Distillates (coal tar)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

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:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

8001-58-9

Cas Number:

8001-58-9

IUPAC Name:

8001-58-9

Constituent 2

Reference substance name:

Creosote

EC Number:

232-287-5

EC Name:

Creosote

IUPAC Name:

232-287-5

Constituent 3

Reference substance name:

A complex combination of monocyclic aromatic hydrocarbons, polycyclic aromatic hydrocarbons, tar acids and tar bases, obtained by the distillation of coal tars produced by the high-temperature destructive distillation of bituminous coal to form coke. It consists of hydrocarbons boiling in the range of approximately 200°C-355°C

IUPAC Name:

A complex combination of monocyclic aromatic hydrocarbons, polycyclic aromatic hydrocarbons, tar acids and tar bases, obtained by the distillation of coal tars produced by the high-temperature destructive distillation of bituminous coal to form coke. It consists of hydrocarbons boiling in the range of approximately 200°C-355°C

Details on test material:

- Name of test material (as cited in study report): Creosote
Creosote WEI-Type B, containing less than 50 ppm BaP
- Substance type: organic
- Physical state: liquid
- Composition

Name CAS-No. Concentration [w%]
RUETGERS Chemicals // Biochem. Inst.Grimmer
=================================================
Naphthalene 91-20-3 5.07 5.39
Fluorene 86-73-7 3.06 3.07
Acenaphthylene 208-96-8 -- 0.09
Acenaphthene 83-32-9 4.09 4.04
Phenanthrene 85-01-8 8.95 8.56
Anthracene 120-12-7 0.45 0.38
Fluoranthene 206-44-0 3.70 3.84
Pyrene 129-00-0 2.49 2.08
Benz[a]anthracene 56-55-3 0.04 0.04
Chrysene 218-01-9 0.03 0.03
Benzo(a)pyrene 50-32-8 <10 ppm ca. 6. ppm
----------------------------------------------------------------

Total of 5 remaining EPA-PAH: --- < 20 pp
----------------------------------------------------------------
(Benzo[b]fluoranthene 205-99-2
Benzo[k]fluoranthene 207-08-9
Indeno[1,2,3-cd]pyrene 193-39-5
Dibenzo[a,h]anthracene 53-70-3
Benzo[ghi]perylene) 191-24-2
===========================================

Method

Target gene:

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

Species / strain

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 table below)
In general six, in one case five concentrations were tested, and – if necessary – adjusted in the second test series.

Controls

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: + S9: benzo(a)pyrene [B(a)P] / - S9: methyl methanesulphonate [MMS]

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>20 - 75 µg/mL

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

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%

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive with metabolic activation

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.