Registration Dossier

Administrative data

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: Guideline study and GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
SAT080033
Batch: Kn-Gi 9328-168
Radiolabelled TM:
SAT 060135 A
Batch: 05BLY097
Radiolabelling:
yes
Remarks:
14C

Test animals

Species:
other: dermatomed pig skin
Strain:
not specified
Sex:
not specified

Administration / exposure

Type of coverage:
open
Vehicle:
other: water or cream formulations with different couplers, mixed with hydrogen peroxide
Duration of exposure:
0.5 hours application time, afterwards test substance was rinsed off.
Sampling of the fluid in the receptor chamber was performed at intervals of 0.5, 1, 2, 4, 6, 12, 24 hours after application
Tape strips and measurement in the skin was performed at 24 hours after application.
Doses:
20mg/cm² (formulations) or 20μl/cm² (aqueous solution), each containing 4 % of the test material
resulting in 800 µg/cm² active substance
No. of animals per group:
Nine intact skin samples (three replicates per pig from three different animals).
Control animals:
no
Details on study design:
1. Dose preparation
Doses were prepared conforming to instructions supplied by the Sponsor. The amount of [14C]-KN 172 added to the preparations was sufficient to guarantee a limit of detection of <0.01% of the applied KN 172 (Section 4.4.5.1). Measurement of the pH of the final formulation preparations was not practicable, due to the small volume of the preparations. The amount of radioactivity in the preparations was confirmed by the analysis of triplicate samples using LSC. The doses were prepared as closeto the time of application as was practicable.

2. Preparation of KN 172 formulation with coupler system 1 with 7.5% H2O2-developer
135μl (equivalent approximately to 6.12mg KN 172) of the [14C]-KN 172 solution (TS00047/002) was added to 1g KN 172 formulation with coupler system 1 (TS00047/003) and mixed. To this mixture 1g developer with 7.5% hydrogen peroxide (TS00052/001) was added and mixed well. The preparation was analysed by LSC for achieved radioactive content and homogeneity. The final preparationcontained 40,802μg total KN 172 /g.

3. Preparation of KN-172 formulation with coupler system 2 with 7.5% H2O2- developer
135μl (equivalent to approximately 6.12mg KN 172) of the [14C]-KN 172 solution (TS00047/002) was added to 1g KN 172 formulation with coupler system 2 (TS00047/004) and mixed. To this mixture 1g developer with 7.5% hydrogen peroxide (TS00052/001) was added and mixed well. The preparation was analysed for achieved radioactive content and homogeneity. The final preparation contained 40,676μg total KN 172 /g.

4. Preparation of 4% KN-172 solution in water
135μl (equivalent to approximately 6.12mg KN 172) of the [14C]-KN 172 solution (TS00047/002) were added to 73.88mg unformulated KN 172 (TS00047/001) and were mixed with water to make up the total weight to 2000mg. The final aqueous solution contained 39,964μg total KN 172 /g.

5. Analytical techniques
5.1 Liquid scintillation counting conditions
Counting period: 6 minutes or to a 0.5% standard deviation of the count
Scintillation fluid: Optiphase ‘Hi-Safe’ 3
Model of LSC: Packard 3100 TR
The limit of quantitation for the receptor fluid using the above procedure was set at 0.006μg/ml (≡ 0.010μg/cm2 ≡ 0.0013%) for Coupler Systems 1 and 2 and 0.008μg/ml (≡ 0.014μg/cm2 ≡ 0.0018%) for the dilution.

6. Assembly of diffusion cells
The type of static glass diffusion cell used in this study fits to an exposed membrane area of 2.54 cm2 and has a receptor chamber volume of approximately 4.5 ml. Discs of approximately 3.3 cm diameter of prepared skin samples were mounted, dermal side down, in such diffusion cells held together with individually numbered clamps and placed in a water bath maintained at 32°C ± 1°C.

7. Measurement of membrane integrity
Membrane integrity was determined by measurement of the electrical resistance across the skin membrane. Skin samples with a measured resistance of <3kΩ (Davies et al, 2004) were regarded as having a lower integrity than normal and not used for exposure to the test materials, due to the possibility of compromised barrier function.

8. Selection of cells and dosing
Cells were selected such that each of the three applications was represented by nine intact skin samples, therefore three replicates per pig from three different animals. Samples from the same three pigs were used for each application.
The receptor chambers of the cells, containing small magnetic stirrer bars, were filled with a recorded volume of receptor fluid (de-gassed physiological saline) and placed in a water bath maintained at a temperature of 32ºC ± 1ºC. KN 172 as a trihydrochloride is suitably soluble in water and this choice of receptor fluid did not limit that the test substance could freely partition into the receptor fluid from the skin and never reached a concentration that would limit its diffusion.
A pre-treatment sample was taken from each receptor chamber for analysis by LSC. The volume reduction in the receptor chamber was immediately compensated by fresh de-gassed receptor fluid.
The achieved application rates used in this study were:
Applied substances Application rate of the final formulation/solution Total amount applied (exposed skin area 2.54cm2)
KN 172 + coupler system 1
+ H2O2 developer 20mg/cm2 ≡ 816μg KN 172/cm2 50.8mg (≡ 2.073mg KN 172)
KN 172 + coupler system 2
+ H2O2 developer 20mg/cm2 ≡ 814μg KN 172/cm2 50.8mg (≡ 2.068mg KN 172)
4% KN 172 solution in
water 20μl/cm2 ≡ 799μg KN 172/cm2 50.8μl (≡ 2.030mg KN 172)
The KN 172 formulations were applied by weight using a suitable positive displacement pipette. Prior to dosing each cell, the pipette and tip were tared. A volume of formulation, equivalent to the target amount applied, was drawn into the pipette and the exterior of the tip wiped clean. The entire pipette was weighed and the weight recorded. After dosing, the entire pipette was weighed again and the difference between the two weights being equal to the achieved applied dose weight. The dose was applied drop-wise over the skin to maximise coverage.
The 4% KN 172 solution was applied by volume, using a positive displacement pipette. The dose was applied drop-wise over the skin to maximise coverage.
The skin penetration in all three test runs was followed for 24 hours. The exposed skin area was left unoccluded throughout.

9. Sampling of receptor fluid
Samples of the receptor fluid (500μL) were taken from the receptor chambers of this static cell system at 0.5, 1, 2, 4, 6, 12, 24 hours after application. The receptor fluid in the chambers were stirred continuously and the volume of fluid in the receptor chamber was maintained by the replacement of a volume of fresh de-gassed receptor fluid, equal to the sample volume, after each sample had been taken.

10. Measurement of mass balance
After the 0.5h sample had been taken, the skin was washed by gently swabbing with a series of 3 natural sponges (approximately 1cm3) pre-wetted with 3% Teepol®L to terminate the exposure. Another 2 sponges, pre-wetted with water, were used to further swab the surface. All the sponges were combined and digested in Soluene 350®. The digests were made up to a recorded volume and a sample taken for analysis.
After the final receptor fluid sample had been taken at the end of the 24h exposure period for LSC analysis, the remaining fluid in the receptor chamber was discarded.
To remove residual receptor fluid from the under surface of the skin, the receptor chamber was again refilled with fresh receptor fluid, which was afterwards discarded. The donor chamber was carefully removed, washed with methanol and a sample of the washing taken for analysis.
The surface of the skin was washed again at 24h (final wash) as described above and the sponge digests analysed. The sponge swabs from this wash were digested in Soluene 350 and a sample of the digest analysed for radioactivity. The results of the analysis of the 0.5h wash, 24h wash and the donor chamber wash were added together and reported as a single value.
To assess the adsorbed amount, successive layers of the stratum corneum were removed by the repeated application of adhesive tape (Scotch 3M Magic Tape, 1.9cm wide), to a maximum of 10 strips (Ramsey et. al. (1994)). Tape stripping was discontinued when it was first observed that the epidermis was beginning to tear away from the underlying dermis. In such cases, the last strip taken was digested with the remaining skin to avoid underestimating residues in the remaining epidermis/dermis compartment. The total number of tape strips was recorded.
The surface of the skin was allowed to dry naturally. Strips of adhesive tape were sequentially pressed onto the skin surface and then carefully peeled off to remove layers of the stratum corneum. The adhesive strips were immersed in methanol to extract any test material. The first 5 strips were extracted individually and the remaining strips were combined into 2 groups (strips 6-8 and 9-10) and each group extracted. The extracts were sequentially numbered and a sample taken for analysis.
The skin was removed from the receptor chamber and the inside of the chamber washed with 2 x 5ml volumes of methanol to remove any penetrated KN 172 that may have adhered to the walls of the chamber. Samples of the wash were analysed and the amount of KN 172 in the wash added to the final amount penetrated.
The dosed area of the remaining skin (2.54cm²) was carefully cut away from the flange area, which has been held under the donor chamber, and digested in Soluene 350 and the digest analysed. The flange area was also digested in Soluene 350 and the digest analysed for inclusion in mass balance determinations.
Details on in vitro test system (if applicable):
Back/flank skin from 6 suckling pigs (aged 6 - 8 weeks) was obtained from an abattoir. Prior to them being scalded, samples of whole skin were excised from the trunk area of each animal and the samples clipped to remove excess hair. Skin was trimmed to a thickness of 0.8-1mm using an electric dermatome. Each skin sample was given an identifying number and stored frozen for 4 weeks, at approximately -20°C, on aluminium foil until required for use.
Intactness of skin was determined prior to use by measurement of membrane integrity via measurement of the electrical resistance across the skin membrane.

Results and discussion

Absorption in different matrices:
Donor chamber (washing 30 min + 24 h; not bioavailable)
- Coupler system 1: 98.1 %
- Coupler system 2: 102 %
- Aqueous solution: 101 %

Tape strips (adsorbed - not bioavailable)
- Coupler system 1: 0.076 %
- Coupler system 2: 0.102 %
- Aqueous solution: 0.026 %

Remaining epidermis/dermis (absorbed - bioavailable)
- Coupler system 1: 0.467 %
- Coupler system 2: 0.487 %
- Aqueous solution: 0.299 %

Receptor fluid + Receptor chamber wash (penetrated - bioavailable)
- Coupler system 1: 0.126 %
- Coupler system 2: 0.131 %
- Aqueous solution: 0.041 %

Total bioavailable
- Coupler system 1: 0.593 %
- Coupler system 2: 0.618 %
- Aqueous solution: 0.340 %
Total recovery:
Coupler system 1: 98.8 %
Coupler system 2: 103 %
Aqueous solution: 101 %
Percutaneous absorptionopen allclose all
Dose:
800 µg/cm²
Parameter:
percentage
Absorption:
0.6 %
Remarks on result:
other: 24 h
Remarks:
Coupler system 1; result referring to active substance
Dose:
800 µg/cm²
Parameter:
percentage
Absorption:
0.6 %
Remarks on result:
other: 24 h
Remarks:
Coupler system 2; result referring to active substance
Dose:
800 µg/cm²
Parameter:
percentage
Absorption:
0.3 %
Remarks on result:
other: 24 h
Remarks:
aqueous solution; result referring to active substance
Conversion factor human vs. animal skin:
no data

Applicant's summary and conclusion

Conclusions:
The results obtained in this study indicate that the bio-availability of KN 172 after dermal exposure under normal exposure conditions to these two coupler system formulations in combination with a hydrogen peroxide developer would be low.
The potential bio-availability of KN 172 was similar for both coupler system formulations.
Virtually the entire dose could be removed from the skin surface after 0.5 hours contact with the formulations by normal washing procedures.
From the formulations, a proportion of the dose remaining in the skin after washing at 0.5 hours continued to penetrate into the receptor fluid, however the penetration process was essentially complete after 6 hours.
Executive summary:

Study design

The percutaneous penetration/dermal absorption of KN 172 from two cream formulations with different couplers, mixed with hydrogen peroxide, and a solution of unformulated KN 172 in water were measured in vitro using dermatomed pig skin. The final applied KN 172 concentration was always 4%. The testing procedure, which employed a static diffusion cell design, conformed to the Regulatory

Guidelines given in Section 2.2. The doses were applied to the skin samples at a dose of 20mg/cm2 (formulations) or 20μl/cm2 (aqueous solution) and left unoccluded. The applications were washed off the skin surface after a 0.5h contact period, with the penetration of KN 172 through the membrane being assessed for 24h, by sampling the fluid in the receptor chamber at intervals of 0.5, 1, 2, 4, 6, 12, 24 hours after application. At the end of the experiment, the distribution of KN 172 in the test system was assessed, which included a tape stripping technique to determine its adsorption to the stratum corneum.

These applications were designed to simulate potential human dermal exposure to KN 172 during normal use.

The percutaneous penetration/dermal absorption was followed using [14C]-labelled KN 172, which was incorporated into the doses prior to application. The samples were analysed by liquid scintillation counting (LSC).

Results

For both coupler systems and the aqueous solution, the vast majority (>98%) of the applied KN 172 was washed from the surface of the skin. The total potentially bioavailable proportion of the applied dose was </=0.62% from the coupler systems and 0.34% from the aqueous solution. The mass balance was regarded as complete with between 98 - 103% of the applied dose being recovered. A summary of the mean results for each test preparation is provided above in the results and discussions section.

Conclusions

The results obtained in this study indicate that the bio-availability of KN 172 after dermal exposure under normal exposure conditions to these two coupler system formulations in combination with a hydrogen peroxide developer would be low.

The potential bio-availability of KN 172 was similar for both coupler system formulations.

Virtually the entire dose could be removed from the skin surface after 0.5 hours contact with the formulations by normal washing procedures.

From the formulations, a proportion of the dose remaining in the skin after washing at 0.5 hours continued to penetrate into the receptor fluid, however the penetration process was essentially complete after 6 hours.