Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Isoprene
EC Number:
201-143-3
EC Name:
Isoprene
Cas Number:
78-79-5
IUPAC Name:
isoprene
Details on test material:
- Name of test material (as cited in study report): Isoprene
- Physical state: Colourless liquid at room temperature
- Analytical purity: 99%
- Lot/batch No.: 0710MO
- Expiration date of the lot/batch: no data
- Molecular weight: 68.12
- Supplier: Aldrich Chemical Company
- Storage condition of test material: ca. 4°C in the dark

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
S9 and microsomal fractions from uninduced B6C3F1 mice liver
Test concentrations with justification for top dose:
0, 0.25 to 25% isoprene (2,500 to 250,000 ppm) in vapour phase
Vehicle / solvent:
- Vehicle(s)/solvent(s) used; DMSO
Controlsopen allclose all
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
other: vinyl chloride (monomer)
Remarks:
with S9/microsome mix
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9/microsome mix

Migrated to IUCLID6: 0.5 µg/plate for strains TA1535 and TA100
Positive control substance:
9-aminoacridine
Remarks:
without S9/microsome mix

Migrated to IUCLID6: 50 µg/plate for strain TA1537
Positive control substance:
2-nitrofluorene
Remarks:
without S9/microsome mix

Migrated to IUCLID6: 1 µg/plate for strain TA98
Positive control substance:
other: 2-(2-Furyl)-3-5(5-nitro-2-furyl) acrylamide (AF-2). 0.05 µg/plate for strain WP2 uvrA (pKM101)
Remarks:
without S9/microsome mix
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- The tester strains were exposed to the test substance in stainless steel vessels.
- The untreated and vapour-phase positive controls were treated under identical conditions to the plates exposed to the test substance.
- The appropriate positive control compounds for confirmation of strain specificity were included using the conventional plate incorporation method.
- An aliquot of 0.1 mL of a 10 hour bacterial culture and 0.5 mL S9/microsome mix (or 0.5 mL 0.1 M phosphate buffer, pH 7.4, for the direct-acting positive controls) were placed in glass vessels. 2 mL of agar containing histidine (0.5 mM), biotin (0.5 mM) and tryptophan (0.5 mM) was then added, the mixture thoroughly shaken and overlaid onto previously prepared glass Petri dishes containing approximately 25 mL minimal agar.
- The seeded plates were placed in stainless steel vessels. The plates were supported, inverted with lids removed, on stainless steel racks inside the vessels, so as to permit circulation of the test substance. The plates containing S9/microsome mix and buffer were placed in separate vessels.
- The vessels were sealed, purged with nitrogen and partially evacuated. Appropriate volumes of the test substance were injected via a PTFE septum.
- The vessels were warmed to 37°C and the contents equilibrated to atmospheric pressure by admitting nitrogen.

DURATION:
- The plates were incubated for ca 48 hours in the vessels at 37°C and then removed under air extraction.
- The lids were replaced and the plates incubated for a further period of ca 24 hours at 37°C to permit the growth of revertant colonies.
- Further sets of plates were prepared for the liquid positive control compounds. An aliquot of 0.1 mL of the positive control solution was added to the plates together with the bacteria, buffer and agar overlay. These plates were not placed in stainless steel vessels and were incubated at 37°C for 72 hours.

NUMBER OF REPLICATIONS: 3




Evaluation criteria:
A positive response is defined as reproducible, dose-related increase in revertant colonies in any one strain. An increase of 2x over background is required for a positive response in Salmonella strains TA98 & TA100 and for E. coli bacteria, and 3x background for Salmonella TA1535 & TA1537.
Statistics:
None performed.

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
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Isoprene vapour exposures above 25% produced significant cytotoxicity based upon a pilot study with exposures up to 100% isoprene.

The concurrent positive controls, including Vinyl Chloride monomer at 2% v/v in vapour phase, demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. Increases obtained with Vinyl Chloride in the presence of mouse S9 were consistently higher than those obtained with mouse microsomes at the same percentage volume in the cofactor mix (i.e. the S9 was more active per unit volume than the microsomes).
Remarks on result:
other: other: Enclosed static vapour phase exposure chambers
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Revertant colony counts appeared to be slightly increased (between 1.3 and 1.7 x untreated control counts) in strains TA1535 and WP2uvrA (pKM101) following exposure to Isoprene at 2.5% v/v in the presence of mouse liver S9, but these were not of sufficient magnitude to be considered evidence of mutagenic activity.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Isoprene was not mutagenic in the Ames bacterial mutagenicity test.
Executive summary:

In an in vitro assessment of the mutagenic potential of Isoprene, histidine-dependent auxotrophic mutants of Salmonella typhimurium, strains TA1535, TA1537, TA98 and TA100, and a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101) were exposed to Isoprene in vapour phase. Revertant colony counts appeared to be slightly increased (between 1.3 and 1.7 x untreated control counts) in strains TA1535 and WP2uvrA (pKM101) following exposure to Isoprene at 2.5% v/v in the presence of mouse S9, but these were not of sufficient magnitude to be considered evidence of mutagenic activity.

Categories Display