1,1,2,2,3,3,4,4-octafluoro-1,4-diiodobutane

Genetic toxicity: in vitro

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

Study period:

10-MAY-1994 to 12-SEP-1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,1,2,2,3,3,4,4-octafluoro-1,4-diiodobutane

EC Number:

206-788-4

EC Name:

1,1,2,2,3,3,4,4-octafluoro-1,4-diiodobutane

Cas Number:

375-50-8

Molecular formula:

C4F8I2

IUPAC Name:

1,1,2,2,3,3,4,4-octafluoro-1,4-diiodobutane

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Di-iodo perfluoro butane
- Physical state: dark red liquid

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

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:

- Plate incorporation method: 313, 625, 1250, 2500, and 5000 µg/plate in the absence and presence of S9 metabolism
- Pre-incubation method:
* Experiment 1: 313, 625, 1250, 2500, and 5000 µg/plate in the absence and presence of S9 metabolism
* Experiment 2: 78.1, 156, 313, 625, and 1250 µg/plate in the absence of S9 metabolism

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.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

(see in Table 1 below)

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

cumene hydroperoxide

other: 2-aminoanthracene

Remarks:

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.

DURATION
- 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

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.

DETERMINATION OF CYTOTOXICITY
- 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.

Statistics:

Regression analysis using the Least squares method, and Student's "t" statistical analysis.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

plate incorporation method

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

plate incorporation method

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

plate incorporation method

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

plate incorporation method

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Remarks:

plate incorporation method

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 1535

Remarks:

pre-incubation method

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9 at >= 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 7

Key result

Species / strain:

S. typhimurium TA 1537

Remarks:

pre-incubation method

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9, at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 8

Key result

Species / strain:

S. typhimurium TA 98

Remarks:

pre-incubation method

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9, at conc >= 1250 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 9

Key result

Species / strain:

S. typhimurium TA 100

Remarks:

pre-incubation method

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9, at >= 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 10

Key result

Species / strain:

S. typhimurium TA 102

Remarks:

pre-incubation method

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9, at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH, effects of osmolality, evaporation from medium, water solubility, precipitation: no data available

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Plate incorporation method: no evidence of toxicity was observed at any dose-level tested with any tester strain, in the absence or presence of S9.
- Pre-incubation method:
* Experiment 1: moderate toxicity was observed at the highest dose-level in the presence of S9 metabolism. In the absence of S9 metabolism, severe toxic effects were observed in tester strain TA1535 at a dose-level of 625 µg/plate and in all tester strains at dose-levels in excess of 625 µg/plate. Indications of toxicity included reduction in revertant numbers, induction of microcolonies and thinning of the background lawn.
* Experiment 2: moderate toxicity was observed at the highest dose-level in all tester strains.

Applicant's summary and conclusion

Conclusions:

Interpretation of results :
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. Due to the toxicity observed at the highest dose, an additional experiment was performed with the pre-incubation method, in the absence of S9 metabolism with a maximum dose-level of 1250 µg/plate and 4 lower dose-levels, separated by 2-fold dilutions. 

The test substance did not induce 2-fold increases in the number of revertant colonies in the plate incorporation of 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.