Naphtha (petroleum), solvent-refined light

Genetic toxicity: in vitro

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other: GLP, guideline study, available as unpublished report, no restrictions, fully adequate for assessment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

Test material information

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 / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

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

Controls

Controls 1

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

other: vinyl chloride (monomer)

Remarks:

with S9/microsome mix

Controls 2

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

Controls 3

Positive control substance:

9-aminoacridine

Remarks:

without S9/microsome mix

Migrated to IUCLID6: 50 µg/plate for strain TA1537

Controls 4

Positive control substance:

2-nitrofluorene

Remarks:

without S9/microsome mix

Migrated to IUCLID6: 1 µg/plate for strain TA98

Controls 5

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 results

Test results 1

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

Test results 2

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.