Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21. Jul. 1997
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
adopted 30. May 2008
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material: tert-butylbenzene
- IUPAC name: tert-Butylbenzene
- Molecular formula: C10H14
- Molecular weight: 134.22 g/mol
- Substance type: Organic
- Physical state: Liquid

Method

Species / strainopen allclose all
Species / strain / cell type:
other: S. typhimurium TA 97a
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
metabolic activation by microsomal enzymes (e.g., benzo(a)pyrene)
Test concentrations with justification for top dose:
5 µL/plate
Vehicle / solvent:
Vehicle: Dimethylsulfoxide (DMSO)
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 4-Nitro-1,2-phenylene diamine, C6H7N3O2; CAS-No.: 99-56-9 and 2-Amino-anthracene, C14H11N; CAS-No.: 613-13-8
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension

DURATION
- Preincubation period: 8 hours at 37 ± 1 °C
- Exposure duration: Experiment 1a (48 hours at 37 ±1 °C), Experiment 1b (48 hours at 37 ±1 °C), Experiment 2 (48 hours at 37 ±1 °C)
- Expression time (cells in growth medium): 24 hours at 37 ±1 °C
- Selection time (if incubation with a selection agent): 48 hours at 37 ±1 °C
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours at 37 ±1 °C

SELECTION AGENT (mutation assays): mutagenic substances: 4-Nitro-1,2-phenylene diamine (CAS#99-56-9), Sodium azide (CAS#26628-22-8), 2-Amino-anthracene(CAS#613-13-8) and Benzo-a-pyrene(CAS#50-32-8)

NUMBER OF REPLICATIONS: 3
Rationale for test conditions:
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.
Evaluation criteria:
The determination of titre should give a number of at least 109 cells/mL, correlating to 100 colonies/plate after dilution.

All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and with-out metabolic activation and nearly all were within the historical control data ranges.
Statistics:
The confirmation tests of the genotype did not show any irregularities. The control of the titre was above the demanded value of 109 bacteria/mL. All of the means of all replicates of the spontaneous revertants (in negative and solvent controls) were within the range of the historical data of the test facility. All numbers of re-vertant colonies of the positive controls were within the range of the historical data of the laboratory, but all were in-creased in comparison with the negative controls, which demonstrated the mutagenic potential of the diagnostic mutagens.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
other: S. typhimurium TA 97a
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 98
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 100
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 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

Applicant's summary and conclusion

Conclusions:
Based on the results of this study it is concluded that tert-Butylbenzene is not muta-genic in the Salmonella typhimurium test strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the present study.
Executive summary:

Three valid experiments were performed.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

The test itemtert-Butylbenzenewas tested in theSalmonella typhimuriumreverse mutation assay with five strains ofSalmonella typhimurium(TA97a, TA98, TA100, TA102 and TA1535).

The test was performed in three experiments in the presence and absence of metabolic activation, with +S9 standing for presence of metabolic activation, and –S9 standing for absence of metabolic activation.

 

Experiment 1a:

In the first experiment,the test item (dissolved in DMSO) was tested up to concentrations of 5 µL/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method.

The test item showed no precipitates on the plates at any of the concentrations.

The bacterial background lawn was not reduced at any of the concentrations, but a relevant decrease (no bacteria growth) in the number of revertants was observed in the bacteria strains TA97a, TA98, TA100 and TA1535 at the highest concentration (5 µL/plate).

The test itemshowed signs of toxicity towards these bacteria strains in both the absence and presence of metabolic activation.

The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

 

Experiment 1b:

Based on the toxicity results of the experiment 1a,the test item was tested up to concentrations of 5 µL/plate in the absence and presence of S9-mix in the bacteria strains TA97a, TA98, TA100 and TA1535 using the plate incorporation method.

The test item showed no precipitates on the plates at any of the concentrations.

The bacterial background lawn was not reduced at any of the concentrations, but a relevant decrease (no bacteria growth) in the number of revertants was observed in the bacteria strains TA97a, TA98, TA100 and TA1535 at the highest concentration (5 µL/plate).

The test itemshowed signs of toxicity towards these bacteria strains in both the absence and presence of metabolic activation.

The results of this experiments showed that the test item caused no increase in the number of revertants in the four bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation. The test item did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation.

 

Experiment 2:

Based on the experiments 1a and 1b,the test item was tested up to concentrations of 5 µL/plate in the absence and presence of S9-mix in all five bacteria strains using the pre-incubation method.

The test item showed no precipitates on the plates at any of the concentrations.

The test itemshowed signs of toxicity towards all bacteria strains in both the absence and presence of metabolic activation in the following concentrations:

 -Bacteria strain TA97a: 5 µL/plate (no bacteria growth was observed, but the bacterial background lawn was present)

Bacteria strain TA98: 5 µL/plate (no bacteria growth was observed, but the bacterial background lawn was present)

-Bacteria strain TA100: 5 µL/plate (no bacteria growth was observed, but the bacterial background lawn was present)

-Bacteria strain TA102: 5 µL/plate (no bacteria growth was observed, but the bacterial background lawn was present)

-Bacteria strain TA1535: 5 µL/plate and 2.5 µL/plate (no bacteria growth was observed, but the bacterial background lawn was present)

 

The results of this experiments showed that the test item caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation. The test item did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation.

 

Based on the results of this study it is concluded thattert-Butylbenzeneis not mutagenic in theSalmonella typhimuriumstrains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.