Coke (coal), naphtha cracking ethylene manuf. by-product

Genetic toxicity: in vitro

Administrative data

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:

start of experiments: 30 June 1997, end of experiments: 14 July 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD, EU)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Carbon black

EC Number:

215-609-9

EC Name:

Carbon black

Cas Number:

1333-86-4

IUPAC Name:

methane

Details on test material:

- Name of test material (as cited in study report): Printex 70
- Substance type: technical product
- Physical state: solid (powder)
- Analytical purity: > 98%
- Impurities (identity and concentrations): not reported
- Purity test date: 22 March 1995 (certificate of analysis date)
- Lot/batch No.: Batch No. AT 9703015A
- Expiration date of the lot/batch: not indicated by the Sponsor
- Stability under test conditions: not reported
- Storage condition of test material: room termperature

Method

Target gene:

Genes for histidine and tryptophan synthesis

Species / strain

Species / strain / cell type:

other: S. typhimurium TA 1535, 1537, 98, 100 and E. coli WP2 and WP2 uvrA

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver-S9 mix (ICN Biomedicals GmbH/Germany)

Test concentrations with justification for top dose:

The extract was tested undiluted, and as 80, 60, 50, 40, and 10% dilutions

Vehicle / solvent:

- Vehicle/solvent: Dimethylsulphoxide (DMSO)
- Justification for choice of solvent/vehicle: not reported

Controls

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Remarks:

water

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

further positive control substances for assays without metabolic activation: 4-NOPD (10-40 µg, for TA 1537, 98), MMS (1 uL; for E. coli strains); for assays with metabolic activation: 2-AA (2.5 ug; for TA 1535, 1537, 98, 100 and E. coli strains).

Migrat

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation; two independent experiments were performed. In experiment I, the plate incorporation method was used; in experiment II, the pre-incubation method was applied.

PREPARATION OF THE TEST SUBSTANCE: 40 grams of the test substance were extracted with 600 mL of toluene in a Soxhlet extractor (the toluene was replenished about 10 times per hour). The toluene was then evaporated and the residue was dried at 140 deg centigrade for two hours. The residue was then taken up in 40 g of dimethylsulfoxide (DMSO).

DURATION
- Preincubation period: 60 minutes at 37 deg C
- Exposure duration: at least 48 hours

NUMBER OF REPLICATIONS:for each strain and dose level, including the controls, three plates were used.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth;

Evaluation criteria:

A test substance was considered mutagenic in this assay, if
- a dose-related and reproducible increase in the number of revertants was found, or if - a substantial and reproducible increase for at least one test concentration was found.
A substantial increase was defined as
- a at least two-fold increase in the number of revertants in TA 100
- a at least three-fold increase in the number of revertants in strains TA1535, TA1537, TA98, WP2 and WP2uvrA.
Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of a potential mutagenic potential, regardless of whether the aforementioned increases were observed or not.

Statistics:

no statistics performed (not required y guideline)

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

maximum 1.4-fold increase in mutation frequency over control

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

maximum 1.7-fold increase in mutation frequency over control

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

maximum 1.2-fold increase in mutation frequency over control

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

maximum 1.2-fold increase in mutation frequency over control

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

maximum 1.3-fold increase in mutation frequency over control

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

maximum 1.4-fold increase in mutation frequency over control

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not reported
- Effects of osmolality: not reported
- Evaporation from medium: not reported
- Water solubility: not reported
- Precipitation: not reported
- Other confounding effects: not reported

RANGE-FINDING/SCREENING STUDIES: The toxicity of the test item extract was determined with strains TA98 and TA100 in a pre-experiment. 8 concentrations (5, 10, 20, 40, 50, 60, 80 and 100%) were tested for toxicity and mutation induction with each 3 plates both in the presence and the absence of metabolic activation. The experimental conditions in this pre-experiment were the same as for the main plate incorporation test. No toxicity was observed with the highest test concentration (100%, corresponding to an equivalent amount of 109 mg Printex 70 per plate).

COMPARISON WITH HISTORICAL CONTROL DATA: yes (spontaneous reversion frequencies for all used strains reported, i.e. TA1535 5-30, TA1537 4-32, TA98 18-63, TA100 79-197, E.coli WP2 23-69 and E. coli WP2uvrA 27-65).


ADDITIONAL INFORMATION ON CYTOTOXICITY:

Remarks on result:

other: strain/cell type: S. typhimurium TA 1535

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation in all Salmonella and E. coli strains tested
negative without metabolic activation in all Salmonella and E. coli strains tested

Printex 70 toluene extract did not induce an increase in mutation frequency in any of the tester strains with and without metabolic activation.

Executive summary:

In order to investigate the potential of Printex 70 to induce gene mutations in bacteria, the plate incorporation test (experiment I) and the pre-incubation test (experiment II) were performed in Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and in E. coli strains WP2 and WP2uvrA. Printex 70 was Soxhlet extracted with toluene and the residue taken up in DMSO. The extract was tested with and without metabolic activation (S9 mix) in triplicate at the following concentrations: 100, 80, 60, 50, 40 and 10%. No cytotoxicity was observed up to the highest concentration tested. No increases in revertant frequencies were detected in any of the tester strains at any dose level either with or without metabolic activation. (increases in revertant frequencies were always below a factor of 2 as compared to the controls). The positive controls were functional.