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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:
29 April 1986 - 29 May 1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was performed according to OECD guidelines and GLP. Deviation from the current guideline: With this test it is not possible to identify certain oxidising mutagens, cross-linking agents and hydrazines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report Date:
1986

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
- With this test it is not possible to identify certain oxidising mutagens, cross-linking agents and hydrazines. Such substances may be detected by E.coli WP2 strains or S. typhimurium TA102 which have an AT base pair at the primary reversion site in stead of GC base pairs which the strains tested in this study have.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Chemical name: Neodecane peroxoic acid 1,1,3,3-tetramethyl butyl ester
- Appearance: Colouless clear liquid
- CAS-reg. no.: 51240-95-0

Method

Target gene:
Histidine operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Each tester strain contains the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cell coat).
gal mutation in the galactose metabolism.
chl : mutation in nitrate reductase.
bio : defective biotin synthesis.
uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene).
Additional strain / cell type characteristics:
other: TA1537 histidine mutation: hisC3076, Frameshift, TA98 histidine mutation: hisD3052/R-factor, Frameshift, TA1535: hisG46, Base-pair substitutions, TA100: hisG46/R-factor, Base-pair substitutions. R-factor =plasmid pKM101 (increases error-prone DNA repair).
Species / strain / cell type:
S. typhimurium TA 1538
Details on mammalian cell type (if applicable):
Each tester strain contains the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cell coat).
gal mutation in the galactose metabolism.
chl : mutation in nitrate reductase.
bio : defective biotin synthesis.
uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene).
Additional strain / cell type characteristics:
other: histidine mutation: hisD3052, Frameshift
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
preliminary test (TA100): 0.1, 0.3, 1.0, 3.3, 10.0, 33.3, 100, 333, 1000, 3330, 5000 µg/plate
Experiment 1, 2, 3, 4: 100, 333, 1000, 3333, 5000 µg/pkate (with and without S9 mix)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol absolute 0.1 ml
- Justification for choice of solvent/vehicle: soluble+stable in vehicle
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethanol absolute 0.1 ml
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see below.
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 48 hours

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: triplicate with independent repeat

NUMBER OF CELLS EVALUATED: revertant colonies (histidine independent) are counted automatically with an Artek model 880 colony counter or manually.

DETERMINATION OF CYTOTOXICITY
- Method: % survival
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the Ames test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance is considered positive (mutagenic) in the Ames test if:
a) It induces at least a 2-fold and statistically significant (student's t-test, p< 0.05) increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. Moreover, the positive response should be dose-rela'ted. If the test substance shows in the first test only a positive response at one or two concentrations, the assay is repeated with doses just below and exceeding those showing positive effects in the first test.
b) The positive response should be reproducible in at least one independently repeated experiment. The preceding criteria are not absolute and other extenuating factors may enter into the final evaluation decision.
Statistics:
See above.

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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Negative in the Ames Salmonella/microsome preincubation test.
Executive summary:

The test substance was tested in the Ames Salmonella/microsome preincubation test up to the limit of toxicity (5 mg/plate). The test substance induced no statistically significant dose-related increase in the numbers of revertant (His+) colonies in each of the five tester strains (TA1535; TA1537; TA1538; TA98 and TA100). These results were confirmed in an independently repeated experiment. The test substance can, therefore, be considered as nonmutagenic in this test system.