Registration Dossier

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:
2012-11-22 to 2012-12-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
butyl 4-hydroxycyclohexanecarboxylate
EC Number:
941-637-2
Cas Number:
1384257-92-4
Molecular formula:
C11H20O3
IUPAC Name:
butyl 4-hydroxycyclohexanecarboxylate
Test material form:
other: liquid

Method

Target gene:
Salmonella: histidine operon
E coli. tryptophane operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
- Type and identity of media: Nutrient broth
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from Aroclor 1254 pretreated rats
Test concentrations with justification for top dose:
1st series: 5.00, 15.8, 50.0, 158, 500, 1580, and 5000 µg/plate
2nd series: 15.8, 50.0, 158, 500, 889, 1580 µg/plate
Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: Sodium azide, 2-Aminoanthracene, 9-Aminoacridine, Daunomycin, 4-Nitroquinoline-N-oxide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 3-5 hours
- Exposure duration: about 2 days


SELECTION AGENT (mutation assays): Histidine, Tryptophane

NUMBER OF REPLICATIONS:
Negative controls 6
Test material 3
Positive controls 3


NUMBER OF CELLS EVALUATED: about 10e9


DETERMINATION OF CYTOTOXICITY
- Method: other: background cytotox

Evaluation criteria:
- valid assay (cf. laboratory historical data)
- no or weak increase in revertant colonies = negative
- clear, dose dependent (over at least two concentrations), and reproducible increase in revertant colonies= positve
Statistics:
not applied

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Precipitation: yes, at 1580 µg/plate with and without metabolic activation
- Other confounding effects: no

Applicant's summary and conclusion

Conclusions:
With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.
Executive summary:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 471. With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.