Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 10, 2012 - August 23, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test method according to EU Method B.17 and OECD Guideline 476. GLP study.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Acetone oxime
EC Number:
204-820-1
EC Name:
Acetone oxime
Cas Number:
127-06-0
Molecular formula:
C3H7NO
IUPAC Name:
acetone oxime
Details on test material:
- Name of test material (as cited in study report): Acetonoxim
- Physical state: Solid
- Analytical purity: 99.6%
- Lot/batch No.: 1000074675
- Expiration date of the lot/batch: 17 February 2014
- Storage condition of test material: Controlled room temperature in a dark storeroom

Method

Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (Rat Liver Homogenate S9 Fraction)
Test concentrations with justification for top dose:
Assay 1, 3-hour treatment with metabolic activation: 5000; 3750; 2500; 1250; 625 and 312.5 μg/mL
Assay 1, 3-hour treatment without metabolic activation: 5000; 3750; 2500; 1250; 625 and 312.5 μg/mL
Assay 2, 3-hour treatment with metabolic activation: 5000; 3750; 2500; 1250; 625 and 312.5 μg/mL
Assay 2, 24-hour treatment without metabolic activation: 5000; 4000; 3000; 2000; 1000; 500; 250 and 125 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Based on the result of the preliminary solubility test the test item was insoluble in Distilled water at 500 mg/mL concentration, but it was soluble in Dimethyl sulfoxide (DMSO) at the same concentrations.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Untreated control
Negative solvent / vehicle controls:
yes
Remarks:
(DMSO 1% v/v)
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
0.15 μg/mL (3h treatment) and 0.1 μg/mL (24h treatment) in DMSO
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
cyclophosphamide
Remarks:
4 μg/mL in DMSO
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium (RPMI-5 medium)

DURATION
- Exposure duration: Assay 1: 3h (with and without metabolic activation); Assay 2: 3h (with metabolic activation), 24h (without metabolic activation)
- Expression time (cells in growth medium, to allow expresion of the TK- mutation ): 3 days
- Selection time (plating for -trifluorothymidine (TFT) resistance): Two weeks

SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: Duplicate cultures were used for each treatment

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was measured by relative survival

OTHER EXAMINATIONS:
- Determination of Survival or Viability:
After the exposure period and the expression time, cells were also diluted to be plated for survival and viability respectively. Microplates were incubated at 37 ºC ± 0.5 °C containing approximately 5% (v/v) CO2 in air for approximately two weeks. Wells containing viable clones were identified by eye using background illumination and counted.

Parameter calculated:
- Relative survival
- Mutant Frequency
- Small and large colony mutant frequencies were calculated.
Evaluation criteria:
The test item was considered to be mutagenic if all the following criteria were met:
1. Statistically significant (p < 0.05) and biologically relevant increases in mutation frequency observed in treated cultures compared to the corresponding negative (solvent) control values at one or more concentrations.
3. Increases in mutation frequency reproducible between replicate cultures and/or between tests (under the same treatment conditions).
4. Significant concentration-relationship indicated by the linear trend analysis (p < 0.05).
5. Mutation frequency at the test concentration showing the largest increase at least 126 mutants per 106 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative (solvent) control value.
Results, which only partially satisfied the acceptance and evaluation criteria, were evaluated on a case-by-case basis.
Statistics:
Statistical significance of mutant frequencies was performed using Microsoft Excel software. The control log mutant frequency (LMF) was compared to the LMF from each treatment dose, based on Dunnett's test for multiple comparisons and the data checked for a linear trend in mutant frequency with treatment dose using weighted regression. The test for linear trend was one-tailed, therefore negative trend was not considered significant. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS: No insolubility was detected in the final treatment medium at the beginning and end of the treatment in any of the experiments. There were no large changes in pH or osmolality after treatment.

RANGE-FINDING/SCREENING STUDIES: No insolubility, but cytotoxicity was observed in the preliminary experiment in one case (24-hour treatment without metabolic activation). The recommended maximum concentration of 5000 μg/mL was selected as highest examined concentration in the main experiments in each case. The lower test concentrations were generally separated by factor of two. More closely spaced concentrations were used in the expected cytotoxic concentration range to cover the concentration range from the maximum cytotoxicity (resulting approximately 10-20% relative survival) to little or no cytotoxicity.

COMPARISON WITH HISTORICAL CONTROL DATA: The positive controls (Cyclophosphamide in the presence of metabolic activation and 4-Nitroquinoline-N-oxide in the absence of metabolic activation) gave the anticipated increases in mutation frequency over the controls and were in accordance with historical data in all assays.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No cytotoxicity was observed in the main test up to 5000 µg/mL.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

In Assay 1 and 2, both with and without metabolic activation, no cytotoxicity of the test item was observed. An evaluation was made using data of all the six examined concentrations. No biologically relevant or statistically significant increase was observed at the evaluated concentrations. No significant dose response to the treatment was indicated by the linear trend analysis.

The experiments were performed using appropriate untreated, negative (solvent) and positive control samples in all cases. The spontaneous mutation frequency of the negative (solvent) controls was in the recommended range in each test. The positive controls gave the anticipated increases in mutation frequency over the controls. The plating efficiencies for the solvent controls at the end of the expression period were accepted in all assays. The evaluated concentration ranges were considered to be adequate. The number of test concentrations met the acceptance criteria. Therefore, all the validity criteria were fulfilled and the study was considered to be valid and to reflect the real potential of the test item to cause mutations in the cultured mouse cells used in this study.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

No mutagenic effect of Acetonoxim was observed either in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.
Executive summary:

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to test the potential of acetone oxime to cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix). Dimethyl sulfoxide was used as the solvent of the test item in this study. The test item was examined up to 5000 μg/mL in the Preliminary Toxicity Test. Based on the results of the preliminary experiment, the following test item concentrations were examined in the mutation assays: Assay 1, 3-hour treatment with and without metabolic activation: 5000; 3750; 2500; 1250; 625 and 312.5 μg/mL; Assay 2, 3-hour treatment with metabolic activation: 5000; 3750; 2500; 1250; 625 and 312.5 μg/mL and 24-hour treatment without metabolic activation: 5000; 4000; 3000; 2000; 1000; 500; 250 and 125 μg/mL In Assays 1 and 2, no insolubility was detected in the final treatment medium at the beginning and end of the treatment in any of the experiments. There were no large changes in pH or osmolality after treatment. In both assays no cytotoxicity of the test item was observed. Therefore, an evaluation was made using data of all the six examined concentrations. No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose response to the treatment was indicated by the linear trend analysis. All the validity criteria were fulfilled and the overall study was considered to be valid. In conclusion, no mutagenic effect of Acetonoxim was observed either in the presence or in the absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.