Registration Dossier

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2005-08-17 to 2005-12-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test procedure in accordance with national standard methods

Data source

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

Materials and methods

Principles of method if other than guideline:
Percutaneous absortion and cutaneous disposition of [14C]-isophorone in vitro in human skin
(U.S. EPA final rule 40 CFR Parts 9 and 799: In vitro dermal absorption  rate testing of certain chemicals of interest to the 
Occupational Safety  and Health Administration)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
14C]-isophorone labeled in position 1.
Specific activity = 53.5 mCi/mmol, dissolved in acetonitrile at 0.3 mCi/ml
Radioactive purity >= 99 %, Lot No. 050425
Radiolabelling:
yes
Remarks:
[14C]-isophorone

Test animals

Species:
human
Sex:
male/female
Details on test animals and environmental conditions:
TEST SYSTEM
- Skin disks from abdominal region of three human cadavers
- Two disks per donor in each of three sets = six disks/set and six  disks/donor
- Two additional disks per donor for stability tests (see *** below)
- Removal of subcutaneous fat layer
- Cutting to a split thickness of 200 to 500 µm
- Barrier function test before exposure: < 0.3% penetration of tritiated  water in 20 minutes
- *** Stability tests: barrier function tests at 0 and 24 hours in the  presence of:   25 mg/ml unlabeled isophorone (1 disk/donor) and   5 % acetonitrile (1 disk/donor)

Administration / exposure

Type of coverage:
occlusive
Vehicle:
other: isopropyl myristate
Duration of exposure:
10 minutes (Set 1), 1 hour (Set 2), 24 hours (Set 3)
Doses:
Skin disks with acceptable barrier function were dosed with 200 µl of the [14C]-isophorone dosing solution on the epidermal surface of each skin
disk.
Details on study design:
EXPOSURE CONDITIONS
- Test substance preparation: Mixing with unlabelled isophorone in  isopropyl myristate to obtain: target concentrations of 25 mg/ml and 15 µCi/ml    (protocol: 25 µCi/ml), actual 10.9 and 9.88 µCi/ml   
Stability of dosing solutions confirmed by HPLC/radioactive detection
- Receptor fluid: balanced salt solution containing antibiotics and 6 %  polyethoxylate, pH 7.45-7.63
- Simultaneous exposure of several disks in Bronaugh flow-through  diffusion cells
- Exposure area approx. 63.6 mm2 (diameter 9 mm)
- Occlusive application of 200 µm dosing solution per disk
- Skin temperature during exposure: approx. 32 °C (30.5-35.0 °C)
SAMPLING
- Collection of receptor fluid (flow rate 1.66-2.19 ml/h) in one vial per disk   
Set 1: application time 10 minutes, receptor fluid collection at 10  minutes   
Set 2: application time 1 hour, receptor fluid collection at 1 hour   
Set 3: application time 24 hours, receptor fluid collection at 1, 2, 3,  4, 5, 6, 7, 8, 12, 16, 20, and 24 hours
- Washing of skin disks, separation into component layers stratum  corneum, epidermis, and dermis
- Digestion of skin layers in 1 ml of 1 N sodium hydroxide
ANALYSIS
- Addition of liquid scintillation cocktail to all samples (surface  washes, receptor fluid collections, skin layer digests, cotton 
swabs used  for cleaning operations)
- Placing in the dark for at least 24 hours at ambient temperature and  scintillation counting.
Details on in vitro test system (if applicable):
see "Details on study design"

Results and discussion

Signs and symptoms of toxicity:
not examined
Dermal irritation:
not examined
Absorption in different matrices:
SKIN INTEGRITY: 
< 0.3 % penetration were observed for all skin disks used  in the experiment with tritiated water. 
After 24 hours of incubation with  25 mg/ml isophorone or 5 % acetonitrile, permeability of water through  the skin disks had 
increased (from 0.114 % to 0.273 % for acetonitrile,  lower levels for isophorone) but was still below 0.3 %.

PERCUTANEOUS ABSORPTION
- Absorption rates:   
10 minutes: 2.04 +- 1.74 µg/cm2 = 12.2 +- 10.4 µg/cm2/h   
1 hour: 0.909 +- 0.836 µg/cm2 = 0.909 +- 0.836 µg/cm2/h   
8 hours: Steady state established. Permeability constant = 5.09E-4 cm/h  (+- 1.79E-4 cm/h) = moderate to fast penetrant.
- Total amount absorbed:   
10 minutes: 2.04 +- 1.74 µg/cm2    
1 hour:    5.14 +- 1.49 µg/cm2   
24 hours:    296 +- 97.1 µg/cm2
CUTANEOUS DISPOSITION  
 ---------------------------------------------------------------------   
Incubation   stratum corneum     epidermis          dermis  
---------------------------------------------------------------------   
10 minutes   0.886 +- 0.814    0.166 +- 0.161   0.457 +- 0.388 µg/cm2    
1 hour      0.683 +- 0.394    0.383 +- 0.240   1.02  +- 0.971 µg/cm2   
24 hours     4.16  +- 2.47     3.54  +- 1.02    7.20  +- 3.75  µg/cm2   
---------------------------------------------------------------------   
In general, the amount of isophorone in each skin layer increased with  increasing exposure time and appeared to accumulate into dermis more than  the epidermis.
Total recovery:
TOTAL RECOVERY (% of total applied dose)
- 10 minutes: 84.4 +- 5.70 (surface wash: 84.4 %)
-  1 hour:    82.6 +- 3.83 (surface wash: 82.6 %)
- 24 hours    78.1 +- 7.37 (surface wash: 74.3 %)   
Most of the applied dose was recoved in the surface wash at the end of the incubation time. The total amount absorbed was less than 5% even after
exposure for 24 hours.
Conversion factor human vs. animal skin:
not reported

Any other information on results incl. tables

no further remarks

Applicant's summary and conclusion

Conclusions:
The amount of isophorone dosed onto the skin disks appeared to simulate an infinite dose under the experimental conditions used. This is inferred
from the observations that the total amount absorbed was less than 5% even after exposure for 24 hours and that the amount of isophorone
absorbed into the receptor fluid reached a stady state.
The short-term absorption rates for isophorone were estimated to be 12.2 +/- 10.44 µg/cm2/hour (following 10 minutes exposure) and 0.909 +/- 0.836 µg/cm2/hour (following 1 hour exposure). The permeability constant (Kp) for isophorone was estimated to be 0.000509 +/- 0.000179 cm/
hour. The Kp value obtained indicates that isophorone is a moderate to fast penetrant based on the classification by Marzulli, et al.
The total amount of isophorone absorbed was determined to be 2.04 +/- 1.74, 5.14 +/- 1.49, and 296 +/- 97.1 µg/cm2, following exposure for 10 minutes, 1 hour, and 24 hours, respectively. The amount absorbed and the amount recovered from the wash fractions accounted for 84.4 +/- 5.70,
82.6 +/- 3.83, and 78.1 +/- 7.37 % of the total applied dose following exposure for 10 minutes, 1 hour, and 24 hours, respectively.
Executive summary:

The objectives of this study were to evaluate the rate and amount of [14C]-isophorone absorbed across human skin after in vitro exposure, to calculate the permeability constant, to determine two short-term absorption rates, and to evaluate the disposition of [14C]-isophorone in the various layers (stratum corneum, epidermis, and dermis) of human skin after in vitro exposure.

Three sets of incubation were conducted for each donor in this study. Human skin samples were incubated for 10 minutes (Set 1), 1 hour (Set 2), and 24 hours (Set 3) with the epidermal surface exposed to [14C]-isophorone in a Bronaugh flow-through diffusion cell under occluded conditions. The amount of [14C]-isophorone absorbed across the skin into the rceptor fluid and the disposition of [14C]-isophorone in the various skin layers following each incubation period were determined by liquid scintillation counting. Receptor fluid was collected during the 24 -hour incubation (Set 3) at multiple time points to facilitate calculation of a rate of absorption and a steady-state permeability constant.

The amount of isophorone dosed onto the skin disks appeared to simulate an infinite dose under the experimental conditions used. This is inferred from the observations that the total amount absorbed was less than 5% even after exposure for 24 hours and that the amount of isophorone absorbed into the receptor fluid reached a stady state.

The short-term absorption rates for isophorone were estimated to be 12.2 +/- 10.44 µg/cm2/hour (following 10 minutes exposure) and 0.909 +/- 0.836 µg/cm2/hour (following 1 hour exposure). The permeability constant (Kp) for isophorone was estimated to be 0.000509 +/- 0.000179 cm/hour. The Kp value obtained indicates that isophorone is a moderate to fast penetrant based on the classification by Marzulli, et al.

The total amount of isophorone absorbed was determined to be 2.04 +/- 1.74, 5.14 +/- 1.49, and 296 +/- 97.1 µg/cm2, following exposure for 10 minutes, 1 hour, and 24 hours, respectively. The amount absorbed and the amount recovered from the wash fractions accounted for 84.4 +/- 5.70, 82.6 +/- 3.83, and 78.1 +/- 7.37 % of the total applied dose following exposure for 10 minutes, 1 hour, and 24 hours, respectively.