Registration Dossier

Administrative data

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:
other: Guideline study conducted according to GLP.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 427 (Skin Absorption: In Vivo Method)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Specific activity: 4.59 GBq/mmol; 124 mCi/mmol
Physical state: Liquid
Radiochemical purity: 96.0 - 99.8%
Solubility (20°C): Water: 0.5 +/- 0.7 µg/I; Methanol: >50 g/ml
The test material was stored below -15°C in the dark.

Non-radiolabelled test substance (supplied by Sponsor)
Purity: 98.8%
Storage: At ambient temperature in the dark.
Expiry date: 31 December 2010

Radiolabelling:
yes

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals and environmental conditions:
Strain as stated in the report: CD-l (albino) [Crl:CD1(ICR)]
Sex: Male
Bodyweight at dosing: 25 - 40 g
Age at dosing: 6-8 weeks
Supplier: Charles River UK Ltd., Margate, Kent, UK
Acclimatization: at least 5 days before the intended date of dosing.
Housing: During acclimatization animals were housed in groups of up to four mice in solid bottomed plastic cages.
After dosing, the animals were housed in sub-groups of 3 in glass metabolism cages (Metabowls®).
Diet ad libitum: VRFl diet manufactured by Special Diet Services
Water ad libitum: tap water

ENVIRONMENTAL CONDITIONS
Room temperature: 21 +/- 2°C.
Relative humidity: 55 - 15%.
Light/dark cycle: alternating 12 hour light/dark.
Air changes: approximately 15 per hour.

Administration / exposure

Type of coverage:
occlusive
Vehicle:
polyethylene glycol
Duration of exposure:
Animals were treated for 6 h.
Doses:
0.5 mg/cm2, corresponding to 1mg/animal.
No. of animals per group:
12 males
Control animals:
no
Details on study design:
RATIONALE FOR THE SELECTION OF THE DOSE ROUTE AND LEVEL
The dermal route was chosen as this is a expected route for human exposure to the test substance. Dose levels were selected upon worst case calculation for dermal exposure of worker.

DOSE PREPARATION AND PRUITY CHECK
14C-labeled test substance was stored in acetonitrile at a concentration of 495.91 µCi/ml (1.58 mg/ml) at or below -15°C. Dose formulations were freshly prepared on the day of administration.
The solvent of 3.2 mg 14C-labeled test substance-Stock solution (1005 µCi) was evaporated and the residue was re-suspended in PEG. Homogeneity of the formulation was kept by stirring at 50°C. Aliquots of the formulation were taken throughout the dosing procedure and pipette tips used for application were washed to accurately determine the applied dose. Purity checks of the dosing formulation were carried out and were >95%.

DETAILS ON EXPOSURE
24 h prior dosing the dorsal skin was clipped free of hair and cleaned with solvent. The dose formulation (0.025 ml) was applied to 2 cm2 of the dorsal skin of lightly anesthetized animals. The treated area was covered by foil and animals were allowed to recover from the anesthesia. The treated area was washed with cotton wool swabs soaked in 1% Tween 80 in distilled water 6 h after treatment. The application site was re-covered with foil to avoid oral exposure and subgroups of three mice were transferred to glass metabolism cages.

EXAMINATIONS
Urine was collected at time intervals of 0 - 6, 6 - 24 h, and subsequently at intervals of 24 h up to sacrifice after 168 hours into cooled receivers. Prior to dosing, pre-dose urine samples were collected for use as backgrounds.
Feces were collected separately into cooled receivers at 24 hour intervals up to 168 hours. Cage washes were performed every 24 h throughout the sample collection period and retained for radioactivity measurement.
Upon sacrifice the gastrointestinal tract (plus contents) was removed. The radioactivity of the carcass, gastrointestinal tract and feces was assessed after homogenization of the respective fraction.
In addition, the treated skin area was dissected and tape stripped to remove the stratum corneum. The initial first two tape strips were collected separately and represented the non-absorbed dose. Subsequent tape strips (3-8) containing the stratum corneum were retained and analyzed in groups of three. After tape stripping, the residual skin membranes and the tape strips were seperately solubilised until digestion was complete (7 days). The weight of the digest was measured and duplicate weighed aliquots were taken for radioassay.
The first 3 swabs used to remove non absorbed test substance after 6 h were pooled for each group of 3 mice and were extracted with methanol by sonication followed by centrifugation. The supernatant was decanted, weighed and duplicate samples were taken for radioassay. The above procedure was repeated twice more.
The protective dressings and foils were pooled for each group of 3 mice and extracted and analyzed as described above.
All samples not analyzed immediately after collection were placed into a freezer at <-15°C until analyzed.

Measurement of radioactivity
Radioactivity was measured by liquid scintillation counting (LSC) with automatic quench correction. Radioactivity below twice the background level was considered to be below the limit of detection. Aliquots of liquid samples were mixed with Ultima Gold scintillator (Packard BioScience) or Monoflow 4 for measurement of radioactivity.
Solid samples were combusted in oxygen using a sample oxidizer. The combustion products were absorbed into CO2 sorbent and mixed with Permafluor E+ scintillator. The efficiency of the oxidizer was found to be above than 95%. Measurements of radioactivity were corrected for oxidizer efficiency.

Results and discussion

Total recovery:
The overall mean recovery of radioactivity was 99.01% of the applied radioactivity. Mean tissue concentrations of radioactivity in carcass, gastrointestinal tract and treated skin corresponded to 2.26%, 0.03% and 0.07% of applied dose.
Percutaneous absorption
Dose:
0.5 mg/cm2
Parameter:
percentage
Absorption:
4.42 %
Remarks on result:
other: 6 h
Remarks:
The absorbable fraction of test substance was calculated as the sum of excreta, tissues, treated skin after stripping and stratum corneum in percent of applied radioactivity.

Any other information on results incl. tables

NON-ABSORBED RADIOACTIVITY

Following the topical application of 0.5 mg/cm2 14C-labeled test substance in PEG to mice under occlusive conditions, approximately 29% of the radioactivity applied was detected in the swabs used to clean the treated skin after 6 h. Another 65.4% of the applied radioactivity was extracted from the protective dressing.

The dose recovered from the treated skin (0.07%) and in the first two tape strips (0.27%) accounted for 0.34% of applied radioactivity. The mean total amount of non-absorbed (sum of the dose removed during the washing procedure and the dose remaining on the skin surface) accounted for 94.32% of applied radioactivity.

ABSORBED RADIOACTIVITY

Radioactivity detected in the stratum corneum corresponded to 0.26 % of the applied radioactivity. Radioactivity in urine, feces and cage washes accounted for 0.90%, 0.71% and 0.20% of applied radioactivity, respectively.

Radioactivity recovered in the gastrointestinal tract (including contents) accounted for 0.03% while 2.26% of applied radioactivity was detected in the carcass. The mean concentrations of radioactivity in carcass, treated skin, and gastrointestinal tract were 0.085, 0.021 and 0.006 µg equivalents/g wet weight, respectively.

The overall mean absorbable proportion of 14C-labelled test substance (sum of direct absorption [excreta, tissues], treated skin and stratum corneum) was 4.42% of applied radioactivity.

Table 1: Distribution of radioactivity detected after topical application of 14C-labelled test substance to male mice in percent of applied radioactive dose.

Sample

Time point/interval (hours after dosing)

6 h

168 h

0 - 168 h

NON-ABSORBD DOSE

Swabs

28.95 +/- 5.62

 

 

Dressing extracts

59.62 +/- 5.60

5.75 +/- 2.17

 

Dose on surface (Strips1,2)

 

0.27 +/-.0.09

 

TOTAL

 

94.59 +/- 2.50

 

DIRECT ABSORPTION (Tissues & Excreta)

Urine total

 

 

0.90 +/- 0.21

Feces total

 

 

0.71 +/- 0.22

Carcass

 

2.26 +/- 2.06

 

Gastrointestinal tract

 

0.03 +/- 0.02

 

Cage wash

 

0.20 +/- 0.03

 

TOTAL

 

 

4.09 +/- 2.33

ABSORBABLE

Skin (after stripping)

 

0.07 +/- 0.02

 

Stratum corneum

 

0.26 +/- 0.16

 

TOTAL ABSORBABLE

Direct Absorption + treated skin + Stratum corneum

 

 

4.42 +/- 2.40

OVERALL RECOVERY

Total

 

 

99.01 +/- 1.95

Applicant's summary and conclusion

Conclusions:
After topical application of 14C-labeled test substance to male CD-1 mice according to OECD guideline 427, the mean total absorbed dose was 4.42 % of the applied radioactivity. Mean tissue concentrations of radioactivity in carcass, gastrointestinal tract and treated skin corresponded to 2.26%, 0.03% and 0.07% of applied dose.
The mean total non-absorbed dose was 94.59 % of the applied radioactivity. The mean overall recovery of radioactivity was 99.01% applied radioactivity.