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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
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other: GLP compliant, near guideline study, available as unpublished report, fully adequate for assessment

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995
Reference Type:
publication
Title:
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.
Author:
Dodd DE, Ledbetter AD, Mitchell AD
Year:
1997
Bibliographic source:
Inhalation Toxicology 9(2):111-31

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
The test material, 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 respinsible for handling, storage, and dilution of the test material. The HFC-227ea was stored at ManTech/RTP at room temperature (approximately 72°F). ManTechiDayton 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.

Method

Target gene:
his
Species / strain
Species / strain / cell type:
other: TA1535, TA1537, TA1538, TA98, TA100
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 homogenate
Test concentrations with justification for top dose:
Preliminary assay, target concentration (IR determined), ppm: 100000 (104730); 200000 (184414); 500000 (531970); 700000 (768139); 900000 (835930)
Mutagenesis assay, target concentration (IR determined), ppm: 450000 (438707); 600000 (598432); 750000 (772997); 900000 (934548)
Vehicle / solvent:
None
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide, 9-aminoacridine, 4-nitro-o-phenylenediamine, 2-anthramine
Details on test system and experimental conditions:
To a sterile glass test tube was added: 0.1 ml of indicator organisms (about 10e+8 bacteria), 0.5 ml of the metabolic activation mixture or buffer, and 2.00 ml of molten 0.615% agar. In addition, 0.1 ml of the appropriate positive control was added to each positive control plate. This top agar mixture was then vortexed gently and poured onto a prelabeled plate containing about 25 ml of minimal glucose agar plus biotin.

Afther the top agar had set, the test material or negative control plates for all five strains that wer to be exposed to one concentratio of the test material (or to air, the negative control) were placed on a shelf on the bottom section of plastic exposure chamber (Modular Incubator Chamber, Billups-Rothernberg, Del Mar, CA) with an internal volume of approximately 1 liter. The chambers, which consisted of an upper and lower section connected by a gasket and a large stainless steel adjustable squeeze clamp, were then closed by connecting and sealing the upper and lower sections.

The exposure chambers were transported from Genesys to ManTech/RTP where they were labeled as to the desired concentrations; then gas was introduced into an inlet port in the bottom section of each chamber, circulated throughout the chamber, and drawn out of an exhaust port, which was also located in the bottom section. Chamber atmosphere samples for infrared (IR) analysis were collected from the exhaust port.

The chambers containing the plates were then returned to Genesys where they were incubated at 37°C for approximately 48 hours. Exposure chambers were not required for plates of bacteria treated with positive controls, which were also incubated at 37°C for approximately 48 hours. Following the exposure period, the plates were removed from the exposure chambers, and the number of histidine independent revertant colonies per each plate was counted using an Artek 982B colony counter, or by hand if the colonies could not be counted accurately with the Artek.

Exposure Method
The test material and dilution air were metered through calibrated flowmeters into the . exposure chambers, and chamber atmosphere samples were coUected, via a gas tight syringe, and injected into the IR instrument for analysis. The test material and the dilution air were adjusted until the desired chamber concentration was obtained. The chamber exhaust was first disconnected, and then the gases were shut off. This was done to prevent diluting the chamber atmosphere. The- inlet and exhaust ports were then sealed with screw clamps. During the mutagenesis assay, two chambers 'per exposure level were required to hold all the petri plates. To ensure that both chambers received the same concentration, ,the gas was introduced to the inlet port of one chamber which was exhau~ted intc;> the inlet of the second chamber. The exhaust from the second chamber was then analyzed. by IR.

Preliminary Range-Finding Assay
A preliminary assay of HFC-227ea was conducted with strain TA100 in the presence and absence of metabolic activation to determine suitable nontoxic dose ranges for the mutagenicity assays. The results of each testing condition, without and with activation, were evaluated separately. Toxic concentrations were defined as those that produced a decrease in the number of colonies, or a clearing in the density of the background lawn, or both.

Mutagenesis Assay
Once a dose range had been established, the substance was assayed utilizing the five tester strains (TA1535, TA1537, TA1538, TA98, and TA100) over 5 dilutions of the test material such that the highest concentration would be one expected to result in toxicity or would be the highest concentration that could be tested. The mutagenesis assay were conducted using three plates per dose level, in the presence and absence of S9 metabolic activation. The procedures used were the same as in the preliminary assay except that, because 30 plates were exposed to each test material concentration (5 strains x 3 plates/concentration x 2 [without and with activation]), two chambers, connected, in series, were used to hold the 30 plates per concentration.
Evaluation criteria:
The data generated were considered acceptable if the controls were within the laboratory's historical ranges and if a sufficient number of nontoxic concentrations were tested to dermine if a test material were capable of inducing a dose-related mutagenic response.
A test material is considered mutagenic for a condition and strain if a dose-related increase in the number of revertants is observed over three concentrations and the highest increase in strains TA1535, TA1537 and TA1538 is equal to three times the solvent control value or the highest increase in strains TA98 and TA100 is equal to two times the solvent control value. A test material is considered negative if the critria for a positive response are not met and the positive control values are within the historical range for the laboratory.
Statistics:
The data were analysed using means and SDs.

Results and discussion

Test results
Species / strain:
other: TA1535, TA1537, TA1538, TA98, TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at the highest concentration tested
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The analysis of the exposure chamber atmospheres indicated that the desired concentrations were achieved within 10%.

In the preliminary assay, the substance was tested in TA100 over 5 concentrations ranging from 104730 to 835390 ppm, and no toxicity or mutagenic response was observed. In the mutagenesis assay, the substance was tested in the five tester strains over 4 concentrations ranging from 438707 to 934548 ppm, the highest concentration that could be obtained under the conditions of testing. In this assay, toxicity, as indicated by a decrease in the number of colonies at the highest concentrations tested, was obtained for all five strains in the absence and presence of activation. In addition, numerous tiny colonies were present at the highest concentrations tested under both testing conditions for all strains except TA1537 in the presence of activation. The tiny colonies were further evidence of toxicity, as they arise from the background lawn when the number of bacteria is reduced such that sufficient histidine is available for addituiinal growth of the nonmutated bacteria that form the backgrund lawn.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion