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Genetic toxicity: in vitro

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in vitro gene mutation study in mammalian cells
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1980 - 1990
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference Type:
A Compilation of Two Decades of Mutagenicity Test Results with the Ames Salmonella typhimurium and L5178Y Mouse Lymphoma Cell Mutation Assay
Seifried HE, Seifried RM, Clarke JJ, Junghans TB and San RHC
Bibliographic source:
Chem. Res. Toxicol. 19: 627-644

Materials and methods

Test guideline
according to
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay

Test material

Details on test material:
Aquired from NCI contractors and analysed for purity at Midwest Research Institute (USA)
appropriate storage condiations as indicated from supplier


Target gene:
thymidine kinase gene
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: Fischer's medium for leukemic cells of mice (Gibco, Grand Island, NY (USA) supplemented with 10% Horse serum and 0.02% pluronic F-60 (BASF Wyandotte Crop)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data, new cultures were started from cryopreserved cultures every three months
Cells were origally obtained from Dr. Donald Clive, Buroughs Wellcome Co. (Research Triangle Park, USA)
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
liver S9 prepared from Arochlor 1254-induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
22, 29, 37, 45 and 52 microgramms/mL (without S9)
18, 24, 31, 37, 44 and 50 microgramms/mL (with S9)
Vehicle / solvent:
Controlsopen allclose all
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
Migrated to IUCLID6: 4.7 microM without metabolic activation
Positive control substance:
Migrated to IUCLID6: 18.6 microM, with metabolic activation
Details on test system and experimental conditions:
Cells at a concentration of 6 x 10exp5/mL (6 x 10 exp6 cells total) were exposed for 4 h to a range of concentrations
from 0.0005 to 10000 microg/mL. The cells were then washed, resuspended in growth medium, and incubated at 37 ( 1 °C for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls.
The doses of chemical selected for testing were within the range yielding approximately 0-90% cytotoxicity.
The mutagenicity assay was performed according to the procedure described by Clive and Spector in 1975 (Mutat. Res. 59: 61 - 180).
A total of 1.2 x 10exp7 cells in duplicate cultures were exposed to the test chemical, positive control, and solvent control for 4 h at 37 ( 1 °C, washed twice with growth medium, and maintained at 37 ( 1 °C for 48 h in log-phase growth to allow recovery and mutant expression. Cells in the cultures were adjusted to 3 x 10exp5/mL at 24 h intervals. They were then cloned (1 x 10exp6 cells/plate for mutant selection and 200 cells/plate for viable count determinations) in soft agar medium containing Fischer’s medium, 20% horse serum, 2 mM sodium pyruvate, 0.02% pluronic F-68, and 0. 23% granulated agar (BBL, Inc., Cockeysville, MD). Resistance to trifluorothymidine (TFT) was determined by adding TFT (final concentration, 3 microg/mL) to the cloning medium for mutant selection. The 100x stock solution of TFT in saline was stored at -70 °C and was thawed immediately before use. Plates were incubated at 37°C in 5% CO2 in air for 10-12 days and then counted with an Artek automated colony counter (Artek 982, DynaTech) or ProtoCol colony counter (Synbiosis, Frederick, MD). Only colonies larger than ca. 0.2 mm in diameter were counted. Mutant frequencies were expressed as
mutants per 10exp6 surviving cells.
The size of mutant mouse lymphoma colonies was also determined using an Artek 982 colony counter/sizer or the ProtoCol colony counter. An internal discriminator was set to step sequentially to exclude increasingly larger colonies in approximate increments of 0.1 mm in colony diameter. The size range used was from ca. 0.2 to 1.1 mm.
Evaluation criteria:
Results were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase. Only doses yielding total growth values of 10% were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response.

Results and discussion

Test resultsopen allclose all
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
Cytotoxicity / choice of top concentrations:
Vehicle controls validity:
Untreated negative controls validity:
not examined
Positive controls validity:
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
A less than 3 fold increase occured at highly toxic concentrations.
Cytotoxicity / choice of top concentrations:
Vehicle controls validity:
Untreated negative controls validity:
not examined
Positive controls validity:

Any other information on results incl. tables

VC = Viable counts (cloning efficiency)

TFT = Counts after TFT-selection

RTG = Relative total growth

Non-Activated Cultures S9-Activated Cultures
Dose (ug/mL) Average TFT Average VC Mut Freq RTG Dose (ug/mL) Average TFT Average VC  Mut Freq RTG
22 33 190 0.4 78 18 36 141 0.5 65
  28 167 0.3 70   30 142 0.4 67
29 29 124 0.4 55 24 55 179 0.6 49
  36 172 0.4 64   39 175 0.4 55
37 35 160 0.4 41 31 63 152 0.8 22
  47 172 0.6 41   66 157 0.8 25
45 51 191 0.6 30 37 73 140 1 15
  40 159 0.5 37   58 173 0.7 18
52 53 151 0.7 17 44 89 145 1.2 8
  64 130 0.9 14   91 139 1.3 9
Solvent 29 179 0.3   50 87 143 1.2 6
Positive 33 79 8.4 23   101 155 1.3 8
Solvent 36 159 0.5  
Positive 107 130 1.7 78

Applicant's summary and conclusion