Registration Dossier

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 03 May 2011 to 08 Oct 2012
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Deviations:
yes
Remarks:
Although the acceptance criteria for recovery could not be achieved (0.85% up to 100% vs. a target of 100% +/- 10%), the determined bioavailable portion was stated acceptable and this deviation had no detrimental influence on the outcome of the study.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Radiolabelling:
no

Administration / exposure

Doses:
Rhodiasolv Iris was applied undiluted and as a 50:50 v/v dilution in corn oil
Details on study design:
APPLICATION OF DOSE: Rhodiasolv Iris (undilated or diluted) was applied on each skin sample, left on the skin for 24 hours and then washed off with Butylacetate.

VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil was used to dilute the registered substance
- Amount(s) applied (volume or weight with unit): 10 µL
- Concentration (if solution): pure or 50:50 v/v in corn oil
- Lot/batch no. (if required): not documented
- Purity: not documented

SAMPLE PREPARATION
- Storage procedure: The samples were stored in tightly closed vesses at about -20°C until analysis
- Preparation details:

ANALYSIS
- Method type(s) for identification: The samples were analysed by gas chromatography coupled to a mass spectrometer
- Validation of analytical procedure: yes
- Limits of detection and quantification: 0. 142 µg/mL

Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin:Human skin (from 5 donors) was provided by Biopredic, Rennes (France)
- Ethical approval if human skin: not documented
- Type of skin: Whole skin (from abdomen), free of any adipose tissue
- Preparative technique: the skin was provided dermatomized
- Thickness of skin (in mm): 0.390 to 0.501 mm
- Membrane integrity check: yes, by measuring the impedance
- Storage conditions: frozen until use
- Justification of species, anatomical site and preparative technique: not documented

PRINCIPLES OF ASSAY
- Diffusion cell: the skin samples were mounted on flow-through diffusion chambers with a diameter of 1. 135 cm
- Receptor fluid: PBS (Phosphate buffered saline)
- Solubility of test substance in receptor fluid: yes
- Static system: no
- Flow-through system: yes. The receptor solution was pumped with a flow rate of 0.8 - 1.1 mL/h
- Test temperature: 32°C +/- 1°C
- Humidity: not documented
- Occlusion: no
- Reference substance(s): Bezoic acid and 2-Ethylhexyl trans-4-methoxycinnamate are used as positive and negative control substances (checks regularly performed at least once a year in the laboratory)
Other:
- Extraction solution: butylacetate
- Skin integrity: The integrity of the skin was demonstrated prior to application and only skin samples within the acceptable range of < or = to 9000 µS/cm were used. In addition, no major impairment of the oskin layer was detectable after incubation with the test item

Results and discussion

Absorption in different matrices:
The quantity and percentages of test item (undiluted and diluted) in the different fractions are summarized in table 7.1.2.1 below.
Total recovery:
For undiluted test item, the recoveries ranged from 0.85% upt to 100%. (see summary table 7.1.2.1)
For the 50:50 diluted test item, the recoveries ranged from 39.2% to 90% (see summary table 7.1.2.1).

- Limit of detection (LOD): 0.142 µg/mL (limit of the lowest calibration sample)
- Quantification of values below LOD or LOQ: The corrected concentration for samples below the LLOQ was 0.142 µg/mL
Percutaneous absorptionopen allclose all
Dose:
undiluted
Parameter:
percentage
Absorption:
1.1 %
Remarks on result:
other: 24 hours
Dose:
50:50 diluted
Parameter:
percentage
Absorption:
1.8 %
Remarks on result:
other: 24 hours

Any other information on results incl. tables

Rhodiasolv Iris was detected in all fractions relevant for dermal absorption, which are the extract of the remaining epidermis and dermis and in the receptor fluid samples. The details are provided in the following summary table:

Table 7.1.2.1: Summary of the amounts of Rhodiasolv Iris (undiluted or diluted) in the different fractions

Rhodiasolv Iris undiluted

Amount of Rhodiasolv Iris in

Expressed asμg/cm2of skin surface mean ± S.D. (n = 12)

Expressed as % of dose mean ± S.D. (n = 12)

Amount applied

8641 ± 1007

100.00 ± 11.65

Receptor fluid

92.9 ± 42.1

1.11 ± 0.56

Outer region of the skin and extracts from the strips

2.31 ± 3.39

0.03 ± 0.03

Remaining Epidermis (after 24 hours)

0.339 ± 0.0648

0.00 ± 0.00

Dermis

0.420 ± 0.167

0.00 ± 0.00

Washing and SN solution (after 24 hours)

3915 ± 3486

46.0 ± 40.81

Recovery

4011 ± 3483

47.1 ± 40.84

Bioavailable portion(receptor fluid + epidermis + dermis)

93.6 ± 42.0

1.12 ± 0.56

Rhodiasolv Iris/corn oil (50:50 v/v)

Amount of Rhodiasolv Iris in

Expressed asμg/cm2of skin surface mean ± S.D. (n = 12)

Expressed as % of dose mean ± S.D. (n = 12)

Amount applied

4622 ± 234

100.00 ± 5.06

Receptor fluid

83.7 ± 46.6

1.78 ± 0.94

Outer region of the skin and extracts from the strips

0.622 ± 0.570

0.01 ± 0.01

Remaining Epidermis (after 24 hours)

0.313 ± 0.0643

0.01 ± 0.00

Dermis

0.513 ± 0.270

0.01 ± 0.01

Washing and SN solution (after 24 hours)

3197 ± 650

68.9 ± 12.51

Recovery

3282 ± 666

70.8 ± 12.71

Bioavailable portion(receptor fluid + epidermis + dermis)

84.5 ± 46.9

1.80 ± 0.94

SN = Solution for conductivity measurement

SD= standard deviation

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions of this study, it can be stated that 93.6 μg/cm² (1.1 +/- 0.6 % of the applied dose) of undiluted Rhodiasolv Iris and 84.5 μg/cm² (1.8 +/- 0.9 % of the applied dose) of diluted Rhodiasolv Iris (50:50 v/v in corn oil) had penetrated the skin and are considered as bioavailable portion.

According to the OECD Guidance notes on Dermal Absorption No. 156, when the variability is high between replicates (i.e S.D >25% of the mean) and group size is > 4 replicates, the addition of a standard deviation to the mean value for absorption is recommended when conducting the risk assessment to be reasonably conservative. In the current study, this would correspond to dermal absorption rounded values of 2% for undiluted Rhodiasolv Iris and 3% for 50:50 diluted Rhodiasolv Iris.
Executive summary:

The test substance Rhodiasolv Iris was investigatedin vitroon its absorption and penetration properties on human skin, either undiluted or 50:50 v/v diluted in corn oil, according to OECD guideline 428.

 

Methods

Two experiments, including 6 replicates each, were performed for each test item under dynamic non-occluded conditions. Thus each test item was tested in 12 replicates.

For each test item (undiluted and diluted), the human skin samples were obtained from five different donors. They were stored frozen until use. The thickness of the skin used was 390 - 501 µm. The blank samples (at 0 hours) were collected immediately after filling the donor chambers at the maximal flow rate of the pump prior to application of the test items. The conductivity across the skin samples of each chamber was determined before treatment and after the last sampling as a measure of skin integrity. The integrity of the skin was demonstrated prior to application and only skin samples within the acceptable range of ≤900μS/cm were used. In addition, no major impairment on the skin layer was detectable after incubation with the test item.

10μL of each test item were applied on each skin sample, left on the skin for 24 hours and then washed off with Butylacetate (extraction solution).

The receptor solution was slowly pumped through the receptor chambers with a flow rate of 0.8 – 1.1 mL/h per hour and fractionated 0.5, 1.0, 2.0, 4.0, 8.0, 12, 16, 23 and 24 hours following the application of the respective test item.

Thestratum corneumwas removed from the skin by stripping 10 times, if possible, and extracted with extraction solution. The epidermis was separated from the dermis using heat separation or only forceps. Both skin compartments were extracted with extraction solution. Analysis for the presence of Rhodiasolv Iris was carried out by means of gas chromatography coupled to a mass spectrometer.

 

Results

The majority of the chambers did not meet the acceptance criteria (100 % 10 %) regarding the mass balance (recovery).

For the undiluted test item, the recoveries ranged from 0.850 % up to 100 %. In non-GLP experiments (data not shown) we could figure out that this was caused mainly by evaporation or dry-out effects. These effects were observed late in the incubation period of 24 h and depend on the microclimate of humidity and air-flow on the surface of the corresponding skin sample. Closing the donor chamber with parafilm gave no better results than the open chamber, in contrast the recovery was lower (mean of 76 % compared to mean of 104 %, n=3; data not shown). Since no possibility was found to control these effects, all chambers were used for evaluation. Despite the variability of the recovery, the penetrated amount (corresponding to the bioavailable portion) could be determined and was found to range only from 23.2μg/cm² up to 169μg/cm² (i.e 0.241 to 2.20 % of the applied dose).

For the diluted test item the evaporation process was not so dominant and especially not so variable most probably due to the corn oil content. But still the recoveries did not meet the acceptance criteria and the values ranged from 39.2 % up to 90.0 %. Again, despite the variability of the recovery, the penetrated amount was less variable with values ranging from 15.0μg/cm² up to 180μg/cm² (i.e 0.34 to 3.71 % of the applied dose).

The mean penetrated amounts were similar, independently from the applied dose (93.6μg/cm² and 84.5μg/cm² for undiluted and diluted test item, respectively), thus indicating that the maximal penetration flux was achieved in both conditions (undiluted or 50:50 v/v diluted). Therefore the results of this study were considered valid to calculate the penetration potential of Rhodiasolv Iris.

 

Conclusion

Under the experimental conditions of this study, it can be stated that 93.6μg/cm² (1.1+/- 0.6% of the applied dose) of undiluted Rhodiasolv Iris and 84.5μg/cm² (1.8+/-0.9 % of the applied dose) of diluted Rhodiasolv Iris (50:50 v/v in corn oil) had penetrated the skin and are considered as bioavailable portion.

According to the OECD Guidance notes on Dermal Absorption No. 156, when the variability is high between replicates (i.e S.D >25% of the mean) and group size is > 4 replicates, the addition of a standard deviation to the mean value for absorption is recommended when conducting the risk assessment to be reasonably conservative. In the current study, this would correspond to dermal absorption rounded values of 2% for undiluted Rhodiasolv Iris and 3% for 50:50 diluted Rhodiasolv Iris.