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Toxicological information

Dermal absorption

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Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2012/06/06-2012/07/30
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study followed OECD 428 protocol with no major deviations and was conducted under GLP. Study is used in weight of evidence/read across approach

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:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): CASRN 74499-35-7
- Substance type: Pure active substance
- Physical state: liquid
- Analytical purity:100%
- Impurities (identity and concentrations): None reported.
- Lot/batch No.: TS07007
- Expiration date of the lot/batch: April 01, 2013
- Radiochemical purity (if radiolabelling): 59.3% (by HPLC, radiodetection)
- Specific activity (if radiolabelling): 8.66MBq/mg (234 uCi/mg)
- Locations of the label (if radiolabelling): ring-labelled
- Expiration date of radiochemical substance (if radiolabelling): N/A-purity checked at the beginning of the study.
- Stability under test conditions: stable
- Storage condition of test material: ambient temperature
Radiolabelling:
yes
Remarks:
14C-ring labelling

Test animals

Species:
other: human cadaver and male rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
in vitro study

Administration / exposure

Type of coverage:
other: permeable tape
Vehicle:
other: Chevron 100 Neutral oil
Duration of exposure:
24h total- perfusate was collected at 1 hr intervals from 0-6hrs and 2 hr intervals from 6-24hrs.
Doses:
- Nominal doses (mg/10 cm^2): A1=0.01; A2=0.1; A3=1.0
- Applied doses (μg/cell): A1=0.6; A2=6.9, A2a= 6.6; A3=65.4, A3a=61.1
- Concentration (μg/cm^3): 102.3, 1148.6, 1105.1, 10908, 10183.2
- Rationale for dose selection: Concentrations selected are based on realistic surrogates to real life exposure (per unit area of exposed skin-mg/cm^2) and expected absorption based on similar material. Due to the lower number of human skin cells with Kp values below the limit of 2.5x10^-3 cm/h at the mid and high dose, another experiment was set up to increase the number of human skin cells for both doses. The repeat doses were assigned A2a and A3a.
No. of animals per group:
in vitro study
Control animals:
yes
Remarks:
3 radioactive control doses for each dose level
Details on study design:
Pieces of the skin membranes (approximately 1.8 x 1.8 cm) were cut and mounted in the diffusion cells between the donor and receptor chamber,7 cells per species each. The cells were placed in the manifolds and connected to the peristaltic pump. For an equilibration period of 1 hour,
perfusate (1:9 ethanol water mix) was pumped through the receptor chamber at a flow rate of about 3 mL/h. Those cells with skin membranes of acceptable Kp values in the integrity test were arranged on the manifold and a 6 μL aliquot of the application solution was applied to the surface of the skin membranes. The donor chambers were covered by a permeable tape (non-occluded conditions). The perfusate was delivered at a flow rate of about 3 mL/h during the testing period and collected 15 intervals between 1-24 hrs. At 24h the surface of the skin membrane was rinsed with mild shower gel solution and tap water and then dried with a cotton swab.
Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source of skin: Male and female (5 donors aged 33-80) human cadaver full thickness skin was obtained from the "Institut fur Pathologie", Kantonsspital, Basel, Switzerland and from Biopredic International, Rennes, France. Male RccHan:WIST (SPF) rat full thickness skin (6 about 9 weeks old) were used.
- Type of skin: human upper leg dorsal; rat dorsal
- Preparative technique: The subcutaneous fat was carefully removed from the full thickness skin and pieces of about 4 x 5 cm were stretched evenly over a cork block, with stratum corneum uppermost. Skin sections of about 400 μm thickness were cut off from the top using a dermatome.
- Membrane integrity check: The integrity of the skin membranes was checked by applying 50 μL tritium water to the skin membrane surface. The permeability coefficient (Kp) of each skin membrane was calculated for the 3 - 6 hours interval. Rat skin membranes with Kp > 3.5·10^-3 cm/h and human skin membranes with Kp > 2.5·10^-3 cm/h were excluded from the subsequent experiment.
- Storage conditions: The skin membranes were wrapped in aluminum foil and stored refrigerated (+5°C) until use.
PRINCIPLES OF ASSAY
- Diffusion cell: PERMEGEAR, Inc., Riegelsville, PA, USA
- Receptor fluid: 1:9 solution of ethanol to water
- Flow-through system:Seven flow-through diffusion cells designated cell 1 through cell 7 were placed in one aluminum manifold connected to a water bath to maintain the temperature of the skin membranes at 31 ± 1°C. The diffusion cells of the second part of the system with the same setup were designated cell 8 through cell 14. Each diffusion cell consisted of a donor and receptor chamber. The area of skin membrane exposed to the donor chamber was 0.64 cm^2. The receptor chambers were connected to a multi-channel peristaltic pump, model IPC-166. The pump speed was adjusted to about 3 mL/h. During the given time intervals, the effluent from the cell was collected directly into vials on a fraction collector, model ISCO retriever IV7.

Results and discussion

Signs and symptoms of toxicity:
not examined
Remarks:
in vitro study
Dermal irritation:
not examined
Remarks:
in vitro study
Absorption in different matrices:
Rat skin membrane:
Within 24 hours, the portion of radiolabeled CASRN 74499-35-7 penetrating through rat skin membranes accounted for 1.01%, 1.10% and 0.92% of the low, mid and high dose, respectively. In the lower skin layers, 14.85%, 18.64% and 17.25% of the applied low, mid and high dose, respectively, were recovered (tape strips III – VII). Together with the amount measured in the perfusate and in the remaining skin membrane after tape stripping, the amount totally penetrated in/through rat skin membrane accounted for 42.08% of the low dose, 55.84% of the mid dose and 59.72% of the high dose. The total amount which penetrated within the period of 24 hours, accounted for 0.01 μg/cm2 at the low dose level, 0.12 μg/cm2 at the mid dose level and 0.94 μg/cm2 at the high dose level. The mean flux, which reflects the penetration rate under steady-state conditions, amounted to <0.001 μg/cm2/h at the low dose level (time interval for steady-state: 1 to 4-6 hours) and to 0.008 μg/cm2/h at the mid dose level (time interval for steady-state: 1 to 6 hours). At the high dose level, the mean flux was calculated to be 0.060 μg/cm2/h (time interval for steady-state: 1 to 6
hours).

Human skin membrane:
Within 24 hours the portion of radiolabeled CASRN 74499-35-7 penetrating through human skin membranes accounted for 0.75%, 0.68% and 0.65% of the low, mid and high dose, respectively. In the lower skin layers (tape strips III – VII), 2.67%, 1.73% and 2.03% of the applied low, mid and high dose, respectively, were recovered. Together with the amount measured in the perfusate and in the remaining skin membrane after tape stripping, the amount totally penetrated in/through human skin membrane accounted for 4.30% of the low dose, 3.03% of the mid dose and 3.09% of the high dose. The total amount which penetrated within this period, accounted for 0.01 μg/cm2 at the low dose level, 0.07 μg/cm2 at the mid dose level and 0.64 μg/cm2 at the high dose level. The mean flux, which reflects the penetration rate under steady-state conditions, amounted to < 0.001 μg/cm2/h at the low dose level (time interval for steady-state conditions: 1 to 6-8 hours) and to 0.005 μg/cm2/h at the mid dose level (time interval for steady-state conditions: 1 to 6 hours). At the high dose level, the mean flux was calculated to be 0.0412 μg/cm2/h (time interval for steady-state conditions: 1 to 6-8 hours).
Total recovery:
For rat skin membrane recovery was 95.89% of the dose for the low dose, 100.35% for the mid dose and 94.74% for the high dose. The total amount of absorbed radioactivity within 24 hours accounted for 42.08% of the low dose, 55.84% of the mid dose and 59.72% of the high dose.

For human skin membrane the recovery was 94.68% for the low dose, 103.15% for the mid dose and 100.73% for the high dose. The total amount of absorbed radioactivity within 24 hours accounted for 4.30% of the low dose, 3.03% of the mid dose and 3.09% of the high dose in human skin membrane.

Applicant's summary and conclusion

Conclusions:
CASRN 74499-35-7 penetrated through the skin membranes at the same rate and extent for both species, and penetrated in the skin membrane at much higher extent in rat than in human.
Executive summary:

The percutaneous penetration of CASRN 74499-35-7 was determined in vitro using split thickness skin membranes from rat and human skin. The skin membranes were set up in flow-through diffusion cells, the formulated [14C] CASRN 74499-35-7 was applied onto the skin membranes and the perfusates were collected at defined time intervals. Three nominal dose levels, i.e. 0.01 mg/10 cm2 (low dose), 0.1 mg/10 cm2 (middle dose) and 1 mg/10 cm2 (high dose) were applied to rat skin of Group Q1 and to human skin of Group Q2, respectively. The total amount of radioactivity recovered in the perfusate was slightly lower in human skin compare to rat skin and constant between doses (at around 1% in rat and 0.7% in human). The flux, at steady-state conditions, increased in a proportional manner with the dose and was found to be slightly lower in human than in rat with a human to rat ratio based on flux ranging between 1:1.5 and 1:1.8. Human to rat ratio based on % absorption shows a clear difference between the two species with rat absorption being 10 to 19-fold higher than human absorption.