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Genetic toxicity: in vitro

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in vitro gene mutation study in bacteria
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: High because a scientifically defensible and guidelined method was used. Critical study for SIDS endpoint. Only study summary reviewed from secondary source.

Data source

Reference Type:
study report

Materials and methods

Test guideline
according to guideline
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
The mutagenicity assays were conducted using the Salmonella plate incorporation assay as described by Marion and Ames, 1983 with certain minor modifications to allow the testing of a volatile liquid.
GLP compliance:
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Constituent 2
Reference substance name:
Details on test material:
- Name of test material (as cited in study report): Ethane,2,2-dichloro-1,1,1-trifluoro, HCFC-123
-Purity: not reported


Species / strain
Species / strain / cell type:
other: Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Test concentrations with justification for top dose:
0, 0.01, 0.02, 0.05, 0.1, 0.25, 0.5 mL liquid/vessel
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
other: Benzyl chloride (TA100 +/- S9-mix); 2-aminoanthracene (TA 1535, TA 1537, TA 1538, TA 98, TA 100 +S9-mix); N-methyl-N'-nitro-N-nitrosoguadinine (TA 100, TA 1535 -S9-mix); acridine mutagen ICR191 (TA1537 - S9-mix); 4-nitro-O-phenyldiamine (TA 100, TA 1538 -
Details on test system and experimental conditions:
The sample of HCFC-123 was assayed twice, both in the presence and absence of a liver S-9 mix prepared from Arochlor 1254-induced Sprague Dawley rats. Three plates per dose were used in each experiment. In addition to the negative (zero exposure) controls, "absolute" negative controls were included in each test to confirm the spontaneous rates for the tester strains.

The S-9 mix contained S-9 fraction, sucrose-tris-EDTA buffer, and co-factor solution. The co-factor solution contained Na2HPO4, KCl, glucose-6-phosphate, NADP (Na salt), and MgCl2. In tests without metabolic activation, the S-9 fraction was replaced with sucrose-tris-EDTA buffer. Treatments with activation were conducted by adding 0.5 mL of the S-9 mix to 0.1 mL of an overnight culture in a sterile bijou bottle. Two mL top agar was added. The force of addition was sufficient to mix the contents. The resulting mixture was then poured rapidly onto the surface of a prepared plate and allowed to gel. Plates were then placed inverted on stainless steel racks inside glass reaction vessels. A sterile cotton swab was placed on a lid at the top of each rack before the lid of the vessel was sealed. Appropriate volumes of HCFC-123 were added to the swab through a port in the lid, and the port was then immediately sealed. Each jar was incubated at 37ºC in the dark for 3 days before the plates were removed for counting.

Revertant colonies were counted using an automated electronic colony counter.

For each experiment, the positive control substances (assay) tested included acridine mutagen, 2-aminoanthracene, daunomycin HCl, 4-nitro-o-phenylenediamine, and N-methyl-N'-nitro-N-nitrosoguanidine. In addition to the "assay" positive controls, a sample of benzyl chloride was also tested as a positive control for the dosing regime used for volatile liquids.

A positive response in an individual experiment was achieved when 1 or both of the following criteria was met: a) a statistically significant dose-related increase in the number of revertant colonies was obtained; and b) a 2-fold or greater increase in the mean number of revertant colonies (over that observed for the concurrent negative control plates) which was statistically significant, was observed at least 1 dose level. A negative result was achieved when: a) there was no statistically significant dose-related increase in the mean number of revertant colonies per plate observed for the test compound; and b) in the absence of any such dose response, no increase in colony numbers was observed (at any test dose) which exceeded 2x the concurrent negative control.

Results and discussion

Test results
Species / strain:
other: Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
observed at 0.5 ml HCFC 123/plate
Vehicle controls validity:
not applicable
Untreated negative controls validity:
Positive controls validity:
Additional information on results:
In 2 replicate experiments, no significant increases in revertant colony numbers were observed in any strain in either the presence or absence of an auxiliary metabolizing system (S-9). In each case, toxic effects were observed at the highest exposure level tested (thinning of background level and/or reductions in colony numbers), indicating that this dose was an effective maximum.

In each experiment, the positive controls responded as expected, indicating that the assay was performing satisfactorily. Similarly, the protocol positive control, benzyl chloride, responded as expected, showing that the dosing regime used was appropriate for assaying a volatile liquid.

Applicant's summary and conclusion

HCFC-123, therefore, gave an unequivocal negative, i.e., non-mutagenic response in all 5 strains of S. typhimurium both in the presence and absenceof S-9, when tested to an exposure level which produced toxicity in each case.