5-ethylidene-8,9,10-trinorborn-2-ene, manufacture of, by-products from

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:

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

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

Test material information

Constituent 1

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

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

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

Reference substance name:

cis-1,2-Divinylcyclobutane

Cas Number:

2422-85-7

Molecular formula:

C8H12

IUPAC Name:

cis-1,2-Divinylcyclobutane

Constituent 5

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

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

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

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

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

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

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

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 / strain

Species / strain 1

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 2

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

Controls

Controls 1

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

Controls 2

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 results

Test results 1

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.

Test results 2

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.

Test results 3

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.

Test results 4

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.

Test results 5

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.