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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to the O.E.C.D. test guideline 471 with GLP compliance.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: Colorless liquid at room temperature.
Details on test material:
As per IUCLID5 Sections 1.1. 1.2. 1.4. and 4.1.

Method

Target gene:
Histidine and trytophan synthesis
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver derived S9 fraction with cofactors.
Test concentrations with justification for top dose:
The dose levels tested were 0.50, 1.5, 5.0, 15, 50 and 150 µg per plate with all Salmonella tester strains in the absence of S9 metabolic activation, 5.0, 15, 50, 100, 150 and 500 µg per plate with all Salmonella tester strains in the presence of S9 metabolic activation and E. coli WP2 uvrA in the absence of S9 metabolic activation and 15, 50, 150, 500, 1500 and 5000 µg per plate with tester strainE. coli WP2 uvrA in the presence of S9 activation.
Vehicle / solvent:
Ethanol
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2 aminoanthracene
Remarks:
The positive control without S9 metabolic activation are tester strain specific and were: 2-nitrofluorene, sodium azide, 9-aminoacridine and methyl methane sulfonate.
Details on test system and experimental conditions:
Salmonella tester strains were from Dr. Bruce Ames’ Master cultures; E. coli tester strains were from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland. Overnight cultures were prepared by inoculating from the appropriate master plate, appropriate frozen permanent stock or with a lyophilized pellet into a flask containing ~30 to 50 mL of Nutrient Broth culture medium. Nutrient Broth was Vogel Bonner salt solution supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder). To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a shaker/incubator programmed to begin shaking at approximately 125 to 175 rpm at 37±2°C approximately 12 to 14 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of greater than or equal to 0.3x109 cells per milliliter. The actual titers were determined by viable count assays on nutrient agar plates.

Aroclor 1254-induced rat liver S9 fraction was used as the metabolic activation system. The S9 fraction was prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254, 500 mg/kg, five days prior to sacrifice. The S9 fraction was prepared by and purchased from Moltox (Boone, NC). Upon arrival at BioReliance, the S9 was stored at 60°C or colder until used. The S9 fraction mix was prepared immediately before its use and contained 10% S9, 5 mM glucose 6 phosphate, 4 mM ß nicotinamide adenine dinucleotide phosphate, 8 mM MgCl2 and 33 mM KCl in a 100 mM phosphate buffer at pH 7.4. The Sham S9 mixture (no metabolic activation), containing 100 mM phosphate buffer at pH 7.4, was prepared immediately before its use.

The test system was exposed to the test article via the preincubation methodology described by Yahagi et al. (1977). One half (0.5) milliliter of S9 mix or sham mix, 100 µL of tester strain (cells seeded) and 25 µL of vehicle or test article dilution were added to 13 X 100 mm glass culture tubes pre-heated to 37±2°C. After vortexing, these mixtures were incubated with shaking for 60±2 minutes at 37±2°C. Following the preincubation, 2.0 mL of selective top agar was added to each tube and the mixture was vortexed and overlaid onto the surface of 25 mL of minimal bottom agar. After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72 hours at 37±2 C. Plates that were not counted immediately following the incubation period were stored at 2- 8 C until colony counting could be conducted.

On the day of its use, minimal top agar, containing 0.8 % agar (W/V) and 0.5 % NaCl (W/V), was melted and supplemented with L histidine, D biotin and L tryptophan solution to a final concentration of 50 µM each. Bottom agar was Vogel Bonner minimal medium E (Vogel and Bonner, 1956) containing 1.5 % (W/V) agar.
Evaluation criteria:
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of testsubstance. Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 3.0 times the mean vehicle control value. Data sets for tester strains TA98, TA100 and E. coli WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 2.0-times the mean vehicle control value.
Statistics:
None

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Positive mutagenic responses were observed with Salmonella tester strains TA1535 and E. coli WP2 uvrA in the presence of S9fraction metabolic activation (8.6 and 4.3-fold maximum increases, respectively). A positive mutagenic response (3.2-fold maximum increase) was observed with tester strain E. coli WP2 uvrA in the absence of S9 metabolic activation.
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive

The test substance induced gene-mutations in Salmonella tester strain TA1535 and E. coli strain WP2 uvrA. The increase in mutant frequency relative to the control background value reach 8.6-fold in tester strain TA1535 without S9 metabolic activation.
Executive summary:

The test substance, Neononanoic acid glycidyl ester was accessed for the potential to induce gene-mutation in bacteria in an O.E.C.D. test guideline 471 conducted by the preincubation method. The test substance induced gene-mutations in Salmonella tester strain TA1535 and E. coli strain WP2 uvrA. The increase in mutant frequency relative to the control background value reach 8.6-fold in tester strain TA1535 without S9 metabolic activation.