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Toxicological information

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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable scientific publication, data suitable for the purpose of read-across.

Data source

Reference
Reference Type:
publication
Title:
Quantitative Mammalian Cell Mutagenesis and a Preliminary Study of the Mutagenic Potential of Metallic Compounds
Author:
Hsie AW, Johnson NP, Couch DB, San Sebastian JR, O'Neill JP, Hoeschele JD, Rahn RO, Forbes NL
Year:
1979
Bibliographic source:
Trace Metals in Health and Disease: 55-69

Materials and methods

Principles of method if other than guideline:
The test substance was tested for its mutagenic potential in the CHO/HGPRT test.
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Titanium tetrachloride
- Analytical purity: No data

Method

Target gene:
HGPRT gene
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
The study described has employed a subclone of CHO-K1 cells designated as CHO-K1-BH4, which was isolated following selection in F12 medium containing aminopterin (10 µM). Cells are routinely cultured in Ham's F12 medium (K. C. Biolagical Co.) containing 5 % heat-inactivated (56 °C, 30 min), extensively dialysed foetal calf serum (K. C. Biological Co.) (medium F12FCM5) in plastic tissue culture dishes (Falcon or Corning Glass Works) under standard conditions of 5% CO2 in air at 37 °C in a 100 % humidified incubator. These cells grow in medium which contains aminopterin as well as in regular medium with 5 or 10 % dialysed foetal calf serum with a population doubling time of 12 to 14 h. Cells are removed with 0.05 % trypsin for subculture, and the number is determined with a Coulter Counter (model B, Coulter Electronics).
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix (acc. to Ames from livers of Aroclor 1254-induced Sprague-Dawley rats; contains (per ml) 30 µmol KCl, 10 µmol MgCl2, 10 µrnol CaCl2, 4 µmol NADP, 5 µmol glu-6-p, 50 µmol phosphate buffer (pH 8.0), and 0.1 ml microsome fraction (3 - 4 mg protein))
Test concentrations with justification for top dose:
no data
Vehicle / solvent:
- Solvent used: DMSO
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: Ethyl methanesulfonate (EMS), N-methyl-N'nitro-N-nitroso-guanidine (MNNG)
Details on test system and experimental conditions:
CHO cells were plated at 0.5 mio cells/25 cm2 bottle in medium F12FCM5. After a 16 - 24-hr growth period (cell number = ~ 1.0 to 1.5 mio cells/plate), the cells were washed twice with saline G, and sufficient serum-free F12 medium was added to bring the final volume to 5 ml after the addition of various amounts of microsome preparation (up to 1 ml) and 50 µl of chemical, usually dissolved in DMSO. Chemicals and/or microsomes are omitted from some plates to provide controls.
For the determination of mutation induction, the treated cells were allowed to express the mutant phenotype in F12 medium for 7 to 9 days, at which time mutation induction reached a maximum which was maintained thereafter (as long as 35 days examined) for several agents [ethyl methanesulfonate (EMS), N-methyl-N' -nitro-N-nitrosoguanidine (MNNG), ICR-19 1, X-ray, and UV light] irrespective of concentration or intensity of the mutagen . Routine subculture was performed at 2-day intervals during the expression period, and at the end of this time the cells were plated for selection in hypoxanthine-free 12FCM5 containing 1.7 pg/ml (10 PM) of TG at a density of 2.0 X 105 celIs/100-rnm plastic dish (Corning or Falcon), which permited 100% mutant recovery in reconstruction experiments. The use of dialyzed serum was found to be particularly important, presumably due to potential competition between hypoxanthine and TG for transport into the cells and for catalysis by WGPRT. After 7 to 8 days in the selective medium, the drug-resistant colonies developed; they were then fixed, stained, and counted. The protocol permited the maximum stable mutation induction by various physical and chemical agents of TG- resistant variants (TGt), >98% of which had highly reduced HGPRT activity. Mutation frequency was calculated based on the number of drug-resistant colonies per survivor at the end of the expression period.
Statistics:
To analyse the nature of the dose-response curve, the data was fit from several different dose ranges to the Iinear model by the method of weighted least-squares.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative