Pitch, coal tar, high-temp., < 1% 4- to 5-membered condensed ring aromatic hydrocarbons

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Pitch, coal tar, high-temp., < 1% 4- to 5-membered condensed ring aromatic hydrocarbons [EC no. 701-305-8] (CTPhtht) showed a positive response in a bacterial reverse mutation assay (Ames test). Furthermore, the substance is classified as mutagenic Cat. 1B due to its benzo[a]pyrene content of up to 0.10 %.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 Jul. - 03 Sep. 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP 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:

Plate incorporation test

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other:

Remarks:

see report, p. 15 for details

Metabolic activation:

with and without

Metabolic activation system:

S9 microsomal fraction was 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:

Pre-experiment: 3.16, 10, 31.6, 100, 316, 1000, 2500, and 5000 µg/plate (TA 98, TA 100: +/-S9)
1st experiment: 31.6, 100, 316, 1000, 2500, and 5000 µg/plate (all tester strains, +/-S9)
2nd experiment: 250, 500, 1000, 2000, 2500, and 5000 µg/plate (all tester strains except TA 1537, +/-S9),
in addition: 125 µg/plate only for TA 98, +S9,
20, 80, 200, 800, 2000, and 5000 µg/plate (TA 1537, +S9),
250, 500, 1000, 2000, 3500, and 5000 µg/plate (TA 1537, -S9)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Test item was suspended in ethanol and diluted prior to use.
- Justification for choice of solvent/vehicle: compatible with survival of bacteria and S9 activity

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Sodium azide, 4-nitro-o-phenylene-diamine (4-NOPD), methyl methane sulphonate (MMS), 2-aminoanthracene (2-AA); see Report p. 15 for details

Details on test system and experimental conditions:

METHOD OF APPLICATION:
in agar (plate incorporation): Materials were mixed in test tube and poured over the surface of a minimal agar plate
- Test solution (at each dose level, solvent control, negative control, or positive control: 100 µl
- S9 mix (for testing with metabolic activation or S9 mix substitution buffer (for testing without metabolic activation): 500 µl
- Bacteria suspension: 100 µl
- Overlaying agar: 2000 µl

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 with or without metabolic activation
and/or
- if a biologically relevant positive response for at least one of the dose groups occurs in at least one tester 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 3x 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 98

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

reproducible, dose-response relationship in both experiments with metabolic activation. Without S9, weakly positive in one test.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

reproducible

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

reproducible, dose-response relationship

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: in all tester strains at test concentrations of >= 1000 µg/plate (1st experiment);
in all tester strains except tester strain TA 1537 at test concentrations of >= 1000 µg/plate (2nd experiment)
in tester strain TA 1537 at a test concentration of >= 1000 µg/plate (2nd experiment, without metabolic activation) and
at a test concentration of >= 800 µg/plate (2nd experiment, with metabolic activation)

Maximum mutation factor: 14.3 (tester strain TA 98 with metabolic activation at a dose of 3500 µg/plate, 1st experiment)

Conclusions:

Interpretation of results:
positive

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Additional information

Additional information from genetic toxicity in vitro:

Mutagenicity of Pitch, coal tar, high-temp., < 1% 4- to 5-membered condensed ring aromatic hydrocarbons [EC no. 701-305-8] (CTPhtht) was tested in a bacterial reverse mutation assay (Ames test) according to OECD TG 471/EU Method B.13/14. CTPhtht proved to be positive in three of five tester strains. Additional cytotoxicity/mutagenicity tests were not conducted, as CTPhtht has been self-classified as mutagenic (Muta. 1B) and carcinogenic (Carc 1B) due to its content of benzo[a]pyrene (≥ 0.1 %), which itself is classified as carcinogenic and mutagenic (Carc. 1B, Muta.1B).

Justification for classification or non-classification

Pitch, coal tar, high-temp., < 1% 4- to 5-membered condensed ring aromatic hydrocarbons [EC no. 701-305-8] (CTPhtht) may contain up to 0.10 % of benzo[a]pyrene (BaP). BaP is known to be mutagenic and is classified as mutagenic Cat. 1B. In an Ames test, CTPhtht was demonstrated to be mutagenic. Based on BaP content, CTPhtht has been classified as mutagenic Cat. 1B by the registrant.