, < 1% feces, 2-3% bile;
AE 1170437-hydroxy GA (3-hydroxyindane GA + hydroxymethyl GA): < 1% urine, 10-13% bile;
AE 1170437-3-hydroxyindane acid: 1-2% urine, < 1-11% feces, 1% bile;
AE 117437-3-hydroxyindane acid epimer: 2-7% urine, 4% feces;
AE 1170437-3-ketoindane acid: < 1-2% urine, < 1-1% feces, 1% bile;
AE 1170437-3-ketohydroxymethyl: 1% feces;
AE 1170437-diaminotriazine: 1-2% urine; < 1% feces;
AE 1170437-hydroxyethyl acid: 3% feces

Metabolic degradation of AE 1170437 is rapid and complete, with between 0% and 10% of the administered dose being recovered as unmetabolised parent. In all experiments, 38 to 66% of the administered dose was excreted as AE 1170437 carboxylic acid, indicating that oxidation is the main route of metabolism for the parent compound. A variety of other oxidation products were also detected. Several glucuronic acid conjugates were observed, particularly in bile.

Distribution of radioactivity among tissues and excreta of rats after administration of [14C] AE 1170437:

Matrix

Percent Dose (averages from all rats/group)

[Indane-3-14C] AE 1170437 (I-LDE-M)

[Triazine-2,4-14C] AE 1170437(T-LDE-M)

[Indane-3-14C] AE 1170437 (I-BCE-M)

[Triazine-2,4-14C] AE 1170437 (T-BCE-M)

Urine

0-6 h

10.4

6.6

4.2

13.5

6-12 h

17.5

17.8

21.3

19.1

12-24 h

8.8

8.4

7.2

14.9

24-48 h

1.3

1.6

4.3

1.3

48-72 h

0.1

0.2

NC

NC

Total urine

38.1

34.6

37.0

48.8

Feces

0-24 h

47.4

56.9

16.1

7.4

24-48 h

14.0

12.7

4.3

2.9

48-72 h

0.8

0.8

NC

NC

Total feces

62.2

70.4

20.4

10.3

Bile

0-1 h

NC

NC

1.0

0.7

1-2 h

3.7

1.8

2-3 h

5.3

2.4

3-4 h

4.9

3.5

 4-6 h

9.0

5.8

6-8 h

6.5

4.7

8-12 h

7.8

7.1

12-24 h

7.6

9.9

24-30 h

0.9

1.4

30-48 h

1.6

0.6

Total bile

48.3

37.9

Expired air

<0.1

<0.1

NC

NC

Tissues

0.2

0.2

4.9

3.9

Cage wash

1.1

1.5

0.1

0.1

Total recovered

101.6

106.7

110.7

101.0

NC = Not collected

Distribution of radioactivity in rat tissues/organs after administration of [14C] AE 1170437:

 

Ppm (µg of residue in AE 1170437 equivalents/g of tissue; averages of all rats/group)

Tissue/organ

[Indane-3-14C] AE 1170437 (I-LDE-M)

[Triazine-2,4-14C] AE 1170437(T-LDE-M)

[Indane-3-14C] AE 1170437 (I-BCE-M)

[Triazine-2,4-14C] AE 1170437 (T-BCE-M)

Bone

0.007

0.007

0.043

0.126

Brain

0.004

0.004

0.012

0.102

Fat

0.015

0.006

0.084

0.263

GI tract

0.084

0.063

6.780

8.015

Heart

0.006

0.006

0.050

0.398

Kidney

0.014

0.012

0.177

0.445

Liver

0.056

0.050

0.411

0.711

Lung

0.006

0.006

0.074

0.357

Muscle

0.004

0.005

0.053

0.160

Skin

0.024

0.025

0.140

0.300

Spleen

0.008

0.006

0.077

0.248

Thyroid gland

0.012

0.025

0.066

0.314

Testes

0.004

0.006

0.025

0.160

Whole blood

0.008

0.009

0.061

0.110

Carcass

0.007

0.010

0.040

0.053

 

CONCLUSION

The uptake and excretion of AE 1170437 was rapid. Following oral administration, greater than 84% of the administered dose was excreted within 24 hours. In general, the majority of the radioactivity was excreted in the feces with fecal:renal excretion ratios ranging from 1:1 to 2:1. No volatile residues were detected, and no mineralization was observed. Residue levels in tissues were highest in GIT and liver. Higher residue levels were found in the residual carcass of animals in the bile cannulation experiments.

Once absorbed, the metabolism of AE 1170437 was rapid, occurring mainly through oxidative pathways. A maximum of 10% of the administered dose was recovered unmetabolized in the feces, and that was likely not absorbed.

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Objective of study:
absorption
distribution
excretion
metabolism
Qualifier:
according to guideline
Guideline:
OECD Guideline 417 (Toxicokinetics)
Version / remarks:
adopted in 2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.7485 (Metabolism and Pharmacokinetics)
GLP compliance:
yes
Radiolabelling:
yes
Remarks:
14C
Species:
rat
Strain:
other: Wistar Hanover
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, NC
- Age at study initiation: approx. 10 weeks
- Weight at study initiation: approx. 200-300 g
- Fasting period before study: Immediately prior to dosing, the rats were fasted for approximately 10 hours.
- Housing: Female rats in the low dose experiments were housed in individual Nalgene rodent metabolic cages (Havard Bioscience, South Natick, MA) which allowed separation and collection of urine, faeces, and respired 14CO2 and volatile metabolites. Male rats in the high dose experiments were housed in individual Nalgene rodent metabolic cages (Havard Bioscience, South Natick, MA) which allowed separation and collection of urine and faeces. Rats in the plasma curve experiments were housed in individual stainless steel cages.
- Individual metabolism cages: yes, except rats in the plasma curve experiments
- Diet: Rodient Diet (PMI Nutrition International, Inc., St. Louis, MO), ad libitum after dosing
- Water: ad libitum after dosing
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 70±5
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: aqueous suspension of 1% (w/w) carboxymethylcellulose + 0.5% Tween® 80 (polyethylene glycol sorbitan monooleate)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Low Dose Experiments:
Individual dosing solutions of [Indane-3-14C] AE1170437 or [Triazine-2,4-14C] AE1170437 were prepared for each rat in 10-ml pear-shaped flasks. A total of five dosing solutions per experiment were prepared.

For the [Indane-3-14C] low dose experiment female (I-LDE-F) group, an aliquot (1.01 mL, 5.00 mg) of the [Indane-3-14C] AE 1170437 stock solution was placed in a 10-mL volumetric flask and an aliquot (16.00 mg) of nonlabelled AE1170437 was added. The solution was dissolved in a total of 10.00 mL of acetonitrile (ACN) and vortex mixed for 2 min. Triplicate aliquots of the solution were diluted and radioassayed. The solvent was removed in vacuo on a rotary-evaporator at ambient temperature, and the residue was stored < -20 °C until used. Immediately prior to dosing, the individual flasks holding the dry residue were removed from the freezer and allowed to warm to ambient temperature. To each residue was added 1.50 mL of aqueous 1% (w/w) carboxymethylcellulose + 0.5% Tween® 80; each flask was vortexed for 2 min and sonicated (Branson Ultrasonics Corp., Model 8510, Danbury, CT) for 2 min to generate a homogeneous suspension.

For the [Triazine-2,4-14C] low dose experiment female (T-LDE-F) group, an aliquot (1.50 mL, 5.00 mg) of the [Triazine-2,4-14C] AE1170437 stock solution was placed in a 10-mL volumetric flask and an aliquot (16.00 mg) of nonlabelled AE1170437 was added. The subsequent steps were identical to that for the I-LDE-F group.

High Dose Experiments:
For the [Indane-3-14C] high dose experiment male (I-HDE-M) group, an aliquot (1.01 mL, 5.00 mg) of the [Indane-3-14C] AE 1170437 stock solution was placed in a 50-mL volumetric flask and an aliquot (1045 mg) of nonlabelled AE1170437 was added. The solution was dissolved in a total of 50.00 mL of ACN and vortex mixed for 2 min and sonicated for 10 min. Triplicate aliquots of the diluted solution were radioassayed. The solvent was removed in vacuo on a rotary-evaporator at ambient temperature, and the residue was stored < -20 °C until used. Immediately prior to dosing, the individual flasks holding the dry residue were removed from the freezer and allowed to warm to ambient temperature. To each residue was added 2.00 mL of aqueous 1% (w/w) carboxymethylcellulose + 0.5% Tween® 80; each flask was vortexed for 2 min and sonicated for 2 min to generate a homogeneous suspension.

For the [Triazine-2,4-14C] high dose experiment male (T-HDE-M) group, an aliquot (1.50 mL, 5.00 mg) of the [Triazine-2,4-14C] AE1170437 stock solution was placed in a 10-mL volumetric flask and an aliquot (1045 mg) of nonlabelled AE1170437 was added. The subsequent steps were identical to that for the I-HDE-M group.

Plasma Curve Experiments:
For the [Indane-3-14C] plasma curve experiment male and female (I-PCE-M and I-PCE-F) groups, an aliquot (1.01 mL, 5.00 mg) of the [Indane-3-14C] AE1170437 stock solution was placed in a 10-mL volumetric flask and an aliquot (16.00 mg) of nonlabelled AE1170437 was added. The subsequent steps were identical to that for the I-LDE-F group.

For the [Triazine-2,4-14C] plasma curve experiment male and female (T-PCE-M and T-PCE-F) groups, an aliquot (1.01 mL, 5.00 mg) of the [Triazine-2,4-14C] AE1170437 stock solution was placed in a 10-mL volumetric flask and an aliquot (16.00 mg) of nonlabelled AE1170437 was added. The subsequent steps were identical to that for the I-LDE-F group.
Duration and frequency of treatment / exposure:
Single exposure by gavage
Dose / conc.:
4.84 mg/kg bw (total dose)
Remarks:
[Indane-3-14C] AE 1170437 Low Dose Experiment - Female (I-LDE-F)
Dose / conc.:
8.85 mg/kg bw (total dose)
Remarks:
[Triazine-2,4-14C] AE 1170437 Low Dose Experiment - Female (T-LDE-F)
Dose / conc.:
558.7 mg/kg bw (total dose)
Remarks:
[Indane-3-14C] AE 1170437 High Dose Experiment - Male (I-HDE-M)
Dose / conc.:
722.8 mg/kg bw (total dose)
Remarks:
[Triazine-2,4-14C] AE 1170437 High Dose Experiment - Male (T-HDE-M)
Dose / conc.:
13.73 mg/kg bw (total dose)
Remarks:
[Indane-3-14C] AE 1170437 Plasma Curve Experiment - Male (I-PCE-M)
Dose / conc.:
13.28 mg/kg bw (total dose)
Remarks:
[Indane-3-14C] AE 1170437 Plasma Curve Experiment - Female (I-PCE-F)
Dose / conc.:
16.96 mg/kg bw (total dose)
Remarks:
[Triazine-2,4-14C] AE 1170437 Plasma Curve Experiment - Male (T-PCE-M)
Dose / conc.:
13.61 mg/kg bw (total dose)
Remarks:
[Triazine-2,4-14C] AE 1170437 Plasma Curve Experiment - Female (T-PCE-F)
No. of animals per sex per dose / concentration:
4 males (I-HDE-M, T-HDE-M, I-PCE-M, T-PCE-M groups)
4 females (I-LDE-F, T-LDE-F, I-PCE-F)
3 females (T-PCE-F)
Control animals:
no
Positive control reference chemical:
not applicable
Details on dosing and sampling:
PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine and faeces (all LDE-F and HDE-M experiments only), cage washes, blood, tissues of animals of all LDE-F and HDE-M experiments (bone, brain, fat, heart, kidney, liver, lung, muscle, skin, spleen, gonads, thyroid), gastrointestinal tract, remaining carcass, expired CO2 and volatile metabolites.
- Time and frequency of sampling: Urine was collected at 6, 12, 24, and 48 hours post-treatment. During the I-LDE-F and T-LDE-F experiments, urine was also collected at 72 hours post-treatment. During the I-HDE-M and T-HDE-M experiments, urine was also collected at 72-, and 96-hours post-treatment.

SAMPLE PREPARATION
The weight of urine at each interval was determined, and triplicate aliquots (0.010 to 0.100 mL) were radioassayed.
Faeces were collected in separate containers at 24 hours post-treatment intervals until sacrifice. Each faeces sample was weighed and homogenized manually using a mortar and pestle. Aliquots of the faecal homogenates were oxidized and radioassayed.
Cage wash: Following completion of each experiment, each metabolism cage was washed with methanol/water (MeOH/H2O; 1:1), and the washes from each cage were separately radioassayed.
Urine and faeces: Collection containers were washed with MeOH/H2O (1:1), and the combined washes from the urine and faeces containers for each rat were radioassayed.
In the plasma concentration experiments blood was collected 5-, 10-, 20-, and 40-min and 1-, 1.5-, 2-, 4-, 6-, 8-, 12-, 24-, 32-, 48-, and 72-hours post-treatment. The weight of blood at each interval was determined, and triplicate aliquots (ca. 0.100 g) were radioassayed.
Tissues and carcass: At the conclusion of each experiment, individual rats were anaesthetized with isofluorane and exsanguinated through cardiac puncture using a 10-ml disposable syringe pre-treated with heparin. Triplicate blood samples were pipetted into combustion pads for oxidation and radioassay. In all of the LDE-F and HDE-M experiments, bone, brain, fat, heart, kidney, liver, lung, muscle, skin, spleen, gonads (testes or uterus), and thyroid were collected and weighed; aliquots of each tissue were oxidized and radioassayed.
The gastrointestinal tract (GIT) of each rat was collected and weighed, and the entire sample was homogenized using liquid nitrogen and an Ultra-Turrax Tissumizer. The homogenized GIT samples were placed in open plastic bags in a walk-in freezer (<-20 °C), and the liquid nitrogen was allowed to evaporate. Triplicate aliquots from each GIT were oxidized and radioassayed.
The remaining carcass was dissolved in 3 N ethanolic potassium hydroxide (KOH) solution (75 g of KOH dissolved in 425 mL of ethanol and 25 mL of H2O). Aliquots (1.0 mL) of the KOH solution were radioassayed.
In an attempt to detect and collect respired 14CO2 and all other volatile compounds in the I-LDE-F and T-LDE-F experiments, the rats were housed in metabolism cages equipped with a flow-through system which allowed for the separation and collection of respired gases. Atmospheric air was drawn through each cage at approximately 2000 mL/min using a vacuum pump. The air leaving each cage was sequentially passed through ethylene glycol (200 mL) to trap organic volatile compounds and 1 N aqueous sodium hydroxide (NaOH) solution (200 mL) to trap carbon dioxide (14CO2). Respired gas collection was discontinued 48 hours post-treatment. Respired gases were not collected during the HDE-M and PCE experiments.

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine, faeces
- Time and frequency of sampling: Quantitation and identification/characterization of radioactive components in urine and faeces were made from composite urine samples. Composite urine and faeces samples were prepared for each time interval described above.
- From how many animals: Composite samples for each time interval were prepared by combining one-half of the urine and faeces, respectively, collected from each rat at a given time interval.
- Method type(s) for identification (HPLC, LC/ESI-MS, NMR): Aliquots (0.010 to 0.100 mL) of the composite urine samples were radioassayed and analysed by high performance liquid chromatography (HPLC). Individual components were isolated by HPLC, and isolated metabolites were analysed by liquid chromatography/electrospray interface - mass spectrometry (LC/ESI-MS).
Aliquots of the composite feces samples were oxidised and radioassayed. A portion of the composited samples was extracted with ACN/H2O (4:1 v/v) using a tissumizer and centrifuged. Washing with fresh solvent was repeated twice, and supernatants and filtered solids, respectively, were combined. The combined supernatants were radioassayed and analysed by HPLC; the filtered solids were radioassayed. Individual components were isolated by HPLC and isolated metabolites were analysed by LC/ESI MS. NMR spectroscopy was performed on individual components isolated from faeces extracts collected from the T-HDE-M dose group.
- Limits of detection and quantification:
Minimum sensitivity (µg/g; ppm) liquid samples: 0.0009 (I-LDE-F, I-PCE-M, I-PCE-F); 0.043 (I-HDE-M); 0.0008 (T-LDE-F, T-PCE-M, T-PCE-F); 0.044 (T-HDE-M)
Minimum sensitivity (µg/g; ppm) solid samples: 0.0023 (I-LDE-F, I-PCE-M, I-PCE-F); 0.135 (I-HDE-M); 0.0027 (T-LDE-F, T-PCE-M, T-PCE-F); 0.140 (T-HDE-M)
Statistics:
All calculations were performed using Microsoft Excel 2002 (10.6841.6839; SP-3).
Preliminary studies:
Not performed.
Type:
absorption
Results:
>57 and 75% was absorbed (high and low dose); females appeared to absorb slightly more material compared to males
Type:
distribution
Results:
residue levels in tissues were highest in the gastrointestinal tract and the liver
Type:
metabolism
Results:
rapid metabolism mainly through oxidative pathways
Type:
excretion
Results:
> 87% within 24 h, fecal:renal excretion is 1:1 for the low dose experiments and 10:1 for the high dose experiments, no volatile residues were detected
Details on absorption:
Low dose experiments (I-LDE-F and T-LDE-F)
The total average radioactive recovery ranged from 93.5-95.5% of the administered dose. The majority of the dose was excreted in faeces and urine: 49.1 and 42.3% in the I-LDE-F and T-LDE-F experiments, respectively, was excreted in faeces, while 43.7 and 49.4% was excreted in urine for a renal:faecal ratio of approximately 1:1. Approximately 1% of the administered dose was excreted as unmetabolised parent compound in the faeces and was therefore considered not to be absorbed. Thus, about 93.5-95.5% of the administered dose have been absorbed via the gastrointestinal tract. The majority of the dose (87.6% and 89.0% in the I-LDE-F and T-LDE-F experiments, respectively) was excreted within 24 hours.

High dose experiments (I-HDE-M and T-HDE-M)
The total average radioactive recovery ranged from 95.4-111.1% of the administered dose. The majority of the dose (100.5 and 89.6% in the I-HDE-M and T-HDE-M experiments, respectively) was excreted in faeces, while 10.3 and 5.5% was excreted in urine resulting in a renal:faecal ratio of approximately 1:10 and 1:16. Approximately 38 and 36% (I-HDE-M and T-HDE-M) of the administered dose was excreted as unmetabolised parent compound in the faeces and was therefore considered not to be absorbed. Thus, about 57.4-75.1% of the administered dose have been absorbed via the gastrointestinal tract. The majority of the dose (96.7% and 88.6% in the I-LDE-F and T-LDE-F experiments, respectively) was excreted within 24 hours.

Plasma curve experiments (I-PCE-M and I-PCE-F)
The average concentration in whole blood of males quickly rose to 2.35 ppm at 40 min post-treatment before beginning to decline. Total radioactive residues were <0.10 ppm by 12 hours post-treatment.
The average concentration in whole blood of females quickly rose to 1.05 ppm at 60 min post-treatment before beginning to decline. Total radioactive residues were at 0.10 ppm by 12 hours post-treatment.

Plasma curve experiments (T-PCE-M and T-PCE-F)
The average concentration in whole blood of males quickly rose to 2.07 ppm at 40 min posttreatment before beginning to decline. Total radioactive residues were below 0.10 ppm by 32 hours post-treatment.
The average concentration in whole blood of females quickly rose to 1.88 ppm at 40 min (0.667 h) posttreatment before beginning to decline. Total radioactive residues were at 0.10 ppm by 48 hours posttreatment.

Plasma curve experiments (overall)
The general curve shape is similar for all four dose groups, with rapid uptake and clearance from the blood. Females appear to absorb slightly more material compared to males. Blood residues smoothly fall off in all dose groups indicating an absence of re-uptake.
Details on distribution in tissues:
Low dose experiments (I-LDE-F and T-LDE-F)
Below 1% of the administered dose remained in the carcass and tissues at sacrifice. The highest average residues in I-LDE-F and T-LDE-F experiments were found in the gastrointestinal tract (0.029 and 0.032 ppm), liver (0.014 and 0.016 ppm), and skin (0.011 and 0.013 ppm).

High dose experiments (I-HDE-M and T-HDE-M)
Below 0.1% of the administered dose remained in the carcass and tissues at sacrifice. The highest average residues in I-HDE-M and T-HDE-M experiments were found in the liver (1.104 and 0.502 ppm) and skin (0.539 and 0.522 ppm).
Details on excretion:
Low dose experiments:
In the I-LDE-F experiment 49.1% was excreted in faeces, while 43.7% was excreted in urine for a renal:faecal ratio of approximately 1:1. The majority of the dose (87.6%) was excreted within 24 hours. A total of six metabolites were detected in urine accounting for between 2% and approximately 26% of the administered dose. Individual metabolites were isolated from composite urine for identification. Five of the isolated metabolites, accounting for >41% of the administered dose, were identified in I-LDE-F urine. A total of six metabolites were detected in faeces extracts accounting for between <1% and approximately 31% of the administered dose. Individual metabolites were isolated from composite faeces extract for identification. Three of the isolated metabolites, accounting for >33% of the administered dose, were identified in the I-LDE-F faeces extracts. A total of approximately 74% of the administered dose was identified in the I-LDE-F experiment.

In the T-LDE-F experiment 49.4% was excreted in urine, while 42.3% was excreted in faeces for a renal:faecal ratio of approximately 1:1. The majority of the dose (89.0%) was excreted within 24 hours. A total of eleven metabolites were detected in urine accounting for between <1% and approximately 29% of the administered dose. Individual metabolites were isolated from composite urine for identification. Six of the isolated metabolites, accounting for >44% of the administered dose, were identified in T-LDE-F urine. A total of eight metabolites were detected in faeces extracts accounting for between <1% and approximately 30% of the administered dose. Individual metabolites were isolated from composite faeces extract for identification. Seven of the isolated metabolites, accounting for approximately 39% of the administered dose, were identified in the T-LDE-F faeces extracts. A total of approximately 83% of the administered dose was identified in the T-LDE-F
experiment.

High dose experiments:
In the I-HDE-M the majority of the dose (100.5%) was excreted in faeces with 10.3% excreted in urine resulting in a renal:faecal excretion ratio of 1:10. The majority of the dose was excreted within 24 hours. A total of 14 metabolites were detected in urine accounting for between <1% and approximately 4% of the administered dose. Individual metabolites were isolated from composite urine for identification. Six of the isolated metabolites, accounting for >8% of the administered dose, were identified in I-HDE-M urine. A total of five metabolites were detected in faeces extracts accounting for between <1% of the administered dose and approximately 49% of the administered dose. Individual metabolites were isolated from composite faeces extract for identification. Four of the isolated metabolites, accounting for approximately 97% of the administered dose, were identified in the I-HDE-M faeces extracts. A total of approximately 105% of the administered dose was identified in the I-HDE-M experiment.

In the T-HDE-M the majority of the dose (89.6%) was excreted in faeces, with 5.5% excreted in urine for a renal:faecal excretion ratio of 1:16. The majority of the dose was excreted within 24 hours. A total of eleven metabolites were detected in urine accounting for between <1% of the administered dose and approximately 2% of the administered dose. Individual metabolites were isolated from composite urine for identification. Eight of the isolated metabolites, accounting for 5% of the administered dose, were identified in T-HDE-M urine. A total of eight metabolites were detected in faeces extracts accounting for between <1% of the administered dose and approximately 46% of the administered dose. Individual metabolites were isolated from composite faeces extract for identification. All eight of the isolated metabolites, accounting for approximately 89% of the administered dose, were identified in the T-HDE-M faeces extracts. A total of approximately 94% of the administered dose was identified in the T-HDE-M experiment.

Plasma curve experiments (overall):
The general curve shape is similar for all four dose groups, with rapid uptake and clearance from the blood. Females appear to absorb slightly more material compared to males. Blood residues smoothly fall off in all dose groups indicating an absence of re-uptake.
Metabolites identified:
yes
Details on metabolites:
unmetabolised AE 1170437 parent: <1% faeces (low dose experiments), 36-38% faeces (high dose experiments);
AE 1170437-carboxylic acid: 26-29% urine (low dose experiments), 2-5% urine (high dose experiments), 30-31% faeces (low dose experiments), 46-49% faeces (high dose experiments);
AE 1170437-dihydroxy: 5-7% urine (low dose experiments), < 1-1% urine (high dose experiments);
AE 1170437-carboxylic acid glucuronic acid conjugate: <1% urine (high dose experiments), 3% faeces (high dose experiments);
AE 1170437-3-hydroxyindane acid: 3% urine (low dose experiments), <1-1% urine (high dose experiments), <1-4% faeces (low dose experiments), <1% faeces (high dose experiments);
AE 1170437-3-hydroxyindane acid epimer: 3% urine (low dose experiments), <1-1% urine (high dose experiments), 1% faeces (low dose experiments), <1% faeces (high dose experiments);
AE 1170437-3-ketoindane acid: 2-3% urine (low dose experiments),<1% urine (high dose experiments), 1% faeces (low dose experiments), <1% faeces (high dose experiments);
AE 1170437-3-ketohydroxymethyl: <1% urine (low dose experiments);
AE 1170437-diaminotriazine: 1% urine (low and high dose experiments), <1% faeces (low and high dose experiments);
AE 1170437-hydroxyethyl acid: 1-2% faeces (low dose experiments), 3-9% faeces (high dose experiments)

Metabolic degradation of AE 1170437 was rapid, with 1% of the administered dose being recovered as unmetabolised parent in the low dose experiments and between 36% and 38% of the administered dose being recovered as unmetabolised parent in the high dose experiments. In all experiments, 49 to 59% of the administered dose was excreted as AE 1170437 carboxylic acid, indicating that oxidation is the main route of metabolism for the parent compound. A variety of other oxidation products were also detected, along with glucuronic acid conjugation.

Table 2: Distribution of radioactivity among tissues and excreta of rats after administration of [14C] AE 1170437

Matrix/Time Point

Percent of administered dose

[Indane-3-14C]

[Triazine-2,4-14C]

I-LDE-F

I-HDE-M

T-LDE-F

T-HDE-M

Urine

 

 

 

 

0 – 6 h

27.3

6.2

38.3

3.1

6 – 12 h

11.0

2.5

7.6

2.0

12 – 24 h

3.7

1.0

2.7

0.8

24 – 48 h

1.1

0.4

0.5

0.2

48 – 72 h

0.6

0.1

0.3

<0.1

72 – 96 h

NC

<0.1

NC

<0.1

Total for urine

43.7

10.3

49.4

5.5

Faeces

 

 

 

 

0 – 24 h

45.6

95.7

40.4

87.8

24 – 48 h

3.1

4.6

1.6

1.6

48 – 72 h

0.4

0.2

0.3

0.2

72 – 96 h

NC

<0.1

NC

<0.1

Total for faeces

49.1

100.5

42.3

89.6

Expired air

<0.1

NC

<0.1

NC

Tissues

0.2

<0.1

0.3

<0.1

Cage Washa

2.7

0.3

1.5

0.2

Total recovered

95.5

111.1

93.5

95.4

NC = The sample was not collected in this experiment.

aCage wash includes the rinses from the metabolism cages and the urine and faeces collection containers.

Table 3: Distribution of radioactivity in rat tissues/organs after administration of [14C] AE 1170437

Tissue/organ

μg of residue (in AE 1170437 equivalents)/g of tissue (ppm)

[Indane-3-14C]

[Triazine-2,4-14C]

[Indane-3-14C]

[Triazine-2,4-14C]

I-LDE-F

T-LDE-F

I-HDE-M

T-HDE-M

Female

Female

Male

Male

Bone

0.003

0.003

0.351

0.155

Brain

0.002

0.003

0.312

0.195

Fat

0.003

0.004

0.403

0.278

GIT

0.029

0.032

0.480

0.321

Heart

0.004

0.004

0.349

0.252

Kidney

0.007

0.009

0.466

0.302

Liver

0.014

0.016

1.104

0.502

Lung

0.005

0.005

0.400

0.355

Muscle

0.003

0.004

0.347

0.196

Skin

0.011

0.013

0.539

0.522

Spleen

0.005

0.006

0.362

0.305

Thyroid Gland

0.008

0.008

0.270

0.121

Gonads (uterus or testes)

0.004

0.004

0.358

0.190

Whole blood

0.007

0.009

0.495

0.344

Carcass

0.004

0.008

 

0.133

PLASMA CURVE EXPERIMENTS

 

Table 4: Total radioactive residues in whole blood from the I-PCE-M group, expressed as μg residue/g blood (ppm), following a single oral dose of [indane-3-14C] AE 1170437 at a rate of 13.73 mg/kg body weight. A) Measured Values and B) Normalized to a 1 ppm dose rate.

A)

 

Total Radioactive Residue in Blood (ppm)

Time Post-treatment (hours)

Rat-1

Rat-2

Rat-3

Rat-4

Average

0.083

0.5228

0.6102

0.6320

0.3839

0.5372

0.167

0.7285

1.6627

1.6077

0.8365

1.2089

0.333

1.2459

2.3753

3.1513

1.1583

1.9827

0.667

1.3980

2.0903

4.3656

1.5278

2.3454

1.000

1.0410

1.6180

2.9872

1.2225

1.7172

1.500

0.6852

1.3277

1.6737

0.9580

1.1612

2.000

0.4504

0.8730

1.1098

0.9843

0.8544

4.000

0.2645

0.4343

0.3985

0.5244

0.4054

6.000

0.1528

0.2592

0.2848

0.3245

0.2553

8.000

0.0946

0.1933

0.1898

0.2081

0.1715

12.000

0.1089

0.0850

0.0890

0.1066

0.0974

24.000

0.0752

0.0677

0.0258

0.0801

0.0622

32.000

0.0896

0.02175

0.0217

0.0404

0.0448

48.000

0.1619

0.0287

0.0144

0.0148

0.0550

72.000

0.0288

0.0348

0.0114

0.0116

0.0216

 

B)

 

Total Radioactive Residue in Blood (ppm)

Time Post-treatment (hours)

Rat-1

Rat-2

Rat-3

Rat-4

Average

0.083

0.0381

0.0444

0.0460

0.0280

0.0391

0.167

0.0531

0.1211

0.1171

0.0609

0.0880

0.333

0.0907

0.1730

0.2295

0.0844

0.1444

0.667

0.1018

0.1522

0.3180

0.1113

0.1708

1.000

0.0758

0.1178

0.2176

0.0890

0.1251

1.500

0.0499

0.0967

0.1219

0.0698

0.0846

2.000

0.0328

0.0636

0.0808

0.0717

0.0622

4.000

0.0193

0.0316

0.0290

0.0382

0.0295

6.000

0.0111

0.0189

0.0207

0.0236

0.0186

8.000

0.0069

0.0141

0.0138

0.0152

0.0125

12.000

0.0079

0.0062

0.0065

0.0078

0.0071

24.000

0.0055

0.0049

0.0019

0.0058

0.0045

32.000

0.0065

0.0020

0.0016

0.0029

0.0033

48.000

0.0118

0.0021

0.0010

0.0011

0.0040

72.000

0.0021

0.0025

0.0008

0.0008

0.0016

 

Table 5: Total radioactive residues in whole blood from the I-PCE-F group, expressed as μg residue/g blood (ppm), following a single oral dose of [indane-3-14C] AE 1170437 at a rate of 13.28 mg/kg body weight. A) Measured Values and B) Normalized to a 1 ppm dose rate.

A)

 

Total Radioactive Residue in Blood (ppm)

Time Post-treatment (hours)

Rat-1

Rat-2

Rat-3

Rat-4

Average

0.083

0.1307

0.1384

0.1286

0.4104

0.2020

0.167

0.5521

0.3560

0.2085

1.0706

0.5468

0.333

0.8656

0.5555

0.4839

1.5570

0.8655

0.667

0.9684

0.9189

0.6117

1.3248

0.9560

1.000

0.9624

0.9952

0.8123

1.4235

1.0484

1.500

0.8625

0.7666

0.8256

1.1663

0.9053

2.000

0.6371

0.6114

0.7025

0.9421

0.7233

4.000

0.2740

0.3487

0.4941

0.4557

0.3931

6.000

0.1636

0.1718

0.5590

0.3119

0.3016

8.000

0.1025

0.0907

0.4996

0.2919

0.2462

12.000

0.0443

0.0479

0.2158

0.1017

0.1025

24.000

0.0149

0.0152

0.0418

0.0237

0.0239

32.000

0.0113

0.0126

0.0215

0.0203

0.0164

48.000

0.0111

0.0102

0.0124

0.0144

0.0120

72.000

0.0062

0.0060

0.0069

0.0084

0.0069

 

B)

 

Total Radioactive Residue in Blood (ppm)

Time Post-treatment (hours)

Rat-1

Rat-2

Rat-3

Rat-4

Average

0.083

0.0451

0.0477

0.0444

0.1415

0.0697

0.167

0.1904

0.1228

0.0719

0.3692

0.1886

0.333

0.2985

0.1915

0.1668

0.5369

0.2984

0.667

0.3339

0.3169

0.2109

0.4568

0.3296

1.000

0.3319

0.3432

0.2801

0.4909

0.3615

1.500

0.2974

0.2643

0.2847

0.4022

0.3122

2.000

0.2197

0.2108

0.2422

0.3249

0.2494

4.000

0.0945

0.1202

0.1704

0.1571

0.1356

6.000

0.0564

0.0593

0.1928

0.1076

0.1040

8.000

0.0353

0.0313

0.1723

0.1007

0.0849

12.000

0.0153

0.0165

0.0744

0.0351

0.0353

24.000

0.0051

0.0052

0.0144

0.0082

0.0082

32.000

0.0039

0.0044

0.0074

0.0070

0.0057

48.000

0.0038

0.00358

0.0043

0.0050

0.0041

72.000

0.0021

0.0021

0.0024

0.0029

0.0024

 

Table 6: Total radioactive residues in whole blood from the T-PCE-M group, expressed as μg residue/g blood (ppm), following a single oral dose of [triazine-2,4-14C] AE 1170437 at a rate of 16.96 mg/kg body weight. A) Measured Values and B) Normalized to a 1 ppm dose rate.

A)

 

Total Radioactive Residue in Blood (ppm)

Time Post-treatment (hours)

Rat-1

Rat-2

Rat-3

Rat-4

Average

0.083

0.0709

0.3057

0.1465

0.2214

0.1861

0.167

0.6378

1.0712

0.3927

0.7541

0.7140

0.333

2.3126

1.9289

0.6586

1.9374

1.7094

0.667

2.7641

2.2971

1.0940

2.1420

2.0743

1.000

2.3146

1.7341

0.7766

1.4057

1.5578

1.500

1.6005

1.4751

1.1899

1.0390

1.3261

2.000

1.1771

1.0776

1.2947

0.7528

1.0756

4.000

0.5429

0.6708

0.9094

0.3084

0.6079

6.000

0.3722

0.6523

0.4568

0.4079

0.4723

8.000

0.1406

0.5700

0.2895

0.3496

0.3374

12.000

0.2101

0.3202

0.1886

0.2650

0.2460

24.000

0.1264

0.3548

0.1356

0.0899

0.1766

32.000

0.0789

0.0515

0.0664

0.0305

0.0568

48.000

0.1262

0.0107

0.1539

0.0105

0.0753

72.000

0.0119

0.0076

0.0112

0.0079

0.0097

 

B)

 

Total Radioactive Residue in Blood (ppm)

Time Post-treatment (hours)

Rat-1

Rat-2

Rat-3

Rat-4

Average

0.083

0.0043

0.0188

0.0090

0.0136

0.0114

0.167

0.0392

0.0658

0.0241

0.0463

0.0438

0.333

0.1420

0.1184

0.0404

0.1189

0.1049

0.667

0.1697

0.1410

0.0672

0.1315

0.1273

1.000

0.1421

0.1065

0.0477

0.0863

0.0956

1.500

0.0983

0.0906

0.0730

0.0638

0.0814

2.000

0.0723

0.0662

0.0795

0.0462

0.0660

4.000

0.0333

0.0412

0.0558

0.0189

0.0373

6.000

0.0228

0.0400

0.0280

0.0250

0.0290

8.000

0.0086

0.0350

0.0178

0.0215

0.0207

12.000

0.0129

0.0197

0.0116

0.0163

0.0151

24.000

0.0078

0.0218

0.0083

0.0055

0.0108

32.000

0.0048

0.0032

0.0041

0.0019

0.0035

48.000

0.0077

0.0007

0.0094

0.0006

0.0046

72.000

0.0007

0.0005

0.0007

0.0005

0.0006

 

Table 7: Total radioactive residues in whole blood from the T-PCE-F group, expressed as μg residue/g blood (ppm), following a single oral dose of [triazine-2,4-14C] AE 1170437 at a rate of 13.61 mg/kg body weight. A) Measured Values and B) Normalized to a 1 ppm dose rate.

A)

 

Total Radioactive Residue in Blood (ppm)

Time Post-treatment (hours)

Rat-1

Rat-2

Rat-3

Average

0.083

0.2208

0.3176

0.0874

0.2086

0.167

0.7092

1.0617

0.4169

0.7293

0.333

1.8478

1.7800

0.9063

1.5114

0.667

2.3843

2.1017

1.1635

1.8832

1.000

2.2114

2.3907

0.8114

1.8045

1.500

1.7842

1.9970

0.6021

1.4611

2.000

1.3466

1.3319

0.6471

1.1085

4.000

0.7542

0.8621

0.6405

0.7523

6.000

0.4438

0.4845

0.3151

0.4145

8.000

0.2264

0.5646

0.2573

0.3495

12.000

0.1078

0.3177

0.1757

0.2004

24.000

0.0267

0.1490

0.7367

0.3041

32.000

0.0205

0.2166

0.3647

0.2006

48.000

0.0151

0.0406

0.0571

0.0376

72.000

0.0104

0.0345

0.0983

0.0477

 

B)

 

Total Radioactive Residue in Blood (ppm)

Time Post-treatment (hours)

Rat-1

Rat-2

Rat-3

Average

0.083

0.0168

0.0241

0.0066

0.0158

0.167

0.0539

0.0806

0.0317

0.0554

0.333

0.1403

0.1352

0.0688

0.1148

0.667

0.1810

0.1596

0.0883

0.1430

1.000

0.1679

0.1815

0.0616

0.1370

1.500

0.1355

0.1516

0.0457

0.1109

2.000

0.1022

0.1011

0.0491

0.0842

4.000

0.0573

0.0655

0.0486

0.0571

6.000

0.0337

0.0368

0.0239

0.0315

8.000

0.0172

0.0429

0.0195

0.0265

12.000

0.0082

0.0241

0.0133

0.0152

24.000

0.0020

0.0113

0.0559

0.0231

32.000

0.0016

0.0164

0.0277

0.0152

48.000

0.0011

0.0031

0.0043

0.0029

72.000

0.0008

0.0026

0.0075

0.0036

Endpoint:
dermal absorption in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 427 (Skin Absorption: In Vivo Method)
Version / remarks:
adopted in 2004
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Environmental Health and Safety Publications Series on testing and Assessment N° 28. Guidance Document for the Conduct of Skin Absorption Studies
Version / remarks:
adopted in 2004
Qualifier:
according to guideline
Guideline:
other: European Commission Guidance Document on Dermal Absorption- Sanco/222/2000 rev. 7
Version / remarks:
adopted in 2004
GLP compliance:
yes
Radiolabelling:
yes
Species:
rat
Strain:
other: Wistar Rj: WI (IOPS HAN)
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: R. Janvier, Le Genest St Isle, France
- Age at study initiation: 7 - 9 weeks
- Weight at study initiation: 259 - 391 g
- Housing: suspended, stainless steel and wire mesh cages
- Individual metabolism cages: yes (Jencon's metabowls Mk III or Radleys metabolism cages)
- Diet: Rodent diet A04C-10, pelleted and irradiated, (S.A.F.E. Scientific Animal Food and Engineering, Augy, France), ad libitum
- Water: filtered and softened tap water, ad libitum
- Acclimation period: at least 13 days in the room, 24 h in the metabolism cages

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 10 to 15
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Duration of exposure:
8 hours
Doses:
- Nominal doses: 0.05, 0.2 and 500 g/L (corresponding to 0.5, 2 and 5000 µg/cm²)
- Actual doses: 0.28-0.31 µg/cm², 1.5 - 1.8 µg/cm² and 5.67 - 6.14 mg/cm²
- Dose volume: approximately 10 µL/cm²
- Rationale for dose selection: neat product and 2 spray dilutions
Please refer to Table 1 under "Any other information on material and methods incl. tables").
No. of animals per group:
4 animals per dose and termination time point
Control animals:
no
Details on study design:
DOSE PREPARATION
- Method for preparation of dose suspensions: Undiluted test substance or spray dilutions with water.
- Method of storage: at approximately -20°C in the dark

APPLICATION OF DOSE:

TEST SITE
- Preparation of test site: The test site was shaved approximately 24 hours prior to dosing. Just prior to dosing the animals were lightly anaesthetized and two plastic protective saddles were secured in place using Cyanoacrylate adhesive to define the site for application of the test substance.
- Area of exposure: dorsal skin, approximately 2 x 6 cm²
- Type of cover / wrap: a perforated plastic cover (to allow ventilation) held in place over the plastic saddle with surgical tape

SITE PROTECTION / USE OF RESTRAINERS FOR PREVENTING INGESTION: yes: (cover was held in place over the plastic saddle with surgical tape

REMOVAL OF TEST SUBSTANCE
- Removal of protecting device: cover was removed
- Washing procedures and type of cleansing agent: Cover and application site were swabbed with freshly prepared 1% v/v Tween 80 in PBS using natural sponge swabs until no more radioactivity was detected on the swabs with a Geiger monitor. Animals that were required to provide samples beyond 8 hours were then fitted with a clean cover to capture any 14C lost by desquamation and replaced in the metabolism cage
- Time after start of exposure: 8 hours

SAMPLE COLLECTION
- Collection of blood: cardiac puncture at different termination time points under anaesthasia (Isofluran)
- Collection of urine and faeces: Urine and faeces were collected separately into receivers at 0 to 8, 8 to 24 and at 24-hour intervals up to sacrifice. At the end of each collection period all debris was removed from the metabolism cage and retained. At each sampling, the cage was carefully washed with distilled water. At study termination the cage was washed with an appropriate organic solvent in addition to water.
- Terminal procedure: The rats were exsanguinated whilst under anaesthesia (Isofluran). The treated skin was swabbed following sacrifice prior to removal. The skin was then shaved (shavings retained), if necessary, prior to tape-stripping to remove the stratum corneum. This procedure involved the application of an adhesive tape (CILS, France) for 5 seconds before the tape was carefully removed against the direction of hair growth. This process was continued until a 'shiny' appearance of the epidermis was evident, indicating that the stratum corneum had been removed.
- Analysis of organs: The treated area of skin was removed and taken for analysis. Further skin samples were also taken for analysis, including the area immediately surrounding the treated skin (approx. 1 cm wide) and a control sample (untreated skin: clearly separated from the application site). The residual carcass was also retained for analysis.

SAMPLE PREPARATION
- Storage procedure: Whenever possible samples were processed as they were collected. Remaining samples were stored at approximately -20°C in the dark until required.
- Preparation details:
Urine: The urine samples were thawed to about room temperature. The weights were recorded and duplicate weighed aliquots added directly to scintillation fluid and analysed.
Cage wash and debris: The cage washings were thawed to about room temperature, and weighed. Duplicate weighed aliquots were taken for analysis. The cage debris (if appropriate) was analysed by a suitable method depending on the nature of the debris.
Faeces: The faeces samples were thawed, then weighed and homogenised in a measured weight of distilled water using a top drive homogeniser (Ultra-Turrax). Triplicate aliquots of the homogenate were combusted directly after drying on Combusto-pads.
Whole blood: The samples of blood were thawed to about room temperature. Triplicate weighed aliquots of cardiac blood were combusted directly after drying of weighed aliquots on Combusto-pads contained in Combusto-cones.
Skin swabs: according to the type of samples the following procedures were used: Swabs 8h and at termination time and surrounding swabs: The swabs of the application site were weighed and were solubilised using Soluene®. Triplicate weighed aliquots were taken for LSC analysis. Swabs X, Y and Z (last three swabs): 2 mL of Soluene and scintillation fluid were directly added to each swab for LSC analysis.
Tape strips: All tape strips were solubilised using tetrahydrofuran. Scintillation fluid was directly added to each tape strip sample for LSC analysis
Skin samples: Skin samples were solubilised separately using alcoholic potassium hydroxide, the total weight determined and duplicate weighed aliquots taken for LSC analysis.
Saddle, gauze, tapes and cover (dressing): The saddle, surgical tape and cover were soaked in an appropriate solvent (acetonitrile). The washings were weighed and duplicate weighed aliquots taken for LSC analysis.
Fur samples: The fur samples removed from the treated skin prior to tape-stripping were soaked in an appropriate solvent (acetonitrile, 2 mL). Scintillation fluid was directly added to each fur sample for LSC analysis
Carcass: The residual carcass was thawed, then weighed and solubilised in alcoholic potassium hydroxide; the total weight determined and duplicated weighed aliquots taken for LSC analysis.

ANALYSIS
- Method type(s) for identification: Liquid scintillation counting
- Limits of detection: twice the background values for blank samples in appropriate scintillation cocktails
Signs and symptoms of toxicity:
no effects
Dermal irritation:
not specified
Absorption in different matrices:
For detailed absorption in different matrices please refer to the result tables included under "Any other information on results incl. tables".
Total recovery:
- Total recovery of groups 1 to 4: 90.6 - 92.7% (500 g/L), 92.7 - 101.4% (0.2 g/L), 99.7 - 107.7% (0.05 g/L)
- Recovery of applied dose acceptable: no (groups 1 - 4, 500 g/L); yes (groups 5 and 6, 0.2 g/L); no (groups 7 and 8, 0.2 g/L); yes (groups 9 -12, 0.05 g/L)
- Results adjusted for incomplete recovery of the applied dose: yes (according to EFSA Guidance on dermal absorption, 2017)
- Limit of detection (LOD): The limit of detection was taken to be twice the background values for blank samples in appropriate scintillation cocktails.
- Quantification of values below LOD or LOQ: no
Time point:
8 h
Dose:
500 g/L
Parameter:
percentage
Absorption:
5.268 %
Remarks on result:
other: mean potentially absorbable percentage
Time point:
8 h
Dose:
500 g/L
Parameter:
percentage
Absorption:
20 %
Remarks on result:
other: mean potentially absorbable percentage, re-evaluated according to EFSA Guidance on dermal absorption (2017)
Time point:
24 h
Dose:
500 g/L
Parameter:
percentage
Absorption:
3.025 %
Remarks on result:
other: mean potentially absorbable percentage
Time point:
24 h
Dose:
500 g/L
Parameter:
percentage
Absorption:
12 %
Remarks on result:
other: mean potentially absorbable percentage, re-evaluated according to EFSA Guidance on dermal absorption (2017)
Time point:
72 h
Dose:
500 g/L
Parameter:
percentage
Absorption:
2.403 %
Remarks on result:
other: mean potentially absorbable percentage
Time point:
72 h
Dose:
500 g/L
Parameter:
percentage
Absorption:
17 %
Remarks on result:
other: mean potentially absorbable percentage, re-evaluated according to EFSA Guidance on dermal absorption (2017)
Time point:
168 h
Dose:
500 g/L
Parameter:
percentage
Absorption:
2.764 %
Remarks on result:
other: mean potentially absorbable percentage
Time point:
168 h
Dose:
500 g/L
Parameter:
percentage
Absorption:
15 %
Remarks on result:
other: mean potentially absorbable percentage, re-evaluated according to EFSA Guidance on dermal absorption (2017)
Time point:
8 h
Dose:
0.2 g/L
Parameter:
percentage
Absorption:
21.254 %
Remarks on result:
other: mean potentially absorbable percentage
Time point:
8 h
Dose:
0.2 g/L
Parameter:
percentage
Absorption:
27 %
Remarks on result:
other: mean potentially absorbable percentage, re-evaluated according to EFSA Guidance on dermal absorption (2017)
Time point:
24 h
Dose:
0.2 g/L
Parameter:
percentage
Absorption:
18.98 %
Remarks on result:
other: mean potentially absorbable percentage
Time point:
24 h
Dose:
0.2 g/L
Parameter:
percentage
Absorption:
28 %
Remarks on result:
other: mean potentially absorbable percentage, re-evaluated according to EFSA Guidance on dermal absorption (2017)
Time point:
72 h
Dose:
0.2 g/L
Parameter:
percentage
Absorption:
11.451 %
Remarks on result:
other: mean potentially absorbable percentage
Time point:
72 h
Dose:
0.2 g/L
Parameter:
percentage
Absorption:
22 %
Remarks on result:
other: mean potentially absorbable percentage, re-evaluated according to EFSA Guidance on dermal absorption (2017)
Time point:
168 h
Dose:
0.2 g/L
Parameter:
percentage
Absorption:
14.866 %
Remarks on result:
other: mean potentially absorbable percentage
Time point:
168 h
Dose:
0.2 g/L
Parameter:
percentage
Absorption:
32 %
Remarks on result:
other: mean potentially absorbable percentage, re-evaluated according to EFSA Guidance on dermal absorption (2017)
Time point:
8 h
Dose:
0.05 g/L
Parameter:
percentage
Absorption:
42.659 %
Remarks on result:
other: mean potentially absorbable percentage
Time point:
8 h
Dose:
0.05 g/L
Parameter:
percentage
Absorption:
48 %
Remarks on result:
other: mean potentially absorbable percentage, re-evaluated according to EFSA Guidance on dermal absorption (2017)
Time point:
24 h
Dose:
0.05 g/L
Parameter:
percentage
Absorption:
28.687 %
Remarks on result:
other: mean potentially absorbable percentage
Time point:
24 h
Dose:
0.05 g/L
Parameter:
percentage
Absorption:
41 %
Remarks on result:
other: mean potentially absorbable percentage, re-evaluated according to EFSA Guidance on dermal absorption (2017)
Time point:
72 h
Dose:
0.05 g/L
Parameter:
percentage
Absorption:
30.208 %
Remarks on result:
other: mean potentially absorbable percentage
Time point:
72 h
Dose:
0.05 g/L
Parameter:
percentage
Absorption:
38 %
Remarks on result:
other: mean potentially absorbable percentage, re-evaluated according to EFSA Guidance on dermal absorption (2017)
Time point:
168 h
Dose:
0.05 g/L
Parameter:
percentage
Absorption:
23.467 %
Remarks on result:
other: mean potentially absorbable percentage
Time point:
168 h
Dose:
0.05 g/L
Parameter:
percentage
Absorption:
29 %
Remarks on result:
other: mean potentially absorbable percentage, re-evaluated according to EFSA Guidance on dermal absorption (2017)

Table 2: Achieved doses

 

High dose (500 g/L nominal)

Intermediate dose (0.2 g/L nominal)

Low dose (0.05 g/L nominal)

Radiochemical dose applied per animal

537.1 to 582.1 kBq

69.3 to 85.6 kBq

13.2 to 15.0 kBq

Compound dose applied per animal

67.98 to 73.68 mg

17.9 to 21.6 µg

3.3 to 3.8 µg

Compound dose applied per cm² *

5.67 to 6.14 mg/cm²

1.5 to 1.8 µg/cm²

0.28 to 0.31 µg/cm²

*The application rate of the three formulations was 10 µL/cm² and the area of application 12 cm².

Table 3: Mean distribution of radioactivity 8, 24, 72 and 168 h after a single topical application of the test material at the highest dose level (500 g/L; results expressed as % of applied dose; n=4 rats/group)

Group

1

2

3

4

Termination time

8 h

24 h

72 h

168 h

 

Mean

SD

Mean

SD

Mean

SD

Mean

SD

SURFACE COMPARTMENT

Skin swabs at 8 h

84.516

3.336

86.305

1.073

86.440

2.637

85.807

1.324

Skin swabs at termination time

-

-

0.925

1.081

0.571

0.519

0.734

0.280

Surrounding swabs

0.169

0.101

0.006

0.006

0.009

0.011

0.000

0.000

Total % swabs

84.685

3.266

87.236

1.349

87.019

2.886

86.541

1.130

Surface dose (Tape-strips 1 & 2)

0.283

0.140

0.952

0.418

0.652

0.357

0.508

0.136

Fur

-

-

-

-

-

-

1.421

0.335

Dressing

0.320

0.059

0.646

0.589

0.997

1.365

1.491

0.570

Total % non absorbed

85.287

3.160

88.834

0.530

88.669

3.323

89.961

1.817

WASHED SKIN COMPARTMENT

Stratum corneuma

1.842

0.855

2.155

0.987

1.894

0.783

1.243

0.582

Treated skinb

1.210

0.942

0.252

0.209

0.062

0.050

0.317

0.238

Surrounding skin

0.695

0.352

0.155

0.090

0.095

0.082

0.440

0.235

Total % at dose site

3.747

1.898

2562

1.208

2.051

0.878

2.001

0.428

SYSTEMIC COMPARTMENT

Urines

0.001

0.002

0.013

0.003

0.010

0.003

0.038

0.012

Faeces

0.019

0.002

0.070

0.019

0.101

0.010

0.202

0.041

Cage wash

0.012

0.015

0.018

0.015

0.000

0.001

0.093

0.067

Total % excreted

0.032

0.014

0.101

0.032

0.112

0.011

0.333

0.071

Cardiac blood

0.000

0.000

0.003

0.001

0.002

0.001

0.002

0.001

Non treated skin

1.020

0.788

0.111

0.060

0.076

0.009

0.246

0.122

Carcass

0.469

0.233

0.249

0.032

0.162

0.009

0.183

0.029

Total % directly absorbed

1.521

0.969

0.463

0.109

0.352

0.013

0. 763

0.218

Total % potentially absorbablec

5.268

2.001

3.025

1.224

2.403

0.880

2.764

0.438

Overall Total % Recovery

90.555

2.075

91.859

1.362

91.072

2.553

92.724

1.888

a: tape-strips excluding 1 & 2 which are considered to be non-absorbed dose

b: skin after tape stripping procedure

c: total % directly absorbed + total at dose site

SD: standard deviation

- = no sample

 

Table 4: Mean distribution of radioactivity 8, 24, 72 and 168 h after a single topical application of the test material at the intermediate dose level (0.2 g/L; results expressed as % of applied dose; n=4 rats/group)

Group

1

2

3

4

Termination time

8 h

24 h

72 h

168 h

 

Mean

SD

Mean

SD

Mean

SD

Mean

SD

SURFACE COMPARTMENT

Skin swabs at 8 h

72.008

4.972

56.794

6.427

75.583

4.192

71.019

9.816

Skin swabs at termination time

-

-

17.819

8.593

2.259

1.030

0.765

0.458

Surrounding swabs

0.124

0.158

0.011

0.009

0.016

0.017

0.008

0.001

Total % swabs

72.133

4.849

74.624

3.989

77.858

4.301

71.792

9.384

Surface dose (Tape-strips 1 & 2)

5.420

2.948

3.949

1.820

1.248

0.743

1.357

1.187

Fur

-

-

-

-

0.223

0.447

0.712

0.545

Dressing

2.573

2.041

0.715

0.271

1.951

3.193

5.368

2.268

Total % non absorbed

80.126

5.577

79.288

3.916

81.280

2.024

79.229

6.971

WASHED SKIN COMPARTMENT

Stratum corneuma

13.877

3.301

10.255

4.339

2.570

1.093

2.466

2.389

Treated skinb

3.702

2.688

0.763

0.439

0.291

0.100

0.611

0.583

Surrounding skin

0.443

0.256

0.174

0.033

0.190

0.054

0.563

0.381

Total % at dose site

18.023

3.521

11.192

4.062

3.052

1.166

3.640

3.204

SYSTEMIC COMPARTMENT

Urines

0.074

0.030

0.664

0.100

1.319

0.148

2.457

1.282

Faeces

0.014

0.029

1.565

0.383

3.354

0.892

5.887

2.511

Cage wash

0.000

0.000

0.122

0.200

0.372

0.247

0.520

0.388

Total % excreted

0.089

0.044

2.352

0.643

5.045

1.188

8.864

4.023

Cardiac blood

0.008

0.016

0.000

0.000

0.000

0.000

0.000

0.000

Non treated skin

0.644

0.108

0.379

0.044

0.460

0.083

0.564

0.109

Carcass

2.491

0.080

5.058

2.749

2.894

0.264

1.797

0.312

Total % directly absorbed

3.232

0.222

7.788

2.617

8.399

1.254

11.226

4.442

Total % potentially absorbablec

21.254

3.573

18.980

5.654

11.451

2.068

14.866

7.517

Overall Total % Recovery

101.380

3.716

98.267

2.092

92.701

1.307

94.094

0.654

a: tape-strips excluding 1 & 2 which are considered to be non-absorbed dose

b: skin after tape stripping procedure

c: total % directly absorbed + total at dose site

SD: standard deviation

- = no sample

 

Table 5: Mean distribution of radioactivity 8, 24, 72 and 168 h after a single topical application of the test material at the low dose level (0.05 g/L; results expressed as % of applied dose; n=4 rats/group)

Group

1

2

3

4

Termination time

8 h

24 h

72 h

168 h

 

Mean

SD

Mean

SD

Mean

SD

Mean

SD

SURFACE COMPARTMENT

Skin swabs at 8 h

56.565

7.240

60.826

13.318

62.481

11.227

64.537

6.532

Skin swabs at termination time

-

-

6.730

4.433

4.244

2.780

3.911

1.515

Surrounding swabs

0.237

0.215

0.028

0.019

0.009

0.008

0.035

0.035

Total % swabs

56.802

7.225

67.585

10.411

66.734

12.042

68.483

5.426

Surface dose (Tape-strips 1 & 2)

3.713

1.617

7.749

7.860

3.012

1.016

1.149

0.280

Fur

-

-

-

-

2.869

2.970

2.137

0.852

Dressing

1.780

2.084

0.746

1.492

4.896

3.981

4.500

1.967

Total % non absorbed

62.295

6.264

76.080

8.188

77.511

6.647

76.269

4.541

WASHED SKIN COMPARTMENT

Stratum corneuma

21.447

3.650

10.978

6.837

7.253

1.700

3.285

0.328

Treated skinb

7.063

6.602

2.402

2.553

0.834

0.218

0.933

0.491

Surrounding skin

1.214

0.702

0.421

0.122

1.675

1.072

0.687

0.060

Total % at dose site

29.725

5.557

13.801

9.319

9.761

2.649

4.904

0.810

SYSTEMIC COMPARTMENT

Urines

0.214

0.070

1.497

0.474

2.646

0.661

2.589

0.727

Faeces

0.000

0.000

2.879

1.147

5.844

1.681

6.430

0.882

Cage wash

0.000

0.000

0.658

0.660

0.115

0.230

0.721

1.191

Total % excreted

0.214

0.070

5.034

2.094

8.605

2.073

9.740

2.041

Cardiac blood

0.000

0.000

0.118

0.125

0.241

0.062

0.000

0.000

Non treated skin

2.575

0.283

1.820

0.177

3.018

0.620

2.814

0.276

Carcass

10.146

2.635

7.913

0.478

8.583

1.251

6.010

1.238

Total % directly absorbed

12.935

2.767

14.886

2.526

20.446

3.178

18.563

3.403

Total % potentially absorbablec

42.659

3.584

28.687

7.656

30.208

4.586

23.467

3.593

Overall Total % Recovery

104.955

4.568

104.767

2.822

107.718

2.691

99.737

4.728

a: tape-strips excluding 1 & 2 which are considered to be non-absorbed dose

b: skin after tape stripping procedure

c: total % directly absorbed + total at dose site

SD: standard deviation

- = no sample

Results (total potentially absorbed) were corrected for the missing material when recovery was below the set limit (mean over all animals < 95%) as well as for variability between animals according to the EFSA Guidance on dermal absorption (2017). Results are shown in the results table "Percutaneous absorption".

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.
Qualifier:
according to guideline
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OECD Environmental Health and Safety Publication Series on testing and Assessment N° 28, Guidance Document for the Conduct of Skin Absorption Studies (March 2004).
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: European Commission Guidance Document on Dermal Absorption- Sanco/222/2000 rev. 7, (March 2004).
Deviations:
no
GLP compliance:
yes
Radiolabelling:
yes
Remarks:
14C
Species:
other: rat + human
Strain:
other: Wistar Rj:WI (IOPS HAN) rats
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: R. Janvier, Le Genest St Isle, France.
- Age at study initiation: 6-10 weeks
- Housing: Together in a wire-mesh bottomed stainless steel cage during the acclimatisation period
- Diet: Pelleted diet (A04 C-10 from SAFE, Rte de StBris, Augy, France), ad libitum
- Water: Filtered tap water from municipal supply, ad libitum
- Acclimation period: At least 5 days; animals killed after acclimatisation period. Dorsal skin was clipped and dermatomed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
open
Vehicle:
water
Duration of exposure:
8 hours
Doses:
- Nominal doses: 0.5, 2, 10, 5000 µg/cm²
- Dose volume: 10 µL/cm²
No. of animals per group:
not applicable
Control animals:
no
Details on study design:
DOSE PREPARATION
- Method for preparation of dose suspensions: Undiluted test substance or spray dilutions with water.
- Method of storage: dark at -20 °C

APPLICATION OF DOSE:
The dose preparation was applied to the split-thickness skin sample with a pipette at the rate of approximately 10 µL/cm² exposed skin.

VEHICLE
- Justification for use and choice of vehicle: water
- Amount(s) applied (volume or weight with unit): 10µL/cm²
- Concentration (if solution): 0.05, 0.2, 1.0, 500 µg/µL


TEST SITE
- Preparation of test site: Rat skin clipped after sacrifice and dermatomed.
- Area of exposure: 1 cm² (diffusion cell)

REMOVAL OF TEST SUBSTANCE
- Washing procedures and type of cleansing agent: Remaining dose material was washed off the skin with freshly prepared 1% v/v Tween 80 in PBS (phosphate buffered saline) using natural sponge swabs.
- Time after start of exposure: 8 hours post application

SAMPLE COLLECTION
- Collection of receptor fluid (flow-through diffusion cell): Receptor fluid was pumped through the receptor chamber at a flow rate of 1.5 mL/h at a temperature of 32±2 °C, and samples were collected at hourly intervals for the duration of the study (24 hours). The solubility of AE 1170437 in the receptor fluid was demonstrated to be sufficient for the study.

SAMPLE PREPARATION
- Storage procedure: Samples were stored at approximately -20 °C in the dark until required.
- Preparation details: The receptor fluid passing through the receptor chamber was collected in glass vials held in a fraction collector. The fraction collector was started after dose application for each group was complete. Samples were then collected hourly for the duration of the experiment (24 hours). At 8 hours post-application, the skin was swabbed with freshly prepared 1% v/v Tween 80 in PBS (phosphate buffer saline) using natural sponge swabs, in order to remove and retain the non-absorbed dose, until no radioactivity was detected with a Geiger-Müller monitor. At the end of the study (24 hours after application), the treated skin cell was swabbed again (as described above) prior to tape-stripping. The tape-stripping procedure involved the application of Monaderm adhesive tape (Monaderm, Monaco) for 5 seconds before the tape was carefully removed against the direction of hair growth. This procedure was continued until a 'shiny' appearance of the epidermis was evident, which indicated that the stratum corneum had been removed. The tape-strips were collected into scintillation vials. The remaining skin was removed and taken for analysis. The receptor fluid remaining in the cell and outlet tubing at the end of the experiment was retained for analysis. The diffusion cell components were also retained and washed. The washings were also analyzed to establish a mass balance.

ANALYSIS
- Method type(s) for identification: Liquid scintillation counting
- Liquid scintillation counting results (cpm) converted to dpm as follows: Total radiocarbon in sample (dpm) = net dpm X total amount of sample/amount of sample assayed.
- Limits of detection and quantification: 0.005%
Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin:
Human skin:
Dermatomed human skin from six different female donors was obtained from a recognized Tissue Bank (Biopredic, Rennes, France) and stored at approximately - 20 °C prior to use. Skin sample thickness ranged between 431 and 545 µm.
Rat skin:
After an acclimatisation period eight male Wistar Rj:WI (IOPS HAN) rats were killed, and dorsal areas of the skin were clipped and removed for use in the study. The skin was dermatomed by use of a mini-dermatome (Decadermatome microsystem motor, Thackray Surgery, Leeds, UK) to obtain samples of 410-580 µm thickness.
- Type of skin: Human abdominal skin, dorsal rat skin
- Preparative technique: Dermatomed.
- Thickness of skin (in mm): human: 0.431-0.545; rat: 0.410-0.580
- Membrane integrity check: Before dose application, the integrity of the skin samples was assessed by measuring the trans-epidermal water loss (TEWL) from the stratum corneum. An evaporimeter probe (Dermalab, Cortex Technology, Hadsund, Denmark) was placed securely on the top of the donor chamber and the amount of water diffusing through the skin was measured in g/hm². Human and rat skin with a TEWL of greater than 40 g/m²/h were considered potentially damaged and were not used. These samples were replaced by new skin fragments which were also tested for integrity before use in the study.
- Storage conditions: at -20 °C
- Justification of species, anatomical site and preparative technique: Human skin has been chosen as the human is the species of interest, and for comparative purposes with the results obtained for rat skin, which is the most preferred species for toxicology studies. Human skin was derived from the abdomen, while rat skin was obtained from the dorsum.

PRINCIPLES OF ASSAY
- Diffusion cell: Flow-through diffusion cell system
- Receptor fluid: Eagle's medium supplemented with 5% bovine serum albumin and gentamycin (50 mg/L) at a pH of 7.3 to 7.4.
- Solubility of test substance in receptor fluid: The solubility of [14C]-labelled test substance in the receptor fluid after 24 hours of incubation was demonstrated at a concentration of approximately 0.6 mg/mL (approximately 1800 times the maximum concentration actually achieved in the receptor fluid during the experiment).
- Flow-through system: Franz's cell modified, Gallas, France
- Test temperature: 32±2 °C
- Occlusion: unoccluded
Time point:
24 h
Dose:
5000 µg/cm²
Parameter:
percentage
Absorption:
0.13 %
Remarks on result:
other: human skin, mean potentially absorbable percentage
Time point:
24 h
Dose:
5000 µg/cm²
Parameter:
percentage
Absorption:
12 %
Remarks on result:
other: human skin, mean potentially absorbable percentage, re-evaluated according to EFSA Guidance on dermal absorption (2017)
Time point:
24 h
Dose:
5000 µg/cm²
Parameter:
percentage
Absorption:
1.39 %
Remarks on result:
other: rat skin, mean potentially absorbable percentage
Time point:
24 h
Dose:
5000 µg/cm²
Parameter:
percentage
Absorption:
14 %
Remarks on result:
other: rat skin, mean potentially absorbable percentage, re-evaluated according to EFSA Guidance on dermal absorption (2017)
Time point:
24 h
Dose:
10 µg/cm²
Parameter:
percentage
Absorption:
2.31 %
Remarks on result:
other: human skin, mean potentially absorbable percentage
Time point:
24 h
Dose:
10 µg/ cm²
Parameter:
percentage
Absorption:
4.1 %
Remarks on result:
other: human skin, mean potentially absorbable percentage, re-evaluated according to EFSA Guidance on dermal absorption (2017)
Time point:
24 h
Dose:
10 µg/cm²
Parameter:
percentage
Absorption:
18.58 %
Remarks on result:
other: rat skin, mean potentially absorbable percentage
Time point:
24 h
Dose:
10 µg/cm²
Parameter:
percentage
Absorption:
39 %
Remarks on result:
other: rat skin, mean potentially absorbable percentage, re-evaluated according to EFSA Guidance on dermal absorption (2017)
Time point:
24 h
Dose:
2 µg/cm²
Parameter:
percentage
Absorption:
1.37 %
Remarks on result:
other: human skin, mean potentially absorbable percentage
Time point:
24 h
Dose:
2 µg/cm²
Parameter:
percentage
Absorption:
14 %
Remarks on result:
other: human skin, mean potentially absorbable percentage, re-evaluated according to EFSA Guidance on dermal absorption (2017)
Time point:
24 h
Dose:
2 µg/cm²
Parameter:
percentage
Absorption:
8.5 %
Remarks on result:
other: rat skin, mean potentially absorbable percentage
Time point:
24 h
Dose:
2 µg/cm²
Parameter:
percentage
Absorption:
13 %
Remarks on result:
other: rat skin, mean potentially absorbable percentage, re-evaluated according to EFSA Guidance on dermal absorption (2017)
Time point:
24 h
Dose:
0.5 µg/cm²
Parameter:
percentage
Absorption:
5.71 %
Remarks on result:
other: human skin, mean potentially absorbable percentage
Time point:
24 h
Dose:
0.5 µg/cm²
Parameter:
percentage
Absorption:
2.4 %
Remarks on result:
other: human skin, mean potentially absorbable percentage, re-evaluated according to EFSA Guidance on dermal absorption (2017)
Time point:
24 h
Dose:
0.5 µg/cm²
Parameter:
percentage
Absorption:
21.51 %
Remarks on result:
other: rat skin, mean potentially absorbable percentage
Time point:
24 h
Dose:
0.5 µg/cm³
Parameter:
percentage
Absorption:
32 %
Remarks on result:
other: rat skin, mean potentially absorbable percentage, re-evaluated according to EFSA Guidance on dermal absorption (2017)
Conversion factor human vs. animal skin:
5000 µg/cm²: 0.09; 0.9 (following re-evaluation according to EFSA Guidance on dermal absorption)
10 µg/cm²: 0.12; 0.11 (following re-evaluation according to EFSA Guidance on dermal absorption)
2 µg/cm²: 0.16; 1.1 (following re-evaluation according to EFSA Guidance on dermal absorption)
0.5 µg/cm²: 0.27; 0.075 (following re-evaluation according to EFSA Guidance on dermal absorption)

Radioactivity, expressed as mean percentage of applied dose ± SD:

 

Test substance concentration (mg/mL)

 

500

1.0

0.2

0.05

Species

Human

Rat

Human

Rat

Human

Rat

Human

Rat

Surface compartment

Skin swabs

90.85±2.98

90.72±3.51

93.43±2.90

68.32±19.89

88.97±3.54

80.75±4.50

84.01±4.82

53.61±7.91

Surface dose (Tape-strips 1&2)

0.15±0.04

1.29±0.39

2.32±1.84

11.38±7.82

1.01±1.12

3.41±2.48

5.70±1.26

20.83±6.49

Donor chamber

0.22±0.34

0.04±0.01

0.03±0.03

0.05±0.08

0.04±0.07

0.12±0.18

<LOQ

0.08±0.18

Total % non-absorbed

91.23±2.86

92.05±3.35

95.78±2.00

79.75±13.96

90.02±3.42

84.27±5.09

89.70±4.04

74.52±9.32

Skin compartment

Skin (a)

0.02±0.02

0.12±0.12

0.38±0.21

0.48±0.18

0.20±0.13

1.04±1.45

0.66±0.68

0.97±0.47

Stratum corneum (b)

0.11±0.04

1.26±0.71

1.78±1.33

17.13±12.07

0.62±0.46

4.69±1.29

4.10±2.34

15.92±8.58

Total % at dose site

0.13±0.06

1.38±0.78

2.16±1.46

17.61±12.05

0.83±0.54

5.72±1.80

4.76±2.99

16.89±8.43

Receptor compartment

Total % directly absorbed (c)

<LOQ

0.02±0.02

0.15±0.01

0.97±0.72

0.54±0.17

2.78±1.08

0.95±0.30

4.62±0.57

Total % potentially absorbed (d)

0.13±0.06

1.39±0.78

2.31±1.46

18.58±12.77

1.37±0.63

8.50±2.31

5.71±2.90

21.51±8.75

Total % recovery

91.36±2.88

93.44±3.84

98.09±2.12

98.33±3.35

91.39±3.70

92.77±4.65

95.41±3.32

96.02±2.64

(a) skin after tape-stripping procedure

(b)  tape-strips excluding no 1 & 2 which are considered non-absorbed

(c)   including receptor fluid (0 to 24h), receptor fluid at termination & receptor chamber

(d)  including receptor fluid (0 to 24h), receptor fluid at termination, receptor chamber & total % at dose site

<LOQ: below limit of quantification (<0.005%)

N.C.: not calculated

Results (total potentially absorbed) were corrected for the missing material when recovery was below the set limit (mean over all animals < 95%) as well as for variability between animals according to the EFSA Guidance on dermal absorption (2017). Results are shown in the results table "Percutaneous absorption".

Description of key information

Absorption (oral): absorption was rapid with maximum plasma concentrations within 60 minutes; approx. 90% via the GI tract (low dose); >57% via the GI tract (high dose)

Distribution: <0.1% up to 4.9% remains in carcass and tissues (tissue-specific distribution of respective metabolites, highest in GI tract, liver, skin, thyroid gland)

Metabolism: rapid metabolism mainly via oxidative pathways, various metabolites have been identified (carboxylic acid derivative, dihydroxy derivative, carboxylic acid glucuronic acid conjugate, 3 -hydroxyindane acid derivative, 3-hydroxyindane acid epimer, 3-ketoindane acid derivative, 3-ketohydroxymethyl derivative, diaminotriazine derivative and hydroxethyl acid derivative)
Excretion (low dose): approx. 50 - 60% in faeces (including 10% unmetabolised parent compound and 40 - 50 % contribution of bile), approx. 40 - 50% in urine; majority of the absorbed dose (84 - 95%) excreted within 24 hours.

Excretion (high dose): approx. 90 - 100% in faeces (including 36 - 38% unmetabolised parent compound), approx. 5 - 10% in urine; majority of the absorbed dose excreted within 24 hours

Dermal absorption rate (re-evaluation according to EFSA Guidance on dermal absorption 2017):

Absorption of human skin in vitro (24 h): 2.4 - 14% (depending on applied dose)

Absorption of rat skin in vitro (24 h): 13 - 32% (depending on applied dose)

Absorption of rat skin in vivo (8 h): 20 - 48% (depending on applied dose)

Absorption of rat skin in vivo (24 h): 12 - 41% (depending on applied dose)

Absorption of rat skin in vivo (72 h): 17 - 38% (depending on applied dose)

Absorption of rat skin in vivo (168 h): 15 - 32% (depending on applied dose)

No conversion factor for absorption of human vs rat skin was derived based on the re-evaluation according to EFSA Guidance on dermal absorption (2017) since at the highest dose (5000 µg/cm²) the absorption rate was 12% for human and 14% for rat skin (in vitro studies).

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential
Absorption rate - dermal (%):
12

Additional information

Discussion on toxicokinetic study results:

In an available toxicokinetics study performed according to OECD TG 417 radioactively-labeled test compound with labels at different positions in the molecule was administered by oral gavage to male rats in a low-dose and a high-dose experiment (Krolski and Nguyen, 2008). In the high-dose experiment the rats were additionally bile cannulated, to assess the proportions excreted in the bile, the urine and unmetabolised in the faeces. Urine samples were collected 6, 12, 24, 48 and 72 hours post-treatment, faeces at 24, 48 and 72 hours post-treatment and bile 1, 2, 3, 4, 6, 8, 12, 24, 30 and 48 hours post-treatment. Additionally, various body tissues (blood, bone, brain, fat, heart, kidney, liver, lung, muscle, skin, testes, thyroid, gastrointestinal tract and remaining carcass) were sampled to determine the distribution of metabolites in different body compartments.

In the low dose experiments the majority of the dose was excreted in the faeces (62.2 -70.4%), while 34.6 - 38.1% was excreted in the urine. Approximately 2 - 8% of the administered dose was excreted as unmetabolised parent compound and therefore considered not to be absorbed. In conclusion, about 90% of the administered dose has been absorbed via the gastrointestinal tract. Similar results were obtained from the bile cannulation experiments. Summing the renal (37.0-48.8%) and biliary (37.9-48.3%) excretion with the residual amount of the dose in the tissues shows a minimum bioavailability of 90.2 - 90.6% of the administered dose being absorbed. In general, the majority of the radioactivity was excreted in the faeces with fecal:renal excretion ratios ranging from 1:1 to 2:1.

Less than 5% of the administered dose remained in the carcass and tissues at sacrifice with the highest amounts in the gastrointestinal tract and the liver, followed by skin and thyroid gland. Slight differences in the amount of radioactivity in the tissues occurred depending on the location of the radioactive label in the test substance, especially in the bile cannulation experiments, reflecting the respective tissue distribution of the individual metabolites. In these experiments up to 4.9% of radioactivity was found in the tissues, in contrast to only 0.2% in the low-dose experiments.

Various metabolites in addition to the unmetabolised parent compound have been identified, namely the carboxylic acid derivative, the dihydroxy derivative, the hydroxy glucuronic acid derivative, the 3-hydroxyindane acid derivative, the 3-hydroxyindane acid epimer, the 3-ketoindane acid derivative, the 3-ketohydroxymethyl derivative, the diaminotriazine derivative and the hydroxethyl acid derivative.

Metabolic degradation of the test compound is rapid and complete; in all experiments, 39 to 67% of the administered dose was excreted as the carboxylic acid derivative, indicating that oxidation is the main route of metabolism for the parent compound. As described above, a variety of other oxidation products and several glucuronic acid conjugates, particularly in the bile were observed.

The majority of the absorbed doses (84.1% in the low-dose and 94.6% in the bile cannulation experiment) was excreted within 24 hours.

 

In a further toxicokinetic study performed according to OECD TG 417 radioactively-labeled test compound with labels at different positions in the molecule was administered by oral gavage to female rats in a low-dose- and to male rats in a high-dose-experiment (Krolski and Nguyen, 2011). In an additional experiment male and female rats with surgically implanted jugular cannulae were given single oral low doses of the different radiolabeled test compound to determine the uptake, blood concentrations and bioavailability of the compound.

Urine samples were collected 6, 12, 24, 48 and 72 hours post-treatment in all groups and additionally at 96 h in the high dose experiment groups. Faeces were collected at 24 hours post-treatment intervals until sacrifice. Additionally, various body tissues (blood, bone, brain, fat, heart, kidney, liver, lung, muscle, skin, gonads, thyroid, gastrointestinal tract and remaining carcass) were sampled after sacrifice to determine the distribution of the test compound and/or metabolites in different body compartments. Metabolites were characterised in urine and faeces via HPLC, LC/ESI-MS and/or NMR analysis. 

In the low dose experiments the majority of the dose (42.3-49.1%) was excreted in faeces, while 43.7-49.4% was excreted in urine for a renal:faecal ratio of approximately 1:1. Approximately 1% of the administered dose was excreted as unmetabolised parent compound in the faeces and was therefore considered not to be absorbed. Thus, about 93.5-95.5% of the administered dose has been absorbed via the gastrointestinal tract. In the high dose experiments the majority of the dose (89.6-100.5%) was excreted in faeces, while 5.5-10.3% was excreted in urine for a renal:faecal ratio of approximately 1:10-1:16. Approximately 36-38% of the administered dose was excreted as unmetabolised parent compound in the faeces and was therefore considered not to be absorbed. Thus, about 57.4-75.1% of the administered dose has been absorbed via the gastrointestinal tract.

Less than 1% ( low dose experiments) or less than 0.1% (high dose experiments) of the administered dose remained in the carcass and tissues at sacrifice with the highest amounts in the gastrointestinal tract and the liver, followed by the skin.

Various metabolites in addition to the unmetabolised parent compound have been identified, namely the carboxylic acid derivative, the dihydroxy derivative, the carboxylic acid glucuronic acid conjugate, the 3-hydroxyindane acid derivative, the 3-hydroxyindane acid epimer, the 3-ketoindane acid derivative, the 3-ketohydroxymethyl derivative, the diaminotriazine derivative and the hydroxethyl acid derivative.

Metabolic degradation of the test compound is rapid. In all experiments, 49 to 59% of the administered dose was excreted as the carboxylic acid derivative, indicating that oxidation is the main route of metabolism for the parent compound. A variety of other oxidation products were also detected, along with glucuronic acid conjugation.

Plasma curve experiments reveal a similar general curve shape for males and females, with rapid uptake and clearance from the blood. Females appear to absorb slightly more material compared to males. Blood residues smoothly fall off in all dose groups indicating an absence of re-uptake.

 

Overall, with regard to oral absorption, results of both toxicokinetic studies demonstrate a rapid and complete uptake of the test substance at low doses (> 90%) while, in contrast, at high doses the oral absorption is incomplete (57 – 75%). Plasma curve experiments after a single oral administration of a low dose reveal a similar general curve shape for males and females, with rapid uptake in the blood. Females appear to absorb slightly more material compared to males. Blood residues smoothly fall off in all dose groups indicating an absence of re-uptake.

Distribution of the test substance is generally low with < 1 up to 4.9% recovered in the carcass and tissues at sacrifice with the highest amounts in the gastrointestinal tract and the liver, followed by skin and thyroid gland after administration of a low dose. Compared to the low dose lower amounts of the test substance remained in the carcass and tissues after administration of a high dose (< 0.1%) with the highest amounts in the liver.

Metabolism is rapid and occurred mainly via oxidation pathways as demonstrated by identification of the carboxylic acid derivative as the main metabolite (38 to 66%) in all experiments. At low doses carboxylic acid derivatives were identified at similar amounts in urine and faeces while at high doses this metabolite was present mainly in the faeces. Additionally, a variety of other oxidation products and glucuronic acid conjugates were identified.

Excretion is demonstrated to be rapid at all dose groups. The majority of the absorbed dose is excreted within 24 hours. With respect to the low dose 40 -50% of the orally administered dose is excreted in the faeces via the bile and the same amount was excreted in the urine resulting in a renal:faecal ratio of approximately 1:1. Approximately, 5 – 10% of the administered high dose is excreted via urine while 90 – 100% is excreted via faeces (including 36 – 38% as unmetabolised parent compound) resulting in a renal:faecal ratio of approximately 1:10 – 1:16. Thus, similar amounts of a low dose are excreted via urine and faeces while this ratio shifts towards the faecal excretion with a high dose. Plasma curve experiments reveal a rapid clearance from the blood in males and females.

Discussion on dermal absorption rate:

In the available in vitro study according to OECD TG 428 the dermal absorption of human and rat skin was compared in a flow-through diffusion cell system (Rascle, 2007). Dermatomed human abdominal skin and dorsal rat skin was applied with 0.5, 2, 10 and 5000 µg/cm² of the radiolabeled test substance. Eight hours after application the test substance remnants were removed, and the penetration over the skin samples monitored in hourly intervals from application until 24 hours posttreatment. After 24 hours tape strippings were performed, and the radioactivity in the surface compartment, the skin compartment and the receptor cell determined. 

The amounts of absorbed radioactivity were 0.13%, 2.31%, 1.37% and 5.71% of the applied doses of 5000, 10, 2 and 0.5 µg/cm² via human skin, and 1.39%, 18.58%, 8.5% and 21.51% via rat skin, with the highest amounts located at the dose site within the skin compartment according to the study report. Following re-evaluation according to EFSA Guidance on dermal absorption (2017) the amounts of absorbed radioactivity were 12%, 4.1%, 14% and 2.4% of the applied doses of 5000, 10, 2 and 0.5 µg/cm² via human skin as well as 14%, 39%, 13% and 32% via rat skin.

It is demonstrated that the penetrability of human skin is comparable to that of rat skin at the highest dose. Thus, based on these values no conversion factor for absorption of human vs rat skin was derived.

 

Additionally, an in vivo study performed according to OECD TG 427 is available for the test substance (Blanck, 2008). The rate and extent of the dermal absorption after a single 8-hour application of the radiolabeled test substance at 5000, 2 and 0.5 µg/cm² to male Wistar rats were investigated in this study. Four groups of 4 animals were treated for each treatment level. Following the 8-hour exposure period, the remaining dose was washed off the skin. Animals of the 4 groups were sacrificed after different post-exposure periods (8, 24, 72 and 168 hours post-application). The tape stripping procedure was included in order to determine the distribution of the radioactivity through the skin.

Mean total recoveries of radioactivity were in the range of 90.6% to 107.7% of the applied dose.

The majority of the radioactivity was removed by swabbing and by removal of the surface dose (first two tape strips). The mean proportion of the applied dose considered to be non-absorbed ranged between 62.3 to 90.0% of the applied dose.

With regard to the highest dose level (5000 µg/cm²) mean amounts of potentially absorbable radioactivity (i.e. systemic compartment and washed skin compartment) at 8, 24, 72 and 168 hours post-application were 5.268, 3.025 and 2.403%, respectively. Corresponding mean potentially absorbable radioactivity of the intermediate dose level (2 µg/cm²) were 21.254, 18.980, 11.451 and 14.866%. In animals of the low dose groups (0.5 µg/cm²) mean potentially absorbable radioactivity was determined to be 42.659, 28.687, 30.208 and 23.467% at 8, 24, 72 and 168 h post-application. Following re-evaluation according to EFSA Guidance on dermal absorption (2017) the amounts of potentially absorbable radioactivity were 20, 12, 17 and 15% for the highest dose group, 27, 28, 22 and 32% for the intermediate dose group as well as 48, 41, 38 and 29% for the lowest dose group at 8, 24, 72 and 168 h post-application, respectively. 

Overall, it is demonstrated that the penetrability of human skin in vitro is comparable to that of rat skin in vitro at the highest dose. Thus, based on these values no conversion factor for absorption of human vs rat skin was derived.