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Diss Factsheets

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:
23 September - 4 November 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, to GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
celery ketone
IUPAC Name:
celery ketone
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Celery ketone
- Substance type: No data
- Physical state: colourless liquid
- Analytical purity: 99.5%
- Impurities (identity and concentrations): No data
- Composition of test material, percentage of components: Celery ketone (99.5%)
- Isomers composition: No data
- Purity test date: 25 August 2003
- Lot/batch No.: 9000505959
- Expiration date of the lot/batch: 24 February 2005
- Stability under test conditions: No data
- Storage condition of test material: room temperature

Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal fraction (S9) metabolising system
Test concentrations with justification for top dose:
Preliminary cytotoxicity test (strains TA 98, 100 and 102 only): 0, 10, 100, 500, 1000, 2500, and 5000 μg/plate
Experiment 1 (direct plate incorporation method): 0, 78.13, 156.3, 312.5, 625 and 1250 μg/plate
Experiment 2 (Pre-incubation method): 0, 14.65, 29.3, 58.59, 117.2 and 234.4 μg/plate (-S9); 0, 58.59, 117.2, 234.4, 468.8, 937.5 and 1875 μg/plate (+S9)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Freely soluble in the vehicleat 50 mg/mL concentration
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
1 μg/plate for TA 1535 and TA 100 without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
50 μg/plate for TA1537 without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
0.5 μg/plate for TA 98 without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
0.5 μg/plate for TA 102 without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-anthramine
Remarks:
2 μg/plate for TA 1535, TA 1537, TA 98 and TA 100 with S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-anthramine
Remarks:
10 μg/plate for TA 102 with S9
Details on test system and experimental conditions:
EXPERIMENT 1
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: Not applicable
- Exposure duration: 48-72 h
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): Not applicable

SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): Not applicable

NUMBER OF REPLICATIONS: 3 (Experiment 1 was carried out twice)

NUMBER OF CELLS EVALUATED: Not applicable

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertant colonies and/or a thinning of the bacterial lawn

OTHER EXAMINATIONS:
- Determination of polyploidy: Not applicable
- Determination of endoreplication: Not applicable

OTHER:


EXPERIMENT 2
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48-72 h
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): Not applicable

SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): Not applicable

NUMBER OF REPLICATIONS: 3 (Experiment 2 was carried out twice)

NUMBER OF CELLS EVALUATED: Not applicable

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertant colonies and/or a thinning of the bacterial lawn

OTHER EXAMINATIONS:
- Determination of polyploidy: Not applicable
- Determination of endoreplication: Not applicable

OTHER:
Evaluation criteria:
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or a 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose level and/or evidence of a dose-relationship constitutes a positive result. Reference to historical data, or other consideratiosn of biological relevance may also be considered.
Statistics:
No data

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
moderate toxicity at 1250 μg/plate without S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
moderate toxicity at 1250 μg/plate without S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
moderate to marked toxicity at 234.4 μg/plate (without S9) and at 1875 μg/plate (with S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
moderate to marked toxicity at 234.4 μg/plate (without S9) and at 468.8 μg/plate and above (with S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
moderate to marked toxicity at 234.4 μg/plate (without S9) and at 937.5 μg/plate and above (with S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
moderate to marked toxicity at 234.4 μg/plate (without S9) and at 937.5 μg/plate and above (with S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
moderate to marked toxicity at 234.4 μg/plate (without S9) and at 468.8 μg/plate and above (with S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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
- Water solubility: No data
- Precipitation: Not observed at any dose level
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: A moderate to marked toxicity was noted at 2500 μg/plate and above (with and without S9) in the preliminary test using strains TA 98, TA 100 and TA 102.

COMPARISON WITH HISTORICAL CONTROL DATA: Numbers of revertants in the vehicle and positive controls were consistent with historical controls

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data
Remarks on result:
other: strain/cell type: TA 1535 (plate incorporation method)
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
In a guideline study (OECD TG 471), to GLP, the test item celery ketone did not show any mutagenic activity in the bacterial reverse mutation assay with Salmonella typhimuium.
Executive summary:

In an OECD guideline study (TG 471), conducted according to GLP, the test item celery ketone was assessed for mutagenicity by reversion to histidine independence in Salmonella typhimurium strains TA 1535, TA1537, TA98, TA100 and TA102. The test item was tested using two independent methods: direct plate incorporation and preincubation. All assays were carried out in triplicate both with and without metabolising fraction S9. Celery ketone did not show mutagenic activity in any of the strains tested both with and without S9.