Plastic, waste, pyrolized, fractionation bottoms

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Plastic, waste, pyrolized, distn., oil

Molecular formula:

Not applicable

IUPAC Name:

Plastic, waste, pyrolized, distn., oil

Test material form:

liquid

Details on test material:

Substance manufactured by the lead registrant according to the third compostion included in the registration dossier of Plastic, waste, pyrolized, fractionation bottoms (Raw Diesel)

Method

Target gene:

Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 97

Additional strain / cell type characteristics:

not specified

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not specified

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not specified

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Metabolic activation: S9

Test concentrations with justification for top dose:

First Experiment: 5 / 1.5 / 0.5 / 0.15 / 0.05 µL/plate
Second Experiment: 5 / 2.5 / 1.25 / 0.63 / 0.31 / 0.16 µL/plate

Vehicle / solvent:

Acetone, CAS No. 67- 64- 1

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Dimethylsulfoxide, Demineralised water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4--Nitro-1,2-phenylene Diamine, 2-Amino-Anthracene,

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Three replicates, with/without S9, for each solvent which was used in the test, incubation for 48 hours at 37 ±1°C.
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
General preparation
Per bacteria strain and concentration, three plates with and three plates without metabolic activation (-S9) were used.
For the top agar 100 mL agar basis was melted in a microwave oven, 10 mL of the histidine-biotin-solution 0.5 mM was added, then the mixture was placed in the water bath at 43 ±1 °C.
Plate incorporation method
The following materials were gently vortexed in a test tube and poured onto the selective agar plates:

• 100 µL test solution at each dose level, solvent (negative control) or reference mu-tagen solution (positive control)
• 500 µL S9 mix (see chapter 6.5.18, for test with metabolic activation) or phosphate buffer (for test without metabolic activation).
• 100 µL bacteria suspension (see chapter 6.4.2, test system, culture of the strains)
• 2000 µL overlay agar (top agar)
The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incubator at 37 ±1 °C.
Pre-incubation method
The following materials were gently vortexed in a test tube and incubated at 37 ±1°C for 20 minutes:
-100 µL test solution at each dose level, solvent (negative control) or reference mu-tagen solution (positive control)
•500 µL S9 mix (see chapter 6.5.18, for test with metabolic activation) or phosphate buffer (for test without metabolic activation).
• 100 µL bacteria suspension (see chapter 6.4.2, test system, culture of the strains)

After the pre-incubation for 20 minutes, 2000 µL top agar was added and the tube was gently vortexed. The mixture was poured onto the selective agar plate.
The plates were closed and left to solidify for a few minutes, then inverted and placed in the incubator at 37 ±1 °C.


FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection):
- Selection time (if incubation with a selective agent):
- Fixation time (start of exposure up to fixation or harvest of cells):
- Method used: agar or microwell plates for the mouse lymphoma assay.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
- Criteria for small (slow growing) and large (fast growing) colonies:

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other:
- Any supplementary information relevant to cytotoxicity:

METHODS FOR MEASUREMENTS OF GENOTOXICIY

TREATMENT AND HARVEST SCHEDULE:

Evaluation criteria:

A substance is considered to be mutagenic, if a reproducible increase with or without metabolic activation of revertant colonies per plate exceeding an increase factor of 2 for the bacteria strains TA97a, TA98, TA100, TA102 and TA1535 compared to vehicle con-trols in at least one strain can be observed and there is a concentration-related increase.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 4

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

True negative controls validity:

valid

Positive controls validity:

valid

Test results 5

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

True negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

The test item TACOIL showed an increase in the number of revertants in the bacteria strains TA97a, TA98 and TA100 in the experiment with metabolic activation.
All vehicle, nearly all negative and nearly all strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
Based on the results of this study it is concluded that TACOIL is mutagenic in the Salmonella typhimurium strains TA97a, TA98 and TA100 in the presence of metabol-ic activation under the experimental conditions in this study.