Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 21 October, 1997 to 20 November, 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
Kanpogyo No. 700, the Japanese Environment Agency, Yakuhatsu No. 1039, the Japanese Ministry of Health and Welfare and 61Kikyoku No. 1014, the Japanese Ministry of International Trade and Industry, 1986
Deviations:
no
Qualifier:
according to
Guideline:
other: JAPAN: Industrial Safety and Health Law
Version / remarks:
Notification No.77, 1988, the Japanese Ministry of Labour
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial forward mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
-Name of test material (as cited in study report): Dihydrofarnesol
-Substance type: colorless oil
-Analytical purity: >99% (subsequent information by sponsor)
-Impurites (identity and concentrations): no information
-Strage condition of test material: in a dark and cool place
-Stability: stable at room temperature

Method

Target gene:
his operon (for S. typhimurium strains)
trp operon (for E.coli strain)
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
Metabolic activation:
with and without
Metabolic activation system:
Cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbitone and β-naphthoflavone
Test concentrations with justification for top dose:
Range-finding study 1 (for all tester strains with and without metabolic activation): 0.31, 1.22, 4.88, 19.5, 78.1, 313, 1250 and 5000 μg/plate
Range-finding study 2 (for TA1535, TA1537, TA98 and TA100 without metabolic activation): 0.076, 0.305, 1.22, 4.88 and 19.5 μg/plate
Main test (for TA1535, TA1537, TA98 and TA100 without metabolic activation): 0.61, 1.22, 2.44, 4.88, 9.77 and 19.5 µg/plate
Main test (for WP2uvrA with and without metabolic activation, TA1535 and TA98 with metabolic activation): 156, 313, 625, 1250, 2500 and 5000 µg/plate
Main test (for TA1537 with metabolic activation): 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 µg/plate
Main test (for TA100 with metabolic activation): 39.1, 78.1, 156, 313, 625 and 1250 µg/plate

Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: Based on the information from the sponsor that the test material was soluble at 5% and more in DMSO.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
Positive control substance: other: 2-aminoanthracene (2AA), 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), 9-aminoacridin (9-AA) , sodium azide (NaN3)
Remarks:
+S9: 2AA (0.5 µg/plate, TA98; 1 µg/plate, TA100; 2 µg/plate, TA1535, TA1537; 10 µg/plate, WP2uvrA) −S9: AF-2 (0.01 µg/plate, TA100, WP2uvrA; 0.1 µg/plate, TA98); 9-AA (80 µg/plate, TA1537); NaN3 (0.5 µg/plate, TA1535)
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation
DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: doubles each in two independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition and test material precipitation
Evaluation criteria:
A test material is considered positive (mutagenic) in the test if:
a) It induces a number of revertant colonies, dose related, greater than two-times the number of revertants induced by the solvent control in any of the test strains, either with or without metabolic activation.
b) The positive response should be reproducible in at least two separated experiments.
Statistics:
Mean values was calculated. The statistical analysis was not used.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
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
Key result
Species / strain:
S. typhimurium TA 1537
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
Key result
Species / strain:
S. typhimurium TA 98
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
Key result
Species / strain:
S. typhimurium TA 100
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
Key result
Species / strain:
E. coli WP2 uvr A
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:
PRECIPITATION
Precipitation on the plates was observed at 2500 and 5000 µg/plate with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
Growth inhibition by the test material was observed in S. typhimurium strains (TA1535, TA1537, TA98 and TA100) with and without metabolic activation. Therefore, as the highest dose level of the test material in the main test, the dose at which the growth inhibition was observed was selected for S. typhimurium strains (TA1535, TA1537, TA98 and TA100).

CONTROL SUBSTANCES:
All positive control substances gave increases in revertants, both with and without metabolic activation, within expected ranges, and solvent control plates gave counts of revertant colonies within the normal range.
Remarks on result:
other: All strains/cell types tested

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative