Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28-03-2019 to 13-05-2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
3a,4,7,7a-tetrahydro-1H-indene
EC Number:
221-260-3
EC Name:
3a,4,7,7a-tetrahydro-1H-indene
Cas Number:
3048-65-5
Molecular formula:
C9H12
IUPAC Name:
3a,4,7,7a-tetrahydro-1H-indene
Constituent 2
Chemical structure
Reference substance name:
4-vinylcyclohexene
EC Number:
202-848-9
EC Name:
4-vinylcyclohexene
Cas Number:
100-40-3
Molecular formula:
C8H12
IUPAC Name:
4-vinylcyclohexene
Constituent 3
Chemical structure
Reference substance name:
Cyclopentadiene
EC Number:
208-835-4
EC Name:
Cyclopentadiene
Cas Number:
542-92-7
Molecular formula:
C5H6
IUPAC Name:
cyclopenta-1,3-diene
Constituent 4
Chemical structure
Reference substance name:
cis-1,2-Divinylcyclobutane
Cas Number:
2422-85-7
Molecular formula:
C8H12
IUPAC Name:
cis-1,2-Divinylcyclobutane
Constituent 5
Chemical structure
Reference substance name:
Ethylbenzene
EC Number:
202-849-4
EC Name:
Ethylbenzene
Cas Number:
100-41-4
Molecular formula:
C8H10
IUPAC Name:
ethylbenzene
Constituent 6
Chemical structure
Reference substance name:
5-vinylnorborn-2-ene
EC Number:
221-259-8
EC Name:
5-vinylnorborn-2-ene
Cas Number:
3048-64-4
Molecular formula:
C9H12
IUPAC Name:
5-vinylbicyclo[2.2.1]hept-2-ene
Constituent 7
Chemical structure
Reference substance name:
Cycloocta-1,5-diene
EC Number:
203-907-1
EC Name:
Cycloocta-1,5-diene
Cas Number:
111-78-4
Molecular formula:
C8H12
IUPAC Name:
cycloocta-1,5-diene
Constituent 8
Chemical structure
Reference substance name:
3a,4,7,7a-tetrahydro-4,7-methanoindene
EC Number:
201-052-9
EC Name:
3a,4,7,7a-tetrahydro-4,7-methanoindene
Cas Number:
77-73-6
Molecular formula:
C10H12
IUPAC Name:
3a,4,7,7a-tetrahydro-4,7-methanoindene,
Constituent 9
Chemical structure
Reference substance name:
3a,4,7,7a-tetrahydro-4-methyl-1H-indene
EC Number:
231-021-5
EC Name:
3a,4,7,7a-tetrahydro-4-methyl-1H-indene
Cas Number:
7413-02-7
Molecular formula:
C10H14
IUPAC Name:
4-methyl-3a,4,7,7a-tetrahydro-1H-indene
impurity 1
Chemical structure
Reference substance name:
Combination of: 6-(cyclohex-3-en-1-yl)bicyclo[2.2.1]hept-2-ene 2-vinyl-1,2,3,4,4a,5,8,8a-octahydro-1,4-methanonaphthalene 6-vinyl-1,4,4a,5,6,7,8,8a-octahydro-1,4-methanonaphthalene 2-vinyl-1,2,3,4,4a,5,8,8a-octahydro-1,4:5,8-dimethanonaphthalene 2,2'-bi(bicyclo[2.2.1]heptane-5,5'-diene) 3a,4,4a,5,8,8a,9,9a-octahydro-1H-5,8-methanocyclopenta[b]naphthalene 4,4a,4b,5,8,8a,9,9a-octahydro-1H-1,4-methanofluorene
Molecular formula:
C13H18 to C14H18
IUPAC Name:
Combination of: 6-(cyclohex-3-en-1-yl)bicyclo[2.2.1]hept-2-ene 2-vinyl-1,2,3,4,4a,5,8,8a-octahydro-1,4-methanonaphthalene 6-vinyl-1,4,4a,5,6,7,8,8a-octahydro-1,4-methanonaphthalene 2-vinyl-1,2,3,4,4a,5,8,8a-octahydro-1,4:5,8-dimethanonaphthalene 2,2'-bi(bicyclo[2.2.1]heptane-5,5'-diene) 3a,4,4a,5,8,8a,9,9a-octahydro-1H-5,8-methanocyclopenta[b]naphthalene 4,4a,4b,5,8,8a,9,9a-octahydro-1H-1,4-methanofluorene
Constituent 10
Chemical structure
Reference substance name:
4,4a,4b,5,8,8a,9,9a-octahydro-1H-1,4-methanofluorene OR 4,4a,4b,5,8,8a,9,9a-octahydro-1H-1,4:5,8-dimethanofluorene
Molecular formula:
C14H18 to C15H18
IUPAC Name:
4,4a,4b,5,8,8a,9,9a-octahydro-1H-1,4-methanofluorene OR 4,4a,4b,5,8,8a,9,9a-octahydro-1H-1,4:5,8-dimethanofluorene
Constituent 11
Chemical structure
Reference substance name:
3a,4,4a,5,8,8a,9,9a-octahydro-4,9:5,8-dimethano-1H-benz[f]indene
EC Number:
230-512-1
EC Name:
3a,4,4a,5,8,8a,9,9a-octahydro-4,9:5,8-dimethano-1H-benz[f]indene
Cas Number:
7158-25-0
Molecular formula:
C15H18
IUPAC Name:
3a,4,4a,5,8,8a,9,9a-octahydro-1H-4,9:5,8-dimethanocyclopenta[b]naphthalene
Constituent 12
Reference substance name:
Tetramers of cyclopentadiene and 1,3-butadiene
Molecular formula:
C16H24 to C20H24
IUPAC Name:
Tetramers of cyclopentadiene and 1,3-butadiene
Test material form:
liquid
Details on test material:
Light brown liquid.
Batch number 776809

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: BIBRA (1987) and Trinova Biochem GmbH (2017)
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : Pre-prepared using standardised in house procedures. Lot no PB/βNF S9 28/10/18. Certificate of efficacy included in study report
- concentration or volume of S9 mix and S9 in the final culture medium : 10% then 0.5mL of mix per plate
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Yes
Test concentrations with justification for top dose:
Plate incorporation method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000µg/plate where top dose is maximum recommended dose in guideline
Pre-incubation method used results from plate incorporation method. Tested doses the same except for TA100 which used 0.5, 1.5, 5, 15, 50, 150, 500 and 1500µg/plate. Selection criteria was to achieve four non-toxic doses and see cytotoxicity at top dose
Vehicle / solvent:
DMSO
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
no
Positive control substance:
benzo(a)pyrene
other: 2-aminoanthracene
Remarks:
With metabolic activation
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate)
- Number of independent experiments : plate incorporation and pre-incubation methods used.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 0.9 to 9.0E9 bacteria/mL
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension;

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48-72hrs
Evaluation criteria:
Dose related increase in mutation frequency, reproducible increases, biological relevance versus historic controls, increase >2x compared to concurrent solvent controls (TA98, 100, uvRA) and >3x fpr TA1535 and 1537 (especiall if outside of historic controls), statistical significance
Statistics:
As per UKEMS (Mahon, 1989) , confirmed usiing Dunnetts regression analysis where statisticlaly significant increases seen.

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:
cytotoxicity
Remarks:
Reduced growth of background lawn from 1500µg/plate (both with and without S9 using pre-incubation method and 1500µg/plate and 500µg/plate with and without S9 respectively in the plate incorporation method.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
Compared to concurrent solvent control: Plate incorporation method 24x and 51x increase with and without metabolic activation respectively , preincubation method 25x and 70x increase with and without metabolic activation respectively.
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Reduced growth of background lawn from 5000µg/plate and 500µg/plate with and without S9 respectively using pre-incubation method and 1500µg/plate and 150µg/plate with and without S9 respectively in the plate incorporation method.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
Compared to concurrent solvent control: Plate incorporation method 35x and 40x increase with and without metabolic activation respectively, preincubation method 23x increase both with and without metabolic activation.
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Reduced growth of background lawn from 1500µg/plate both with and without S9 respectively using pre-incubation method and 150µg/plate both with and without S9 respectively in the plate incorporation method.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
Compared to concurrent solvent control: Plate incorporation method 6.5x and 4.6x increase with and without metabolic activation respectively, preincubation method 5.3x and 13x increase with and without metabolic activation respectively.
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Reduced growth of background lawn from 500µg/plate both with and without S9 respectively using pre-incubation method and 500µg/plate and 50µg/plate with and without S9 respectively in the plate incorporation method.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
Compared to concurrent solvent control: Plate incorporation method 16x and 4.6x increase with and without metabolic activation respectively, preincubation method 13x and 5.3x increase with and without metabolic activation respectively.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Reduced growth of background lawn from 1500µg/plate both with and without S9 respectively using pre-incubation method and 5000µg/plate and 150µg/plate with and without S9 respectively in the plate incorporation method.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
Compared to concurrent solvent control: Plate incorporation method 8.3x and 25x increase with and without metabolic activation respectively, preincubation method 3.6x and 43x increase with and without metabolic activation respectively.
Additional information on results:
- Signs of toxicity: Cytotoxicity was taken to be a signficant reduction in the background lawn of more than 50%. Cytotoxicity seemed higher in the pre-incubation assay compared to the plate incorporation assay.
One statistically significant value was noted (WP2uvrA at 5µg/plate without S9). However, this was within the historical control range for the strain and was therefore considered not of biological relevance.

Applicant's summary and conclusion

Conclusions:
Not mutagenic in bacterial reverse mutation assay
Executive summary:

In a guideline and GLP bacterial reverse mutation assay, the UVCB substance THI-TM showed no evidence of mutagenic potential both with and without metabolic activation up to cytotoxic concentrations.