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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
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, near guideline study, available as unpublished report, fully adequate for assessment

Data source

Referenceopen allclose all

Reference Type:
study report
Report date:
Reference Type:
Genotoxicity testing of the Halon replacement candidates trifluoroiodomethane(CF3I) and 1,1,1,2,3,3,3-heptafluoropropane(HFC227ea) using the Salmonella typhimurium and L5178Y mouse lymphoma mutation assays and the mouse micronucleus test.
Dodd DE, Ledbetter AD, Mitchell AD
Bibliographic source:
Inhalation Toxicology 9(2):111-31

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
The test material, 1,1,1,2,3,3,3-heptafluoropropane (HFC-227ea, molecular weight 170; CAS Number 431-89-0), a colorless gas, was received in a steel gas container from ManTech/Dayton on April 7, 1994 then transferred to Allen Ledbetter, ManTech/RTP, who was responsible for handling, storage, and dilution of the test material. The HFC-227ea was stored at ManTech/RTP at room temperature (approximately 72°F). ManTech/Dayton documented the strength, purity, and composition of the test material and provided a Material Safety and Data Sheet (MSDS) from Great Lakes Chemical Corporation for HFC-227ea.


Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Cell culture
L5178Y mouse lymphoma cells, clone 3.7.2C, provided by Dr. Donald Clive, Burroughs Wellcome Co., Research Triangle Park, NC, are stored in liquid nitrogen at Genesys.
The cells were grown as a suspension culture in FIOHP medium, cleansed of homozygous (tk-I-) cells with medium containing 0.1 μg/ml methotrexate, as described by Mitchell et al., 1988, and used as target cells for chemical exposure.
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 homogenate
Test concentrations with justification for top dose:
Nominal concentrations:
Preliminary assay: 100000; 200000; 300000; 400000; 500000; 600000; 700000; 800000; 900000 ppm
Mutagenesis assay: 100000; 300000; 500000; 700000; 900000 ppm

IR determined concentrations:
Preliminary assay: 63,967, 147,112; 204,380, 269,853, 344,792; 378,811; 423,733; 458,673; 538,535 ppm
Mutagenesis assay: 84,765; 274,259; 408,759; 518,570; 568,484 ppm
Vehicle / solvent:
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
other: hycanthone and cyclophosphamide
Details on test system and experimental conditions:
Exposure of cell cultures
For testing this volatile material in the preliminary concentration range-finding and mutagenesis assays, three sterile 15 ml round-bottom glass blood tubes, sealed with red rubber serum stoppers, were prepared for each concentration level: a tube for the culture tested without activation, a tube for the culture tested with activation, and a sham tube that contained medium only (no cells or S9) which was used to estimate post-exposure infrared (IR) analysis of the concentration of test material in the other two tubes.

To provide a maximum available volume for the test material, each culture contained approximately 2.5 x 10e+6 cells in 5 ml of F(10HP) for cultures tested without exogenous metabolic activation, or in 1.5 ml S9 mix plus 3.5 ml F(5HP) for cultures tested with metabolic activation. 'Therefore, at least 10 mI/tube was available for the volatile test material. After the cultures had been placed in the tubes at Genesys and the stoppers replaced, the tubes to be exposed to HFC-227ea were transported to ManTech/RTP where, using a syringe, a predetermined volume of air was withdrawn from each tube and an equal volume of the pure test material was added. (This was not necessary for the negative control [air] or the positive controls; the latter were added directly to the cell cultures before the tubes were sealed.) The three tubes per test material concentration were then returned to Genesys, and exposure was initiated by placing them in a roller drum, and rotating them (40 rpm) for 4 hours at 37°C.

After the exposure period, the cultures containing cells tested without and with S9 were transferred from the blood tubes to 15 ml plastic centrifuge tubes, for subsequent steps in the assays. The sham tubes were allowed to cool to room temperature prior to analysis of the concentrations of the test materials by IR.

Preliminary Range-Finding Assay
One range-finding assay of HFC-227ea was conducted, with and without metabolic activation, to determine the most effective concentrations of HFC-227ea to use in the mutagenesis assay. A series of 9 nominal (calculated theoretical) concentrations of HFC-227ea were used in the preliminary assay. The procedures followed were the same as for the mutagenesis assays (described below) except that the cells were not cloned.
For each culture, growth of the cells in suspension (SG) was calculated each day by dividing the cell concentration at the end of that time period by the initial cell concentration. Total suspension growth (TSG) was calculated by multiplying day one SG by day two SGi relative suspension growth (RSG) was calculated by dividing TSG of each culture by the average TSG of the medium controls. The results from these experiments were then evaluated to select concentrations for mutagenesis testing.

Mutagenesis assay
After exposure of the cultures as described above, the cells were centrifuged at low speed (-250 x g) for 5 minutes and the supernatant removed. The cells were rinsed at least twice by resuspension and centrifugation in F(10HP) medium and then resuspendedin 10 m1 of F(10HP) for growth during a two-day expression period.

During the expression period, the cell density was determined each day, and cells were diluted as necessary to maintain an optimum growth rate. On the second day of each assay, cultures were selected for cloning and 3 x 10e+6 cells were removed from each of the test material, negative and positive control cultures to be cloned. An aliquot of 1000 cells was then obtained from each of these cultures by serial dilution and was cloned to determine cloning efficiency. After adding 1 μg/ml of TFf, the remainder of the 3 x 10e+6 cells from each culture were cloned to determine mutant frequency. The soft-agar cloning medium was allowed to gel at room temperature for 15-20 minutes, then the dishes containing the cells were placed in a humidified CO2 incubator and incubated at 30 C for 14 days.

Colonies in the mutant count and cloning efficiency dishes were counted, and the colonies in the mutant count plates were sized using an Artek 982B semi-automatic colony counter with a high resolution video camera. For each culture, the absolute cloning efficiency (CE) was calculated by dividing the number of colonies in the cloning efficiency dishes by the number of cells cloned to measure cloning efficiency; relative cloning efficiency (RCE) was calculated by dividing the CE of each culture by the average CE of the negative (medium) controls, and relative total growth (RTG) was obtained by multiplying RSG by RCE. The mutation frequency (MF) for each culture was calculated by dividing the number of mutant colonies by the number of cells plated and multiplying by the reciprocal of the cloning efficiency. The average mutation frequency of the solvent control cultures was subtracted from that of each treated sample to express each result as an induced mutation frequency (IMF).
Evaluation criteria:
The results were evaluated according to the categories of responses utilized by the U.S. EP A Gene- Tox Workgroup (Mitchell et al., in preparation), as follows:
++ : Strong positive response with evidence of a dose-response and an induced muta¬tion frequency of at least 100 x 10e-6 at a relative total growth (RTG) ~ 20%.
+ : Positive response with evidence of a dose-response and an induced mutation frequency of at least 70 x 10e-6 at a RTG ≥ 10%
- : Negative response for which toxicity is evidenced by a RTG of 10-20%, and the positive control mutation frequency demonstrates that there are no inherent problems with the assay.
= : Negative response with no toxicity, and the positive control mutation frequency demonstrates that there are no inherent problems with the assay.
E : Equivocal response in which positive and negative results are obtained in repeated experiments, and no reason is found to give greater weight to the positive or the negative result.
# : Not-testable. The test material can not be tested to sufficiently high concentration to obtain a conclusive result in the MLA because of limited solubility, acidic pH shifts, the test material's dissolving plastic, etc.
The data were analysed using means and SDs.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
Positive controls validity:
Additional information on results:
HFC-227ea was tested over a nominal concentration range of 100,000 to 900,000 ppm, which resulted in IR determined concentrations of up to 538,535 and 568,484 ppm, respectively, in the preliminary concentration range-finding and the mutagenesis assay.

Because in the preliminary assay HFC-227ea was tested over a range of nine concentrations, up to the maximum concentration that could be obtained under the conditions of testing, with no evidence of toxicity, the mutagenesis assay was conducted over a similar range of concentrations, but with only five, more widely spaced, concentrations.

No concentration of HFC-227ea was mutagenic. Therefore, the results obtained in testing HFC-227ea in the L5178Y/TK+/- mouse lymphoma cell mutagenesis assay are evaluated as =, which is defined as a negative response with no toxicity in which the positive control mutation frequency demonstrates that there were no inherent problems with the assay.
Remarks on result:
other: all strains/cell types tested
Migrated from field 'Test system'.

Applicant's summary and conclusion