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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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, guideline, fully adequate for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): isooctene
- Physical state: clear liquid
- Analytical purity: 99.80%
- Lot/batch No.: 16.06.2008
- Expiration date of the lot/batch: July 2009
- Storage condition of test material: room temperature
- Sponsor's ID number: 0649/82533

Method

Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain characteristics:
other: histidine-dependent auxotrophic mutants
Metabolic activation:
with and without
Metabolic activation system:
S9 from phenobarbital and 5,6-benzoflavone induced rat liver
Species / strain:
E. coli WP2 uvr A pKM 101
Additional strain characteristics:
other: tryptophan-dependent mutant
Metabolic activation:
with and without
Metabolic activation system:
S9 from phenobarbital and 5,6-benzoflavone induced rat liver
Test concentrations with justification for top dose:
Limit test - up to 5000 µg/plate (and a series of ca half-log10 dilutions)
Vehicle:
Dimethyl formamide (DMF)
Controlsopen allclose all
Negative controls:
yes
Remarks:
untreated
Solvent controls:
yes
Remarks:
DMF
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: sodium azide, 9-aminoacridine, 2-nitrofluorene and 4-nitroquinoline-N-oxide
Remarks:
without S9
Negative controls:
yes
Remarks:
untreated
Solvent controls:
yes
Remarks:
DMF
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene and benzo(a)pyrene
Remarks:
with S9
Details on test system and conditions:
Test 1: Aliquots of 0.1 mL of the test substance solutions (seven concentrations up to 5000 µg/plate), positive control or negative control were placed in glass vessels. The negative control was the chosen vehicle, DMF. Untreated controls were also prepared. S9 mix (0.5 mL) or 0.1 M pH 7.4 phosphate buffer (0.5 mL) was added, followed by 0.1 mL of a 10-hour bacterial culture and 2 mL of agar containing histidine (0.05 mM), biotin (0.05 mM) and tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 mL minimal agar. Each Petri dish was individually labelled with a unique code, identifying the contents of the dish. Three Petri dishes were used for each treatment. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S9 mix and sodium phosphate buffer. All plates were incubated at approximately 37°C for ca 72 hours. After this period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer).
Test 2: As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test. The pre-incubation procedure is not suitable for DMF, which is toxic under such conditions. The variation used was, therefore, an increase in the S9 content of the S9 mix from 10% v/v to 20% v/v. In the absence of S9 mix, the test procedure was the same as in test 1. The maximum concentration chosen was again 5000 µg/plate, but only five concentrations were used.
Evaluation criteria:
If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls, with some evidence of a positive dose-response relationship, it is considered to exhibit mutagenic activity in this test system. If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system.
Statistics:
No statistical analysis 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
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes

Any other information on results incl. tables

No evidence of mutagenic activity was seen at any concentration of Isooctene in either mutation test.

The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Isooctene showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.
Executive summary:

In an in vitro assessment of the mutagenic potential of Isooctene, 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 Isooctene diluted in dimethyl formamide (DMF). DMF was used as a negative control. Untreated controls were also included.

Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone.  Both tests were standard plate incorporation assays.

Concentrations of Isooctene up to 5000 µg/plate were tested. Other concentrations used were a series of ca half-log10dilutions of the highest concentration. No signs of toxicity were observed towards the tester strains in either mutation test.

No evidence of mutagenic activity was seen at any concentration of Isooctene in either mutation test.

The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. The mean revertant colony counts for the vehicle and untreated controls were within or close to the 99% confidence limits of the current historical control range of the laboratory.

It is concluded that Isooctene showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.