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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
In a bacterial reverse mutation assay (Ames test), AOH was tested to be positive with but not without metabolic activation. Furthermore, AOH is self-classified as mutagenic Cat. 1B due to its BaP content. The substance is identified as mutagen for further use in the chemical safety assessment. Besides the Ames test, no other genetic toxicity studies have been performed as AOH is classified as carcinogenic Cat. 1B according to CLP regulation.
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:
03 Aug. - 06 Sep. 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium 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, 1000, 2500, and 5000 µg/plate (TA 98, TA 100, +/-S9)
1.0, 3.16, 10, 31.6, 100, 316, and 1000 µg/plate (TA 1535, TA 1537, - S9)
10, 31.6, 100, 316, 1000 and 2500 µg/plate (TA 1535, TA 1537, + S9)
3.16, 10, 31.6, 100, 316, and 1000 µg/plate (TA 102, +/-S9)
2nd experiment: with variations based on results of 1st experiment (Report, p. 11)
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: in agar (plate incorporation)

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).
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
reproducible in both tests
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at >= 31.6 µg/pl. (-S9); at >= 316 µg/pl. (+S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA 98, TA 100, TA 1537
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
reproducible in both tests
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
not or weakly cytotoxic in the mutagenic concentration range
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA 102
Metabolic activation:
with
Genotoxicity:
ambiguous
Remarks:
weak positive effect, not reproducible
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
not in the relevant mutagenic concentration range
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA 98
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
dose response positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
not cytotoxic in the relevant concentration range
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Summary:

The positive effects noted without S9 were weak and not reproducible in either experiment (see below).

In the presence of S9 clear dose-related increases in the mutation rates were found in TA 98 and 1537 and a weak positive correlation in TA 100 and TA 102, reproducible at non-cytotoxic concentrations in both experiments. The most responsive tester strain was TA 98.

[Mutation factors (+S9) = 3 - 14x above background with maximum in TA 98]

In either experiment, no biological relevant increases in the mutation rate were seen in TA 1535.

Experiment I:

No relevant increases in TA 98, TA 100, TA 1537, and TA 102 (-S9);

relevant increases in TA 98, TA 100, and TA 1537 (>= 10 µg/plate, +S9)

Experiment II:

Relevant increases in TA 98 (>= 15 µg/plate, -S9);

relevant increases in TA 98, TA 100, TA 1537, and TA 102 (>= 5 - 500 µg/plate, +S9)

Dose-response relationship of the reversion rate was observed with each tester strain.

Conclusions:
Interpretation of results (migrated information):
positive
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Additional information

Additional information from genetic toxicity in vitro:

Data for assessing the mutagenicity of the substance distillates (coal tar) heavy oils (anthracene oil high (> 50 ppm) BaP, AOH) is only available from one mutagenicity test. Additional tests have not been performed as AOH is classified as Carc. 1B (Regulation (EC) No. 1272/2008) and as Muta. 1B due to its benzo[a]pyrene (BaP) content of up to 1.5 % (see below).

For AOH, a bacterial reverse mutation assay (Ames test) was performed. In this test, five different strains of S. typhimurium were tested with and without metabolic activation. No mutagenic response was observed without metabolic activation. Positive results were obtained with metabolic activation for four of the five bacterial strains tested. One strain (TA 1535) did not show any biologically relevant increase in the mutation rate neither with nor without metabolic activation.

Based on this experimental result and due to the BaP content of AOH, the substance is identified as mutagen.

Justification for classification or non-classification

The substance distillates (coal tar) heavy oils (anthracene oil high (> 50 ppm) BaP, AOH) is self-classified as mutagenic Cat. 1B due to its content of benzo[a]pyrene (up to 1.5 %). Benzo[a]pyrene is classified as mutagenic Cat. 1B (Regulation (EC) No 1272/2008, Annex VI, Table 3.1), and the generic concentration limit of a mutagen Cat. 1B in a mixture triggering classification of the mixture itself as mutagenic Cat. 1B is ≥ 0.1 % (Regulation (EC) No 1272/2008, Table 3.5.2).