1,6-Octadiene, 8-methoxy-

Genetic toxicity: in vitro

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: This study was a screening study and not intended to meet any test guidelines. Sufficient data is available for the interpretation of results.

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)

Deviations:

yes

Remarks:

only 3 strains used

Principles of method if other than guideline:

This was a screening study.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

Approx. 96.07% cis, and 3.58% trans isomers as estimated by gas chromatography with flame ionization detection.

Method

Target gene:

histidine gene for Salmonella typhimurium
tryptophan gene for E. coli WP2uvrA

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not specified

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9

Test concentrations with justification for top dose:

1, 3.3, 10, 33, 100, 333, 1000, 3333, and 5000 μg/plate

Vehicle / solvent:

DMSO

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Controls 2

Positive control substance:

benzo(a)pyrene

Remarks:

TA98 with S-9

Migrated to IUCLID6: 2.5 μg/plate

Controls 3

Positive control substance:

other: 2-aminoanthracene; 2.5 μg/plate, 25 μg/plate

Remarks:

TA100, E Coli WP2uvrA with S-9

Controls 4

Positive control substance:

2-nitrofluorene

Remarks:

TA98 without S-9

Migrated to IUCLID6: 5 μg/plate

Controls 5

Positive control substance:

sodium azide

Remarks:

TA100 without S-9

Migrated to IUCLID6: 10 μg/plate

Controls 6

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

E coli WP2uvrA without S-9

Migrated to IUCLID6: 0.4 μg/plate

Details on test system and experimental conditions:

Source and Storage of Tester Strains:
The Salmonella typhimurium and E.coli tester strains used in this study (TA98, TA100, and E.coli WP2uvrA) were acquired from Moltox Inc., Boone, North Carolina. Frozen permanent stocks were prepared by growing fresh overnight cultures, adding DMSO (0.09 mL/mL of culture) and freezing appropriately vialed aliquots. Frozen permanent stocks of the tester strains were stored in liquid nitrogen vapor or at least at -70 °C.

Confirmation of Tester Strain Genotype:
Tester strain cultures were checked for the following genetic markers concurrent with their use in the mutagenicity assay. The presence of the rfa wall mutation was confirmed by demonstration of the sensitivity of the cultures to crystal violet. The presence of the pKM101 plasmid was confirmed for cultures of tester strain TA98 and TA100 by demonstration of resistance to ampicillin.

Culturing and Harvest:
Overnight cultures for use in all testing procedures, were inoculated by transferring an aliquot of the frozen tester strain to a flask containing culture medium. Inoculated flasks were placed in a shaker/incubator (37 ± 2°C) so that the overnight cultures were in late log phase when density monitoring began. To ensure that cultures were harvested in late log phase, the length of incubation was determined by spectrophotometric monitoring of culture density. Cultures were harvested once a predetermined density was reached, which ensured that cultures had reached a density of approximately 1 x 10^9 cells per mL and that the cultures had not overgrown. Overgrown (stationary) cultures may exhibit decreased sensitivity to some mutagens. Cultures were removed from incubation when the target density was reached and were held at 5 ± 3°C until used in the assay.

Tester Strain Media:
All tester strain media and experimental reagents were acquired from Moltox Inc., Boone, North Carolina. The broth used to grow overnight cultures of the tester strains were Vogel-Bonner salt solution (Vogel and Bonner, 1956) supplemented with 2.5% (w/v) Oxoid Nutrient Broth #2 (dry powder). Bottom agar (25 mL per 15 x 100 mm petri dish) was Vogel-Bonner minimal medium E (Vogel and Bonner, 1956) supplemented with 1.5% (w/v) agar and 0.2% (w/v) glucose. Top (overlay) agar was prepared with 0.7% agar (w/v) and 0.5% NaCl (w/v) and supplemented with 10 mL of 0.5 mM histidine/biotin solution per 100 mL agar for selection of histidine revertants or 10 mL of 0.5 mM tryptophan solution per 100 mL agar for selection of tryptophan revertants. For an agar overlay, 2.0 mL of the supplemented top agar was used.

Vehicle Controls:
Vehicle controls were plated for all strains in the absence or presence of S-9 mix as appropriate and constituted the solvent used for the test material.

Sterility Controls:
The most concentrated test article dilution, buffer mixes and S-9, were checked for sterility.

Positive Control Materials:
The positive controls were of reagent grade or better.

Mutagenicity Screening Assay:
The assay was performed using tester strains TA98, TA100, and WP2uvrA in the presence and absence of S-9. Nine concentrations of the test material were evaluated along with the appropriate positive and negative controls. The test material was evaluated up to a concentration of 5000 mg/plate. The concentrations selected under these conditions were 5000, 3333, 1000, 333, 100, 33, 10, 3.3, and 1 mg/plate in the presence and absence of S-9.

For the assay with activation, the bacteria (0.1 ml), test article (0.1 ml of the appropriately diluted test material or DMSO solvent ), and the S-9 mix (0.5 ml) were placed into sterile tubes and incubated at 37oC for approximately 20 minutes. 2 ml of top agar (supplemented with trace amounts of histidine and biotin or tryptophan) were then added, mixed, and poured onto minimal glucose agar plates. For the non-activation assay, S-9 mix was omitted and replaced by 0.5 ml of 0.2M phosphate buffer, pH 7.4. The top agar was allowed to solidify and plates were incubated for approximately 52 ± 4 hours in an incubator at 37oC. This assay was conducted by exposing all three strains to negative controls (3 plates/dose), positive controls (3 plates/dose) and the above concentrations of test article (3 plates/dose), in both the presence and absence of S-9 activation.

Evaluation criteria:

The following criteria had to be met for the mutagenicity assay to be considered valid.

Tester Strain Integrity:
To demonstrate the presence of the rfa mutation, the S. typhimurium tester strain cultures had to exhibit sensitivity to crystal violet. To demonstrate the presence of the pKM101 plasmid R- factor, tester strain cultures of TA98 and TA100 had to exhibit resistance to ampicillin as described above.

Negative Control Values:
Based on historical control data, all tester strain cultures had to exhibit characteristic numbers of spontaneous revertants per plate in the negative controls (vehicle). The mean revertants per plate had to be within the following ranges (inclusive): TA98, 10-50, TA100, 80-200, and WP2uvrA, 10-54.

Positive Control Values:
Each mean positive control value had to exhibit at least a 3.0-fold increase over the respective mean negative control value (vehicle) for each tester strain.

Toxicity:
A minimum of three non-toxic dose levels was required to evaluate assay data. In the event that less than three non-toxic dose levels were achieved, the affected portion of the assay was repeated with an appropriate change in dose levels.

Statistics:

For a test article to be judged positive for TA98 or WP2uvrA a concentration-related increase in mean revertants must be equal to or greater than 3.0 times the mean negative control value (vehicle). Similarly, for strain TA100, data sets were judged positive if the concentration-related increase in mean revertants was equal to or greater than 2.0 times the mean negative control value (vehicle).

Results and discussion

Test results

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

1-Methoxy 2,7-octadiene was assayed in Salmonella typhimurium strains TA98, TA100, and E. coli WP2uvrA at concentrations of 5000, 3333, 1000, 333, 100, 33, 10, 3.3, and 1 μg/plate in the presence and absence of S-9. In the absence of S-9, TA98 and TA100 (100-5000 μg/plate) showed signs of toxicity. Also, in this assay, E. coli WP2uvrA exhibited cytotoxicity from 333 mg/plate up to the maximal concentration tested (5000 mg/plate). No precipitation was observed on any of the plates in either the presence or absence of S-9. In the presence of S-9, cytotoxicity was present in all strains at 333 mg/plate and higher.

No increases in revertant counts satisfying the criteria for a positive response (3- fold) were observed. The positive and negative control responses satisfied the criteria for assay acceptability.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

1-Methoxy 2,7-octadiene was concluded to be negative in the bacterial reverse mutation screening assay using Salmonella typhimurium tester strains TA98, TA100, and E. coli W2uvrA under the experimental conditions used.