Anthracene oil

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genetic toxicity of anthracene oil (BaP < 50 ppm, AOL) is assessed based on tests with anthracene oil itself as test material and with the closely structure-related supporting substance creosote. AOL showed a weak mutagenic potential in a bacterial reversed mutation assay (Ames test) with and without metabolic activation. In an in vitro chromosomal aberration study with creosote, only negative results were found with and without metabolic activation. In a third test (in vitro gene mutation study in mammalian cells) using creosote as test material, a weak mutagenic response was observed, but only with metabolic activation. Experiments without metabolic activation did not show a mutagenic response.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 Aug. - 06 Sep. 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Principles of method if other than guideline:

Two replicate experiments: plate incorporation assay and preincubation test

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): anthracene oil (< 50 ppm BaP)
- Lot/batch No. of test material: ATE No. 10443
- Stability under test conditions: no measured data; based on chemical structure assumed to be stable
- Storage condition of test material: room temperature, exclusion of light

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Microsomal fraction prepared from induced livers of male Wistar rats, induced with phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw) orally (3x)

Test concentrations with justification for top dose:

1st experiment: 3.16, 10, 31.6, 100, 316, and 1000 µg/plate (TA 98, TA 100, -S9)
1.0, 3.16, 10, 31.6, 100, 316, and 1000 µg/plate (TA 1535, TA 1537, TA 102, - S9)
3.16, 10, 31.6, 100, 316, 1000 and 2500 µg/plate (+ S9)
2nd experiment: with variations based on results of 1st experiment (Report, p. 11, p. 19)
3rd experiment: 200, 400, 500, 600, 750, and 1000 (only with TA 100, +S9)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: compatible with survival of bacteria and S9 activity

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: see Report p. 15

Details on test system and experimental conditions:

METHOD OF APPLICATION:
1st experiment: in agar (plate incorporation)
2nd and 3rd experiment: pre-incubation variant

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth/colony formation

Evaluation criteria:

Considered as mutagenic
- if a clear and dose-related increase in the number of revertants occurs in at least one tester strain with or without metabolic activationand/or
- if a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.

An increase is considered relevant
- if in TA 100 and TA 102 mutation rate is at least twice as high as the rate of the solvent control;
- if in TA 98, TA 1535, and TA 1537 the mutation rate is at least 3 fold higher than that of the solvent control.

Statistics:

According to the OECD guidelines, the biological relevance is the criterion for the interpretation of the results: a statistical evaluation was not considered necessary under this premise (report p. 21).

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

in 2nd and 3rd test: reproducible at cytotoxic concentrations (MF = 2.2 and 2.3)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at ≥ 200 µg/pl. (+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxic at higher concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA 98, TA 1535, TA 1537, TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

reproducible in both tests

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxic at higher concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

No mutagenic activity was observed in any test (plate incorporation and pre-incubation assay) at non-cytotoxic concentrations. In the pre-incubation assay, a mutagenic response was only observed in tester strain TA 100 with metabolic activation and at cytotoxic concentrations.

Experiment I (plate incorporation assay): No mutagenic activity was observed at all concentrations with and without metabolic activation.

Experiment II (pre-incubation assay): Relevant increase in the number of revertants in TA 100 but only at cytotoxic concentrations (500 µg/pl., cytotoxic, +S9) with MF = 2.0

Experiment III (pre-incubation assay): Relevant increases in revertants in TA 100 but only at cytotoxic concentrations (≥ 200 µg/plate, cytotoxic, +S9) with MF = 2.2 and 2.3.

Conclusions:

From the five tester strains tested, only one (TA 100) showed a weak positive response with metabolic activation at already cytotoxic concentrations.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

Only one cultivation instead of two was conducted per test series (no duplicates).

GLP compliance:

no

Type of assay:

other: in vitro human lymphocytes chromosome aberration test

Specific details on test material used for the study:

- Creosote WEI-Type B
- Name of test material (as cited in study report): Creosote Spéciale 14130

Species / strain / cell type:

lymphocytes: human

Metabolic activation:

with and without

Metabolic activation system:

microsomes derived from Arochlor1254-induced rat liver (male SD rats, 500 mg/kg i.p. 5 d prior to sacrifice)

Test concentrations with justification for top dose:

0.05, 0.1, 0.15, 0.25 mg/ml
concentrations of 0.15 and 0.25 mg/ml showed a reduction of the mitotic index of about 56 % and between 90.5 and 82.6 %, respectively.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: yes, no further information

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 0.15 and 0.25 mg/mL; the two highest concentrations exhibited a reduction of the mitotic index of about 50% and between 90.5 and 82.6%, respectively

Vehicle controls validity:

valid

Positive controls validity:

valid

Summary of cytogenetic In-Vitro-Test: Chromosomal Analysis

		Vehicle control		0.05 mg/ml		0.1 mg/ml		0.15 mg/ml		0.25 mg/ml
cytotoxicity		no		no		no		Yes (MI ~ -50%)		Yes (MI < -80%)
Metaphases scored (1^sttest / 2^ndtest)		100 / 100	100 / 100	100 / 100	100 / 100	100 / 100	100 / 100	- / 100	- / 100	19 / 97	38 / 38
		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
chromatid aberrations	Gaps (TG)^1)^2)	1 / 3	3 / 2	1 / 0	3 / 1	1 / 2	2 / 0	- / 1	- / 2	0 / 4	2 / 0
	Breaks (TB)^2)	0 / 0	3 / 1	0 / 1	1 / 2	1 / 1	0 / 4	- / 1	- / 1	0 / 0	1 / 0
	interchanges	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0
Isochromatid aberrations	Gaps (SG)^1)2)	0 / 2	1 / 0	1 / 0	0 / 0	1 / 1	0 / 0	- / 0	- / 0	0 / 1	0 / 1
	Breaks (SB)^2)	1 / 1	2 / 1	2 / 1	0 / 1	0 / 1	0 / 1	- / 4	- / 6	0 / 4	0 / 0
	interchanges	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0
uncoiled chromatin (UC)^2)		0 / 0	0 / 0	0 / 0	0 / 0	0 / 0	1 / 0	- / 0	- / 0	0 / 0	0 / 0
mitotic index		4.2 / 5.4	4.6 / 5.4	5.4 / 6.0	4.4 / 5.0	4.2 / 4.6	4.6 / 4.2	- / 2.4	- / 3.0	0.4 / 1.4	0.8 / 1.2
polyploidy		0 / 0	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0	- / 0	- / 0	0 / 0	0 / 0
endo reduplication		0 / 0	0 / 0	0 / 0	0 / 0	0 / 0	0 / 0	- / 0	- / 0	0 / 0	0 / 0
Mean number of aberrations per cells		0.01 / 0.01	0.05 / 0.02	0.02 / 0.02	0.01 / 0.03	0.01 / 0.02	0.00 / 0.05	- / 0.05	- / 0.07	0.00 / 0.04	0.03 / 0.00
CH^2test results		--	--	0.34 / 0.34	1.85 / 0.21	0.00 / 0.34	4.08 / 1.33	- / 1.85	- / 0.69	0.19 / 1.94	0.15 / 0.77

MI = Mitotic index; 

¹⁾not included in total aberration frequency

²⁾TB, TG, SB, SB, UC = Abbreviations used in tables of the test report

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The selected test concentrations clearly covered a cytotoxic range, with about 50 % depression of the mitotic index at the additional concentration of 0.15 mg/ml, as compared to vehicle control and the lower test levels.

Under the test conditions, the creosote WEI Type B (containing <50 ppm BaP) did not induce chromosome aberrations in human lymphocytes in culture in the presence and absence of metabolic activation. Based upon the CHI-square test (p <0.01), no statistical significance was found as compared with the vehicle control. Metabolic activation had no noticeable influence on the aberration spectrum and yield. The positive controls showed significant increases in aberration frequencies.

Conclusions:

Creosote WEI Type B (containing < 50 ppm BaP) did not induce chromosome aberrations under the conditions of the test performed in human lymphocytes in culture in the presence and absence of metabolic activation. Metabolic activation had no noticeable influence on the aberration spectrum and yield.

Executive summary:

A creosote containing less than 50 ppm BaP was tested according to the standard test guidelines OECD 473 (1983) and EEC Directive 84/449, B.10 (1984). Blood had been obtained from healthy non-smoking volunteers, and lymphocytes were proliferated and stimulated through cultivation with phytohaemagglutinin (48 h). Exposure time was 3 h, post-exposure time 22 h. Cell growth was stopped by addition of colchicine (2 h incubation). In a preliminary test, the cytotoxic potential of the creosote was estimated (mitotic index without S9, based on 500 cells per concentration). A confirmatory second independent main study was performed without variation of culture parameters (except a further test concentration: see below).

In the second test series, a further test concentration (0.15 mg/ml) was introduced as 3rd of 4 concentrations, because the highest one previously selected proved to be too cytotoxic in the 1st test series.

Besides examination of chromosomal aberrations, the mitotic index was concurrently determined for each concentration tested. Gaps were recorded, but not included in the final result of aberration frequency. Under the test conditions, the creosote WEI Type B (containing <50 ppm BaP) did not induce chromosome aberrations in human lymphocytes in culture in the presence and absence of metabolic activation. Based upon the CHI-square test (p <0.01), no statistical significance was found as compared with the vehicle control. Metabolic activation had no noticeable influence on the aberration spectrum and yield. The positive controls showed significant increases in aberration frequencies.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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 special 14130 (WEI-Type B, 1991) consists predominantly of polycyclic aromatic hydrocarbons (PAH) ranging in size from two up to five fused rings. The target substance anthracene oil (anthracene oil with < 50 ppm benzo[a]pyrene (BaP), AOL) is as well composed of a broad range of PAH but predominantly consisting of two to four aromatic rings.
The nature of both substances and their constituents are considered to be sufficiently similar that in vitro cytogenic effects (chromosomal aberration in mammalian cells) are equivalent. Therefore, the source substance is suited as supporting substance with regard to in vitro cytogenicity and data resulting from the source substance can be used for characterising the in vitro genotoxic potency of the target substance anthracene oil.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source test material creosote (special 14130, WEI-Type B) 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 (PAH) with only minor levels of other components (phenols, N-compounds approx. < 10 %). Accumulated concentrations of PAH with two and three rings are approx. 50 % with two-ring aromatic building the larger fraction. Analytical data for four-ring PAH is incomplete. Taking information from other creosotes into account, it is assumed that accumulated percentage of four- and five-ring PAH is < 10 % with individual concentrations of carcinogenic five-ring PAH being < 50 ppm. The water solubility of creosote is relatively low. It is determined by the solubility properties of its constituents.
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 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. 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
In in vitro chromosomal aberration studies in mammalian cells, test materials are directly applied to the test system, in this case dissolved in a suitable solvent. Upon application, components of complex mixtures like UVCBs can directly exert their adverse effects. Effects will be determined by the individual constituents of the mixtures and in combination will specify the effect of the complete mixture. If substances are similar, the genetic toxicity will be comparable.
The general composition of the source and target substance is similar. Relevant constituents are two-ring up to four- to five-ring PAH. Two- and three-ring PAH are present in the source substance creosote WEI-Type B and in the target substance anthracene oil in comparable amounts, just the fraction of two-ring aromatics being somewhat higher in creosote and the fraction of three-ring aromatics being somewhat higher in AOL. Concentrations of PAH with carbon frames containing more than three rings may be somewhat higher in anthracene oil, while the concentrations of (carcinogenic) five-ring PAH like e.g. benzo[a]pyrene are very low (< 50 ppm).
Overall, some differences in the PAH composition of both materials are considered not to result in significantly modified genotoxic effects of the source and the target material. The range of PAH is similar. Due to similar metabolic activation pathways, PAH will result in similar final toxicants in chromosomal aberration assays with metabolic activation. Within a range, cytotoxic effects caused by the source and the target substance are considered to be comparable. For these reasons, it is considered justified to use in vitro genetic toxicity data of creosote in order to characterise genotoxic 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: creosote spéciale 14130, WEI-Type B_1991;
Reference: Weill 1991

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 0.15 mg/mL and 0.25 mg/mL; the two highest concentrations exhibited a reduction of the mitotic index of about 50 % and between 90.5 and 82.6 %, respectively

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: the test results of the source substance are adopted for the target substance anthracene oil

Conclusions:

The test (source) material creosote WEI Type B (containing < 50 ppm BaP) did not induce chromosome aberrations under the conditions of the test performed in human lymphocytes in culture in the presence and absence of metabolic activation. Metabolic activation had no noticeable influence on the aberration spectrum and yield.
The test results of the source substance Creosote WEI Type B (containing < 50 ppm BaP) are adopted for the target substance anthracene oil.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian cell gene mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Creosote
- Supplier: HGD S.A., France; Creosote WEI-Type B, containing less than 50 ppm BaP

Target gene:

Thymidine-kinase(TK) competent cells --> TK-deficient cells (= mutation to allow survival)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Mouse lymphoma L5178Y cells (TK+/-), stock from ATCC/Maryland, USA (code: CRL 9518)

Metabolic activation:

with and without

Metabolic activation system:

microsomes derived from phenobarbital/ß-naphthoflavone induced rat liver (male SD rats), metabolic activity verified in separate testing for specific enzyme activity as well as in bacterial mutation assay.

Test concentrations with justification for top dose:

2.5 - 100 µg/mL (details see below)
In general six, in one case five concentrations were tested, and - if necessary - adjusted in the second test series.

Experiment 1: with metabolic activation: 40.0, 30.0, 20.0, 10.0, 5.00 and 2.50 µg/mL;
without metabolic activation: 50.0, 40.0, 20.0, 10.0, 5.00 and 2.50 µg/mL;
Experiment 2: with metabolic activation: 40.0, 30.0, 20.0, 13.3 and 8.89 µg/mL;
without metabolic activation: 100, 75.0, 50.0, 25.0, 12.5 and 6.25 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension
- Cell density at seeding (if applicable): 0.15E6 or 0.5E6

DURATION
- Exposure duration: experiment 1: 3 hrs with and without metabolic activation
experiment 2: 3 hrs with metabolic activation
24 hrs without metabolic activation
- Expression time (cells in growth medium): no data
- Selection time (if incubation with a selection agent): no data

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- Any supplementary information relevant to cytotoxicity: cytotoxicity was examined in preliminary experiments. At a concentration of 100 µg/mL, 100 % mortality of cells was observed.

Evaluation criteria:

Criteria for outcome of assay:
For a test item to be considered mutagenic in this assay, it is required:
(ii)   the mutant frequency at one or more doses is statistically significantly greater than that of the negative control;
(iii)  there is a significant dose-relationship as indicated by the linear trend analysis;
(iv)  any increase in mutant frequency should significantly lie outside the historical control range.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at > 20 - 75 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at > 40 - 100 mg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: weak mutagenic potential

Report TABLE 18 - SUMMARY TABLE

Dose-level	WITHOUT S9 (Treatment time: 3 hours), SOLVENT: DMSO
(mg/ml)	%RS	RTG	MF°	P	Proportion small colony mutants
0.00 2.50 5.00 10.0 20.0 40.0 50.0 MMS 10.0	100 91 91 71 76 75 37 75	100 79 87 90 86 54 21 44	58.11 72.37 56.58 55.38 65.04 113.1 69.60 296.8	- NS NS NS NS ** NS -	0.32 0.53 0.47 0.44 0.42 0.50 0.46 0.48
Linear trend				*

Dose-level	WITHOUT S9 (Treatment time: 24 hours), SOLVENT: DMSO
(mg/ml)	%RS	RTG	MF°	P	Proportion small colony mutants
0.00 6.25 12.5 25.0 50.0 75.0 100 MMS 5.00	100 83 71 86 61 39 0 48	100 99 91 101 64 33 · 54	55.05 54.14 72.63 74.30 80.52 75.86 · 444.2	- NS NS NS NS NS -	0.25 0.17 0.21 0.26 0.28 0.26 · 0.23
Linear trend				*

Report TABLE 19 - SUMMARY TABLE

Dose-level	WITH S9 (Treatment time: 3 hours), SOLVENT: DMSO
(mg/ml)	%RS	RTG	MF°	P	Proportion small colony mutants
0.00 2.50 5.00 10.0 20.0 30.0 40.0 B(a)P 2.00	100 100 77 65 58 38 30 50	100 69 55 36 26 23 18 28	94.02 145.8 166.1 204.8 211.2 198.0 184.1 560.6	- NS ** ** ** ** ** -	0.28 0.27 0.28 0.35 0.44 0.45 0.44 0.37
Linear trend				***

Dose-level	WITH S9 (Treatment time: 3 hours), SOLVENT: DMSO
(mg/ml)	%RS	RTG	MF°	P	Proportion small colony mutants
0.00 8.89 13.3 20.0 30.0 40.0 B(a)P 2.00	100 58 51 31 26 32 43	100 53 39 22 17 30 35	63.59 121.2 146.7 216.9 254.7 167.2 519.6	- ** ** ** ** ** -	0.16 0.27 0.25 0.38 0.35 0.33 0.36
Linear trend				***

°   = Figures displayed are mutation frequencies per million surviving cells

 ·  = Insufficient viable cells recovered after treatment incubation period

NS = Not statistically significant

*   = Statistically significant at P<5%

** = Statistically significant at P<1%

Conclusions:

When tested in an in vitro mammalian cell gene mutation assay according to OECD TG 476 (TK locus of mouse lymphoma L5178Y cells), creosote WEI Type B (containing < 50 ppm BaP) did only show a mutagenic response (statistically significant increase in mutation frequency) in the presence of a metabolic activation system. Without metabolic activation, the response was negative. The mutation potential of the applied creosote appears to be low based on the test results.

Executive summary:

A creosote containing less than 50 ppm BaP was tested according to standard guidelines OECD 476 (1997). The established microtiter method was applied. An exposure time was chosen of 3 and 24 h in the absence and 2x 3 h in the presence of a metabolic system. Cytotoxicity and solubility were examined in preliminary experiments. Two independent main test series were conducted, each in duplicate for each test concentration. In general six, in one case five concentrations were tested, and – if necessary – adjusted in the second test series. The highest concentrations exhibited moderate cytotoxicity. Based on 100-% mortality of cells at 100 µg/mL, 50 to 75 µg/mL were the highest possible concentrations for testing. Creosote <50 ppm BaP showed a weak positive mutagenic activity in the presence of metabolic activation.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

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 (WEI-Type B, 2002) consists predominantly of polycyclic aromatic hydrocarbons (PAH) ranging in size from two up to five fused rings. The target substance anthracene oil (anthracene oil with < 50 ppm benzo[a]pyrene (BaP), AOL) is as well composed of a broad range of PAH but predominantly consisting of two to four aromatic rings.
The nature of both substances and their constituents are considered to be sufficiently similar that in vitro mutagenic effects in mammalian cells are equivalent. Therefore, the source substance is suited as supporting substance with regard to in vitro genetic toxicity and data resulting from the source substance can be used for characterising the in vitro mutagenic potency of the target substance anthracene oil.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source test material creosote (WEI-Type B) 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 (PAH) with only minor levels of other components (phenols, N-compounds approx. < 10 %). Accumulated concentrations of PAH with two and three rings are approx. 25 % with two-ring aromatic building the larger fraction. Four ring PAH accumulate to about 6 % with the accumulated concentration of carcinogenic five-ring PAH being < 50 ppm. The water solubility of creosote is relatively low. It is determined by the solubility properties of its constituents.
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 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. 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
In in vitro mammalian cell gene mutation assays, test materials are directly applied to the test system, in this case dissolved in dimethyl sulfoxide. Upon application, components of complex mixtures like UVCBs can directly exert their adverse effects. Effects will be determined by the individual constituents of the mixtures and in combination will specify the effect of the complete mixture. If substances are similar, the genetic toxicity will be comparable.
The general composition of the source and target substance is similar. Relevant constituents are two-ring up to four- to five-ring PAH. Two- and three-ring PAH are present in the source substance creosote WEI-Type B and in the target substance anthracene oil in comparable amounts, just the fraction of two-ring aromatics being somewhat higher in creosote and the fraction of three-ring aromatics being somewhat higher in AOL. Concentration of PAH with carbon frames containing more than three rings may be somewhat higher in anthracene oil, while the concentrations of (carcinogenic) five-ring PAH like e.g. benzo[a]pyrene are very low (< 50 ppm).
Overall, some differences in the PAH composition of both materials are considered not to result in significantly modified genotoxic effects of the source and the target material. The range of PAH is similar. Due to similar metabolic activation pathways, PAH will result in similar final toxicants in gene mutation assays with metabolic activation. Within a range, mutagenic effects caused by the source and the target substance are considered to be comparable. For these reasons, it is considered justified to use data of in vitro mammalian cell gene mutation assays obtained with creosote in order to characterise gene mutation 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: creosote spéciale 14130, WEI-Type B_1991;
Reference: Brightwell 2002

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at > 20 - 75 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at > 40 - 100 mg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: weak mutagenic potential

Remarks:

the test result of the source substance is adopted for the target substance anthracene oil

Conclusions:

When tested in an in vitro mammalian cell gene mutation assay according to OECD TG 476 (TK locus of mouse lymphoma L5178Y cells), the test (source) material creosote WEI Type B (containing < 50 ppm BaP) did only show a mutagenic response (statistically significant increase in mutation frequency) in the presence of a metabolic activation system. Without metabolic activation, the response was negative. The mutation potential of the applied creosote appears to be low based on the test results.
The test result of the source substance creosote WEI Type B (containing < 50 ppm BaP) is adopted for the target substance anthracene oil.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The in vivo genetic toxicity was investigated in a mammalian erythrocyte micronucleus test with creosote as test material. No increase in micronuclei formation was observed, demonstrating the test substance as being not genotoxic in vivo.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

other: mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

- Creosote WEI-Type B (benzo[a]pyrene < 50 ppm)
- Name of test material (as cited in study report): Creosote Spéciale 14130

Species:

mouse

Strain:

other: OF1 (IOPS Caw)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Iffa-Crédo, 69592 L´Arbresle France
- Age at study initiation: Young adults, 6 - 8 weeks of age
- Weight at study initiation: 28.4 - 34.2 g (m), 22.8 - 30.3 g (f)
- Assigned to test groups randomly: yes

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: olive oil
- Justification for choice of solvent/vehicle: no data
- Concentration of test material in vehicle: 220 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw

Duration of treatment / exposure:

see below: time points

Frequency of treatment:

single dose

Post exposure period:

Time points of sampling: 24, 48, 72 h after treatment

Dose / conc.:

2 200 other: mg/kg bw (actual dose received)

Remarks:

limit test

No. of animals per sex per dose:

5 per time point

Control animals:

yes, concurrent vehicle

Positive control(s):

- Substance: cyclophosphamide
- Route of administration: i.p.
- Doses / concentrations: a single application of 100 mg/kg bw

Tissues and cell types examined:

- Tissue: bone marrow
- Number of animals: all animals
- Number of cells: 1000 per animal
- Type of cells: erythrocytes in bone marrow
- Parameters: numbers and types of structural aberrations; polychromatic/normochromatic erythrocytes ratio; mitotic index

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Acute toxicity; a preliminary test with 220, 1100, 2200, and 11000 mg/kg creosote and two males and two females per group preceded the main study. No mortality but clear clinical signs of toxicity at 2200 mg/kg. One hundred per cent mortality within 3 days for all animals at the highest dose.

DETAILS OF SLIDE PREPARATION: May-Grünwald-Giemsa staining

Statistics:

Arithmetic means, Student´s test, indicator of significance p < 0.01

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

clinical signs of toxicity

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

No significant increases of the number of polychromatic erythrocytes bearing micronuclei were observed in treated animals at any time interval. A marked decrease in the ratio of polychromatic to normochromatic erythrocytes was observed after 72-h exposure, indicating a significant cytotoxic treatment-related effect on erythrocytes formation.

The positive control substance produce significant increases in the incidence of micronucleated cells and clear depression in the cellular ratio of poly- to normochromatic erythrocytes.

Table for Micronucleus Test In Vivo (MN)

	Positive control group	Vehicle control	Limit dose [2200 mg/kg]
Number of cells evaluated per 10 animals (group)	10,000	10,000/time point	10,000/time point

Sampling time (h)				24	24	48	72		24	48	72
Number of erythrocytes	normochromatic			Not specified
	polychromatic			Not specified
	polychromatic with micronuclei	male		31.2 ± 4.0	0.4 ± 0.5	1.2 ± 0.8	0.6 ± 0.9		0.4 ± 0.5	0.4 ± 0.5	0.4 ± 0.5
		female		30.6 ± 8.0	1.2 ± 1.1	0.2 ± 0.4	0.2 ± 0.4		0.2 ± 0.4	0.4 ± 0.9	0.2 ± 0.4
Ratio of erythrocytes	polychromatic / normochromatic			Not specified
	polychromatic with micronuclei / normochromatic (males + females combined)			0.57 ± 0.13	1.03 ± 0.25	0.78 ± 0.23	1.00 ± 0.16		0.84 ± 0.14	0.71 ± 0.11	0.62 ± 0.07

Conclusions:

The test material creosote spéciale 14130, WEI-Type B (benzo[a]pyrene content < 50 ppm) did not induce clastogenic/chromosomal defects in developing erythrocytes in vivo in a mammaliam erythrocyte micronucleus test according to OECD TG 474. Thus, no mutagenic potential was observed for the test substance under the conditions of this test.

Executive summary:

Creosote spéciale WEI Type B [containing less than 50 ppm B(a)P] was tested for its capability to induce clastogenic effects in red blood cells isolated from bone marrow after three time points i.p. post-application. The study was conducted according to OECD guideline 474 (1983) EEC Directive 84/449, B.12 (1984). Based on the results of a preliminary toxicity study, a limit test at 2200 mg/kg (single dose, oral) was carried out. Following treatment with creosote, possibly clastogenic and toxic effects during development of erythroblasts into polychromatic red blood cells were examined at three time intervals after application on 10 animals each (5 m, 5 f). The ratio of polychromatic (immature) to normochromatic (mature) cells was followed as measure of cytotoxicity.

Under the conditions of in vivo testing, Creosote spéciale 14130, WEI Type B, was negative for the induction of clastogenic/chromosomal defects in developing erythrocytes in vivo, thus showing no mutagenic potential.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

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 special 14130 (WEI-Type B, 1991) consists predominantly of polycyclic aromatic hydrocarbons (PAH) ranging in size from two up to five fused rings. The target substance anthracene oil (anthracene oil with < 50 ppm benzo[a]pyrene (BaP), AOL) is as well composed of a broad range of PAH but predominantly consisting of two to four aromatic rings.
The nature of both substances and their constituents are considered to be sufficiently similar that effects resulting from in vivo mammalian cell cytogenicity studies (erythrocyte micronucleus test) are equivalent. Therefore, the source substance is suited as supporting substance with regard to in vivo cytogenicity in somatic mammalian cells and data resulting from the source substance can be used for characterising genotoxic effects on mammalian erythrocytes caused by the target substance anthracene oil.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source test material creosote (special 14130, WEI-Type B) 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 (PAH) with only minor levels of other components (phenols, N-compounds approx. < 10 %). Accumulated concentrations of PAH with two and three rings are approx. 50 % with two-ring aromatic building the larger fraction. Analytical data for four-ring PAH is incomplete. Taking information from other creosotes into account, it is assumed that accumulated percentage of four- and five-ring PAH is < 10 % with individual concentrations of carcinogenic five-ring PAH being < 50 ppm. The water solubility of creosote is relatively low. It is determined by the solubility properties of its constituents.
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 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. 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
In an in vivo mammalian erythrocyte micronucleus test, test materials are applied by gavage, eventually dissolved in a suitable solvent. After resorption from the gastrointestinal tract, metabolic activation, and distribution within the body, adverse effects can be provoked on somatic cells and their components. Effects of complex mixtures like UVCBs will be determined by the individual constituents of the mixtures and in combination will specify the effects of the complete mixture. If substances are similar, the resulting effects concerning genetic toxicity will be comparable.
The general composition of the source and target substance is similar. Relevant constituents are two-ring up to four- to five-ring PAH. Two- and three-ring PAH are present in the source substance creosote WEI-Type B and in the target substance anthracene oil in comparable amounts, just the fraction of two-ring aromatics being somewhat higher in creosote and the fraction of three-ring aromatics being somewhat higher in AOL. Concentrations of PAH with carbon frames containing more than three rings may be somewhat higher in anthracene oil, while the concentrations of (carcinogenic) five-ring PAH like e.g. benzo[a]pyrene are very low (< 50 ppm).
Overall, some differences in the PAH composition of both materials are considered not to result in significantly modified genotoxic effects of the source and the target material. The range of PAH is similar. Due to similar metabolic activation pathways, PAH will result in similar final toxicants in the mammalian erythrocyte micronucleus test. Within a range, cytotoxic effects caused by the source and the target substance are considered to be comparable. For these reasons, it is considered justified to use in vivo genetic toxicity data of creosote in order to characterise in vivo genotoxic 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: creosote spéciale 14130, WEI-Type B_1991;
Reference: Weill 1991

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

clinical signs of toxicity

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: the test result of the source substance is adopted for the target substance anthracene oil

Conclusions:

The test (source) material creosote spéciale 14130, WEI-Type B (benzo[a]pyrene content < 50 ppm) did not induce clastogenic/chromosomal defects in developing erythrocytes in vivo in a mammaliam erythrocyte micronucleus test according to OECD TG 474. Thus, no mutagenic potential was observed for the test substance under the conditions of this test.
The test result of the source substance creosote spéciale 14130, WEI-Type B is adopted for the target substance anthracene oil.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Data for assessing the genetic toxicity of anthracene oil (AOL) itself is only available from one mutagenicity test in bacterial cells. In addition, data from three assays with creosote will be used in the hazard assessment based on following reasons.

Anthracene oil and creosote are closely related tar oils. They are produced in a similar process (fractionated distillation of coal tar using overlapping conditions). Consequently, composition of both substances is similar. Major components are mid-range PAH (naphthalene to pyrene). Individual differences in distillation conditions and in starting material may cause gradual variations in qualitative and quantitative composition. But the nature of constituents and the individual components coincide (PAH) and the composition of both substances is similar. Genotoxic effects of both substances will arise from the PAH present and they are considered to be approximately the same for both substances. Thus, creosote can be used as supporting substance for the evaluation of the mutagenic/cytogenetic potential of anthracene oil.

Genetic toxicity in vitro

In vitro genotoxicity data for anthracene oil itself is available only from one bacterial reverse mutation study (Ames test, OECD TG 471) performed under GLP conditions. In this test, five different strains of S. typhimurium were tested with and without metabolic activation at five or six test material concentrations. Only in one tester strain (TA 100), a weak mutagenic response was observed, but only with metabolic activation and at cytotoxic concentrations.

With creosote, two in vitro genetic toxicity studies were performed (a mammalian chromosome aberration test (OECD TG 473) with human lymphocytes and a mammalian cell gene mutation assay (OECD TG 476) using mouse lymphoma cells). The studies were carried out under GLP conditions with and without metabolic activation using four and six test material concentrations, respectively, the highest concentrations being cytotoxic. The chromosome aberration tests proved to be negative at all test concentrations with and without metabolic activation. The mammalian cell gene mutation assay showed a weak mutagenic response only with metabolic activation. Tests without metabolic activation were negative (no statistically significant increase in mutation frequency).

Genetic toxicity in vivo

An in vivo genetic toxicity study (mammalian erythrocyte micronucleus test - OECD TG 474) was performed under GLP conditions with creosote as test material. Mice were treated by gavage (2200 mg/kg bw/day) and erythrocytes were isolated from bone marrow 24, 48, and 72 hours after treatment. At no time point, a cytotoxic response was observed. There was no increase in the incidence of micronucleated cells. A marked increase in the ratio of polychromatic to normochromatic cells after 72-h exposure indicated a significant cytotoxic treatment-related effect on erythrocytes formation.

Conclusion

In two out of three in vitro genetic toxicity tests with anthracene oil and with creosote, respectively, a weak mutagenic/cytotoxic response was observed, indicating a weak mutagenic potential in vitro. But an in vivo erythrocyte micronucleus study in mammalian somatic cells with the supporting substance creosote did not provide any evidence of a genotoxic potential.

Taking into account the combined evidence of the tests performed, the overall genetic toxicity of anthracene oil is evaluated to be negative. In two in vitro standard assays, only a weak/ambiguous mutagenic response was observed. In two other tests including an in vivo mouse micronucleus assay, test results were negative not confirming an in vivo mutagenic potency of the test substance.

Justification for classification or non-classification

The mutagenic potential eventually detected in in vitro standard assays conducted with tar oils containing BaP levels of ≤ 50 ppm has not been confirmed in an in vivo mammalian erythrocyte micronucleus assay.

Anthracene oil (BaP ≤ 50 ppm) is supposed to reflect similar mutagenic properties. It is evaluated not to be a germ cell mutagen. Thus, classification according to Regulation (EC) No 1272/2008 (CLP regulation) is not required.