Registration Dossier

Administrative data

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study meets generally accepted scientific standards, well documented, acceptable for assessment

Data source

Reference
Reference Type:
publication
Title:
Dermal Absorption of Polycyclic Aromatic Hydrocarbons in the Blood-Perfused Pig Ear
Author:
Van Rooij JGM, Vinke E, De Lange J, Bruijnzeel PL, Bodelier-Bade MM, Noordhoek J, Jongeneelen FJ
Year:
1995
Bibliographic source:
J. Appl. Toxicol., 15, 193-200

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
Skin model/in-vitro blood perfusion model: by de Lange et al. 1991, J. Pharmacol. Toxicol. Methods, 27, 71-77
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Coal tar
- Substance type: organic
- Physical state: liquid, viscous
- Composition and analytical limits

Coal tar, industrial, from ACSZ/NL which contained the test PAH in the following concentrations:

Content Detection limit
in coal tar [%] [pmol/ml blood]
=====================================
fluorene 2.1 53.0
phenanthrene 6.8 1.7
anthracene 3.7 2.0
fluoranthene 4.0 51.0
pyrene 2.1 2.7
benzo[b]fluoranthene 0.9 7.8
benzo[k]fluoranthene 0.4 0.3
BaP 0.9 6.3
indeno[123-cd]pyrene 0.6 8.3
dibenzo[ah]anthracene 0.4 16.5
=====================================
Radiolabelling:
no

Test animals

Species:
pig
Strain:
other: domestic
Sex:
not specified
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Slaughterhouse: no details
- Weight at study initiation: 75 – 100 kg
-


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


IN-LIFE DATES: From: To:

Administration / exposure

Type of coverage:
open
Vehicle:
unchanged (no vehicle)
Duration of exposure:
average perfusion time: 250 min
Doses:
~11 mg tar/cm2 [100 %]

Content Single PAH dose
in coal tar [%] [µg/cm2]
===========================================
fluorene 2.1 230
phenanthrene 6.8 750
anthracene 3.7 410
fluoranthene 4.0 440
pyrene 2.1 230
benzo[b]fluoranthene 0.9 90
benzo[k]fluoranthene 0.4 44
BaP 0.9 90
indeno[123-cd]pyrene 0.6 66
dibenzo[ah]anthracene 0.4 44
===========================================
Total ~22 % ~2400 µg/cm2
No. of animals per group:
5 pig ears per treatment
Control animals:
no
Details on study design:
DOSE PREPARATION
not applicable

APPLICATION OF DOSE: topical to the ear

VEHICLE: not applicable

TEST SITE
- Preparation of test site: no particular action
- Area of exposure: 6 x 4 cm2
- Type of cover / wrap if used: none



REMOVAL OF TEST SUBSTANCE
- Perfusion: until perfusion pressure became too high (> 70 mm Hg), max. 250 min.
- Removal of protecting device: not relevant
- Washing procedures and type of cleansing agent: not relevant, termination of test
- Time after start of exposure: max. 250 min


SAMPLE COLLECTION
- Collection of blood: 200 min, for pyrene: complete kinetics with 10 – 11 time intervals up to 200 – 250 min


ANALYSIS
Blood samples (10 ml): Reversed-phase HPLC after 3-fold extraction with n-hexane, evaporation of the solvent
and re-dissolution of the residues in methanol (2 ml)
- Limits of detection and quantification: see above under dose

The method for analysis of the 10 PAHs has an average coefficient of variation of 14.4 %.
Recovery ranged from 45 % for dibenzo[a]anthracene to 97 % for anthracene.


STATISTICS:
Paired t-test to determine whether the dermal absorption of the various PAH was statistically different from pyrene absorption.
Details on in vitro test system (if applicable):
In-vitro blood perfusion model:
Five ears from domestic pigs were used to study the uptake of 10 PAH in blood after coal-tar application.
The treatment started after 30-min pre-perfusion with oxygenated blood.Perfusion at 30 °C with heparinized pig blood collected from the pigs.
Coal tar was applied to a skin area of 6x4 cm2 with an average dose of 11 mg/cm2.
Simultaneously, functional control measurements were conducted to assure the physiological integrity of the organ, including blood pressure,
blood flow, and glucose uptake.

Results and discussion

Total recovery:
see also above "Details of Study Design"
- Limit of detection (LOD): see under "Doses"
Conversion factor human vs. animal skin:
no data

Any other information on results incl. tables

Initial absorption rates of 10 PAHs from coal tar applied to the perfused pig ear

Absorption fluxes at 200 min p.a.

pmol/(h*cm2)

ng/(h*cm2)

fluorene

430

approx. 71

phenanthrene     

580

approx. 103

anthracene          

110

approx. 19.5

fluoranthene

105

approx. 21

pyrene  

60

approx. 12

benzo[b]fluoranthene

3

approx. 0.8

benzo[k]fluoranthene

1

--

BaP

3

approx. 0.8

indeno[123-cd]pyrene

1

--

dibenzo[ah]anthracene

1

--

The mean absorption fluxes [pmol/(h*cm2)] at 200 min after application of coal tar varied strongly between the 10 PAH.

Furthermore, variation between ears was high, too: for pyrene, for example, the flux ranged between 6 and 155 pmol/(h*cm2) and the cumulative uptake after 200 min was between 26 and 193 pmol/cm2. This was not caused by differences in dosing: applied amounts were high overdoses, because 0.2 % of each PAH was absorbed through the skin after 200 min.

The relative cumulative uptake (in relation to pyrene) was 0.01 for indeno[123-cd]pyrene to 12 for phenanthrene, for each PAH except fluoranthene, statistically different from pyrene uptake (p= 0.01). The inter-ear variation was relatively small when the absorbed amount was related to pyrene, indicating that the relative absorption fluxes of the various PAH were quite constant.

Applicant's summary and conclusion