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Genetic toxicity: in vitro

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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Amines, C10-14-tert-alkyl
EC Number:
701-175-2
Cas Number:
68955-53-3
Molecular formula:
C10H23N to C14H31N
IUPAC Name:
Amines, C10-14-tert-alkyl
Details on test material:
- Name of test material (as cited in study report): PRIMENE 81-R
- Physical state: liquid
- Lot/batch No.: 804554082

Method

Target gene:
HGPRT
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: The cells were routinely maintained in Ham's F-12 nutrient mix (GIBCO, Grand Island, New York) supplemented with 5% (v/v) heat-inactivated (56°C, 30 minutes), dialyzed fetal bovine serum (GIBCO), antibiotics and antimycotics (penicillin G, 100 units/ml; streptomycin sulfate, 0.1 mg/ml; fungizone, 25 µg/ml; GIBCO) and an additional 2 mM L-glutamine (GIBCO).
The selection medium used for the detection of HGPRT- mutants was Ham's F-12 nutrient mix without hypoxanthine, supplemented with 10 µM 6 thioguanine (GIBCO) and 5% serum and the above-mentioned antibiotics.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 liver homogenates prepared from Aroclor 1254-induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
Preliminary Toxicity Assay
0 (solvent control), 3.13, 6.25, 12.5, 18.75, 25, 37.5, 50, 75, and 100 µg/ml

Initial Mutagenicity Assay
100, 110, and 120 µg/ml

Confirmatory Mutagenicity Assay
50 to 110 µg/ml in the absence of S9 and 50 to 160 µg/ml in the presence of S9

Repeat Confirmatory Mutagenicity Assay in the Presence of S9
0 (solvent control), 25, 50, 75, 100, 125, 150, 175, 200, 225, and 250 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: appropriate to dissolve the test material
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Ethyl methanesulfonate was used as the positive control for the non-activation system (without S9 factor). The positive control for assays performed with S9 (activation system) was 20-methylcholanthrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: Cells in logarithmic growth phase were trypsinized and placed in medium containing 5% serum at a standard density of 3.0 x 106 cells/T-75 flask approximately 24 hours prior to treatment.
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 4 hours

STAIN (for cytogenetic assays): methanol/crystal violet

NUMBER OF REPLICATIONS: duplicate

NUMBER OF CELLS EVALUATED: 1 x 10e6 surviving cells

DETERMINATION OF CYTOTOXICITY
- Method: other: ability of the treated cells to form colonies

Evaluation criteria:
For an assay to be acceptable, the mutant frequency in positive controls should have been significantly higher than the solvent controls. An additional criteria, was that the mutant frequency in the solvent controls should have been within reasonable limits of the laboratory historical control values and literature values. The test chemical was considered positive if it induced a statistically significant, dose related, reproducible increase in mutant frequency. The final interpretation of the data took into consideration such factors as the mutant frequency and cloning efficiencies in the solvent controls.
Statistics:
The frequency of mutants per 106 clonable cells was statistically evaluated using a weighted analysis of variance; weights were derived from the inverse of the mutation frequency variance. The actual plate counts are assumed to follow a Poisson distribution therefore the mean plate count was used as an estimate of variance (Kirkland, 1989).
If the analysis of variance was significant at alpha = 0.05, a Dunnett's t-test was conducted (Winer, 1971), comparing each treated group and the positive control to the solvent control (alpha = 0.05, one-sided). Linear dose-related trend tests were performed if any of the pairwise comparisons of test material with the solvent control yielded significant differences.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
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: none
- Effects of osmolality: none


RANGE-FINDING/SCREENING STUDIES: The treated cultures in the absence of S9 showed toxicity with relative cell survival (RCS) values ranging from 7.7 to 108.6%. In the presence of S9 activation, moderate to no toxicity was observed with RCS values ranging from 34.1 to 102.0%. Based upon the results of this assay, concentration levels of 0 (solvent control), 25, 50, 70, 80, 90, 100, 110, and 120 µg/ml were selected for the initial gene mutation assay in the absence of S9 and 0 (solvent control), 25, 50, 80, 100, 110, 120, 130, and 140 µg/ml in the presence of S9.

COMPARISON WITH HISTORICAL CONTROL DATA: were within the range of the laboratory historical background

ADDITIONAL INFORMATION ON CYTOTOXICITY: In order to achieve the guideline stated acceptable level of cytotoxicity (10-20% survival compared to the solvent control) in the presence of S9 a third gene mutation assay was performed. Concentration levels in this assay were 0 (solvent control), 25, 50, 75, 100, 125, 150, 175, 200, 225, and 250 µg/ml.
Remarks on result:
other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative

The results of the in vitro Chinese hamster ovary cell/hypoxanthine-guanine-phosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay with the test material indicate that under the conditions of this study, the test article was non-mutagenic when evaluated in the absence or presence of an externally supplied metabolic activation (S9) system.
Executive summary:

The test material was evaluated in the in vitro Chinese hamster ovary cell/hypoxanthine-guanine-phosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay. The genotoxic potential of the test material was assessed in two independent assays in the absence of an externally supplied metabolic activation (S9) and three independent assays in the presence of S9. The concentrations ranged from 25 to 120 µg/ml in the absence of S9 and from 25 to 250 µg/ml in the presence of S9. The highest concentration was based on the cytotoxicity of the test material to the cells. The adequacy of the experimental conditions for detection of induced mutation was confirmed by employing positive control chemicals, ethyl methanesulfonate for assays in the absence of S9 and 20-methylcholanthrene for assays in the presence of S9. Solvent control cultures were treated with the solvent used to dissolve the test material (i.e.ethanol). The results of thisin vitro CHO/HGPRT forward gene mutation assay with the test material indicate that under the conditions of this study, the test article was non-mutagenic when evaluated in the absence or presence of S9.