Cyclopropanecarboxylic acid, [(3S,4R,4aR,6S,6aS,12R,12aS,12bS)-3-[(cyclopropylcarbonyl)oxy]-1,3,4,4a,5,6,6a,12,12a,12b-decahydro-6,12-dihydroxy-4,6a,12b-trimethyl-11-oxo-9-(3-pyridinyl)-2H,11H-naphtho[2,1-b]pyrano[3,4-e]pyran-4-yl]methyl ester

Dermal absorption

Administrative data

Endpoint:

dermal absorption in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From: 2014-07-07 To: 2015-09-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 427 (Skin Absorption: In Vivo Method)

Version / remarks:

2004

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.7600 (Dermal Penetration)

Version / remarks:

1998

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OECD Guidance Document No. 28 for the conduct of skin absorption studies

Version / remarks:

2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

[(3S,4R,4aR,6S,6aS,12R,12aS,12bS)-3-(cyclopropanecarbonyloxy)-6,12-dihydroxy-4,6a,12btrimethyl-11-oxo-9-(pyridin-3-yl)-1,2,3,4,4a,5,6,6a,12a,12b-decahydro-11H,12Hbenzo[f]pyrano[4,3-b]chromen-4-yl]methylcyclopropanecarboxylate

EC Number:

815-966-6

Cas Number:

915972-17-7

Molecular formula:

C33H39NO9

IUPAC Name:

[(3S,4R,4aR,6S,6aS,12R,12aS,12bS)-3-(cyclopropanecarbonyloxy)-6,12-dihydroxy-4,6a,12btrimethyl-11-oxo-9-(pyridin-3-yl)-1,2,3,4,4a,5,6,6a,12a,12b-decahydro-11H,12Hbenzo[f]pyrano[4,3-b]chromen-4-yl]methylcyclopropanecarboxylate

Test material form:

solid

Details on test material:

- Analytical purity: 96.1 %
- Appearance: Solid yellowish
- Batch: COD-001545

Specific details on test material used for the study:

RADIOLABLELED TEST MATERIAL
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 1055-0201

RADIOLABELLING INFORMATION
- Radiochemical purity: > 95 %
- Specific activity of AI: 3.7 MBq/mg
- Specific activity: 16.7 MBq/g test substance solution
- Locations of the label: pyranone-6-C14

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: freezer
- Homogeneity: given

FORMULATION CONCENTRATE
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: FD-140131-0011
- Composition: Formulation; active substance: nominal content : 50.0 g/L; analyzed content : 48.2 g/L
- Density: 1.025 g/cm3

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient
- Homogeneity: given
- Storage stability: expiry date: 2016-02-27
- Physical state / appearance: liquid/yellowish, clear

BLANK FORMULATION (WITHOUT TEST SUBSTANCE)
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: FD-140624-0028
- Density: 1.027 g/cm³

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient
- Homogeneity: given
- Storage stability: expiry date: 2016-02-27

NON-LABELED TEST MATERIAL
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: COD-001545
- Purity: 97.3 % (tolerance +/-1.0 %)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient, hygroscopic; keep away from humidity
- Homogeneity: given
- Storage stability: expiry date: 2016-01-31
- Physical state / appearance: solid/yellowish

STABILITY, HOMOGENEITY AND CONCENTRATION CONTROL ANALYSES OF THE TEST-SUBSTANCE PREPARATIONS
Stability of the testsubstance preparation: The stability of the test substance in the test-substance preparations over the test period was verified by analyses.
Concentration control and homogeneity analysis of the test-substance preparation: The concentration and the homogeneous distribution of the test substance in the test-substance preparations were confirmed by analyses.

See also Table 1 and 2.

Radiolabelling:

yes

Test animals

Species:

rat

Strain:

other: Crl: WI (Han)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, 97633 Sulzfeld, Germany
- Age at study initiation: about 10 weeks
- Weight at study initiation: about 290–365 g prior to dosing
- Housing: During acclimatization animals housed in groups (up to five animals) in Polysulfonate cages (H-Temp [PSU]) supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm2); during the experiment they were kept individually in all-glass metabolism cages type Metabowl (Jencons Leighton Buzzard, U.K.), labeled with the project number, animal number, dose and time of application.
- Diet: Kliba lab diet (mouse / rat “GLP”) meal. Origin:Provimi Kliba SA, 4303 Kaiseraugst, Switzerland ad libitum prior to and during the experiment
- Water: tap water ad libitum
- Acclimation period: at least 8 days before the beginning of the experimental phase; during the acclimatization period, the animals were accustomed to the environmental conditions of the study and to the diet

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24 °C
- Humidity: 30 – 70 %
- Air changes: 15 air changes per hour
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2014-07-07 To: 2014-12-04

Administration / exposure

Type of coverage:

semiocclusive

Remarks:

To protect the test area, a glass saddle was placed around the spacer and the whole application site was covered with a permeable (gauze) dressing and a semi occlusive adhesive bandage above

Vehicle:

other: Pure radiolabeled test substance was taken and was added to the blank formulation mixed with tap water at a ratio of 1:100 (v:v) and 1:200 (v:v) for the mid and low dose, respectively.

Duration of exposure:

Group A B C
Duration of exposure [h] 8 8 8
Skin wash after [h] 8 8+24 8+120
Skin strip after [h] 8 24 120
Sacrifice after [h] 8 24 120
Number of animals 4 4 4

Doses:

14C-test substance in the formulation concentrate was applied at three concentrations.

For the calculation of the dermal dose, it was considered that 100 μL of the test-substance preparation was applied to a total area of about 10 cm².

Dermal application was selected in order to simulate user specific exposure scenarios. According to the relevant guidelines, experiments shall be performed using doses expected in field exposure. Here, doses were selected to cover a broad range of potential exposure during use.

See also Table 3.

No. of animals per group:

4 animals/group

Control animals:

no

Details on study design:

TEST-SUBSTANCE PREPARATIONS
High dose:
To obtain the desired specific activity, a respective aliquot of the solution of the radiolabeled test substance was taken and the organic solvent (acetonitrile) was evaporated to dryness. An appropriate amount of the formulation concentrate was added to the dried residue. Based on the current data, the nominal concentration of the test substance in the formulation concentrate was 49.5 mg/g (corresponding to 50.8 mg/mL). The nominal specific activity in the test substance preparation was 9.8 MBq/g (corresponding to 10.0 MBq/mL). The applied mean radioactive dose per animal was 0.97 MBq.

Mid dose and low dose:
Due to the low concentration of active ingredient in the 1:100 (v:v) and 1:200 (v:v) aqueous spray dilutions, pure radiolabeled test substance was taken and was added to the blank formulation mixed with tap water at a ratio of 1:100 (v:v) and 1:200 (v:v) for the mid and low dose, respectively. Therefore appropriate amounts of the stock solution of the radiolabeled test substance were evaporated to dryness and then mixed with an appropriate amount of the previously prepared blank formulation mixed with tap water. The results of the measured radioactivity of the first run of the mid dose showed a declined amount of radioactivity in the spray dilution 1 with correlated inhomogeneity between samples of the test-substance preparation taken before and after application. Aditionally, recoveries were insufficient recoveries for analysed samples of this dose group. Therefore this experiment was repeated and the first preparation is not outlined in the report in detail. Based on the assumption that the inhomogeneity and incompete recovery was caused by absorption of the test substance to the glass surface, the surface of the glassware was inertisized by pretreatment with Dichlorodimethylsilane before it was used for the test-substance preparations of the low dose and the repetition of the mid dose. Since only radiolabeled test substance was used for both concentrations the amount of test substance was calculated by the measured amount of activity in the spray dilutions.

Based on the current data, the measured concentrations of the test substance in the formulation concentrate preparations were 0.48 mg/g (corresponding to 0.48 mg/mL) for the mid dose and 0.27 mg/g (corresponding to 0.27 g/mL) for the low dose. The measured specific activities in the test-substance preparations were 1.78 MBq/g (corresponding to 1.78 MBq/mL) and 1.0 MBq/g (corresponding to 1.0 MBq/mL) for the mid and the low dose, respectively.

The applied mean radioactive dose per animal was 0.2 MBq for the mid dose and 0.1 MBq for the low dose.

All preparations were stirred, the preparations of the mid and low dose additionally ultrasonicated in order to produce homogeneous preparation. At least before start and at the end of the application, samples were taken to determine the amount of radioactivity in the preparations and to demonstrate the correct concentrations of the test substance, its homogeneity and its radiochemical purity.

ANALYSES/MEASUREMENTS
Homogeneity/Concentration control and stability analyses
The amounts of radioactivity of 14C-test substance in the formulation concentrate preparations were determined in samples that were taken at least before and after the application at the laboratory for Biokinetics. The analyses of these samples allow to demonstrate the homogeneity, correctness of the concentration and the stability of the test substance in the test-substance preparation.

Stability in vehicle: The stability in the vehicle was investigated by Radio-HPLC and HPLC/UV.
Homogeneity and correctness of the concentrations in the vehicle: The homogeneity and correctness of the concentrations were verified by LSC and HPLC.

Methods of analysis
The analyses of the test-substance preparations were performed by HPLC (HP 1100 system and HP 1300 system) according to the following conditions:
Column: Luna C18, 250 mm x 3 mm
Eluent: A: HPLC water
B: acetonitrile
Flow: 0.5 mL/min, 50 % B, isocratic
Detection: UV-extinction 230 nm HPLC Radioactivity Monitor LB 509 (cell: YG-75)

Weight
The body weight was determined on the day of application prior to dosing. The weights of all samples were determined, for skin wash samples, application material and tape strips, weights were determined with the added amount of Soluene®-350 (Perkin Elmer).

Total radioactivity in biological material and samples used for washing and protecting the application site
Analyses and measurements were performed according to following work up procedures:
After weighing, aliquots of liquid samples (urine, plasma and cage wash) were mixed with scintillation cocktail (Hionic Fluor, Perkin Elmer) and analyzed for radioactivity without any additional treatment.
Soluene®-350 (Perkin Elmer) was added to blood cells. The samples were incubated at room temperature and isopropanol was added afterwards. Then the samples were bleached with perhydrol solution (30%). After further incubation Hionic Fluor cocktail (Perkin Elmer) was added and the samples were measured by LSC.
The samples of terminal skin wash (cotton swabs with washing solution), skin wash (cotton swabs with washing solution), application material after exposure (glass saddle, rubber ring and bandage), and after post-observation period (bandage) as well as tape strips were placed in appropriate containers and Soluene®-350 (Perkin Elmer) was added (100-200 g to the application material after exposure, 50-100 g to the application material after post-observation, 10-20 g to the tape strips, 15-30 g to the terminal skin wash and 40-60 g to the skin wash after exposure). The samples were extracted with Soluene®-350 (Perkin Elmer) for several days at room temperature. Then scintillation cocktail (Hionic Fluor, Perkin Elmer) was added to aliquots of the extracts and the samples were counted in a LSC.
Feces and carcass were suspended in ultrapure water and were homogenized using a WARING Blender. Aliquots of the suspensions were dried by lyophylisation, dissolved in Soluene®-350, filled up with isopropanol, bleached with perhydrol solution (30%) and were incubated afterwards. Hionic Fluor was added and the samples were measured by LSC.
Skin from the application site and surrounding skin was cut into three pieces each. The pieces were placed into scintillation vials and Soluene®-350 (Perkin Elmer) was added (3 to 4 mL). The samples were incubated and isopropanol was added afterwards. Then, bleaching was performed with perhydrol solution (30%) followed by incubation. Hionic Fluor cocktail (Perkin Elmer) was added and the samples were measured by LSC.

EXPERIMENTAL PROCEDURE
Application of the test material 24 hours before treatment, the back of the animals was thoroughly clipped and then the application site was washed with acetone. On the day of dosing, a spacer was glued to the skin with tissue glue. The test substance preparation was applied to the application site (dosing volume: 10 μL/cm²; treated area: about 10 cm²). The pipette used was weighed before and after application. To protect the test area, a glass saddle was placed around the spacer and the whole application site was covered with a permeable (gauze) dressing and a semi occlusive adhesive bandage above.

Balance/excretion
After treatment, animals were placed in metabolism cages. Excreta was collected over the respective complete exposure period (8 h) and – where appropriate – additionally after 24 and at intervals of 24 hours up to a maximum of 120 h.

Washing procedure:
After the 8 hour exposure period, the gauze and bandages as well as the rubber ring and the glas saddle were removed from the animals. The skin of the animals was washed with a mild soap solution [Texapon ® N 70 (Sodium-laurylethersulfate, Cognis, Germany)] and rinsed with water.
The washing procedure was:
- Cotton swabs dampened with water and liquid soap
- Area inside the glass saddle washed using a circular motion
- Cotton swabs dampened with water
- Area inside the glass saddle wiped using a circular motion
- The wiping was repeated several times with new swabs and finally the area was dried with at least one dry cotton swab
For animals with a post-observation period, a new gauze and bandage was applied after the 8 h skin wash and an additional skin wash (according to the 8 hours skin wash) was performed before sacrifice. The washing solutions and the gauze with bandage were collected separatly from those at the end of exposure for determination of residual radioactivity.

Tape stripping:
Scotch tape strips (stripping was performed once with two tape strips in parallel for the high dose (to strip the complete application area) and twice with two tape strips in parallel for the mid and low dose) were taken from the application site after the final surface wash to check for further easily removable test material. The tape strips were pooled and analyzed.

Mortality
A check for moribund and dead animals was conducted at least once daily.

Clinical signs
A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented for each animal.

Results and discussion

Signs and symptoms of toxicity:

no effects

Dermal irritation:

not specified

Absorption in different matrices:

Mean recoveries of radioactivity from all dose groups were in the range of 92.96 to 99.30 % of the total radioactivity applied. The largest proportion of radioactivity was recovered from the skin washes for all dose groups.

High dose:
At all sampling time points, the largest proportion of radioactivity was recovered from the skin wash. The mean radioactive recoveries in skin wash was 88.72% (sacrifice after 8 h), 82.72% (sacrifice after 24 h) and 81.54% (sacrifice after 120 h), respectively. Second skin washes contained 1.17 and 0.15% of the administered radioactivity for the dose groups being sacrificed after 24 and 120 h, respectively.
Mean radioactivity in the protective cover of the exposure period ranged from 1.42 – 9.61% of the radioactivity applied. Mean radioactivity in the protective cover of the post exposure periods were 4.88 (24 h) and 6.00% (120 h) of the radioactivity applied. In the animals sacrificed directly after the 8 h exposure period, the application site and the surrounding skin contained means of 2.07 and 0.82% of the applied radioactivity. For the animals with post-observation periods of 24 h and 120 h, the mean radioactivity at the application site amounted to 1.26% and 0.48% of the radioactivity applied, respectively. The amounts in the skin surrounding the application site were 0.76 and 0.27% of the radioactivity applied. Skin strips, performed with scotch tapes after sacrifice accounted for means of 0.25%, 0.07% and 0.03% of the applied radioactivity for the animals of the 8 h, 24 h and 120 h sacrifice group.
The mean amounts of radioactivity absorbed (including excreta, cage wash, carcass, blood cells and plasma) were 0.37%, 0.69% and 1.22% of the applied dose at 8 h, 24 h and 120 h after beginning of the 8 h exposure period, respectively. These results together with the amounts in the skin (application site and surrounding skin) and the values of the second skin wash imply that at this dose level less than 30% of the radioactivity remaining with the skin after end of exposure penetrates through the skin during the post-observation period. The radioactivity absorbed was excreted via urine and faeces with higher amounts in faeces at later time points. The highest tissue concentrations of radioactivity were found in carcass for the dose groups being sacrificed after 8 and 24 hours and in blood cells for the dose group being sacrificed after 120 hours. Individual samples of blood cells with activities below background (that was unusual high, with a value of 108.5 cpm) yielded a tissue concentration of formally 0.00 μg Eq/g. Correspondingly, this fact leads (for individual samples) to artificial low blood/plasma ratios. However, since the absolute values of background and the residues in these samples are low, the observed background variability is negligible and have no impact on the overall data assessment of the study.

Mid dose:
At all sampling time points, the largest proportion of radioactivity was recovered from the skin wash. The mean radioactive recoveries in skin wash were 79.47% (sacrifice after 8 h), 78.43% (sacrifice after 24 h) and 78.63% (sacrifice after 120 h), respectively. Second skin washes contained 2.18 and 0.97% of the administered radioactivity for the dose groups being sacrificed after 24 and 120 h, respectively.
Radioactivity in the protective cover of the exposure period ranged from 3.87 to 5.87% of the radioactivity applied. Mean radioactivity in the protective cover of the post exposure periods were 1.09 (24 h) and 3.99% of the radioactivity applied (120 h). In the animals sacrificed directly after the 8 h exposure period, the application site and the surrounding skin contained means of 9.74 and 0.12% of the applied radioactivity. For the animals with post-observation periods of 24 h and 120 h, the radioactivity at the application site amounted to 8.74% and 5.52% of the radioactivity applied, respectively. The mean amounts in the skin surrounding the application site were 0.10 (120 h) 0.17% of the radioactivity applied (24 h). Skin strips, performed with scotch tapes after sacrifice accounted for means of 0.23%, 0.16% and 0.19% of the applied radioactivity for the animals in the 8 h, 24 h and 120 h sacrifice group.
The mean amounts of radioactivity absorbed (including excreta, cage wash, carcass, blood cells and plasma) were 2.89%, 1.87% and 3.48% at 8 h, 24h and 120 h after beginning of the 8 h exposure period, respectively. These results together with the amounts in the skin (application site and surrounding skin) and the values of the second skin wash imply that at this dose level less than 15% of the radioactivity remaining on the skin after end of exposure penetrates through the skin during the post-observation period. The radioactivity absorbed was excreted via urine and feces with higher amounts in feces at later time points. The highest tissue concentrations of radioactivity were found the in blood cells.


Low dose:
At all sampling time points, the largest proportion of radioactivity was recovered from the skin wash. The mean radioactive recoveries in skin wash were 80.07% (sacrifice after 8 h), 73.49% (sacrifice after 24 h) and 74.81% (sacrifice after 120 h). Second skin washes contained means of 2.14 and 1.06% of the administered radioactivity for the dose groups being sacrificed after 24 and 120 h, respectively.
Mean radioactivity in the protective cover of the exposure period ranged from 1.38 to 5.43% of the radioactivity applied. Mean radioactivity in the protective cover of the post exposure period was 1.75 (24 h) and 4.49% of the radioactivity applied (120 h). In the animals sacrificed directly after the 8 h exposure period, the application site and the surrounding skin contained means of 10.45 and 0.12% of the applied radioactivity. For the animals with post-observation periods of 24 h and 120 h, the mean radioactivity at the application site amounted to 7.29% and 5.73% of the radioactivity applied, respectively. The mean amounts in the skin surrounding the application site were 0.07 (120 h) and 0.09% of the radioactivity applied (24 h). Skin strips, performed with scotch tapes after sacrifice accounted for means of 0.43%, 0.15% and 0.16% of the applied radioactivity for the animals in the 8 h, 24 h and 120 h sacrifice group.
The mean amounts of radioactivity absorbed (including excreta, cage wash, carcass, blood cells and plasma) were 4.25%, 4.67% and 5.55% at 8 h, 24 h and 120 h after beginning of the 8 h exposure period, respectively. These results together with the amounts in the skin (application site and surrounding skin) and the values of the second skin wash imply that at this dose level less than 15% of the radioactivity remaining on the skin after end of exposure penetrated through the skin during the post-observation period. The radioactivity absorbed was excreted via urine and feces with higher amounts in feces at later time points. The highest tissue concentrations of radioactivity were found in the blood cells.


See also Table 1 and Table 2.

Total recovery:

- Total recovery: 93.25 - 98.83 % (2.5 µg/cm²); 96.31 - 98.52 % (5.0 µg/cm²); 92.96 - 99.30 % (500 µg/cm²)
- Recovery of applied dose acceptable: Yes

Percutaneous absorption

Percutaneous absorption 1

Time point:

8 h

Dose:

500 µg/cm² [target dose]

Parameter:

percentage

Remarks:

mean

Absorption:

0.37 %

Percutaneous absorption 2

Time point:

24 h

Dose:

500 µg/cm² [target dose]

Parameter:

percentage

Remarks:

mean

Absorption:

0.69 %

Percutaneous absorption 3

Key result

Time point:

120 h

Dose:

500 µg/cm² [target dose]

Parameter:

percentage

Remarks:

mean

Absorption:

1.22 %

Percutaneous absorption 4

Time point:

8 h

Dose:

5.0 µg/cm² [target dose]

Parameter:

percentage

Remarks:

mean

Absorption:

2.89 %

Percutaneous absorption 5

Time point:

24 h

Dose:

5.0 µg/cm² [target dose]

Parameter:

percentage

Remarks:

mean

Absorption:

1.87 %

Percutaneous absorption 6

Key result

Time point:

120 h

Dose:

5.0 µg/cm² [target dose]

Parameter:

percentage

Remarks:

mean

Absorption:

3.48 %

Percutaneous absorption 7

Time point:

8 h

Dose:

2.5 µg/cm² [target dose]

Parameter:

percentage

Remarks:

mean

Absorption:

4.25 %

Percutaneous absorption 8

Time point:

24 h

Dose:

2.5 µg/cm² [target dose]

Parameter:

percentage

Remarks:

mean

Absorption:

4.67 %

Percutaneous absorption 9

Key result

Time point:

120 h

Dose:

2.5 µg/cm² [target dose]

Parameter:

percentage

Remarks:

mean

Absorption:

5.55 %

Conversion factor human vs. animal skin:

not applicable

Any other information on results incl. tables

Table 1: The mean percentages of absorbed dose

mean percentage and ± SD of                       ^14 C-test substance in test substance radioactivity absorbed
target dose [μg/cm²] exposure time [h]	sacrifice time [h]	500	5.0	2.5
8	8	0.37±0.11	2.89±2.19	4.25±1.65
8	24	0.69±0.45	1.87±0.68	4.67±0.60
8	120	1.22±0.31	3.48±1.09	5.55±1.08

Table 2: Mean excretion and retention of radioactivity after a single dermal application of ¹⁴C-BAS 440 I in BAS 440 01 I to rats at nominal dose levels of 500, 5.0 and 2.5 μg/cm ². Results expressed in % of dose administered

Target dose [μg/cm²]	500	500	500	5.0	5.0	5.0	2.5	2.5	2.5
Exposure time [h]	8	8	8	8	8	8	8	8	8
Sacrife time [h]	8	24	120	8	24	120	8	24	120
Dose admin. [mg/cm²]	0.49	0.49	0.49	0.0048	0.0048	0.0048	0.0026	0.0026	0.0026
Urine -8	0.02	0.02	0.02	0.11	0.07	0.12	0.15	0.15	0.17
Urine -24	---	0.05	0.04	---	0.07	0.09	---	0.12	0.18
Urine -48	---	---	0.03	---	---	0.05	---	---	0.06
Urine -72	---	---	0.02	---	---	0.02	---	---	0.04
Urine -96	---	---	0.01	---	---	0.01	---	---	0.02
Urine -120	---	---	0.01	---	---	0.01	---	---	0.02
Subtotal Urine	0.02	0.06	0.13	0.11	0.14	0.30	0.15	0.27	0.48
Feces -8	0.00	0.00	0.01	0.03	0.02	0.01	0.02	0.03	0.03
Feces -24	---	0.25	0.17	---	0.55	0.62	---	0.74	0.57
Feces -48	---	---	0.25	---	---	0.85	---	---	0.94
Feces -72	---	---	0.16	---	---	0.36	---	---	0.46
Feces -96	---	---	0.15	---	---	0.34	---	---	0.21
Feces -120	---	---	0.07	---	---	0.18	---	---	0.09
Subtotal Feces	0.00	0.25	0.81	0.03	0.57	2.34	0.02	0.77	2.29
Cage wash	0.00	0.01	0.03	0.00	0.00	0.11	0.00	0.00	0.00
Blood cells	0.00	0.00	0.01	0.08	0.05	0.05	0.13	0.08	0.06
Plasma	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
Carcass	0.35	0.36	0.25	2.67	1.12	0.68	3.95	3.56	2.72
Percentage absorbed	0.37	0.69	1.22	2.89	1.87	3.48	4.25	4.67	5.55
Surrounding skin	0.82	0.76	0.27	0.12	0.17	0.10	0.12	0.09	0.07
Protective cover expos.	4.44	1.42	9.61	3.87	5.87	5.16	3.52	5.43	1.38
Protective cover post expos.	n.s.	4.88	6.00	n.s.	1.09	3.99	n.s.	1.75	4.49
Application site	2.07	1.26	0.48	9.74	8.74	5.52	10.45	7.29	5.73
Skin wash	88.72	82.72	81.54	79.47	78.43	78.63	80.07	73.49	74.81
2nd skin wash	n.s.	1.17	0.15	n.s.	2.18	0.97	n.s.	2.14	1.06
Skin strip	0.25	0.07	0.03	0.23	0.16	0.19	0.43	0.15	0.16
Total	96.65	92.96	99.30	96.31	98.52	98.02	98.83	94.99	93.25