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Dermal absorption

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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:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to other study
Reference
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:
guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Principles of method if other than guideline:
Comparative study using rat and human skin specimens in a static diffusion-cell model
GLP compliance:
yes
Specific details on test material used for the study:
IDENTITY OF TEST MATERIAL
- Name of test material (as cited in study report): Creosote; AWPA P1-P13 Creosote (North American Creosote Composite Test Material)

RADIOLABELLING INFORMATION (if applicable)
- Radiochemicals: the test material used was spiked with eight 14-C radiolabelled PAH that are main constituents of the creosote (see below)
- Locations of the label: various (see below)
- Amount of radiochemicals: the radioactivity of each marker component added to creosote was related to the composition of the creosote in order to represent compound abundance in the mixture.
- Radiolabelled components (marker chemicals) and position of the radiolabel
- Naphthalene – Benzene – UL – 14C
- 2-Methylnaphthalene – 8 – 14C
- Biphenyl – UL – 14C
- Anthracene – 1,2,3,4,4A,9A – 14C
- Phenanthrene – 9 – 14C
- Fluoranthene – 3 – 14C
- Pyrene – 4,5,9,10 – 14C
- Benzo[a]pyrene – 7 – 14C
- Sum of marker chemicals (radiolabelled substances) in creosote: ~ 43 %
Radiolabelling:
yes
Species:
other: rat and human skin biopsies
Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Duration of exposure:
8 h
Doses:
- Nominal doses: 10 µL creosote/cm² (10.7 mg/cm²)
- Size of test site: 0.64 cm²
No. of animals per group:
Number of samples per group: 6 for either species, rat and humans
Control animals:
no
Details on study design:
DOSE PREPARATION
The radiolabelled materials (8 PAH) were dissolved in toluene and aliquots were combined such that the specific activity corresponded to the target level and the percentage of the different PAH was the same as in the creosote material. After evaporation of the sample to dryness by nitrogen convection, 5 mL of creosote was added and the sample was mixed and sonicated.

TEST SITE (SKIN SAMPLES) PREPARATION
Samples of rat and human skin that were stored frozen were allowed to thaw at room temperature. Full thickness skin was dermatomed to approx. 450 μm using a Padgett Electro Dermatome® (Padgett Instruments, Inc., Kansas City, MO). The skin sample was then placed onto an aluminium pan, with its code embossed or written on the pan, and stored refrigerated at 1 - 10 °C until readied for use.
Glass [static] diffusion cells (Report, Fig. 1) were used in this study. The skin membrane was first mounted, stratum corneum uppermost, onto the top of the receptor chamber, which was filled with receptor fluid (e.g. saline for equilibration with 50 % (v/v) ethanol in deionized water solution for test). The donor (top) chamber was then placed over the skin section and clamped in place. The in vitro diffusion cells had an exposure area of 0.64 cm².
The receptor fluid was continuously stirred throughout the exposure using a magnetic stir bar.
The integrity of each membrane was assessed by measurement of electrical impedance prior to application of test substance.
Following dosing, the donor chamber opening was occluded with Parafilm® for the duration of the exposure period.
Details on in vitro test system (if applicable):
RAT SKIN:
The shaved skin area was excised, placed on an aluminium pan with the ID written on the pan, held briefly on wet ice, and then frozen at approximately -20 °C until prepared for use.

HUMAN SKIN:
Samples of human cadaver skin from the National Disease Research Interchange (NDRI, Philadelphia, PA, USA), were stored frozen at approx. -20 °C until prepared for use.
Dermal irritation:
not specified
Absorption in different matrices:
Findings for rat skin:
After 8 hours, 15.1 % (SD ± 3.64 %) of the total dose applied (based on radioactivity measured) was absorbed (present in receptor fluid), and 19.2 % (SD ± 6.82 %) remained in/on the tape-stripped skin, amounting to a total absorbable dose of 34.3 % (SD ± 6.84 %). The total unabsorbed dose was 44.0 % (SD ± 5.98 %) (skin wash, donor chamber, tape strips)._____________________________
Findings for human skin:
After 8 hours, 3.38 % (SD ± 1.03 %) of the total dose applied (based on radioactivity measured) have been absorbed (present in receptor fluid), and 0.86 % (SD ± 0.26 %) remained in/on the tape-stripped skin, amounting to a total absorbable dose of 4.24 % (SD ± 1.07 %). The total unabsorbed dose was 79.7 % (SD ± 4.08 %) (skin wash, donor chamber, tape strips).
(see Report, Table 2, p. 19 and Attachment) _________________________
Total recovery:
Findings for rat skin: 78.3 % ± 2.44 %;
Findings for human skin: 83.9 % ± 3.68 %;
(see Report, Table 2, p. 19 and Attachment);
Mass balance:
The recoveries of the applied doses ranged from 78.3 % (rat skin) to 83.9 % (human skin). Although this was outside of the target boundary (100 % ± 10 %), it is plausible that chemical instability and subsequent volatilisation from the wash, from the skin section during tape-stripping, and/or from the tape strip sections prior to solvent extraction may have occurred. As both recoveries for human and rat experiments are close together and no particular losses of radioactivity that were different in either case could be suspected, it is assumed that unacceptable biases had been unlikely in any of the test assays.
Key result
Time point:
8 h
Dose:
10.7 mg/cm²
Parameter:
percentage
Absorption:
15.1 %
Remarks on result:
other: rat skin sample: absorbed dose
Key result
Time point:
8 h
Dose:
10.7 mg/cm²
Parameter:
percentage
Absorption:
3.4 %
Remarks on result:
other: human skin sample: absorbed dose
Key result
Time point:
8 h
Dose:
10.7 mg/cm²
Parameter:
percentage
Absorption:
34.3 %
Remarks on result:
other: rat skin sample: absorbable dose (including tape-stripped skin)
Key result
Time point:
8 h
Dose:
10.7 mg/cm²
Parameter:
percentage
Absorption:
4.2 %
Remarks on result:
other: human skin sample: absorbable dose (including tape-stripped skin)
Conversion factor human vs. animal skin:
Based on the ratio of total absorbable doses: 4.24 % (human) / 34.3 % (rat) = ~ 0.12.
Based on the ratio of absorbed doses (receptor fluid): 3.4 % (human) / 15.1 % (rat) = ~ 0.225.

Key observation of mean data(Report p. 13):

• Over the course of the 8-hour exposure period, radioactivity increased almost linearly over time, but penetrated through rat skin approx. 4.3-times faster (85.3 µg equiv/(cm²*h) than through human skin (19.7 µg equiv/(cm²*h) (see Attachment).

• Total penetration of radioactivity at the end of the exposure was 4.4-fold greater for rat skin (665.8 µg equiv/cm²) than for human skin (149.7 µg equiv/cm²): ==> Conversion factor 0.225

• The total absorbable dose, receptor fluid plus any dose remaining in the tape-stripped skin (excluding the stratum corneum), was 8-fold greater for rat skin (34.3 %) than for human skin (4.24 %): ==> Conversion factor 0.12

• Washing of the skin removed 12.8 % and 70.3 % of the applied creosote test substance from rat and human skin, respectively, which reflected the greater rate and extent of total penetration for rat skin.

• A significant portion of the unabsorbed dose for rat skin (44 %) was contained in the stratum corneum (23.6 %); a minor portion of the unabsorbed dose for human skin (79.7 %) was contained in the stratum corneum (5.33 %).

Note: The lower conversion factor human vs. rat of 0.12 is adopted which includes both, species-specific penetration kinetics through and binding capacities to the skin types.

Taking account of the in-vivo results in rats, which showed that dermal exposure to the same creosote dose for 8 hours resulted in a maximum absorbable portion of 14.8 % of the applied dose (see other study record: Fasano 2007a), it is expected that not more than 14.8 * 0.12 % = ca. 1.8 % of a dermal dose will be absorbed through human skin within and after 8 h of exposure.

Conclusions:
The total absorbable dose, receptor fluid plus any dose remaining in the tape-stripped skin, was 8-fold greater for rat skin (34.3 %) than for human skin (4.24 %) in vitro.
Executive summary:

In this comparative kinetic in-vitro study, the penetration of creosote through rat and human skin specimens as well as the adsorption of creosote equivalents after an 8 h exposure period were to be examined under identical conditions. The objective was to extrapolate from rat in-vivo results (see other summary: Fasano 2007a) to the human in-vivo situation by making use of the comparison of rat-human in-vitro data.

The test material was a complex mixture (US creosote P1-P13) that was spiked with radiolabelled key constituents of creosote: A set of 8 target chemicals had been selected, which overall amounted to about 43 % (w/w) of creosote. For detection, 14C-radio-labeled compounds were chosen as most sensitive indicators. The radioactivity of each compound was such as to properly reflect the composition of creosote.

An in-vitro static diffusion cell served as technical device, which had an effective diffusion area of 0.64 cm².

The procedure correspondeds to acknowledged methods, e.g. OECD guideline 428.

Results and discussion

The direct comparison of the migration kinetics of radiolabelled marker components applied in creosote into and through rat and human skin in vitro revealed significant quantitative differences between both species with respect to the passage rate as well as the adsorptive uptake into the skin layers: The mean rate of cross-transfer after 8 h was more than 4 times lower in human than in rat skin. Although the variation was considerable, in human specimens from ca. 100 - ca. 200 µg equiv/(cm²*h) at 8 h sampling time, in rat specimens from ca. 500 - ca. 820 µg equiv/(cm²*h), the difference was highly significant.

In the initial phase of 2 h, the rate through rat skin appears to be more marked than the initial phase in human skin. The ratio within that time interval was about 6.5 to 7.0 rather than 4.3 (see Report Fig. 2, p. 23).

This indicates that the entry of creosote components into human skin is delayed to a relatively higher extent than in rat skin.

This is also evidenced by the fact that a much higher portion of unabsorbed material remained on human skin (ca. 80 % vs. ca. 44 % in rat), associated with less radioactivity adsorbed to the stratum corneum (ca. 5.3 % vs. ca. 24 % in rat) and to the tape-stripped skin (ca. 0.9 vs. ca. 19 % in rat) (see Report Table 2, p. 19).

Finally, due to the pronounced difference in the skin-bound, potentially bioavailable radioactivity, the total absorbable dose, receptor fluid plus any dose remaining in the tape-stripped skin, was 8-fold greater for rat skin (34.3 %) than for human skin (4.24 %).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 427 (Skin Absorption: In Vivo Method)
Principles of method if other than guideline:
Other guidances and guidelines have been taken into account:
• European Commission Guidance Document on Dermal Absorption. Sanco/222/2000 rev 7 (2004);
• OECD Guidance Document for the Conduct of Skin Absorption Studies. OECD Environmental Health and Safety Publication Series on Testing and Assessment No. 28. (2004);
• MAFF Japan, Agricultural Chemicals Laws and Regulations, Japan (II), (59 Nousan Number 4200) (1985)
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Creosote
EC Number:
232-287-5
EC Name:
Creosote
Cas Number:
8001-58-9
Molecular formula:
not applicable
IUPAC Name:
Creosote
Test material form:
liquid
Details on test material:
- Substance type: organic
Specific details on test material used for the study:
IDENTITY OF TEST MATERIAL
- Name of test material (as cited in study report): Creosote; AWPA P1-P13 Creosote (North American Creosote Composite Test Material)

RADIOLABELLING INFORMATION (if applicable)
- Radiochemicals: the test material used was spiked with eight 14-C radiolabelled PAH that are main constituents of the creosote (see below)
- Locations of the label: various (see below)
- Amount of radiochemicals: the radioactivity of each marker component added to creosote was related to the composition of the creosote in order to represent compound abundance in the mixture.
- Radiolabelled components (marker chemicals) and position of the radiolabel
- Naphthalene – Benzene – UL – 14C
- 2-Methylnaphthalene – 8 – 14C
- Biphenyl – UL – 14C
- Anthracene – 1,2,3,4,4A,9A – 4C
- Phenanthrene – 9 – 14C
- Fluoranthene – 3 – 14C
- Pyrene – 4,5,9,10 – 14C
- Benzo[a]pyrene – 7 – 14C
- Sum of marker chemicals (radiolabelled substances) in creosote: ~ 43 %
Radiolabelling:
yes
Remarks:
see Specific details on test material

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Lab. NC/USA
- Age at study initiation: 8 - 10 weeks
- Weight at study initiation: 340 ± 19 g
- Fasting period before study: not fasted
- Individual metabolism cages: yes
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: quarantine

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 26 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Duration of exposure:
8 h and 469 h (21 d)
Doses:
Nominal doses: 10 µL/cm² (10.7 mg/cm²);
Dose volume: 105 µL on a test site of 10.5 cm² (= 112.35 mg/test site)
No. of animals per group:
4 in pre-testing (recovery of target chemicals in blood plasma)
8 in the dermal main absorption study (distribution and recovery of radioactivity)
Control animals:
no
Details on study design:
DOSE PREPARATION
- Method for preparation of dose: The radiolabelled materials (PAH) were dissolved in toluene and aliquots were combined such that the specific activity corresponded to the target level and the percentage of the different PAH was the same as in the creosote material. After evaporation of the sample to dryness by nitrogen convection, 5 mL of creosote was added and the sample was mixed and sonicated.

APPLICATION OF DOSE:
- Spiked creosote aliquote added into an O-ring unit

TEST SITE (main test)
- Preparation of test site: Dorso-lumbar surface, clipped free of hair and washed with an aqueous solution of 2 % Ivory Soap 24 h before treatment
- Glass O-ring appliance glued to the clipped area on the back using Instant Krazy Glue Gel adhesive
- Area of exposure: 10.5 cm² (internal surface of the O-ring appliance) [radius: ~1.83 cm]
- % coverage: approx. 5 % (estimated from 2/3 of body weight)
- Type of cover / wrap if used: Coban™ body wrap

TEST SITE (supplemental study with t = 496 h post-exposure)
- Procedure as previous main test but using a silicone O-ring not glued to the skin

REMOVAL OF TEST SUBSTANCE / SAMPLE COLLECTION
After 8 hours (= 0 h post-exposure), the organic trapping content was removed from the skin site and placed into acetonitrile, and the application site was then washed using at least 3 cycles of one natural sponge soaked in a 2 % Ivory® Soap solution (Report p. 21). Washings and sponge pieces were collected for LSC.

Urine and faeces: 0-8 hour exposure period, and for surviving rats 8-12, 12-24, and every 24 hours thereafter until sacrifice

Exhaled air: drawn through a 2N NaOH trap (14CO2) and an ethylene glycol trap (14C-volatiles) in series during the 0-8 hour exposure period, and for surviving rats 8-12, 12-24, and every 24 h thereafter until radioactivity in sample aliquots was ≤ LOD.

Residual feed and cage washings were collected as needed. At the end of the in-life phase, the metabolism cages were rinsed with a dilute soap solution followed by an acetone rinse. The rinse was placed in a suitable container and retained for analysis.

Blood plasma, organs (lung, liver, kidney, heart), the carcass, and the skin site were collected and preserved at the indicated time intervals (Report p. 22).

SAMPLE PREPARATION
- Aliquots of whole blood were combusted.
- Aliquots of plasma were added directly to Ultima Gold™ XR liquid scintillant.
- Aliquots of red blood cells were combusted.
- Faeces were homogenised in water. Aliquots were combusted.
- Residual feed was homogenised in water. Aliquots were combusted.
- Carcasses were homogenised with water. Aliquots were combusted.
- Tissues were minced. Aliquots were combusted.
- Urine, cage wash, sodium hydroxide (14CO2), and ethylene glycol (14C) were not processed further. Aliquots were added directly to Ultima Gold™ XR liquid scintillant.
- The application skin site and sponge pieces were digested in Soluene®-350. Aliquots were added directly to Hionic-Fluor™ liquid scintillant.

COMBUSTION
- Aliquots of whole blood, red blood cells, faeces, residual feed, carcass homogenate, and tissues were combusted using a Packard Tri-Carb Automatic Sample Oxidizer. The resultant 14CO2 generated was collected in a suitable absorbent scintillation system.

ANALYSIS
- Method type(s) for identification: GC-MS, Liquid scintillation counting
- LSC: All samples were analysed in a Packard liquid scintillation counter for total radioactivity. Samples were counted for 10 min or until 160,000 disintegrations were accumulated (0.5 %, 2σ), whichever came first.

Results and discussion

Signs and symptoms of toxicity:
no effects
Dermal irritation:
not specified
Absorption in different matrices:
After an 8-hour exposure period, ca. 6.3 % of the applied dose (radioactivity) had been absorbed. Ca. 2.13 % of the absorbed dose was found in urine, 0.026 % in faeces, 0.7 % in the cage wash, and 3.1 % in the carcass. Liver, kidney, and lungs contained 0.185 %, 0.073 %, and 0.006 %, respectively (see Report, Table 4, p. 35 - Attachment Fasano 2007a).
Total recovery:
The mass balances were satisfactory with recoveries of more than 92 % of the applied radioactivity.
Percutaneous absorption
Key result
Time point:
8 h
Dose:
10.7 mg/cm²
Parameter:
percentage
Absorption:
>= 7.9 - <= 14.8 %
Remarks on result:
other: low value: minimum without stratum corneum / high value: maximum including label of stratum corneum
Conversion factor human vs. animal skin:
not derivable from this study.

Any other information on results incl. tables

Results of GC analysis in blood(non-labelled target chemicals):

The concentration of the 12 selected chemicals was found to be below the limit of detection (individual LODs see "Any other information on materials and methods ... above) in all serial plasma samples from all collection time points during and following an 8 h exposure to a single finite application of the creosote test substance. These results suggest that all 12 target chemicals disappeared very quickly, were metabolised upon first pass through the skin and likely have negligible bioavailability (Report p. 28). See also distribution of radioactivity (below).

Results of LSC analysis in organs and body fluids(Report Tab. 4 / Attachment):

After 8 h (exposure phase), 3.14 % of the applied dose was found in the carcass and > 0.2 % in liver and even lower in other organs, while only marginal label was detectable in blood (> 0.05 %). In the post-exposure period over 21 days, radioactivity in the carcass and organs decreased to below quantification limits, while increasing in the urine and faeces. This provides evidence that creosote components do not tend to bioaccumulate but are excreted rapidly (see Attachment).

Percutaneous absorption(Report, Tab. 4 / Attachment):

After 8 h (exposure time): 6.34 % [SD ± 0.81 %] of the applied dose had been absorbed, tape-stripped skin and tape strips of the stratum corneum (SC) contained 1.55 ± 0.30 % and 6.89 ± 2.74 %, respectively. SC turnover may reduce absorption, but is considered to be potentially available. Hence, the maximum amount that can be absorbed is 14.8 ± 3.38 %, the sum of the three compartments (= absorbable dose + SC tape strips).

Note: The author considered the dose fraction bound to the upper horny layer of the epidermis (stratum corneum) as being unavailable for absorption. Hence, the absorbable dose is supposed to consist of the fractions of the absorbed dose (6.34%) plus the residual fraction bound to the skin layer after tape stripping (without SC) (1.55%), in total 7.9% as minimum. The real absorption rate is expected to lie in between.

Given this, contrary to specifications in Report, Tab. 4, the total unabsorbable dose after 8 h amounts to about 80 % of the dose applied, with the majority of the remainder accumulated in the wash (~ 59 %) and on the O-ring (~ 18 %), while only some 2 % was found in the body wrap. Evaporative loss is minimal (see charcoal trap).

The findings after 21 days (496 h post-exposure) are less clear: In the first long-term experiment, the amount absorbed apparently was far too high: The results from the 21 d recovery phase could be refuted as artefact due to incomplete removal of unabsorbed material from the skin site directly after the 8 h exposure.

Therefore, a supplemental 21 d study was carried out: However, also the second run may have been confounded, in this case by the very high dose fraction that adsorbed to the body wrap: 32 % already was removed during the cleaning procedure at termination of exposure period, while only about 22 % was left in the skin wash. This conflicts with the findings of the first 8-h-experiment (only 2 % in body wrap, but 59 % in wash) and raises the suspicion that a substantial part of the applied dose escaped from potential absorption. Hence, the low absorption rate of 8.85 % (SD ± 1.57%) of the applied dose may be unreliable.

Irrespective of this deficiency, it is evident that after 21 d practically no radioactivity remained in the treated skin compartments (see Tab. 4. "Dosed skin" and "Tape strips"). The decrease in label in the horny layer (SC) over 21 days may be attributable to physiological SC turnover or to skin permeation into the blood system. It is assumed that all SC-bound material has been absorbed. The most reliable maximum absorption rate is that one derived from the fractional distribution of radioactivity in the main 8 h study, in total 14.8 % of the dose applied.

Applicant's summary and conclusion

Conclusions:
A maximum of 14.8 % of creosote can be absorbed through rat skin within and after an exposure period of 8 hours, based on the analysis of a representative fraction of creosote. Furthermore, comprehensive analytical data show that creosote is very unlikely to bioaccumulate.