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Endpoint:
basic toxicokinetics, other
Remarks:
in vivo and in vitro
Type of information:
experimental study
Remarks:
information comes from a comprehensive report summarising properties, toxicological/ecotoxicological effects, and effects on humans and the environment of a variety of polycyclic aromatic hydrocarbons
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Review, peer-reviewed data
Reason / purpose for cross-reference:
reference to same study
Objective of study:
other: various objectives
Principles of method if other than guideline:
Review on PAHs summarising data peer-reviewed by a group of international experts
GLP compliance:
no
Specific details on test material used for the study:
PAH mostly consisting of two to four condensed aromatic ring systems are relevant components of anthracene oil. Their properties and toxicokinetic behaviour are used for the characterisation of the toxicokinetic properties of anthracene oil.
Species:
other: various
Route of administration:
other: oral, dermal, inhalation
Type:
absorption
Results:
PAH are absorbed through the pulmonary tract, the gastrointestinal tract, and the skin. Absorption rate from lung depends on type of PAH. Gastrointestinal absorption is rapid in rodents with metabolites returning to the intestine via biliary excretion.
Type:
distribution
Results:
PAH are widely distributed throughout the organism after administration by any route and are found in almost all internal organs, but particularly in those rich in lipids.
Type:
metabolism
Results:
Metabolism is via intermediary epoxides that are further transformed by rearrangement or hydration to phenols or dihydrodiols. Secondary oxidation yield tetrols. Hydroxylated metabolites can be conjugated with sulphuric or glucuronic acid, or glutathione.
Type:
excretion
Results:
PAH metabolites and their conjugates are excreted via the urine and faeces. Conjugates may be hydrolysed by gut flora after biliary excretion and be reabsorbed. With increasing size, excretion into faeces increases. PAH seem not to persist in the body.

PAH are absorbed through the pulmonary tract, the gastrointestinal tract, and the skin. The rate of absorption from the lungs depends on the type of PAH, the size of the particles, on which they are absorbed, and the composition of the adsorbent. PAH adsorbed onto particulate matter are cleared from the lungs more slowly than free hydrocarbons. Absorption from the gastrointestinal tract occurs rapidly in rodents, but metabolites return to the intestine via biliary excretion. Studies with 32P-postlabelling of percutaneous absorption of mixtures of PAH in rodents showed that components of the mixtures reach the lungs, where they become bound to DNA. The rate of percutaneous absorption in mice varies according to the compound.

PAH are widely distributed throughout the organism after administration by any route and are found in almost all internal organs, but particularly those rich in lipids. Intravenously injected PAH are cleared rapidly from the bloodstream of rodents, but can cross the placental barrier and have been detected in foetal tissues.

The metabolism of PAH to more water-soluble derivatives, which is a prerequisite for their excretion, is complex. In general, parent compounds are converted into intermediate epoxides (a reaction catalysed by cytochrome P450-dependent mono-oxygenases), which are further transformed by rearrangement or hydration to yield phenols or diols. Secondary oxidation of diols results in tetrols, which can themselves be conjugated with sulfuric or glucuronic acids or with glutathione. Most metabolism results in detoxification, but some PAH are activated to DNA-binding species, principally diol epoxides, which can initiate tumours.

PAH metabolites and their conjugates are excreted via the urine and faeces, but conjugates excreted in the bile can be hydrolysed by enzymes of the gut flora and reabsorbed.

It can be inferred from the available information on the total human body burden that PAH do not persist in the body and that turnover is rapid. This inference excludes those PAH moieties that become covalently bound to tissue constituents, in particular nucleic acids, and are not removed by repair.

Endpoint:
basic toxicokinetics, other
Remarks:
in vivo and in vitro
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The source materials are individual polycyclic aromatic hydrocarbons. Uptake by humans and/or other living organisms will depend on the properties of the individual PAH. Being member of the same chemical class of substances, toxicokinetics and metabolism of individual PAH will have some common features, but can in detail differ to some extent determined by their size, molecular weight/structure, physico-chemical and distribution properties. The target substance anthracene oil (benzo[a]pyrene < 50 ppm; AOL) is composed of a broad range of PAH differing in size and degree of condensation.
Distribution and toxicokinetic properties of anthracene oil will be characterised by the range of PAH that are components of anthracene oil. Data resulting from experiments with individual PAH can therefore be used as weight of evidence in order to characterise the toxicokinetic properties of the target substance anthracene oil.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemicals are individual PAH. The source reference is a comprehensive review compiled by technical experts that cover besides other topics general toxicokinetic features of PAH. Individual PAH test materials are not specified, but due to the review process by experts, results are considered to be valid and specific for the individual test substances as well as for PAH as a chemical class of substances.
The target material anthracene oil is a UVCB substance produced by the distillation of coal tars extracting the approximate distillation range from ca. 300 °C to 400 °C. 10 % to 95 % of the total product distil over between ca. 300 °C and 375 °C. The substance is a brown pasty or liquid material consisting of a complex and within limits variable combination of polycyclic aromatic hydrocarbons. PAH contained in AOL range from naphthalene up to pyrene and benzofluorenes. Two- and three-ring aromatics amount to about 50 % (typical concentration) with two-ring aromatics forming the smaller fraction. PAH with four rings accumulate to about 10 %. The water solubility of AOL is low being limited by the solubility properties of its constituents.

3. ANALOGUE APPROACH JUSTIFICATION
Under ambient conditions or during processing of the substance, environmentally available or volatile components of anthracene oil may be released. These will be PAH with up to four fused rings. After intake by living organisms, these substances will be distributed within the organism, metabolised, and excreted. Toxicokinetics and metabolism of anthracene oil as such will thus be characterised by its individual constituents. Therefore, it is considered justified that data obtained for individual PAH, especially two- to four-ring PAH, are used as supporting information for read-across between the source substances (PAH) and the target substance anthracene oil.
Reason / purpose for cross-reference:
read-across source
Principles of method if other than guideline:
Read-across to the preceding entry:
Source substance: polycyclic aromatic hydrocarbons (PAH), generic mixture;
Reference: WHO 1998
Type:
absorption
Results:
PAH are absorbed through the pulmonary tract, the gastrointestinal tract, and the skin. Absorption rate from lung depends on type of PAH. Gastrointestinal absorption is rapid in rodents with metabolites returning to the intestine via biliary excretion.
Type:
distribution
Results:
PAH are widely distributed throughout the organism after administration by any route and are found in almost all internal organs, but particularly those rich in lipids.
Type:
metabolism
Results:
Metabolism is via intermediary epoxides that are further transformed by rearrangement or hydration to phenols or dihydrodiols. Secondary oxidation yield tetrols. Hydroxylated metabolites can be conjugated with sulphuric or glucuronic acid, or glutathione.
Type:
excretion
Results:
PAH metabolites and their conjugates are excreted via the urine and faeces. Conjugates may be hydrolysed by gut flora after biliary excretion and be reabsorbed. With increasing size, excretion into faeces increases. PAH seem not to persist in the body.
Conclusions:
The results presented above were obtained in studies with several supporting (source) substances (various PAH). These results are adopted as weight of evidence for the target substance anthracene oil (AOL).
Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: study meets generally accepted scientific principles, identification of primary metabolic steps, limited assessment possible
Objective of study:
metabolism
Qualifier:
no guideline available
Principles of method if other than guideline:
Early metabolism study based on scientific principles. Aim of the study was to identify key metabolic steps of biotransformation.
GLP compliance:
no
Specific details on test material used for the study:
- No data on test material
Radiolabelling:
no
Species:
rat
Strain:
other: "white"
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Weight at study initiation: approx. 300 g
Route of administration:
other: oral feed or gavage
Vehicle:
other: gavage: fine suspension of the TS in dilute starch solution
Details on exposure:
DIET PREPARATION
- Diet: no data about the way of preparation
- concentration in diet: 1 %

VEHICLE
- Gavage: fine suspension of the TS in dilute starch solution
- Concentration in vehicle: 100 mg/mL
Duration and frequency of treatment / exposure:
4 - 5 exposure days (estimated), daily treatment, application in diet;
12 exposure days (estimated), treatment on alternate days, application by gavage
Dose / conc.:
4.1 other: g (application in diet); total dose for all six animals and total exposure duration
Remarks:
approx. 0.7 g/rat; daily estimated dose: 0.15 g/day/rat
Dose / conc.:
1.8 other: g (application by gavage); total dose for all three animals and total exposure duration
Remarks:
0.6 g/rat; a dose of 0.1 g/rat was administered on alternate days
No. of animals per sex per dose / concentration:
6 animals (application in diet)
3 animals (application by gavage)
Details on dosing and sampling:
SAMPLING
Urine and feces were daily collected separately. But only the urine was collected for analysis and isolation.

SAMPLE PROCESSING and ANALYTCS
Urine samples were made alkaline and extracted with ether. After acidification and resting for 1 hour, a precipitate formed. The total was extracted with dichloromethane. The combined extracts were concentrated to a small volume by distillation and evaporated to dryness under vacuum. The residue was treated with hot ethanol, the solution filtered and cooled, while a precipitate formed (pale yellow needles), melting point: 268 °C.
The identity of the compound was established through comparison with phys.-chem. data of a reference substance, re-synthesis and characteristic reactions.
Type:
absorption
Results:
no information
Type:
distribution
Results:
no information
Type:
metabolism
Results:
the only metabolite identified is naphthalic anhydride. This anhydride is readily formed from naphthalene-1,8-dicarboxylic acid (naphthalic acid), which strongly indicates that the 5-membered ring of acenaphthene undergoes oxidative cleavage in the rat.
Type:
excretion
Results:
no information
Details on absorption:
no data
Details on distribution in tissues:
no data
Details on excretion:
no data
Metabolites identified:
yes
Details on metabolites:
the only metabolite identified is naphthalic anhydride. This anhydride is readily formed from naphthalene-1,8-dicarboxylic acid (naphthalic acid), which strongly indicates that the 5-membered ring of acenaphthene undergoes oxidative cleavage in the rat.

The primary metabolite that is excreted into the urine of rats was not identified, i.e. naphthalic acid or conjugates of it that are hydrolysed upon acidification. Whatever the nature of the compound(s) excreted into the urine, the study strongly suggests that the 5-membered carbon ring of acenaphthene undergoes oxidative fission in the rat.

No quantitative conclusions can be drawn from the work, because no complete analysis on the metabolite profile was performed (urine partly examined, faeces dismissed).

The identity of the isolated compound was established through comparison with phys.-chem. data of a reference substance, re-synthesis and characteristic reactions. Thus, the metabolite, which appeared on acidification of the urine, was identified as naphthalic anhydride, the anhydride that readily forms from naphthalene-1,8-dicarboxylic acid (naphthalic acid).

Conclusions:
This early study provides strong evidence that the primary metabolic reaction of acenaphthene in rats starts with the oxidative cleavage of the 5-membered ring in acenaphthene. The primary metabolite that is excreted into the urine of rats was not identified, but the substance identified (naphthalic anhydride) indicates the excretion of naphthalic acid or conjugates of it that are hydrolysed upon acidification.
No quantitative conclusions can be drawn from the work, because no complete analysis on the metabolite profile was performed (urine partly examined, faeces dismissed).
Endpoint:
basic toxicokinetics in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The source test material is acenaphthene a polycyclic aromatic hydrocarbon. It consists of a three-ring system and is beside others a constituent of the target substance. The target substance anthracene oil (benzo[a]pyrene < 50 ppm, AOL) is composed of a broad range of PAH predominantly consisting of two up to four aromatic rings.
Toxicokinetic properties of anthracene oil will be characterised by the range of PAH that constitute its composition. Therefore, results obtained from a metabolism study with the AOL constituent acenaphthene can be used as weight of evidence in order to characterise the toxicokinetic properties of anthracene oil.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source material is the substance acenaphthene. It is composed of two annulated benzene rings that are connected by two bridging methylene moieties forming a third non-aromatic five-membered ring. Analytical purity is not specified. But in the context of the report, it can be assume that the test material used is unambiguously related to the test results and that the study results are specific for the test substance acenaphthene.
The target material anthracene oil is a UVCB substance produced by the distillation of coal tars extracting the approximate distillation range from ca. 300 °C to 400 °C. 10 % to 95 % of the total product distil over between ca. 300 °C and 375 °C. The substance is a brown pasty or liquid material consisting of a complex and within limits variable combination of polycyclic aromatic hydrocarbons. PAH contained in AOL range from naphthalene up to pyrene and benzofluorenes. Two- and three-ring aromatics amount to about 50 % (typical concentration) with acenaphthene being present in concentrations of ca. 2.2 % (typical concentration). PAH with four rings accumulate to about 10 %.

3. ANALOGUE APPROACH JUSTIFICATION
Properties of the target substance anthracene oil will be determined by the properties of the PAH that are constituents of anthracene oil. Under environmental conditions or during processing of the substance, environmentally available or volatile components of anthracene oil can be released. These will be PAH (mainly compounds consisting of two to four rings). After intake by living organisms, these substances will be distributed within the organism, metabolised, and excreted. Toxicokinetics and metabolism of anthracene oil as such will thus be characterised by its individual constituents. In combination, they will specify the toxicokinetic fate of anthracene oil as a whole. Therefore, it is justified to use data determined for individual PAH that are constituents of anthracene oil, in this case acenaphthene, to characterise the toxicokinetic fate and behaviour of anthracene oil itself.
Reason / purpose for cross-reference:
read-across source
Principles of method if other than guideline:
Read-across to preceding entry:
Source test material: acenaphthene (generic, commercial product);
Reference: Chang 1943
Type:
absorption
Results:
no information
Type:
distribution
Results:
no information
Type:
metabolism
Results:
the only metabolite identified is naphthalic anhydride. This anhydride is readily formed from naphthalene-1,8-dicarboxylic acid (naphthalic acid), which strongly indicates that the 5-membered ring of acenaphthene undergoes oxidative cleavage in the rat.
Type:
excretion
Results:
no information
Details on absorption:
see above
Details on distribution in tissues:
see above
Details on excretion:
see above
Details on metabolites:
see above (source study record)
Conclusions:
This early study provides strong evidence that the primary metabolic reaction of acenaphthene in rats starts with the oxidative cleavage of the 5-membered ring in acenaphthene. The primary metabolite that is excreted into the urine of rats was not identified, but the substance identified (naphthalic anhydride) indicates the excretion of naphthalic acid or conjugates of it that are hydrolysed upon acidification.
This data is adopted as weight of evidence for the target substance anthracene oil.
Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
absorption
excretion
metabolism
Qualifier:
no guideline followed
Principles of method if other than guideline:
metabolic study
GLP compliance:
not specified
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name of test material (as cited in study report): phenanthrene
- Source of test material: commercial supplier
- Analytical purity: ≥ 99.3 % by GC (after re-purification of the commercial product)
Radiolabelling:
no
Species:
rat
Strain:
not specified
Sex:
male
Route of administration:
oral: gavage
Vehicle:
other: DMSO and corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Test material was dissolved/suspended in either dimethylsulfoxid (DMSO) or corn oil. Applicated doses were 50 µg test material in 0.1 mL DMSO or 0.2 mL corn oil.
Duration and frequency of treatment / exposure:
single dose
Dose / conc.:
50 other: µg/animal, single dose (corresponds to approx. 0.20 - 0.25 mg/kg bw)
Remarks:
application by gavage in DMSO (0.1 mL) and corn oil (0.2 mL)
No. of animals per sex per dose / concentration:
males: 8
Control animals:
no
Details on dosing and sampling:
DOSING
50 µg per animal (single dose) ==> approx. 0.20 - 0.25 mg/kg bw.
by gavage in DMSO (0.1 mL) and corn oil (0.2 mL)

SAMPLING
Urine and faeces were collected separately, single determinations and pooled over 8 animals.
Sampling intervals: 24, 48, and 72 h

SAMPLE PROCESSING
- Urine samples (50 ml) were acidified (12 ml 10 N HCl). 100 ml toluene and indeno(1,2,3-cd)fluoranthene as internal standard were added. The mixture was refluxed for 2 h to cleave conjugated metabolites. The toluene phase was washed and evaporated to a 1-ml volume.
Phenols in the extract were methylated by addition of methanol and diazomethane (16 h at room temperature). Phenolethers were obtained after further clean-up, addition of cyclohexane, and separation on a LH-20-Sephadex column with propan-2-ol. The fraction containing phenolethers was directly analysed by capillary GC or GC/MS.
- Faeces were saponified with 0.1 N KOH for 2 h in the presence of toluene and subsequently, after acidification with HCl (to 2-N conditions) treated as described above for urine.
Type:
absorption
Results:
The detection of about 4 % of phenanthrene in the faeces means that at least approx. 96 % of the applied dose was absorbed. But about 90 % of the dose administered was not recovered.
Type:
metabolism
Results:
1-, 2-, 3-, 4-, and 9-OH-phenanthrene were formed by oxidation at three different molecular regions (1,2- and 3,4-position = non-K-region; 9,10-position = K-region). Major metabolites were 1- and 2-OH-phenanthrene (60 % of excreted hydroxylated fraction).
Type:
excretion
Results:
About 37 % and 63 % of the excreted fraction appeared in the urine and faeces, respectively. The total recovery of phenanthrene (96 %) plus OH-derivatives (4 %) was found to be only about 10.5 % with 4.1 % in the urine and 6.4 % in the faeces.
Details on absorption:
see above
Details on distribution in tissues:
no data
Details on excretion:
see above
Metabolites identified:
yes
Details on metabolites:
Major metabolites were 1- and 2-OH-phenanthrene (about 60 % of total excreted hydroxylated fraction). 3-, 4-, and 9-OH-phenanthrene were minor. The hydroxylated metabolites (total approx. 3.8 %) arise from oxidation at 3 different molecular regions (1,2- and 3,4-position = non-K-region; 9,10-position = K-region).
Dihydroxy-dihydrophenanthrenes may have escaped from determination.
Low overall recovery (only about 10%) indicates that probably other water-soluble metabolites were formed not detected under the experimental conditions (i.e. glutathione conjugates and related mercapturic acids).

Following oral administration of 50 µg phenanthrene, the urinary and faecal excretion of unconverted phenanthrene and OH-derivatives decreased considerably within 3 days: by > 84 % for phenanthrene and by > 90 % for total OH-phenanthrenes, related to the day-1 excretion rate. The total recovery of phenanthrene plus OH-derivatives in urine and faeces was found to be only about 10.5 % with 4.1 % in the urine and 6.4 % in the faeces. Thus, within 3 days, about 37 % of the excreted fraction appeared in the urine, while 63 % were found in faeces. Approx. 3.8 % comprised of the fraction that was excreted in hydroxylated form.

The spectrum of hydroxyphenanthrenes comprised 1-, 2-, 3-, 4-, and 9-OH-phenanthrene, which arise from oxidation at 3 different molecular regions (1,2- and 3,4- position = non-K-region; 9,10-position = K-region). The major part resulted in the generation of 1- and 2-OH-phenanthrene with about 60 % of total excreted hydroxylated fraction. Thus, the oxidation at the 1,2-position was predominant in normal rats under those experimental conditions (see Report Tab. 2).

It has to be kept in mind that dihydroxy-dihydrophenanthrenes may have escaped from quantitative determination due to limited transformation into primary phenols under experimental conditions.

The amount of phenanthrene found in the urine (approx. 0.5 %) is likely to originate from mercapturic acid, which reverses to the parent compound under the analytical procedures applied.

In the mass balance, about 90 % of the dose administered was not recovered. This indicates that probably other water-soluble metabolites are formed that were not recorded under the experimental conditions, i.e. glutathione conjugates and related mercapturic acids.

The detection of only about 4 % of phenanthrene in the faeces means that at least approx. 96 % of the applied dose was absorbed.

Conclusions:
In the rat (non-induced), hydroxylated metabolites accounted for approx. 4 % of the admininistered dose. The vast majority of approx. 90% is likely attributable to water-soluble conjugates.
Executive summary:

In the normal rat (non-induced), the hydroxylated metabolites (phenol derivatives and dihydrodiols) accounted only for about 4 % of the dose initially administered. This is an indication for that probably most of the phenanthrene was metabolised to different intermediate or ultimate products that could not be captured by the method employed. The nature of the ultimate metabolites that are excreted, in particular, the kind of conjugation and GSH-related compounds, could not be elucidated by this technique.

The metabolite pattern gave some evidence of a predominance of 1,2-oxidation in apparently non-pretreated rats (compare in vitro). But the method used here regards only phenanthrene itself, free primary metabolites such as phenols and dihydrodiols and their sulfates and glucuronides. Even dihydrodiols are not recorded quantitatively since they are not completely converted into the corresponding phenols under saponification conditions. Especially in case of K-region dihydrodiols, the yield of phenols resulting from alkaline and acidic treatment is poor and was found to be only 36 % in the case of trans-9,10-diOH-9,10-dihydrophenanthrene in a series of experiments.

But approx. 90 % of a mass deficit in the mass balance are likely to also account for a considerable amount of other not identified water-soluble, GSH-dependent conjugates (see Overall remark).

Endpoint:
basic toxicokinetics in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The source test material is phenanthrene a polycyclic aromatic hydrocarbon. It consists of a three-ring system and is beside others a major constituent of the target substance. The target substance anthracene oil (benzo[a]pyrene < 50 ppm, AOL) is composed of a broad range of PAH predominantly consisting of two up to four aromatic rings.
Toxicokinetic properties of anthracene oil will be characterised by the range of PAH that constitute its composition. Therefore, results obtained from an in-vitro metabolism study with the AOL constituent phenanthrene can be used as weight of evidence in order to characterise the toxicokinetic/metabolism properties of anthracene oil.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source material is the substance phenanthrene. It was purchased from a commercial source and the analytical purity of the test substance after re-purification is reported to be ≥ 99.3 %. Hence, the study results are related to effects caused by phenanthrene.
The target material anthracene oil is a UVCB substance produced by the distillation of coal tars extracting the approximate distillation range from ca. 300 °C to 400 °C. 10 % to 95 % of the total product distil over between ca. 300 °C and 375 °C. The substance is a brown pasty or liquid material consisting of a complex and within limits variable combination of polycyclic aromatic hydrocarbons. PAH contained in AOL range from naphthalene up to pyrene and benzofluorenes. Two- and three-ring aromatics amount to about 50 % (typical concentration). Main constituent is phenanthrene present in a typical concentration of ca. 25 to 31 % (composite sample 7). PAH with four rings accumulate to about 10 %.

3. ANALOGUE APPROACH JUSTIFICATION
Properties of the target substance anthracene oil will be determined by the properties of the PAH that are constituents of anthracene oil. Under environmental conditions or during processing of the substance, environmentally available or volatile components of anthracene oil can be released. These will be PAH (mainly compounds consisting of two to four rings). After intake by living organisms, these substances will be distributed within the organism, metabolised, and excreted. Toxicokinetics and metabolism of anthracene oil as such will thus be characterised by its individual constituents. In combination, they will specify the toxicokinetic fate of anthracene oil as a whole. Therefore, it is justified to use data determined for individual PAH that are constituents of anthracene oil, in this case phenanthrene, to characterise the toxicokinetic fate and behaviour of anthracene oil itself.
Reason / purpose for cross-reference:
read-across source
Principles of method if other than guideline:
Read-across to preceding entry:
Source test material: phenanthrene (generic, commercial product);
Reference: Grimmer et al. 1991
Type:
absorption
Results:
The detection of about 4 % of phenanthrene in the faeces means that at least approx. 96 % of the applied dose was absorbed. But about 90 % of the dose administered was not recovered.
Type:
metabolism
Results:
1-, 2-, 3-, 4-, and 9-OH-phenanthrene were formed by oxidation at three different molecular regions (1,2- and 3,4-position = non-K-region; 9,10-position = K-region). Major metabolites were 1- and 2-OH-phenanthrene (60 % of excreted hydroxylated fraction).
Type:
excretion
Results:
About 37 % and 63 % of the excreted fraction appeared in the urine and faeces, respectively. The total recovery of phenanthrene (96 %) plus OH-derivatives (4 %) was found to be only about 10.5 % with 4.1 % in the urine and 6.4 % in the faeces.
Details on absorption:
see above
Details on distribution in tissues:
no data
Details on excretion:
see above
Details on metabolites:
see above (source study record)
Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
metabolism
Qualifier:
no guideline available
Principles of method if other than guideline:
in-vitro metabolism study using liver microsomes of non-pretreated rats and of rats pretreated with various PAH and related compounds
GLP compliance:
not specified
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name of test material (as cited in study report): phenanthrene
- Source of test material: commercial supplier
- Analytical purity: ≥ 99.3 % by GC (after re-purification of the commercial product)
Radiolabelling:
no
Species:
rat
Strain:
not specified
Route of administration:
other: in-vitro incubation
Vehicle:
acetone
Details on exposure:
DOSING SOLUTIONS
- test substance was dissolved in acetone at a concentration of 100 nmol (17.8 µg) in 20 µL.
Duration and frequency of treatment / exposure:
15 minute(s)
Dose / conc.:
17.8 other: µg test substance per assay (microsomal incubation)
Remarks:
test substance was dissolved in 20 µL acetone
No. of animals per sex per dose / concentration:
males: 3-4 (microsomal preparations)
Control animals:
no
Details on dosing and sampling:
DOSING
100 nmol (17.8 µg) test substance dissolved in 20 µL acetone were incubated with 2 mL of the microsomal preparation (resulting test concentration approx. 9 mg/L).

SAMPLE PROCESSING
The incubations were stopped by addition of 10 mL acetone and 5 mM EDTA. After evaporation of organic solvent and dilution with water, 20 µg benzo[ghi]perylene was added as internal standard. Unconverted phenanthrene and metabolites were extracted at pH 3 (acetic acid) with ethyl acetate (2x).
Recovery of 2-OH-phenanthrene and trans-9,10-dihydroxy-9,10-diOHphenanthrene proved to be > 91 % when checked in a separate incubation mixture (blank).
The residues of the extract were evaporated almost to dryness, dissolved in propan-2-ol and chromatographed on a LH-20-Sephadex column with propan-2-ol. The fraction containing phenanthrene and the metabolites was silylated after evaporation and analysed by GC and GC/MS.
Type:
metabolism
Results:
The incubation of phenanthrene with liver microsomes from untreated rats resulted in the formation of exclusively trans-9,10-dihydroxy-9,10-dihydrophenanthrene (K-region oxidation). After phenobarbital induction, 9,10-oxidation was further increased
Type:
metabolism
Results:
Other inducers (PCBs, higher PAH) also induced the oxidation at the 1,2- and 3,4-position (non-K-region). Simultaneously, the yield of the K-region 9,10-dihydrodiol was also enhanced. Thus, the 9,10-oxidation remained dominant in all variants of treatment
Metabolites identified:
yes
Details on metabolites:
The incubation of phenanthrene with liver microsomes from untreated rats resulted in the formation of exclusively trans-9,10-dihydroxy-9,10-dihydrophenanthrene (K-region oxidation).
Most inducers (PCBs and higher PAH) additionally induced the oxidation at the non-K-region at the 1,2- and 3,4-position. Simultaneously, the yield of the K-region 9,10-dihydrodiol was also enhanced. Thus, the 9,10-oxidation remained dominant in all variants of treatment.
Phenobarbital induction of rat liver microsomes almost only caused a further marked increase in 9,10-oxidation.

The incubation of phenanthrene with liver microsomes from untreated rats resulted in the formation of exclusively trans-9,10-dihydroxy-9,10-dihydrophenanthrene (K-region oxidation).

Most inducers (PCBs and higher PAH) additionally induced the oxidation at the non-K-region as indicated by the formation of trans-1,2-dihydroxy-1,2-dihydrophenanthrene and - to a lesser extent - trans-3,4-dihydroxy-3,4-dihydrophenanthrene, while simultaneously also enhancing the yield of the K-region 9,10-dihydrodiol (Reference Tab. 1). Thus, the 9,10-oxidation remained dominant in all variants of treatment.

Phenobarbital as inducer almost only caused further marked induction of 9,10-oxidation.

[Note: In early studies, Sims (1970) also identified 1,2-dihydrodiols at about the same extent as 9,10-dihydrodiols along with some 3,4-dihydrodiols in in-vitro incubations using liver homogenate from non-induced male rats (Chester Beatty strain).]

Conclusions:
The method was only able to identify hydroxylated intermediates including dihydrodiols through silylation.
Contrary to the in-vivo observations (see entry Grimmer et al. 1991), in-vitro results indicated that the K-region oxidation (9,10-position) is predominant using microsomes obtained from untreated rats. The nature of the ultimate metabolites that are excreted, in particular, the kind of conjugation, could not be elucidated by this technique.
Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The source test material is phenanthrene a polycyclic aromatic hydrocarbon. It consists of a three-ring system and is beside others a major constituent of the target substance. The target substance anthracene oil (benzo[a]pyrene < 50 ppm, AOL) is composed of a broad range of PAH predominantly consisting of two up to four aromatic rings.
Toxicokinetic properties of anthracene oil will be characterised by the range of PAH that constitute its composition. Therefore, results obtained from an in-vitro metabolism study with the AOL constituent phenanthrene can be used as weight of evidence in order to characterise the toxicokinetic properties of anthracene oil.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source material is the substance phenanthrene. It was purchased from a commercial source and the analytical purity of the test substance after re-purification is reported to be ≥ 99.3 %. Hence, the study results are related to effects caused by phenanthrene.
The target material anthracene oil is a UVCB substance produced by the distillation of coal tars extracting the approximate distillation range from ca. 300 °C to 400 °C. 10 % to 95 % of the total product distil over between ca. 300 °C and 375 °C. The substance is a brown pasty or liquid material consisting of a complex and within limits variable combination of polycyclic aromatic hydrocarbons. PAH contained in AOL range from naphthalene up to pyrene and benzofluorenes. Two- and three-ring aromatics amount to about 50 % (typical concentration). Main constituent is phenanthrene present in a typical concentration of ca. 25 to 31 % (composite sample 7). PAH with four rings accumulate to about 10 %.

3. ANALOGUE APPROACH JUSTIFICATION
Properties of the target substance anthracene oil will be determined by the properties of the PAH that are constituents of anthracene oil. Under environmental conditions or during processing of the substance, environmentally available or volatile components of anthracene oil can be released. These will be PAH (mainly compounds consisting of two to four rings). After intake by living organisms, these substances will be distributed within the organism, metabolised, and excreted. Toxicokinetics and metabolism of anthracene oil as such will thus be characterised by its individual constituents. In combination, they will specify the toxicokinetic fate of anthracene oil as a whole. Therefore, it is justified to use data determined for individual PAH that are constituents of anthracene oil, in this case phenanthrene, to characterise the toxicokinetic fate and behaviour of anthracene oil itself.
Reason / purpose for cross-reference:
read-across source
Principles of method if other than guideline:
Read-across to preceding entry:
Source test material: phenanthrene (generic, commercial product);
Reference: Jacob et al. 1982
Type:
metabolism
Results:
The incubation of phenanthrene with liver microsomes from untreated rats resulted in the formation of exclusively trans-9,10-dihydroxy-9,10-dihydrophenanthrene (K-region oxidation). After phenobarbital induction, 9,10-oxidation only was further increased.
Type:
metabolism
Results:
Other inducers (PCB, higher PAH) also induced the oxidation at the 1,2- and 3,4-position (non-K-region). Simultaneously, the yield of the K-region 9,10-dihydrodiol was also enhanced. Thus, the 9,10-oxidation remained dominant in all variants of treatment.
Details on metabolites:
see above (source study record)
Conclusions:
The results presented above were obtained in a study with the reference (supporting) substance phenanthrene. These results are adopted as weight of evidence for the target substance anthracene oil (AOL).
Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
metabolism
Qualifier:
no guideline followed
Principles of method if other than guideline:
Investigation of fluoranthene metabolites in serum; comparison of these metabolites with metabolites obtained with microsomal fractions of rat hepatocytes
GLP compliance:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name of test material (as cited in study report): fluoranthene
- Source: no data, commercial product
- Purity: no data
Radiolabelling:
no
Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: no data
- Age at study initiation: young adults
- Weight at study initiation: 200 - 220 g
Vehicle:
corn oil
Duration and frequency of treatment / exposure:
24 hour(s)
Dose / conc.:
1 000 mg/kg bw (total dose)
Remarks:
applied as single dose
No. of animals per sex per dose / concentration:
Males: 3
Control animals:
yes, concurrent vehicle
Positive control reference chemical:
not required
Details on dosing and sampling:
METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: blood
- Time and frequency of sampling: at 3, 6, and 24 h
- From how many animals: 3 animals; samples not pooled
- Method type(s) for identification: HPLC using a C18 separation column with an acetonitrile-water gradient as mobile phase, UV and fluorescence detection.

TREATMENT OF SAMPLES / CLEAVAGE OF CONJUGATES
- Blood: the samples were centrifuged at 150 g for 10 min. The serum was extracted with 5-fold excess of ethylacetate, dried over anhydrous sodium sulphate and evaporated to dryness.The initial volumes were replaced with methanol.
Statistics:
n.a.
Type:
metabolism
Results:
The metabolic pattern gave evidence of 2,3-oxidation, because 2,3-dihydro-2,3-diOH-fluoranthene (only very small peak) as well as 3-OH-fluoranthene were identified. Besides these peaks, 1-OH-fluoranthene was recorded.
Metabolites identified:
yes
Details on metabolites:
Identified metabolites: 2,3-dihydro-2,3-dihydroxyfluoranthene (2,3-dihydrodiol), 3-hydroyfluoranthene, 1-hydroxyfluorathene; the metabolic pattern gave evidence of 2,3-oxidation.

The metabolic pattern gave evidence of 2,3-oxidation, because 2,3-dihydro-2,3-dihydroxy-fluoranthene (only very small peak) as well as 3-OH-fluoranthene were identified. Besides these peaks, 1-OH-fluoranthene was recorded.

After 3 and 6 h, an unidentified more polar peak appeared, which after 24 h decreased, while at the same time another unidentified, more polar peak emerged almost at the solvent front. No quantification and mass balancing is possible from the available data base.

Absorption: no information

Clinical signs of toxicity: no

Conclusions:
Only limited qualitative analytical data were presented. The basic object of this study was to draw a link between the mutagenic activity and the metabolic profile. Therefore, high dosage and a short exposure time was applied.
No quantitative evaluation of the HPLC data is provided. The study gives only limited information about the formation of key intermediates, one of which being 2,3-dihydro-2,3-dihydroxy-fluoranthene. There is no information on absorption, excretion, and elimination kinetics.
Endpoint:
basic toxicokinetics in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The source test material is fluoranthene a polycyclic aromatic hydrocarbon. It consists of four fused rings and is beside others a main constituent of the target substance. The target substance anthracene oil (benzo[a]pyrene < 50 ppm, AOL) is composed of a broad range of PAH predominantly consisting of two up to four aromatic rings.
Toxicokinetic properties of anthracene oil will be characterised by the range of PAH that constitute its composition. Therefore, results obtained from an in-vivo metabolism study with the AOL constituent fluoranthene can be used as weight of evidence in order to characterise the toxicokinetic properties of anthracene oil.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source material is the substance fluoranthene. This PAH is composed of three peripheral six-membered benzene rings around a central five-membered ring. It is theoretically constructed by connecting the five-membered ring of an indane/indene moiety to the 1-, 6-position of naphthalene. The test material was purchased from a commercial source, but its analytical purity is not specified. In the context of the report, it can be assume that the test material used is unambiguously related to the test results and that the study results are specific for the test substance fluoranthene.
The target material anthracene oil is a UVCB substance produced by the distillation of coal tars extracting the approximate distillation range from ca. 300 °C to 400 °C. 10 % to 95 % of the total product distil over between ca. 300 °C and 375 °C. The substance is a brown pasty or liquid material consisting of a complex and within limits variable combination of polycyclic aromatic hydrocarbons. PAH contained in AOL range from naphthalene up to pyrene and benzofluorenes. Two- and three-ring aromatics amount to about 50 % (typical concentration). PAH with four rings accumulate to about 10 % with fluoranthene being present in a typical concentration of ca. 7 % (composite sample 7).

3. ANALOGUE APPROACH JUSTIFICATION
Properties of the target substance anthracene oil will be determined by the properties of the PAH that are constituents of anthracene oil. Under environmental conditions or during processing of the substance, environmentally available or volatile components of anthracene oil can be released. These will be PAH (mainly compounds consisting of two to four rings). After intake by living organisms, these substances will be distributed within the organism, metabolised, and excreted. Toxicokinetics and metabolism of anthracene oil as such will thus be characterised by its individual constituents. In combination, they will specify the toxicokinetic fate of anthracene oil as a whole. Therefore, it is justified to use data determined for individual PAH that are constituents of anthracene oil, in this case fluoranthene, to characterise the toxicokinetic fate and behaviour of anthracene oil itself.
Reason / purpose for cross-reference:
read-across source
Principles of method if other than guideline:
Read-across to the preceding entry:
Source substance: fluoranthene (generic, commercial product);
Reference: Polcaro C et al. 1988
Type:
metabolism
Results:
The metabolic pattern gave evidence of 2,3-oxidation, because 2,3-dihydro-2,3-dihydroxy-fluoranthene (only very small peak) as well as 3-OH-fluoranthene were identified. Besides these peaks, 1-OH-fluoranthene was recorded.
Details on metabolites:
see above (source study record)
Conclusions:
The results presented above were obtained in a study with the supporting (source) substance fluoranthene. These results are adopted as weight of evidence for the target substance anthracene oil (AOL).
Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Objective of study:
metabolism
Qualifier:
no guideline followed
Principles of method if other than guideline:
metabolic in vitro study
GLP compliance:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name of test material (as cited in study report): fluoranthene
- Source of test material: no data, commercial product
- Analytical purity: > 99%
Radiolabelling:
no
Species:
other: rat and human
Strain:
Sprague-Dawley
Sex:
male
Details on test animals or test system and environmental conditions:
Source of liver microsomes:
Rats: Arochlor-induced
Human: 11 donors of liver samples (normal)
Route of administration:
other: incubation of liver microsomes in-vitro
Vehicle:
acetone
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test substance was applied dissolved in acetone
Duration and frequency of treatment / exposure:
2.5 to 3 hour(s)
Dose / conc.:
125 other: nmol/mL
Remarks:
minimal dose, corresponds to approx. 25 µg/mL
Dose / conc.:
1 000 other: nmol/mL
Remarks:
maximal dose, corresponds to approx. 200 µg/mL
No. of animals per sex per dose / concentration:
Rats: no data
Humans: 11 subjectts
Control animals:
yes, concurrent vehicle
Details on dosing and sampling:
DOSING
- 125 - 1000 nmol/mL, approx. 25 - 200 µg/mL

SAMPLE PROCESSING
- Termination of microsomal incubation: after 2.5 - 3 h, the incubations were stopped by addition of ethylacetate (followed by extraction) or by loading the microsomal mixture directly onto a solid phase extraction C18-BondElut disposable column, followed by washing and elution (see below).
- Processing of ethylacetate extracts: extracts were combined and dried over anhydrous sodium sulphate. The dried extracts were concentrated under a stream of nitrogen or on a Seed-vac lyophiliser and then dissolved in water:methanol (9:1) for reverse-phase HPLC.
- Processing of solid phase columns: loaded C18-BondElut columns were washed with water and the bound material was eluted with 3 mL each of methanol:water (1:1) and methanol. The eluates were combined and concentrated under a stream of dry nitrogen or on a Seed-vac lyophiliser and then dissolved in water:methanol (9:1) for reverse-phase HPLC.

ANALYTICS
- Standard HPLC: reverse-phase HPLC with diode array UV detection followed by MS analysis
- Special HPLC: HPLC using chiral stationary-phase columns for separation the enantiomeric 2,3-dihydro-2,3-dihydroxyfluoranthenes (determination of absolute configuration)
HPLC using chiral stationary-phase columns for enantiomeric resolution of the secondary dihydrodiol-1,10b-epoxides (compound 3 and 4 in Scheme I below)
- Determination of sterical configuration: application of the benzoate exciton chirality rules to the CD spectra of the 4-(dimethylamino)benzoylesters of the dihydrodiols
Type:
metabolism
Results:
The metabolic profile from microsomes of human liver and induced rat liver were qualitatively similar. 2,3-Oxidation was predominant primarily resulting in 2,3-dihydro-2,3-dihydroxyfluoranthene (dihydrodiol) as enantiomeric mixture of optical isomers.
Type:
metabolism
Results:
The 2R,3R enantiomer was formed by rat liver microsomes in 75 - 78 % enantiomeric excess, while human liver microsomes produced this enantiomer in only 6 - 12 % excess. The dihydrodiol was subsequently transformed to the anti 1,10b epoxide.
Metabolites identified:
yes
Details on metabolites:
The metabolic profiles from human liver and induced rat liver microsomes were qualitatively similar. 2,3-Oxidation was predominant, primarily resulting in 2,3-dihydro-2,3-trans-dihydroxyfluoranthene (dihydrodiol). The dihydrodiol is further metabolised to the syn- and anti-2,3-trans-dihydrodiol 1,10b-epoxides. These are fairly stable and can hydrolyse to the 1,2,3,10b-tetrahydrotetrols.

The metabolic pathways that could be elucidated by the sophisticated analytical procedures are summarised in the flow scheme from the report that is attached below.

The major primary metabolite formed in both rat and human systems that could be analysed was trans-2,3-dihydro-2,3-dihydroxyfluoranthene (trans-2,3-dihydrodiol) (2) obtained as enantiomeric mixture. The 2R,3R enantiomer could be isolated and its configuration determined. This diol is formed by rat liver microsomes in 75 - 78 % enantiomeric excess, while human liver microsomes produced this enantiomer of the diol in only 6 - 12 % excess.

In all 11 human liver microsome samples, the 2,3-dihydrodiol (2) (enantiomeric mixture) accounted for 70 - 90 % of the metabolites in a given sample. All but one sample produced the anti-2,3-dihydro-2,3-dihydroxyfluoranthene 1,10b-epoxide (3). No syn-diol epoxide (4) was formed but only to very minor extent in one sample. Only one sample exclusively produced fluoranthene-2,3-dione, but did not yield any diol epoxide.

Conclusions:
The study confirms that the main metabolic pathway of fluoranthene proceeds via the formation of 2,3-dihydro-2,3-dihydroxyfluoranthene.
Executive summary:

This study provides a survey on the metabolic profile arising from fluoranthene in the presence of human liver microsomes. No ADME data are available.

The study confirms that the main metabolic pathway of fluoranthene proceeds via the formation of 2,3-dihydro-2,3-dihydroxyfluoranthene. It also succeeds in elucidating the absolute configuration of the key intermediates. Note: The anti-diol epoxide (3) as well as the fluoranthene-2,3-dione are those metabolites, which proved to be toxic and mutagenic in the Ames test.

It is unclear whether this pattern is complete, i.e. if other intermediates were formed but that possibly escaped from analytical recording under the experimental procedure.

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The source test material is fluoranthene a polycyclic aromatic hydrocarbon. It consists of four fused rings and is beside others a main constituent of the target substance. The target substance anthracene oil (benzo[a]pyrene < 50 ppm, AOL) is composed of a broad range of PAH predominantly consisting of two up to four aromatic rings.
Toxicokinetic properties of anthracene oil will be characterised by the range of PAH that constitute its composition. Therefore, results obtained from an in-vitro metabolism study with the AOL constituent fluoranthene using liver microsomes can be used as weight of evidence in order to characterise the toxicokinetic properties of anthracene oil.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source material is the substance fluoranthene. This PAH is composed of three peripheral six-membered benzene rings around a central five-membered ring. It is theoretically constructed by attaching the five-membered ring of an indane/indene moiety to the 1-, 6-position of naphthalene. The test material was purchased from a commercial source and the analytical purity of the test substance is reported to be > 99 %. Hence, the study results are related to effects caused by fluoranthene.
The target material anthracene oil is a UVCB substance produced by the distillation of coal tars extracting the approximate distillation range from ca. 300 °C to 400 °C. 10 % to 95 % of the total product distil over between ca. 300 °C and 375 °C. The substance is a brown pasty or liquid material consisting of a complex and within limits variable combination of polycyclic aromatic hydrocarbons. PAH contained in AOL range from naphthalene up to pyrene and benzofluorenes. Two- and three-ring aromatics amount to about 50 % (typical concentration). PAH with four rings accumulate to about 10 % with fluoranthene being present in a typical concentration of ca. 7 % (composite sample 7).

3. ANALOGUE APPROACH JUSTIFICATION
Properties of the target substance anthracene oil will be determined by the properties of the PAH that are constituents of anthracene oil. Under environmental conditions or during processing of the substance, environmentally available or volatile components of anthracene oil can be released. These will be PAH (mainly compounds consisting of two to four rings). After intake by living organisms, these substances will be distributed within the organism, metabolised, and excreted. Toxicokinetics and metabolism of anthracene oil as such will thus be characterised by its individual constituents. In combination, they will specify the toxicokinetic fate of anthracene oil as a whole. Therefore, it is justified to use data determined for individual PAH that are constituents of anthracene oil, in this case fluoranthene, to characterise the toxicokinetic fate and behaviour of anthracene oil itself.
Reason / purpose for cross-reference:
read-across source
Principles of method if other than guideline:
Read-across to the preceding entry:
Source substance: fluoranthene (generic, commercial product);
Reference: Day BW et al. 1982
Type:
metabolism
Results:
The metabolic profile from microsomes of human liver and induced rat liver were qualitatively similar. 2,3-Oxidation was predominant primarily resulting in 2,3-dihydro-2,3-dihydroxyfluoranthene (dihydrodiol) as enantiomeric mixture of optical isomers.
Type:
metabolism
Results:
The 2R,3R enantiomer was formed by rat liver microsomes in 75 - 78 % enantiomeric excess, while human liver microsomes produced this enantiomer in only 6 - 12 % excess. The dihydrodiol was subsequently transformed to the anti 1,10b epoxide.
Details on metabolites:
see above (source study record)
Conclusions:
The study confirms that the main metabolic pathway of fluoranthene proceeds via the formation of 2,3-dihydro-2,3-dihydroxy-fluoranthene (trans 2,3-dihydrodiol). Subsequently, the anti 1,10b-epoxide is formed from the dihydrodiol. This metabolic pattern corresponds to the major metabolic pathway of other polycyclic aromatic hacrocarbons.
Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
absorption
excretion
metabolism
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: application of pyrene by various routes and observation of elimination/excretion of original substance and metabolites in urine and faeces
- Parameters analysed / observed: pyrene, hydroxypyrene
GLP compliance:
not specified
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name of test material (as cited in study report): pyrene
- Source of test material: commercial supplier
- Analytical purity: ≥ 99.1 % (after re-purification of the commercial product)
Radiolabelling:
no
Species:
rat
Strain:
not specified
Sex:
not specified
Route of administration:
oral: gavage
Vehicle:
not specified
Duration and frequency of treatment / exposure:
single dose
Dose / conc.:
50 other: µg/animal, single dose (corresponds to approx. 0.25 mg/kg bw)
No. of animals per sex per dose / concentration:
8 (no information on sex)
Control animals:
no
Details on dosing and sampling:
DOSING
50 µg per animal (single dose) ==> approx. 0.25 mg/kg bw.

SAMPLING
Urine and feces were collected separately, pooled over 8 animals.
Sampling intervals: 24, 48, and 72 h

SAMPLE PROCESSING
- Urine samples (50 ml) were acidified (12 ml 10 N HCl) and 100 ml toluene was added. The mixture was refluxed for 2 h to cleave conjugated metabolites. The toluene phase was washed and evaporated to a 1-ml volume.
Phenols in the extract were methylated by addition of methanol and diazomethane (16 h at room temperature). Phenolethers were obtained after further clean-up, addition of cyclohexane, and separation on a LH-20-Sephadex column with propan-2-ol. The fraction containing phenolethers was directly analysed by capillary GC or GC/MS.
- Faeces were saponified with 0.1 N KOH for 2 h in the presence of toluene and subsequently, after acidification with HCl (to 2-N conditions) treated as described above for urine.
Type:
absorption
Results:
The detection of about 7 % of pyrene in the faeces means that at least approx. 93 % of the applied dose were absorbed. But at least 40 % of the dose administered was not recovered.
Type:
metabolism
Results:
Main metabolite was 1-OH-pyrene, which is partly transformed to two dihydroxypyrenes in a secondary oxidation step. The nature of the ultimate metabolites that are excreted, in particular the kind of conjugation, could not be elucidated by this technique.
Type:
metabolism
Results:
Since 1- and 4-methoxypyrene could not be distinguished with the GC method in this study, it could not be excluded that pyrene is not only excreted as 1-OH-pyrene but also as 4,5-dihydroxy-4,5-dihydropyrene which forms 4-OH-pyrene upon acidification.
Type:
excretion
Results:
The total recovery (excretion) of pyrene plus 1-OH-pyrene in urine and faeces was found to be 53.4 % (50% after 24 hrs) with 7.2 % pyrene and 46.2 % 1-OH-pyrene. Amount in urine was only about 0.4 %.
Details on absorption:
see above
Details on distribution in tissues:
no data
Details on excretion:
see above
Metabolites identified:
yes
Details on metabolites:
1- and 4-hydroxypyrene were identified as metabolites of pyrene. Due to the method applied, conjugates were cleaved during the clean-up process and could not be identified as well as vicinal dihydroxy-dihydropyrenes (4,5-dihydroxy-4,5-dihydropyrene).

Following oral administration of 50 µg pyrene, the excretion was almost complete after 3 days: The total recovery of pyrene plus 1-OH-pyrene in urine and faeces was found to be 53.4 % with 7.2 % pyrene and 46.2 1-OH-pyrene. About 50 % of the dose, i.e. about 94 % of the excreted compounds appeared in the urine and faeces already after 24 hours.

The detection of about 7 % of pyrene in the faeces means that at least approx. 93 % of the applied dose were absorbed. Significantly less than 1 % of the excreted amount, about 0.4 %, was recovered as pyrene plus 1-OH-pyrene in the urine. In the mass balance, at least 40 % of the dose administered was not recovered. No statistical intraspecies evaluation of excretion could reasonably be carried out, because no single-animal data were generated.

Note: Since 1- and 4-methoxypyrene could not be distinguished with the GC method in this study, it could not be excluded that pyrene is not only excreted as 1-OH-pyrene but also as 4,5-dihydroxy-4,5-dihydropyrene, which forms 4-OH-pyrene upon acidification (the formation of 4-OH-pyrene was verified in a separate experiment). 4-Pyrenylmercapturic acid possibly occurring in the faeces would also result in 4-OH-pyrene after alkaline hydrolysis. But approx. 40 % of a mass deficit in the mass balance are not explained.

Conclusions:
In the normal rat (uninduced), the main metabolite is 1-OH-pyrene, which is partly transformed to two dihydroxypyrenes in a secondary oxidation step. The nature of the ultimate metabolites that are excreted, in particular the kind of conjugation, could not be elucidated by this technique.
Endpoint:
basic toxicokinetics in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The source test material is pyrene a polycyclic aromatic hydrocarbon. It consists of four fused aromatic rings and is beside others a main constituent of the target substance. The target substance anthracene oil (benzo[a]pyrene < 50 ppm, AOL) is composed of a broad range of PAH predominantly consisting of two up to four aromatic rings.
Toxicokinetic properties of anthracene oil will be characterised by the range of PAH that constitute its composition. Therefore, results obtained from an in-vivo toxicokinetic study with the AOL constituent pyrene can be used as weight of evidence in order to characterise the toxicokinetic properties of anthracene oil.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source material is the substance pyrene. It was purchased from a commercial source and the analytical purity of the test substance after re-purification is reported to be ≥ 99.1 %. Hence, the study results are related to effects caused by pyrene.
The target material anthracene oil is a UVCB substance produced by the distillation of coal tars extracting the approximate distillation range from ca. 300 °C to 400 °C. 10 % to 95 % of the total product distil over between ca. 300 °C and 375 °C. The substance is a brown pasty or liquid material consisting of a complex and within limits variable combination of polycyclic aromatic hydrocarbons. PAH contained in AOL range from naphthalene up to pyrene and benzofluorenes. Two- and three-ring aromatics amount to about 50 % (typical concentration). PAH with four rings accumulate to about 10 % with pyrene being present in a typical concentration of ca. 3 % (composite sample 7).

3. ANALOGUE APPROACH JUSTIFICATION
Properties of the target substance anthracene oil will be determined by the properties of the PAH that are constituents of anthracene oil. Under environmental conditions or during processing of the substance, environmentally available or volatile components of anthracene oil can be released. These will be PAH (mainly compounds consisting of two to four rings). After intake by living organisms, these substances will be distributed within the organism, metabolised, and excreted. Toxicokinetics and metabolism of anthracene oil as such will thus be characterised by its individual constituents. In combination, they will specify the toxicokinetic fate of anthracene oil as a whole. Therefore, it is justified to use data determined for individual PAH that are constituents of anthracene oil, in this case pyrene, to characterise the toxicokinetic fate and behaviour of anthracene oil itself.
Reason / purpose for cross-reference:
read-across source
Principles of method if other than guideline:
Read-across to the preceding entry:
Source substance: pyrene (generic, commercial product);
Reference: Jakob J et al. 1989
Type:
absorption
Results:
The detection of about 7 % of pyrene in the faeces means that at least approx. 93 % of the applied dose were absorbed. But at least 40 % of the dose administered was not recovered.
Type:
metabolism
Results:
Main metabolite was 1-OH-pyrene, which is partly transformed to two dihydroxypyrenes in a secondary oxidation step. The nature of the ultimate metabolites that are excreted, in particular the kind of conjugation, could not be elucidated by this technique.
Type:
metabolism
Results:
Since 1- and 4-methoxypyrene could not be distinguished with the GC method in this study, it could not be excluded that pyrene is not only excreted as 1-OH-pyrene but also as 4,5-diOH-4,5-dihydropyrene, which forms 4-OH-pyrene upon acidification.
Type:
excretion
Results:
The total recovery (excretion) of pyrene plus 1-OH-pyrene in urine and faeces was found to be 53.4 % (50% after 24 hrs) with 7.2% pyrene and 46.2% 1-OH-pyrene. Amount in urine was only about 0.4%.
Details on absorption:
see above
Details on distribution in tissues:
no data
Details on excretion:
see above
Metabolites identified:
yes
Details on metabolites:
see above (source study record)
Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
metabolism
Qualifier:
no guideline available
Principles of method if other than guideline:
in-vitro metabolism study using liver microsomes of non-pretreated rats and of rats pretreated with various mono-oxygenase inducers
GLP compliance:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name of test material (as cited in study report): pyrene
- Source of test material: commercial supplier
- Analytical purity: > 99.5 % (GC, HPLC, and MS) after re-purification
Radiolabelling:
no
Species:
rat
Strain:
not specified
Sex:
not specified
Route of administration:
other: in-vitro incubation
Duration and frequency of treatment / exposure:
30 minute(s)
Dose / conc.:
100 other: nmol/2 mL
Remarks:
approx. 10 mg/L (in vitro incubation test with liver microsomes)
Details on dosing and sampling:
DOSING
100 nmol/2 mL (approx. 10 mg/L)

METABOLITE CHARACTERISATION STUDIES
- Samples: microsomal incubation mixture
- Time and frequency of sampling: at the end of the experiment
- From how many animals: not applicable, in vitro test
- Method type(s) for identification: GC and GC/MS

TREATMENT OF SAMPLES / CLEAVAGE OF CONJUGATES
- Microsoma incubation mixture: The incubations were stopped by addition of acetone. After evaporation of organic solvent and dilution with water, unconverted pyrene and metabolites were extracted at pH 3 (acetic acid) with ethyl acetate (2x). The residues of the extract were chromatographed on a LH-20-Sephadex column with propan-2-ol. The fraction containing pyrene and the metabolites was silylated after evaporation and analysed by GC and GC/MS.
- Recovery: recovery of 1-OH-pyrene proved to be > 95 % when checked in a separate incubation mixture.
Type:
metabolism
Results:
Metabolic transformation with liver microsomes resulted in the formation of one dihydrodiol, one phenol (1-OH-Py, major metabolite, non-K-region oxidation), two diphenols and traces of a triol; the diphenols are formed by secondary oxidation of 1-OH-Py.
Type:
metabolism
Results:
After induction of rat livers with phenobarbital, the 4,5-dihydrodiol became the main metabolite (K-region oxidation). Induction with higher PAH (> 4 rings) resulted in predominance of 1,2-oxidation (non-K-region).
Metabolites identified:
yes
Details on metabolites:
The incubation of pyrene with liver microsomes from untreated rats resulted in the formation of one dihydrodiol, one phenol, two diphenols, and traces of a triol: 1-OH-pyrene is the major metabolite in the normal rat (non-K-region oxidation).
The two dihydroxypyrenes are formed by secondary oxidation of 1-OH-pyrene, which was shown in experiments with 1-OH-pyrene as substrate. One of the diphenols is identical to 1,6-diOH-pyrene, while the position of the second OH-group in the other diphenol could not definitely be concluded. But it is likely to be 1,8-diOH-pyrene, as described by Harper (1957).
[Reference: Harper (1957): The metabolism of pyrene. Brit. J. Cancer, 11, 499-507]
4,5-Dihydroxy-4,5-dihydropyrene was produced in amounts of about 1/3 of the 1-OH-derivatives. After induction of cytochrome P450 with Phenobarbital or PCB, the 4,5-dihydrodiol became the main metabolite, which corresponds to K-region oxidation.
After induction with higher PAH (while phenanthrene and pyrene failed as inducers), cytochrome-P448 stimulation is favoured resulting in the predominance of 1,2-oxidation (non-K-region).
Traces of triols seem to result from secondary oxidation of the dihydrodiols rather than from the phenols as no triols could be detected after incubation with 1-OH-pyrene. As epoxides are converted into phenols during the isolation procedure, it is not possible to distinguish between either compound with this technique.
The nature of the ultimate metabolites that are excreted, in particular the kind of conjugation, could not be elucidated by this technique.
Conclusions:
Metabolism of pyrene: Oxidation occurs primarily in the non-K-region (major metabolite 1-OH-pyrene) when using microsomes from non-induced rats. K-region oxidation (4-,5-position) is only about 1/3. After microsomal induction with higher PAH, only 1,2-oxidation (non-K-region) is increased, while after induction with phenobarbital or PCB, the 4,5-dihydrodiol became the main metabolite, which corresponds to K-region oxidation.
Epoxides and ultimate conjugates could not be detected with the technique used.
Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The source test material is pyrene a polycyclic aromatic hydrocarbon. It consists of four fused aromatic rings and is beside others a main constituent of the target substance. The target substance anthracene oil (benzo[a]pyrene < 50 ppm, AOL) is composed of a broad range of PAH predominantly consisting of two up to four aromatic rings.
Toxicokinetic properties of anthracene oil will be characterised by the range of PAH that constitute its composition. Therefore, results obtained from an in-vitro metabolism study with the AOL constituent pyrene can be used as weight of evidence in order to characterise the toxicokinetic properties of anthracene oil.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source material is the substance pyrene. It was purchased from a commercial source and the analytical purity of the test substance after re-purification is reported to be > 99.5 %. Hence, the study results are related to effects caused by pyrene.
The target material anthracene oil is a UVCB substance produced by the distillation of coal tars extracting the approximate distillation range from ca. 300 °C to 400 °C. 10 % to 95 % of the total product distil over between ca. 300 °C and 375 °C. The substance is a brown pasty or liquid material consisting of a complex and within limits variable combination of polycyclic aromatic hydrocarbons. PAH contained in AOL range from naphthalene up to pyrene and benzofluorenes. Two- and three-ring aromatics amount to about 50 % (typical concentration). PAH with four rings accumulate to about 10 % with pyrene being present in a typical concentration of ca. 3 % (composite sample 7).

3. ANALOGUE APPROACH JUSTIFICATION
Properties of the target substance anthracene oil will be determined by the properties of the PAH that are constituents of anthracene oil. Under environmental conditions or during processing of the substance, environmentally available or volatile components of anthracene oil can be released. These will be PAH (mainly compounds consisting of two to four rings). After intake by living organisms, these substances will be distributed within the organism, metabolised, and excreted. Toxicokinetics and metabolism of anthracene oil as such will thus be characterised by its individual constituents. In combination, they will specify the toxicokinetic fate of anthracene oil as a whole. Therefore, it is justified to use data determined for individual PAH that are constituents of anthracene oil, in this case pyrene, to characterise the toxicokinetic fate and behaviour of anthracene oil itself.
Reason / purpose for cross-reference:
read-across source
Principles of method if other than guideline:
Read-across to the preceding entry:
Source substance: pyrene (generic, commercial product);
Reference: Jacob J et al. 1982
Type:
metabolism
Results:
Metabolic transformation with liver microsomes resulted in the formation of one dihydrodiol, one phenol (1-OH-Py, major metabolite, non-K-region oxidation), two diphenols and traces of a triol; the diphenols are formed by secondary oxidation of 1-OH-Py.
Type:
metabolism
Results:
After induction of rat livers with phenobarbital, the 4,5-dihydrodiol became the main metabolite (K-region oxidation). Induction with higher PAH (> 4 rings) resulted in predominance of 1,2-oxidation (non-K-region).
Details on metabolites:
see above (source study record)
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
Reason / purpose for cross-reference:
reference to other study
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
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
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
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.
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.
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.

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.

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.
Endpoint:
dermal absorption in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The source test material creosote (US type P1/P13) consists predominantly of polycyclic aromatic hydrocarbons ranging in size from two up to five fused rings. The target substance anthracene oil (benzo[a]pyrene < 50 ppm, AOL) is as well composed of a broad range of PAH but predominantly consisting of two to four aromatic rings.
The nature of the matrix and the constituents of both substances are considered to be sufficiently similar that dermal absorption processes will proceed in a similar way. Therefore, the source substance is suited as supporting substance and data resulting from the source substance can be used as weight of evidence for characterising dermal absorption processes of the target substance anthracene oil.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source material creosote (US type P1/P13) is a condensation product in the distillation of coal tars that have been obtained in the high temperature carbonisation of bituminous coal. The material is a UVCB substance forming a dark brown oily liquid. It is only partly volatile and consists of a complex mixture of polycyclic aromatic hydrocarbons with no or only a minimal content of other components (phenols, nitrogen containing compounds < 4 %). Two- and three-ring aromatics amount to about 40 % (typical concentration) with two-ring aromatics forming the smaller fraction. PAH with four and more rings accumulate to about 14 %. Five-ring PAH are only present in low concentration (below 0.3 %). The water solubility of creosote is relatively low. It is determined by the solubility properties of its constituents.
For the skin absorption experiments, individual radiolabelled PAH were added to unlabelled creosote taking into account the quantitative composition of the PAH in creosote. Labelled substances comprised three two-ring, two three-ring, and three four-and five ring PAH providing a mean for determining the contribution of different relevant constituents of creosote in dermal absorption/penetration.
The target material anthracene oil (< 50 ppm BaP, AOL) is a UVCB substance as well produced by the distillation of coal tars extracting the approximate distillation range from ca. 300 °C to 400 °C. 10 % to 95 % of the total product distil over between ca. 300 °C and 375 °C. The substance is a brown pasty or liquid material consisting of a complex and within limits variable combination of polycyclic aromatic hydrocarbons. The distillation range excludes mostly low molecular weight aromatic hydrocarbons (especially one-ring and to a lower extent two-ring aromatics) as well as polycyclic aromatic hydrocarbons composed of more than four to five rings depending on the respective boiling points of the individual aromatic substances. Two- and three-ring aromatics amount to about 50 % (typical concentration) with two-ring aromatics forming the smaller fraction. PAH with four and more rings accumulate to about 10 % with pyrene and benzofluorenes representing the highest molecular weight PAH found in AOL. The water solubility of AOL is low being limited by the solubility properties of its constituents.

3. ANALOGUE APPROACH JUSTIFICATION
Upon contact with skin, substances can be absorbed to skin and in subsequent processes can penetrate skin. Absorption and penetration will depend on the individual substances and on the concentration and matrix, in which the substances are applied to skin.
In this study, the total percentage of absorption (radioactivity) is measured involving all the radiolabelled components in creosote. Substances investigated include compounds that are major constituent of anthracene oil as well. Seven of the eight radiolabelled components of creosote are also present in anthracene oil (exception benzo[a]pyrene). But concentrations in anthracene oil and creosote are somewhat different. Concentration of two- and three-ring PAH up to acenaphthene are higher in creosote than in anthracene oil, while concentrations of the remaining three- and four-ring PAH are higher in anthracene oil. The effect of the matrix is assessed to be similar for both materials, as consistency (slightly viscous liquids) is quite similar. The concentration of the different substances within the materials applied is considered to have only a minor effect, because creosote was overdosed in the study and exhaustion of the different components is of no concern.
It is known from in vitro studies (VanRoiij et al. 1995, Sartorelli et al. 1999) that smaller size PAH are absorbed at a higher rate than larger size PAH. Therefore, it is to be expected that overall absorption of anthracene oil will be lower compared to creosote due to the higher concentration of larger size PAH. Taking into account differences, measured for the absorption of different individual PAH, absorption differences between anthracene oil and creosote can be accounted for. Taking these differences into account, absorption data obtained with creosote can be used to approximate absorption properties of anthracene oil. For these reasons, it is considered justified to use skin absorption data of creosote as weight of evidence in order to characterise skin absorption effects of anthracene oil.
Reason / purpose for cross-reference:
read-across source
Principles of method if other than guideline:
Read-across to preceding entry:
Source test material: US Creosote P1/P13;
Reference: Fasano 2007
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.
Total recovery:
The mass balances were satisfactory with recoveries of more than 92 % of the applied radioactivity.
Key result
Time point:
8 h
Dose:
10.7 mg creosote/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
Remarks:
the test result of the source substance is adopted as weight of evidence for the target substance anthracene oil
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 %).

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The source test material creosote (US type P1/P13) consists predominantly of polycyclic aromatic hydrocarbons ranging in size from two up to five fused rings. The target substance anthracene oil (benzo[a]pyrene < 50 ppm, AOL) is as well composed of a broad range of PAH but predominantly consisting of two to four aromatic rings.
The nature of the matrix and the constituents of both substances are considered to be sufficiently similar that dermal absorption processes will proceed in a similar way. Therefore, the source substance is suited as supporting substance and data resulting from the source substance can be used as weight of evidence for characterising dermal absorption processes of the target substance anthracene oil.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source material creosote (US type P1/P13) is a condensation product in the distillation of coal tars that have been obtained in the high temperature carbonisation of bituminous coal. The material is a UVCB substance forming a dark brown oily liquid. It is only partly volatile and consists of a complex mixture of polycyclic aromatic hydrocarbons with no or only a minimal content of other components (phenols, nitrogen containing compounds < 4 %). Two- and three-ring aromatics amount to about 40 % (typical concentration) with two-ring aromatics forming the smaller fraction. PAH with four and more rings accumulate to about 14 %. Five-ring PAH are only present in low concentration (below 0.3 %). The water solubility of creosote is relatively low. It is determined by the solubility properties of its constituents.
For the skin absorption experiments, individual radiolabelled PAH were added to unlabelled creosote taking into account the quantitative composition of the PAH in creosote. Labelled substances comprised three two-ring, two three-ring, and three four-and five ring PAH providing a mean for determining the contribution of different relevant constituents of creosote in dermal absorption/penetration.
The target material anthracene oil (< 50 ppm BaP, AOL) is a UVCB substance as well produced by the distillation of coal tars extracting the approximate distillation range from ca. 300 °C to 400 °C. 10 % to 95 % of the total product distil over between ca. 300 °C and 375 °C. The substance is a brown pasty or liquid material consisting of a complex and within limits variable combination of polycyclic aromatic hydrocarbons. The distillation range excludes mostly low molecular weight aromatic hydrocarbons (especially one-ring and to a lower extent two-ring aromatics) as well as polycyclic aromatic hydrocarbons composed of more than four to five rings depending on the respective boiling points of the individual aromatic substances. Two- and three-ring aromatics amount to about 50 % (typical concentration) with two-ring aromatics forming the smaller fraction. PAH with four and more rings accumulate to about 10 % with pyrene and benzofluorenes representing the highest molecular weight PAH found in AOL. The water solubility of AOL is low being limited by the solubility properties of its constituents.

3. ANALOGUE APPROACH JUSTIFICATION
Upon contact with skin, substances can be absorbed to skin and in subsequent processes can penetrate skin. Absorption and penetration will depend on the individual substances and on the concentration and matrix, in which the substances are applied to skin.
In this study, the total percentage of absorption (radioactivity) is measured involving all the radiolabelled components in creosote. Substances investigated include compounds that are major constituent of anthracene oil as well. Seven of the eight radiolabelled components of creosote are also present in anthracene oil (exception benzo[a]pyrene). But concentrations in anthracene oil and creosote are somewhat different. Concentration of two- and three-ring PAH up to acenaphthene are higher in creosote than in anthracene oil, while concentrations of the remaining three- and four-ring PAH are higher in anthracene oil. The effect of the matrix is assessed to be similar for both materials, as consistency (slightly viscous liquids) is quite similar. The concentration of the different substances within the materials applied is considered to have only a minor effect, because creosote was overdosed in the study and exhaustion of the different components is of no concern.
It is known from in vitro studies (VanRoiij et al. 1995, Sartorelli et al. 1999) that smaller size PAH are absorbed at a higher rate than larger size PAH. Therefore, it is to be expected that overall absorption of anthracene oil will be lower compared to creosote due to the higher concentration of larger size PAH. Taking into account differences measured for the absorption of different individual PAH, absorption differences between anthracene oil and creosote can be accounted for. Taking these differences into account, absorption data obtained with creosote can be used to approximate absorption properties of anthracene oil. For these reasons, it is considered justified to use skin absorption data of creosote as weight of evidence in order to characterise skin absorption effects of anthracene oil.
Reason / purpose for cross-reference:
read-across source
Principles of method if other than guideline:
Read-across to preceding entry:
Source test material: US Creosote P1/P13;
Reference: Fasano 2007
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)._____________________________
Total recovery:
Findings for rat skin: 78.3 % ± 2.44 %;
Findings for human skin: 83.9 % ± 3.68 %;
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
Remarks:
the test result of the source substance is adopted as weight of evidence for the target substance anthracene oil
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
Remarks:
the test result of the source substance is adopted as weight of evidence for the target substance anthracene oil
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)
Remarks:
the test result of the source substance is adopted as weight of evidence for the target substance anthracene oil
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)
Remarks:
the test result of the source substance is adopted as weight of evidence for the target substance anthracene oil
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
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:
study well documented, meets generally accepted scientific principles, acceptable for assessment
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
Specific details on test material used for the study:
- Name of test material (as cited in study report): coal tar
- Physical state: liquid, viscous
Radiolabelling:
no
Species:
pig
Strain:
other: domestic
Sex:
not specified
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Slaughterhouse: no details
- Weight of animals: 75 - 100 kg
- Experimental conditions: only skin samples (ear) of the animals were used in the in-vitro dermal absorption experiment
Type of coverage:
open
Vehicle:
unchanged (no vehicle)
Duration of exposure:
average perfusion time: 250 min
Doses:
~11 mg coal tar/cm² [100 %]
============================
Content in coal tar / Single PAH dose

fluorene: 2.1 % / 230 µg/cm² ;
phenanthrene: 6.8 % / 750 µg/cm² ;
anthracene: 3.7 % / 410 µg/cm² ;
fluoranthene: 4.0 % / 440 µg/cm² ;
pyrene: 2.1 % / 230 µg/cm² ;
benzo[b]fluoranthene: 0.9 % / 90 µg/cm² ;
benzo[k]fluoranthene: 0.4 % / 44 µg/cm² ;
benzo[a]pyrene: 0.9 % / 90 µg/cm² ;
indeno[123-cd]pyrene: 0.6 % / 66 µg/cm² ;
dibenzo[ah]anthracene: 0.4 % / 44 µg/cm² ;

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, neat substance tested

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)
- 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
- Limits of detection:
[pmol/ml blood]
===============================
fluorene 53.0
phenanthrene 1.7
anthracene 2.0
fluoranthene 51.0
pyrene 2.7
benzo[b]fluoranthene 7.8
benzo[k]fluoranthene 0.3
benzo[a]pyrene 6.3
indeno[123-cd]pyrene 8.3
dibenzo[ah]anthracene 16.5
===============================

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 was carried out at 30 °C with heparinized pig blood collected from the pigs.
Coal tar was applied to a skin area of 6 x 4 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.
Total recovery:
It is not reasonable to estimate total recovery in relation to an applied dose, because the test material was highly overdosed. But owing to the high dosage, the measured fluxes can be supposed to be maximum absorption rates.
==============================
Initial absorption rates of 10 PAHs from coal tar applied to the perfused pig ear:

(Absorption flux at 200 min p.a.)
----------------------------------------------------
Substance: [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 / -- ;
benzo[a]pyrene: 3 / approx. 0.8;
indeno[123-cd]pyrene: 1 / -- ;
dibenzo[ah]anthracene: 1 / --
==============================
Key result
Time point:
200 min
Dose:
11 mg coal tar (230 µg fluorene)/cm²
Parameter:
rate
Absorption:
ca. 71 other: ng/(h*cm²)
Remarks on result:
other: absorption rate for fluorene
Key result
Time point:
200 min
Dose:
11 mg coal tar (750 µg phenanthrene)/cm²
Parameter:
rate
Absorption:
ca. 103 other: ng/(h*cm²)
Remarks on result:
other: absorption rate for phenanthrene
Key result
Time point:
200 min
Dose:
11 mg coal tar (410 µg anthracene)/cm²
Parameter:
rate
Absorption:
ca. 19.5 other: ng/(h*cm²)
Remarks on result:
other: absorption rate for anthracene
Key result
Time point:
200 min
Dose:
11 mg coal tar (440 µg fluoranthene)/cm²
Parameter:
rate
Absorption:
ca. 21 other: ng/(h*cm²)
Remarks on result:
other: absorption rate for fluoranthene
Key result
Time point:
200 min
Dose:
11 mg coal tar (230 µg pyrene)/cm²
Parameter:
rate
Absorption:
ca. 12 other: ng/(h*cm²)
Remarks on result:
other: absorption rate for pyrene
Conversion factor human vs. animal skin:
no applicable

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

--

benzo[a]pyrene

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.

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The source test material coal tar consists predominantly of polycyclic aromatic hydrocarbons ranging in size from two to five fused ring systems. The target substance anthracene oil (benzo[a]pyrene < 50 ppm, AOL) is as well composed of a broad range of PAH but predominantly consisting of two to four aromatic rings.
The nature of the matrix and constituents of both substances are considered to be sufficiently similar that dermal absorption processes will proceed in a similar way. Even if the composition of the source substance is somewhat different from the composition of the target substance, the source substance is considered suited as supporting substance and data resulting from the source substance can be used as weight of evidence for characterising the behaviour of the target substance anthracene oil in dermal absorption processes.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source material coal tar is a condensation product obtained in the high temperature (> 700 °C) destructive distillation of coal. The material is a UVCB substance forming a dark-brown to black viscous liquid denser than water. It is only partly volatile and consists of a complex mixture of condensed ring aromatic hydrocarbons containing hundreds of individual compounds. Minor amounts of phenolic compounds and aromatic nitrogen bases may be included. The composition reported specifies only the ten PAH (three- to five-ring systems) that were determined in the skin absorption experiment ranging from fluorene over phenanthrene/anthracene and pyrene to dibenzo[ah]anthracene and benzo[a]pyrene. Concentrations of two-ring PAH are not specified. Accumulated concentration of the three-ring PAH specified is ca. 13 %, while four- and five-ring PAH amount to ca. 6 % and ca. 3 %, respectively. The water solubility of coal tar is relatively low. It is determined by the solubility properties of its constituents.
For the in-vitro skin absorption experiment, coal tar as such was applied to the perfused pig ear. The parameters measured, however, were flux and 200 min. cumulative uptake in the perfusion blood of the ten individual PAH specified above.
The target material anthracene oil is a UVCB substance as well, produced by the distillation of coal tars extracting the approximate distillation range from ca. 300 °C to 400 °C. 10 % to 95 % of the total product distil over between ca. 300 °C and 375 °C. The substance is a brown pasty or liquid material consisting of a complex and within limits variable combination of polycyclic aromatic hydrocarbons. The distillation range excludes mostly low molecular aromatic hydrocarbons (especially one-ring and to a lower extent two-ring aromatics) as well as polycyclic aromatic hydrocarbons composed of more than four to five rings depending on the respective boiling points of the individual aromatic substances. The three-ring aromatics specified for AOL amount to about 45 % (typical concentration). PAH with four rings accumulate to about 10 % with pyrene and benzofluorenes representing the highest molecular weight PAH found in AOL. The water solubility of AOL is low being limited by the solubility properties of its constituents.

3. ANALOGUE APPROACH JUSTIFICATION

Upon contact with skin, substances can be absorbed to skin and in subsequent processes can penetrate skin. Absorption and penetration will depend on the individual substances and on the concentration and matrix, in which the substances are applied to skin.
In this in-vitro study, the absorption rates of ten individual PAH have been determined that were quantitatively specified as components of coal tar. Coal tar was applied to perfused pig ears and flux and cumulative uptake after 200 min of individual PAHs was determined in the perfusion blood. Five of the ten PAH analysed are also constituents of anthracene oil (fluorene, phenanthrene, anthracene, fluoranthene, pyrene).
The matrix (coal tar and anthracene oil) and the concentration of the components in the matrix is somewhat different. But the differences are considered not to have a relevant effect on the skin absorption/penetration process of individual substances. The application of neat coal tar results in a high local overdose with regard to the individual substances present in coal tar. Thus, the fluxes measured are supposed to represent maximum absorption rates due to the high dosage. The dermal absorption is mainly governed by the skin properties and the properties of individual substances rather than by diffusion limitations in the matrix of the material applied. This is also true for anthracene oil when it is applied to skin.
Overall, absorption of individual components to skin and penetration is considered to be sufficiently similar for both materials that absorption data obtained by an in-vitro skin absorption experiment with coal tar can be used to approximate absorption properties of anthracene oil. For these reasons, it is considered justified to use skin absorption/penetration data obtained with the source substance coal tar as weight of evidence to characterise skin absorption/penetration properties of anthracene oil.
Reason / purpose for cross-reference:
read-across source
Principles of method if other than guideline:
Read-across to preceding entry:
Source test material: coal tar, industrial;
Reference: VanRooij et al. 1995
Total recovery:
It is not reasonable to estimate total recovery in relation to an applied dose, because the test material was highly overdosed. But owing to the high dosage, the measured fluxes can be supposed to be maximum absorption rates.
Key result
Time point:
200 min
Dose:
11 mg coal tar (230 µg fluorene)/cm²
Parameter:
rate
Absorption:
ca. 71 other: ng/(h*cm²)
Remarks on result:
other: absorption rate for fluorene
Remarks:
the test result of the source substance is adopted as weight of evidence for the target substance anthracene oil
Key result
Time point:
200 min
Dose:
11 mg coal tar (750 µg phenanthrene)/cm²
Parameter:
rate
Absorption:
ca. 103 other: ng/(h*cm²)
Remarks on result:
other: absorption rate for phenanthrene
Remarks:
the test result of the source substance is adopted as weight of evidence for the target substance anthracene oil
Key result
Time point:
200 min
Dose:
11 mg coal tar (410 µg anthracene)/cm²
Parameter:
rate
Absorption:
ca. 19.5 other: ng/(h*cm²)
Remarks on result:
other: absorption rate for anthracene
Remarks:
the test result of the source substance is adopted as weight of evidence for the target substance anthracene oil
Key result
Time point:
200 min
Dose:
11 mg coal tar (440 µg fluoranthene)/cm²
Parameter:
rate
Absorption:
ca. 21 other: ng/(h*cm²)
Remarks on result:
other: absorption rate for fluoranthene
Remarks:
the test result of the source substance is adopted as weight of evidence for the target substance anthracene oil
Key result
Time point:
200 min
Dose:
11 mg coal tar (230 µg pyrene)/cm²
Parameter:
rate
Absorption:
ca. 12 other: ng/(h*cm²)
Remarks on result:
other: absorption rate for pyrene
Remarks:
the test result of the source substance is adopted as weight of evidence for the target substance anthracene oil
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:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 428 (Skin Absorption: In Vitro Method)
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD (1996): Dermal delivery and percutaneous absorption: in-vitro method. OECD guideline for testing chemicals – Proposal for new guideline. OECD, Paris
GLP compliance:
not specified
Specific details on test material used for the study:
Mixtuer of 13 PAH (all substances tested): Composition
Name CAS-No. Mol. Mass Applied dose
[nmol/cm²] [ng/cm²]
==============================================================
Naphthalene 91-20-3 128.2 160.0 20,507
Acenaphthene 83-32-9 154.2 120.0 18,505
Fluorene 86-73-7 166.2 231 3,840
Anthracene 120-12-7 178.2 15.1 2,691
Phenanthrene 85-01-8 178.2 12.1 2,157
Pyrene 129-00-0 202.3 9.3 1,881
Benz[a]anthracene 56-55-3 228.3 8.5 1,940
Chrysene 218-01-9 228.3 6.5 1,485
Benzo[b]fluoranthene 205-99-2 252.3 16.5 4,163
Benzo[k]fluoranthene 207-08-9 252.3 6.3 1.589
Benzo[a]pyrene 50-32-8 252.3 6.1 1,539
Dibenz[a,h]anthracene 191-24-2 278.4 7.5 2,088
Benzo[ghi]perylene 53-70-3 276.3 6.9 1,906
==============================================================
Radiolabelling:
no
Species:
monkey
Strain:
other: Ceropithecus aetops
Sex:
not specified
Details on test animals or test system and environmental conditions:
Not details reported, animals were from a medical facility producing poliovirus vaccine
Type of coverage:
other: open diffusion cell
Vehicle:
other: 1.) Lubricating oil: commercial lubricator for engines (PAHs below detection limits) and 2.) Artificial sweat was simulated by using 2.5 g NaH2PO4, 0.2 g triolein, 2 drops of Tween 85 per 1 litre water, pH 5.2 with HCl
Duration of exposure:
static up to 72 h
Doses:
Mixture of PAH:
=============================
Substance: applied dose [ng/cm²];

Acenaphthene: 18,505;
Fluorene: 3,840;
Anthracene: 2,691;
Phenanthrene: 2,157;
Pyrene: 1,881
=============================
Note: Additional lower and higher molecular weight PAH not relevant for anthracene oil have been included in the study.
Control animals:
no
Details on study design:
DOSE PREPARATION
- Method for preparation of dose suspensions: 13 PAH were dissolved/dispersed in either aceton or lubricating oil in such concentrations that after addition to the diffusion cell, doses were as specified under Test material or Doses
- Method of storage: no data

APPLICATION OF DOSE:
- PAH mixture: 1.) 30 µl PAH acetone solution were placed in the diffusion cell, and the acetone was allowed to evaporate. Then a few drops of artificial sweat were added.
2.) PAH mixture dispersed in lubricating oil was placed into the diffusion cell
TEST SITE
- Preparation of test site: no data
- Area of exposure: 1.77 cm²

REMOVAL OF TEST SUBSTANCE
- Removal of protecting device: diffusion cell was dismantled at the end of the test and the skin sample was disposed as waste
- Washing procedures and type of cleansing agent: no washings
- Time after start of exposure:

SAMPLE COLLECTION
- Receiving liquid from diffusion cells: ten 1 ml samples were taken after initiation of skin contact at various intervals from 20 min to 72 h (see Report Fig. 1-6)

SAMPLE PREPARATION
- Preparation details: Purification of media samples with acetonitrile in the presence of NaCl

ANALYSIS
- Method type(s) for identification: reverse-phase HPLC; Column: LC-PAH Supelchem column (25 cm long, 4.6 mm i.d., 5 µm grain size), Eluent: acetonitrile-water gradient; Detector: programmable excitation and emission wavelength spectro-fluorimeter Shimadzu RF 551
- Limits of detection [nmol/L]: naphthalene 25.8, acenaphthene 1.67, fluorene 7.5, phenanthrene 2.80, anthracene 0.56, pyrene 0.49, benzo[a]anthracene 1.32, and chrysene 0.45.
- Coeffcient of variation of the analytical method: 10.8 %
Details on in vitro test system (if applicable):
SKIN PREPARATION
- Source and type of skin: Full-thickness skin from abdomen of Ceropithecus aetops
- Membrane integrity check: Barrier integrity of skin specimens was demonstrated by measuring 3H-H2O penetration after treatment with acetone.
- Storage conditions: Skin samples were frozen and stored for a few days.

PRINCIPLES OF ASSAY
- Diffusion cell: Static diffusion cell (FDC 400, Crown Glass, N.J. USA), exposure area 1.77 cm² (diameter 1.5 cm).
- Receptor fluid: (volume not specified): saline/bovine serum albumin solution (4 % BSA) containing gentamycin sulphate as antibiotic.
The PAH mixture was applied
1. in 30 µl acetone followed by addition of artificial sweat after solvent evaporation (6 diffusion cells)
2. in lubricating oil (7 diffusion cells)
- Static system: yes
- Test temperature: diffusion cell kept at 37 °C resulting in a skin temperature of 32 °C
- Occlusion: no
Absorption in different matrices:
Kinetic key data from in-vitro studies on monkey skin specimens (exposure medium lubrication oil or artificial sweat/acetone):
===============================
Average steady state absorption rate [ng/(h*cm²)]
-----------------------------------------------------
Substance: Lubricating oil / Acetone/sweat

Acenaphthene: 34 / 147;
Fluorene: 6 / 24;
Anthracene: 2.1 / 9.4;
Phenanthrene: 1.0 / 5.7;
Pyrene: 0.3 / 7.8
Results for the additional PAH tested in the study are not reported.
-----------------------------------------------------
Absorption rates of anthracene oil components are calculated from steady state absorption rate, exposure time (8 h corresponding to a work shift) and dose.
===============================
Key result
Dose:
18.5 µg/cm²
Parameter:
percentage
Absorption:
ca. 6.4 %
Remarks on result:
other: substance acenaphthene; absorption period 8 hrs, absorption from artificial sweat/acetone, calculated from steady-state flux after a lag phase of ca. 3 hrs
Key result
Dose:
3.84 µg/cm²
Parameter:
percentage
Absorption:
ca. 5 %
Remarks on result:
other: substance fluorene; absorption period 8 hrs, absorption from artificial sweat/acetone, calculated from steady-state flux after a lag phase of ca. 5 hrs
Key result
Dose:
2.69 µg/cm²
Parameter:
percentage
Absorption:
ca. 2.8 %
Remarks on result:
other: substance anthracene; absorption period 8 hrs, absorption from artificial sweat/acetone, calculated from steady-state flux after a lag phase of ca. 13 hrs
Key result
Dose:
2.16 µg/cm²
Parameter:
percentage
Absorption:
ca. 2.1 %
Remarks on result:
other: substance phenanthrene; absorption period 8 h, absorption from artificial sweat/acetone, calculated from steady-state flux after a lag phase of ca. 11 hrs
Key result
Dose:
1.88 µg/cm²
Parameter:
percentage
Absorption:
ca. 3.3 %
Remarks on result:
other: substance pyrene; absorption period 8 hrs, absorption from artificial sweat/acetone, calculated from steady-state flux after a lag phase of ca. 25 hrs

TEST RESULTS are expressed as

1.    specific steady-state absorption flux F [nmol/(cm²*h)],

2.    a penetration constant Kp [cm/h], a dose-independent parameter which results from division of F by the dose and

3.    the lag time [h]. 

After the lag time, the increase in concentration on the receptor side was nearly linear for some 70 h except for naphthalene, which did not accumulate any more after 30 to 40 h. 

Tab. 1: Kinetic key data from in-vitro studies on monkey skin specimens: specific steady-state absorption flux F [nmol/(cm²*h)], penetration constant Kp [cm/h], and the lag time [h]

 

Lubricating oil

Acetone/
sweat

Lubricating oil  

Acetone/
sweat

Lubricating oil  

Acetone/
sweat

 

Average steady-state absorption rate [ng/(h*cm²]

Average Kp
[cm/h]

Average
lag-time [h]

Naphthalene

35

129

1.87

6.31

4.9

1.2

Acenaphthene

34

147

1.72

7.80

8.4

2.3

Fluorene

6

24

1.64

6.56

5.7

4.2

Anthracene

2.1

9.4

0.93

3.97

17.6

12.9

Phenanthrene

1.0

5.7

0.5

2.63

15.2

11.0

Pyrene

0.3

7.8

0.17

4.13

13.4

24.5

Benz[a]anthracene

n.d.

3.2

n.d.

1.72

--

27.1

Chrysene

0.3

0.8

0.22

0.57

26.1

23.8

Benzo[b]fluoranthene

n.d.

0.9

n.d.

0.09

n.d.

22.5

Benzo[k]fluoranthene

n.d.

0.4

n.d.

0.09

n.d.

23.8

Benzo[a]pyrene

n.d.

0.4

n.d.

0.23

n.d.

31.2

Dibenzo[a,h]anthracene

n.d.

n.d.

n.d.

n.d

n.d.

n.d.

Benzo[g,h,i]perylene

n.d.

n.d.

n.d.

n.d

n.d.

n.d.

n.d. = not detected in receptor fluid (see Report, Tab. 2 + 3)

 

PASSAGES estimated from Fig. 1, 2, 4, and 6 and Tab. 1):

 

OIL MATRIX    

ACETONE/SWEAT

 

Passage in % dose after 24 h

Naphthalene*)

1.5

5

Acenaphthene

2.0

15.0

Fluorene

1.7

10.0

Anthracene

1.2

3.0

Phenanthrene

0.4

2.5

Pyrene

0.5

1.8

Chrysene

0.1

0.25

*) For naphthalene after 10 h /data from Report, Fig. 1- 4, 6 and 7 and Tab. 1)

The average skin absorption of aromatic components was significantly delayed when incorporated in an oil matrix, at factors from about 3 to 7 for individual substances as compared to application in acetone-artificial sweat solution. For benz[a]anthracene, benzo[b]- and benzo[k]fluoranthene as well as benzo[a]pyrene it was possible to demonstrate a passage through the skin only when applied in acetone/sweat mixture (Report, Tab. 3, and p. 530). No passage was found for dibenz[a,h]anthracene and benzo[g,h,i]perylene.
   

Executive summary:

Materials and methods

This in-vitro diffusion system is a suitable technique to demonstrate penetration through the skin, in particular for comparative purposes (species differences and/or different exposure scenarios). In this case, reasons for selection of monkey: similarity of percutaneous penetration through human and monkey skin, based on published data.

Results and discussion

The penetration constant Kp values of compounds resulted in a significant slower passage from the oil matrix than from the acetone/sweat combination (p 0.05) (although the SD was very high, see Tab. 2 and 3): for naphthalene, acenaphthene, fluorene, anthracene and phenanthrene, the penetration was about 4 to 5 times less in the oil matrix than in the solvent/sweat combination. For pyrene and chrysene the factor was in the order of 3. This is also reflected in the significant extended lag-time for the oil matrix. After the lag-time, the increases in concentration on the receptor side were nearly linear for some 70 h except for naphthalene, which did not accumulate any more after 30 to 40 h (see Report Fig. 6). Chrysene was found to be below or at the border of detection limit, while a passage for the other PAH with 4 rings could only be demonstrated when they were applied in acetone/sweat mixture.

Endpoint:
dermal absorption in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The source test materials are individual polycyclic aromatic hydrocarbons (PAH) applied as mixture in an in-vitro skin penetration study. Dermal absorption characteristics will depend on the properties of the individual PAH. The target substance anthracene oil (benzo[a]pyrene < 50 ppm, AOL) is composed of a broad range of PAH differing in size and degree of condensation.
Dermal absorption of anthracene oil will be characterised by the range of PAH that are components of anthracene oil. Five of the major PAH constituents contained in AOL are also present in the PAH mixture of the skin penetration study. Therefore, results from the skin penetration experiment with individual PAH are considered suited to be used as weight of evidence for characterising the properties of the target substance anthracene oil in dermal absorption processes.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemicals are individual PAH. The specific analytical purity of individual chemicals is not reported. But they are assumed to be obtained from commercial sources and to be of sufficient quality for the intended experimental purpose. Results are reported specified for the different PAH used. Hence, the results reported are considered to be valid and to specify the effects caused by the individual test substances.
The target material anthracene oil is a UVCB substance, produced by the distillation of coal tars extracting the approximate distillation range from ca. 300 °C to 400 °C. The substance is a brown pasty or liquid material consisting of a complex and within limits variable combination of polycyclic aromatic hydrocarbons. The distillation range excludes mostly low molecular aromatic hydrocarbons (especially one-ring and to a lower extent two-ring aromatics) as well as polycyclic aromatic hydrocarbons composed of more than four to five rings depending on the respective boiling points of the individual aromatic substances. The three-ring aromatics specified for AOL amount to about 45 % (typical concentration). PAH with four rings accumulate to about 10 % with pyrene and benzofluorenes representing the highest molecular weight PAH found in AOL. The water solubility of AOL is low being limited by the solubility properties of its constituents.

3. ANALOGUE APPROACH JUSTIFICATION
Upon contact with skin, substances can be absorbed to skin and in subsequent processes can penetrate skin. Absorption and penetration will depend on the individual substances and on the concentration and matrix, in which the substance is applied. Substances investigated in the in-vitro skin penetration study include, besides others, substances that are also present in anthracene oil (predominantly three- and four-ring PAH). Even if the matrix is different (artificial sweat/mineral oil versus anthracene oil itself), skin absorption and penetration is assumed to be sufficiently similar that absorption data obtained by an in-vitro skin absorption experiment with a mixture of individual PAH can be used to approximate absorption properties of anthracene oil. For these reasons, it is considered justified to use skin absorption/penetration data of individual PAH that are constituents of anthracene oil in combination as weight of evidence in order to characterise skin absorption/penetration effects of anthracene oil as such.
Reason / purpose for cross-reference:
read-across source
Principles of method if other than guideline:
Read-across to preceding entry:
Source test material: Mixture of PAH (in vitro percutaneous penetration assay);
Reference: Sartorelli et al. 1999
Absorption in different matrices:
Kinetic key data from in-vitro studies on monkey skin specimens (exposure medium lubrication oil or artificial sweat/acetone):
===============================
Average steady state absorption rate [ng/(h*cm²)]
-----------------------------------------------------
Substance Lubricating oil / Acetone/sweat

Fluorene: 6 / 24;
Anthracene: 2.1 / 9.4;
Phenanthrene: 1.0 / 5.7;
Pyrene: 0.3 / 7.8
Results for the additional PAH tested in the study are not reported.
-----------------------------------------------------
Absorption rates of wash oil components are calculated from steady state absorption rate, exposure time (8 h corresponding to a work shift) and dose.
===============================
Key result
Dose:
18.5 µg/cm²
Parameter:
percentage
Absorption:
ca. 6.4 %
Remarks on result:
other: substance acenaphthene; absorption period 8 hrs, absorption from artificial sweat/acetone, calculated from steady-state flux after a lag phase of ca. 3 hrs
Remarks:
the test result of the source substance is adopted as weight of evidence for the target substance anthracene oil
Key result
Dose:
3.84 µg/cm²
Parameter:
percentage
Absorption:
ca. 5 %
Remarks on result:
other: substance fluorene; absorption period 8 hrs, absorption from artificial sweat/acetone, calculated from steady-state flux after a lag phase of ca. 5 hrs
Remarks:
the test result of the source substance is adopted as weight of evidence for the target substance anthracene oil
Key result
Dose:
2.69 µg/cm²
Parameter:
percentage
Absorption:
ca. 2.8 %
Remarks on result:
other: substance anthracene; absorption period 8 hrs, absorption from artificial sweat/acetone, calculated from steady-state flux after a lag phase of ca. 13 hrs
Remarks:
the test result of the source substance is adopted as weight of evidence for the target substance anthracene oil
Key result
Dose:
2.16 µg/cm²
Parameter:
percentage
Absorption:
ca. 2.1 %
Remarks on result:
other: substance phenanthrene; absorption period 8 h, absorption from artificial sweat/acetone, calculated from steady-state flux after a lag phase of ca. 11 hrs
Remarks:
the test result of the source substance is adopted as weight of evidence for the target substance anthracene oil
Key result
Dose:
1.88 µg/cm²
Parameter:
percentage
Absorption:
ca. 3.3 %
Remarks on result:
other: substance pyrene; absorption period 8 hrs, absorption from artificial sweat/acetone, calculated from steady-state flux after a lag phase of ca. 25 hrs
Remarks:
the test result of the source substance is adopted as weight of evidence for the target substance anthracene oil

Description of key information

Anthracene oil (benzo[a]pyrene < 50 ppm; AOL) is composed of a broad range of PAH. These PAH are absorbed rapidly through the pulmonary tract, the gastrointestinal tract and to a much lesser extent through skin.

PAH are widely distributed throughout the organism after administration by any route and are found in almost all internal organs.

Metabolism is complex resulting mostly in hydroxylated species, which in part may further be metabolised by additional hydroxylation. Metabolites can be conjugated to sulfuric or glucuronic acids or with glutathione. Excretion is via urine, bile and faeces. Conjugates excreted in bile can be hydrolysed in the gut and reabsorbed.

Absorption of complex PAH mixtures like tar oils through human skin will be not more than 2 % within and after 8 h of exposure. Permeation through rat skin is much more pronounced (ca. 8 fold).

Key value for chemical safety assessment

Bioaccumulation potential:
low bioaccumulation potential
Absorption rate - dermal (%):
2

Additional information

Toxicokinetics

Anthracene oil (AOL) is mainly composed of two- to four-ring PAH. Constituents with typical concentration above ca. 2 % w/w are naphthalene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, and pyrene. These substances amount to about 50 to 55 % of total anthracene oil (typical concentration). They, together with other minor PAH, will primarily determine the toxicokinetic properties of anthracene oil. The toxicokinetics of anthracene oil can largely be characterised based on information determined for its PAH constituents, even if there are some differences depending on their individual properties (molecular structure, log Pow, water solubility, vapour pressure).

Absorption:

In general, anthracene oil PAH constituents will be absorbed rapidly through the pulmonary tract, the gastrointestinal tract, and to a lesser extent through skin (see WHO 1998, Grimmer et al. 1991, Jacob et al. 1989). The rate of absorption from the lungs depends on the type of PAH, the size of the particles, on which they are absorbed, and the composition of the adsorbent. PAH adsorbed onto particulate matter are cleared from the lungs more slowly than free hydrocarbons (WHO 2003).

Degree of absorption may be different for individual substances. Gastrointestinal absorption in rodents has been reported to be 80 to over 90 % (Grimmer et al. 1991, Jacob et al. 1989). Effective absorption by the different routes is also evidenced by the observation of systemic toxicity following exposure by the different routes.

Distribution:

PAH are widely distributed throughout the organism after administration by any route and are found in almost all internal organs, but particularly those rich in lipids. Intravenously injected PAH are cleared rapidly from the bloodstream of rodents, but can cross the placental barrier and have been detected in foetal tissues (WHO 1998).

Metabolism:

The metabolism of PAH to more water-soluble derivatives, which is a prerequisite for their excretion, is complex. Major metabolic pathway of typical PAH is oxidation at an aromatic ring (a reaction catalysed by cytochrome P450-dependent mono-oxygenases) forming a cyclic epoxide in a first step followed by ring cleavage/rearrangement resulting in a phenol, or followed by epoxide hydroxylation yielding a dihydrodiol derivative. In following steps, conjugation by glutathione, sulphate or glucuronic acid may occur. Alternatively or in addition, a second oxidation at another position of the aromatic system is possible resulting in dihydrodiol epoxides or in tetrahydrotetrols. Secondary oxidation can also be followed by conjugation. Most metabolism results in detoxification, but some PAH are activated to DNA-binding species, principally diol epoxides, which can initiate tumours.

For phenanthrene, metabolites detected were hydroxy-phenanthrenes (1-, 2-position ca. 60 % of total OH-derivatives, 3-, 4-, and 9-position minor). Dihydrodiols were not detected and may have escaped determination (Grimmer et al. 1991). In an experiment with liver microsomes from untreated rats (Jacob et al. 1982), trans 9,10-dihydrodiol was identified (K-region oxidation) indicating that other hydroxy-derivatives of phenanthrene can be formed as recovered in the study of Grimmer et al. Metabolites of fluoranthene identified by Polcaro et al. (1988) were 2,3-dihydro-2,3-dihydroxyfluoranthene (2,3-dihydrodiol), 3-hydroyfluoranthene, and 1-hydroxyfluorathene. In addition, a further minor metabolite, the 2,3-dihydro-2,3-dihydroxyfluoranthene 1.10b-epoxide, was detected by Day et al. (1992) formed by subsequent oxidation. The main metabolite of pyrene formed in-vivo was 1-OH-pyrene. Due to the technique applied, other possible metabolites (4-OH-pyrenes and dihydrodiols) as well as ultimate metabolites (conjugates) could not be detected. In in-vitro experiments with liver microsomes (Jacob et al. 1982), additional metabolites were detected (di- and trihydroxypyrenes, dihydrodiols).

Some PAH constituents in anthracene oil have special structural features with regard to their aromatic nature. Acenaphthene and fluorene possess besides aromatic carbon atoms also aliphatic carbon atoms. These are methylene groups bridging the two phenyl rings. These structural elements are also metabolised by oxidation leading to (intermediate) hydroxyl derivatives. In acenaphthene, oxidation of the two methylene groups results finally in the 1,8-dicarboxylic acid identified as the anhydride of the acid (Chang 1943). The carboxylic acid is formed by subsequent oxidation of the primary hydroxyl derivative.

Excretion:

In general, PAH metabolites and their conjugates are excreted via the urine and faeces, but conjugates excreted in the bile can be hydrolysed by enzymes of the gut flora and be reabsorbed. It can be inferred from the available information on the total human body burden that PAH do not persist in the body and that turnover is rapid. This inference excludes those PAH moieties that become covalently bound to tissue constituents, in particular nucleic acids, and are not removed by repair.

Excretion of PAH constituents of anthracene oil will change from excretion to urine for small PAH to faeces for larger size PAH (increased lipophilicity) depending on the nature and conjugation of metabolites. For naphthalene, 83 % of the radioactivity administered was recovered in urine and 6 % in faeces after 72 hours (Bakke 1985). Recovery of phenanthrene including hydroxyl derivatives was only 10.5 % within three days with 4.1 % in the urine and 6.4 % in faeces (ratio ca. 4 : 6). About 90 % of the dose could not be accounted for indicating that probably other water soluble metabolites are formed that were not recorded under the experimental conditions of the study (Grimmer 1991).

Dermal absorption

In synopsis of observations from comparative in-vivo and in-vitro studies (human vs. rodent) on spiked creosote (Fasano 2007a,b), there is evidence that only ca. 3 % to 4 % of a dermal dose will be absorbed through human skin specimens within and after 8 hours of exposure (Fasano 2007b). For rat skin, absorption was ca. 15 to 34 %. The conversion factor human vs. rat skin was found to be 0.12 (4/34), which means that the dermal dose absorbable within 8 hours is about 8-fold higher in rat than in human skin. Taking into account the maximum absorption of creosote in the in-vivo skin absorption experiment with rats (14.8 %), the maximum human skin absorption is calculated to be below 2 % (14.8 x 0.12).

Data for creosote cannot be transferred directly to anthracene oil. Studies of VanRooij et al. 1995 and Sartorelli et al. 1999 show that lower molecular weight PAH are absorbed through skin faster than higher molecular weight PAH. Two-ring and some smaller three-ring PAH are present in creosote in higher concentrations than in anthracene oil. On the other hand, three- and four ring PAH are more abundant in anthracene oil than in creosote. Overall it is considered that differences in both materials do not require an adjustment of the percentage for skin absorption of humans, especially as this value is already a worst-case estimate.

 

References:

WHO (2003). HEALTH RISKS OF PERSISTENT ORGANIC POLLUTANTS FROM LONG-RANGE TRANSBOUNDARY AIR POLLUTION, JOINT WHO/CONVENTION TASK FORCE ON THE HEALTH ASPECTS OF AIR POLLUTION. WHO Regional Office for Europe, World Health Organization 2003

Bakke J et al. (1985). Catabolism of pre-mercapturic acid pathway metabolites of naphthalene to naphthols and methylthio-containing metabolites in rats. Proc. Natl. Acad. Sci. USA Vol. 82 (3), pp. 668-671