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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
Study period:
10-MAY-1994 to 29-JUL-1994
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This GLP-compliant study was conducted according to OECD guideline 471.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
according to
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
according to
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
GLP compliance:
Type of assay:
bacterial reverse mutation assay

Test material

Test material form:
other: solid
Details on test material:
- Name of test material (as cited in study report): Di-iodo perfluoro hexane
- Physical state: light violet solid


Target gene:
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
two batches (94/5 and 94/7) of liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone
Test concentrations with justification for top dose:
313, 625, 1250, 2500, and 5000 µg/plate in the absence and presence of S9 metabolism, in both experiments
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: trichlorotrifluoroethane (ALGOFRENE 113), labelled CFC 113; due to the toxicity of the solvent, in the pre-incubation assay the test substance solutions were added to the incubation mixture in a volume of 25 µL. For the plate incorporation method, 100 µL of the test substance solution were used in the preparation of each plate, therefore a maximum concentration of 5000 µg/plate was used in the toxicity test.
- Justification for choice of solvent/vehicle: the test substance was found to be soluble in trichlorotrifluoroethane at a concentration of 200 mg/mL.
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
(see in Table 1 below)
Positive control substance:
sodium azide
cumene hydroperoxide
other: 2-aminoanthracene
Sodium azide was prepared in distilled water, while the other positive control substances were prepared in dimethylsulfoxide (DMSO).
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and pre-incubation
- Type and identity of media:
* Nutrient broth: Oxoid Nutrient Broth No 2 was prepared at a concentration of 2.5% in distilled water and autoclaved prior to use. This was used for the preparation of liquid cultures of the tester strains.
* Nutrient agar: Oxoid Nutrient Broth No 2 (25 g) and Difco Bacto-agar (15 g) was added to 1 L of distilled water and autoclaved. The solution was then poured into 9 cml plastic Petri dishes and allowed to solidify and dry before use. These plates were used for the non-selective growth of the tester strains. Incubations on Nutrient Agar were for approximately 24 or 48 hrs.
* Minimal Agar: minimal medium agar was prepared as 1.5% Difco Bacto-agar in Vogel-Bonner Medium E, with 2% Glucose, and poured into 9 cm plastic Petri dishes.
* Top Agar: "Top Agar" (overlay agar) was prepared as 0.6% Difco Bacto-agar + 0.5% NaCl in distilled water. This solution was autoclaved, and stored. Prior to use 10 mL of a sterile solution of 0.5 mM Biotin + 0.5 mM Histidine was added to 100 mL of the top agar.

- Pre-incubation period: 30 min at 37°C
- Exposure duration: 72 hrs at 37°C
- Expression time (cells in growth medium): 72 hrs

SELECTION AGENT: Histidine-deficient agar

NUMBER OF REPLICATIONS: Three replicate plates were used at each test point, and two independent experiments were performed. If a positive result was obtained in any tester strain, a confirmatory experiment was performed under the same experimental conditions. If, however, negative results were obtained in the first experiment, the confirmatory experiment was performed using the pre-incubation method.

- Method: the test substance was assayed using the plate incorporation method, at a maximum dose-level of 5000 µg/plate and at 4 lower dose-levels spaced at approximately half-log intervals: 50, 158, 500, and 1580 µg/plate.
Evaluation criteria:
For the test substance to be considered mutagenic, 2-fold (or more) increases in mean revertant numbers must be observed at 2 consecutive dose-levels or at the highest practicable dose-level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose-levels. The effect must be reproduced in an independent experiment.
Regression analysis using the Least squares method, and Student's "t" statistical analysis.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
Untreated negative controls validity:
Positive controls validity:
Additional information on results:
- Effects of pH, effects of osmolality, evaporation from medium, water solubility, precipitation: no data available

No evidence of toxicity was observed at any dose-level tested with any tester strain, in the absence or presence of S9, at up to the maximum recommended test concentration, in both the plate incorporation and the pre-incubation methods.
Remarks on result:
other: all strains/cell types tested
Migrated from field 'Test system'. Remarks: plate incorporation method

Applicant's summary and conclusion

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The test substance did not induce reverse mutation in Salmonella typhimurium under the reported experimental conditions.
Executive summary:

The test substance was examined for mutagenic activity by assaying for reverse mutation to histidine prototrophy in the prokaryotic organism Salmonella typhimurium according to OECD guideline 471 and in compliance with good laboratory practices (GLP).


The five tester strains TA1535, TA1537, TA98, TA100, and TA102 were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. Test substance solutions were prepared using trichlorotrifluoroethane.


In the toxicity test, the test substance was assayed at a maximum dose-level of 5000 µg/plate and 4 lower dose-levels spaced at approximately half-log intervals.

No toxicity was observed in any tester strain even at the highest dose-level testes. The same maximum dose-level was selected for the principal assays.


Two experiments were performed, one using a plate incorporation method, the other using a pre-incubation method. The test substance was assayed at a maximum dose-level of 5000 µg/plate and 4 lower dose-levels, separated by 2-fold dilutions: 313, 625, 1250 and 2500 µg/plate. 

The test substance did not induce 2-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose-level, in any tester strain, in the absence or presence of S9 metabolism.


It was concluded that the test substance did not induce reverse mutation in Salmonella typhimurium under the reported experimental conditions.