Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
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:
1997
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Specific details on test material used for the study:
Clear colourless liquid
Purity: 97.2%

Method

Target gene:
histidine
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1537
Details on mammalian cell type (if applicable):
Source: Trinova Biochem GmbH, Germany; Master culture from Dr. Bruce N. Ames
Histidine mutation: hisC3076
Mutation type: Frameshift

Contains the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cellcoat)
gal : mutation in the galactose metabolism
chl : mutation in nitrate reductase
bio : defective biotin synthesis
uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene)
Species / strain / cell type:
S. typhimurium TA 98
Details on mammalian cell type (if applicable):
Source: Trinova Biochem GmbH, Germany; Master culture from Dr. Bruce N. Ames
Histidine mutation: hisC3052
Mutation type: Frameshift

Contains the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cellcoat)
gal : mutation in the galactose metabolism
chl : mutation in nitrate reductase
bio : defective biotin synthesis
uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene)
Species / strain / cell type:
S. typhimurium TA 1535
Details on mammalian cell type (if applicable):
Source: Trinova Biochem GmbH, Germany; Master culture from Dr. Bruce N. Ames
Histidine mutation: hisG46
Mutation type: Base-pair substitutions

Contains the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cellcoat)
gal : mutation in the galactose metabolism
chl : mutation in nitrate reductase
bio : defective biotin synthesis
uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene)
Species / strain / cell type:
S. typhimurium TA 100
Details on mammalian cell type (if applicable):
Source: Trinova Biochem GmbH, Germany; Master culture from Dr. Bruce N. Ames
Histidine mutation: hisG46/R-factor
Mutation type: Base-pair substitutions

Contains the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cellcoat)
gal : mutation in the galactose metabolism
chl : mutation in nitrate reductase
bio : defective biotin synthesis
uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene)
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Source: Trinova Biochem GmbH, Germany; Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK

The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes (S9 homogenate) obtained from Trinova Biochem GmbH, Giessen, Germany and prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
Test concentrations with justification for top dose:
Preliminary dose-range finding test: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate
First experiment (Direct plate assay): 52, 164, 512, 1600 and 5000 μg/plate
Second experiment (Pre-incubation assay): 17, 52, 164, 512, 1600, 2500 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Controls
Untreated negative controls:
yes
Remarks:
Ethanol
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191: Positive control for TA1537 in the absence of metabolic activation 2-aminoanthracene (2AA): Positiv control for all tester strains in the presence of metabolic activation
Remarks:
Saline = physiological saline (Eurovet Animal Health, Bladel, The Netherlands) DMSO = dimethyl sulfoxide (Merck, Darmstadt, Germany)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)
- Cell density at seeding (if applicable): 109 cells/mL

DURATION
First experiment: Direct Plate Assay
- Exposure duration: 48 ± 4 h, in the dark
- Temperature: 37.0 ± 1.0°C

Second experiment: Pre-incubation Assay
- Preincubation period: 30 ± 2 minutes
- Exposure duration: 48 ± 4 h, in the dark
- Temperature: 37.0 ± 1.0°C

NUMBER OF REPLICATIONS: 3

Evaluation criteria:
DETERMINATION OF MUTAGENICITY:
A test item is considered negative (not mutagenic) in the test if:
- The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent control.
- The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
- The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent control.
- In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

DETERMINATION OF TOXICITY:
- To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of tHe revertant colonies were observed.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The negative and 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.

Any other information on results incl. tables

FIRST EXPERIMENT: DIRECT PLATE ASSAY

- Precipitation of 1,1’(1,1,3-trimethylpropane-1,3-diyl)bis(cyclohexane) on the plates was observed at the start and at the end of the incubation period at concentrations of 1600 μg/plate and upwards.

 

- Observations made in the determination of the toxicity of the test item: No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed. In tester strain TA1537 (absence of S9-mix), a fluctuation in the number of revertant colonies below the laboratory historical control data range was observed at the mid dose of 512 μg/plate. However, since no dose-relationship was observed, this reduction is not considered to be caused by toxicity of the test item. It is more likely this reduction is caused by an incidental fluctuation in the number of revertant colonies.

 

- Observations made in the determination of the mutagenicity of the test item: In the direct plate test, no increase in the number of revertants was observed upon treatment with 1,1’(1,1,3-trimethylpropane-1,3-diyl)bis(cyclohexane) under all conditions tested.

 

 

SECOND EXPERIMENT: PRE-INCUBATION ASSAY

- Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 512 μg/plate and above and at 1600 μg/plate at the end of the incubation period.

 

- Observations made in the determination of the toxicity of the test item: There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.

 

- Observations made in the determination of the mutagenicity of the test item: In the pre-incubation test, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

Applicant's summary and conclusion

Conclusions:
All bacterial strains showed negative responses over the entire dose-range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments. The negative and 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
Executive summary:

All bacterial strains showed negative responses over the entire dose-range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments. The negative and 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.