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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study. Original study report not available.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1998

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Method of Ames (1975)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Test substance: 5-Ethylidene-2-norbornene (CAS# 16219-75-3)
- Purity: 99.4%
- Source: Union Carbide Corporation, South Charleston, West Virginia,

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
S9 liver homogenate, prepared from Aroclor l254-induced, Sprague-Dawley male rats, was purchased from Microbiological Associates, Bethesda, MD. For tests with metabolic activation, 0.5 ml of S9 mix containing 50 microL of S9 was added per plate.
Test concentrations with justification for top dose:
0.001-0.1 mg/plate
Controls
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylenediamine, sodium azide, 2-aminoanthracene, and 9-aminoacridine (depending on strain)
Details on test system and experimental conditions:
ENB was tested in triplicate at each of 5 doses.  Either 0.5 mL sodium phophate buffer or S9/bacteria mix was added to the tubes containing  2 mL top agar, 100 uL bacterial strain and 100 uL of ENB, control substance, or solvent.  The contents of the tubes were poured on agar plates which were allowed to harden.  Plates were then incubated for 48-72 hours at 37C in a darkened incubator. Revertant colonies were counted by automatic colony counter. 
Evaluation criteria:
The test substance was considered positive if it produced at least a two-fold increase in the number of revertant colonies compared to the negative control, and there is evidence for a dose-related increase in the number of revertant colonies.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: yes in all strains except TA1535 without metabolic activation. Only in TA100 with metabolic activation.
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No indication of mutagenicity was observed at any of the tested doses, either by evidence of a dose-response relationship or a doubling of the number of colonies over the solvent control.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

A preliminary toxicity/initial mutagenicity assay was conducted with 10 doses over a range of 0.01 to 90 mg/mL using strain TA100 with and without S-9. Toxicity was exhibited at dosages in the range of 1-99 mg/plate. Dosages of 0.1 and 0.3 mg/plate allowed only sparse growth. The dosages used in the definitive study were chosen to span the range 0.001-0.1 mg/plate. None of the five strains, with or without induced rat liver S-9, exhibited reversion frequencies substantially different from spontaneous controls in this assay.

In the test without metabolic activation, toxicity was observed at 0.l mg/plate with all strains except TAl535. In the test with metabolic activation, toxicity was observed at 0.1 mg/plate in both strains TA100 and TA1538. All strains exhibited a positive mutagenic response with the positive controls tested both with and without S9 metabolic activation.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test substance was not mutagenic with or without metabolic activation in this test system.
Executive summary:

In an Ames test ENB did not produce a mutagenic response in the Salmonella/microsome mutagenicity assay when tested with or without metabolic activation.