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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
Study period:
1998-02-24 - 1998-04-20
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP-compliant study comparable to OECD TG 471 with minor restriction, no negative control (medium alone) was investigated. The reliable study is acceptable for assessment,

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report date:
1998

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted on 21st July 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
429-460-4
EC Name:
-
Cas Number:
7078-98-0
Molecular formula:
C21 H26 O
IUPAC Name:
2,6-di-tert-butyl-4-(phenylmethylidene)cyclohexa-2,5-dien-1-one

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
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from Aroclor 1254 induced liver of male Wistar rats
Test concentrations with justification for top dose:
6.67,10,0, 33.3, 66.7, 100, 333, 667, 1000, 3330 and 5000 µg per plate (dose range-finding study)
10.0, 33.3, 100, 333, 1000 and 5000 µg per plate (main study and independent repeat study)
Vehicle / solvent:
VEHICLE
- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: relatively non-toxic, standard vehicle for studies of this type
- Concentrations: Stock solution 50 mg/mL (mutagenicity assay), 100 mg/mL (dose rangefinding study)

ASSAY
The assay was conducted with three doses of test item in both the presence and absence of S9 mix along with concurrent vehicle and positive controls using three plates per dose.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S9 (TA1535, TA100): sodium azide; (WP2 uvrA): 4-nitroquinoline-N-oxide; (TA98): 2-nitrofluorene, (TA1537): ICR-191; with S9 (TA 100, WP2 uvrA, TA 1535, TA 1537): 2-aminoanthracene; (TA 98): benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Expression duration: 52 ± 4 hours (at 37 ± 2°C)
- Expression time (cells in growth medium): see exposure duration
- Selection time (if incubation with a selection agent): see exposure duration
- Fixation time (start of exposure up to fixation or harvest of cells): Plates which were not evaluated immediately following the incubation period were held at 5 ± 3°C until evaluation.

SELECTION AGENT (mutation assays)
- S. typhimurium strains: histidine/biotin solution for selection of histidine revertants
- E. coli strain: tryptophan solution for selection of tryptophan revertants

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED:
- Number of colonies (counting revertant colonies): colony counter (positive controls) and manually (vehicle controls and test item concentrations)

DETERMINATION OF CYTOTOXICITY
- Method: Bacterial Background Lawn Evaluation

OTHER: Experimental phase: 1998-02-24 – 1998-03-27; 2 independent experiments were conducted
Evaluation criteria:
Criteria for a positive response:

For a test item to be considered positive, it had to produce at least a 2-fold increase in the mean revertants per plate of at least one of the tester strains TA98, TA100 and WP2uvrA over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article.

For a test item to be considered positive, it had to produce at least a 3-fold increase in the mean revertants per plate of at least one of the tester strain TA1535 and TA1537 over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test item.
Statistics:
Due to international guidelines a statistical evaluation of the results is recommended. However, no evaluated statistical procedure can be recommended for analysis of data from the bacterial assays at this time.

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 / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS:
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Precipitation: yes (> 333 µg test item/mL)

COMPARISON WITH HISTORICAL CONTROL DATA:
The observed reversion rates were not different from the historical control range (and positive control range).

ADDITIONAL INFORMATION ON CYTOTOXICITY (Dose Rangefinding Study, data generated in Experiment 19219-A):
Doses to be tested in the mutagenicity assay were selected based on the results of the dose rangefinding study conducted on the test article using tester strains TA100 and WP2UvrA in both the presence and absence of S9 mix (one plate per dose). Ten doses of test item, from 6.67 to 5000 µg per plate, were tested. No cytotoxicity was observed in either the presence or absence of S9 mix as evidenced by no decrease in the number of revertants per plate. The bacterial background lawns were evaluated as normal up to the 1000 µg per plate dose. Lawns above this dose were obscured by precipitate.

OTHER:
Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative