5-octylbicyclo[2.2.1]hept-2-ene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 March 2018 to 5 April 2018 (Experimental start to Experimental completion)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Materia, Inc.
- Batch/Lot No.of test material: RP367_ONB-D
- Expiration date of the batch/Lot: 9 August 2018
- Purity test date: 9 August 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room Temperature (Ambient), in original container as supplied by the Sponsor. Kept at room temperature, protected from light (as a precautionary measure)
- Stability under test conditions: Assumed stable for the duration of the study
- Solubility and stability of the test substance in the solvent/vehicle: Soluble in Dimethyl sulphoxide (DMSO) at 50 µL/mL (to permit a limit concentration of 5 µL/plate). Stability of the test item in DMSO not performed, assumed stable for the duration of the study
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Formulated with DMSO at 50 µL/mL

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Initial Toxicity Assay: (0) 0.0015, 0.005, 0.015, 0.05, 0.15, 0.2, 1.5 and 5 (µL/plate).
Confirmatory Mutation test: (0) 0.16, 0.31, 0.63, 1.25, 2.5 and 5 (µL/plate).

In accordence with OECD 471, the highest tested concentration of 5 µL/plate is the recommended maximum concentration for soluble non-cytotoxic substances in the absence and presence of metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was found to emulsify in distilled water (stock A, 50 µL/mL) and therefore not dosable within this test system. The test item was soluble in DMSO (stock B, 50 µL/mL), DMSO was therefore selected as the vehicle for treatment. A volume of 100 µL of the test item from stock B (50 µL/mL) was added to 2 mL of top agar, to assess precipitation. No precipitation was observed at the recommended maximum test concentration of 5 µL/plate. Hence, 5 µL/plate was selected as the highest concentration to be tested in the initial toxicity-mutation test both in the absence and presence (5% v/v S9 mix) of metabolic activation.

Untreated negative controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2--Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): The cell densities (OD at 660 nm) of all tester strain were within the required range to produce cultures with approximately 1 - 2 x 109 bacteria/mL, demonstrating that appropriate numbers of bacteria were plated.

DURATION
- Preincubation period: not specified
- Exposure duration: 48 hours incubation at 37 ± 1°C,

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: other: Cytotoxicity was characterised by inhibition of the background bacterial lawn and/or a reduction in number of revertant colonies.

Rationale for test conditions:

In accordence with stardard test conditions as per OECD 471.

Evaluation criteria:

Assay Evaluation Criteria:
A result was considered positive if a concentration-related increase over the range tested and/or a reproducible increase at one or more test concentration in the number of revertant colonies per plate in at least one strain with or without metabolic activation.

Strains TA1535, TA1537
Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0 times the mean negative control value.

Strains TA98, TA100 and TA102
Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0 times the mean negative control value.

Statistical analysis was used as an aid in the evaluation of a dose response.

A response that did not meet all three of the above criteria (magnitude, concentration-responsiveness, reproducibility) was not evaluated as positive.
Negative results obtained in the initial toxicity-mutation test were confirmed by a second trial, using the same method as specified above, with an alteration in concentration spacing.

Statistics:

Simple linear regression analysis was performed for TA1537, TA1535, TA98, TA100 and TA102, separately, to assess the dose dependent nature of any increase in revertant colonies.

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

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 nor precipitates, but tested up to recommended limit concentrations

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 nor precipitates, but tested up to recommended limit concentrations

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 nor precipitates, but tested up to recommended limit concentrations

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 nor precipitates, but tested up to recommended limit concentrations

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None
- Effects of osmolality: None
- Precipitation: None

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: yes
- Negative (solvent/vehicle) historical control data: yes

Conclusions:

From the results of this study, it is concluded that 5-octyl-2-norbornene is non-mutagenic to any of the five strains of Salmonella typhimurium, viz., TA1537, TA1535, TA98, TA100, and TA102, when tested under the specified experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)