Tetradecafluorohexane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 28-November 21, 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

AF0150 Lot number: ZZ15031 (400 mg/vial)

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Preliminary Test (Dose Range-Finding): TA98, TA100 and Wp2uvrA were incubated with AF0150 at the dose range from 0.4 to 20 mg/plate (total 8 doses) in the absence and presence of S9 activation. No cytotoxicity (decreased number of revertant colonies, or thinning/disppearance of the bacterial background lawn) was observed at all dose levels. The maximal dose, 20 mg/plate, was selected for mutagenicity assay.

Vehicle / solvent:

Water

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

other: 2-aminoanthracene; ICR-191

Details on test system and experimental conditions:

Preliminary Test (Dose Range-Finding): TA98, TA100 and Wp2uvrA were incubated with AF0150 at the dose range from 0.4 to 20 mg/plate (total 8 doses) in the absence and presence of S9 activation. No cytotoxicity (decreased number of revertant colonies, or thinning/disppearance of the bacterial background lawn) was observed at all dose levels. The maximal dose, 20 mg/plate, was selected for mutagenicity assay.
AF0150 and Positive Control Preparations: AF0150 (400 mg fill) was reconstituted with 10 ml sterile deionized water to a final concentration of 40 mg/ml. For low dose groups (0.4-2 mg/plate), 4 mg/ml AF0150 stock solution was made by dilution of 40 mg/ml. All AF0150 stock solutions were used within 30 minutes alter reconstitution. Positive control chemicals included 2- aminoanthracene, 2-nitrofluorene, sodium azide, ICR-191, 4-nitroquinoline-N-oxide [Preparation methods were not indicated].
Mutagenicity Assay: The five bacterial strains were pre-incubated in culture tubes (tightly capped) with AF0150 at the doses of 1-20 mg per plate in the absence and presence of rat liver S9 for 2 hours at 37°C. The overlay (top) agar (selection agar medium) containing histidine/biotin or tryptophan was then added to the tubes. The agar and preincubation mixtures were overlaid onto petri dishes containing minimal bottom agar, in triplicate for all dose levels and controls (negative and positive). Following incubation of the petri dishes for 48 hours at 37°C, the number of revenant colonies on each petri dish was counted manually (for AF0150 and negative control plates) or automatically (with automated colony counter for positive control plates).

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

AF0150 at the doses of 1-20 mg/plate had no significant cytotoxicity and did not increase revenants in all 5 bacterial strains covered both C-C and A-T point mutations in the absence and presence of a rat liver S9. Positive control compounds (2-aminoanthracene, 2-nitrofluorene, sodium azide, ICR-191, 4-nitroquinoline-N-oxide) significantly increased reverse mutation in all bacterial strains.

In addition, bubbles were observed in the top agar at doses of 4 mg AF0150 per plate and above in both the presence and absence of S9 mix in dose range finding study.

Table 1.Mitotic Index (MI, %) and Chromosomal Aberration in PCE (CA-PCE, %)
of AF0150-Treated Human LvmnhocvteIn vitro

Treatment	Initial Assay				Confirmatory Assay
	-S9		+S9		-S9		+S9
	MI	CA-PCE	MI	CA-PCE	MI	CA-PCE	MI	CA-PCE
2 mg/mi *	5.4	0.5	4.1	0	2.6	0.5	5.4	0.5
3 mg/m1	4.5	1.0	3.9	0	2.8	0	2.9	1.0
4 mg/mi	6.5	0	4.8	0.5	2.6	0.5	4.2	1.0
5 mg/m1	3.4	0	3.3	1.0	2.9	0	4.1	0
CPt			1.6	32			3.6	12
MMCt	0.8	62			1.8	34
H2O	6.4	1.5	4.5	0	3.5	0	4.2	0

 

* AF0150 concentration in cultured human lymphocytes.

tCP (cyclophosphamide, with S9 activation) and MMC (mitomycin C, without S9 activation) were positive controls.

Conclusions:

AF0150, which active ingredient is tetradecafluorohexane, is not mutagenic.

Executive summary:

AF150 at the dose of 1-20 mg/plate did not result in significant increase in bacterial reverse mutation. However, there was also no evidence of significant cytotoxicity at the highest dose, 20 mg/plate in terms of changes on revertant numbers or bacterial background lawn. This suggests that the dose selection may not be sufficient.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 7-September 12, 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

AF0150 Lot Number: ZZ16054 (400 mg/vial)

Species / strain / cell type:

lymphocytes: Human

Details on mammalian cell type (if applicable):

Blood sample (vein) was taken from healthy adult male into a heparinized tube. Whole blood was cultured in RPMI 1640 medium containing 15% fetal bovine serum (FBS), antibiotics, L-glutamine and 1% PHA-M (phytohemagglutinin M) for 2 days before treatment with AF0150 or control compounds. For the initial assays, the culture were treated for 3 hours in the presence or absence metabolic activation (S9), washed and then incubated in fresh culture medium until harvested at 22 hours alter inititation of treatment. For confirmatory assay without S9, the culture was treated for 19.3 hours, washed and then incubated in fresh culture medium until harvested at 22 hours of initiation of treatment. For confirmatory assays with S9, the culture was treated for 3 hours in the medium without FBS, washed and then incubated in fresh complete culture medium. In all the assays, Colcemid (0.1 uWm1) was added to the cell culture at 2 hours prior to cell harvesting for a metaphase-arresting.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

AF0150 and Positive Control Preparations: AF0150 (400 mg fill) was reconstituted with 10 ml SWFI to a final concentration of 40 mg/ml. For low dose groups (0.4-2 mg/plate), 4 mg/m1 AF0150 stock solution was made by dilution of 40 mg/ml. All AF0150 stock solutions were used within 30 minutes alter reconstitution. Mitomycin C (MMC) and cyclophosphamide (CP) were dissolved in water and used as positive controls. AF0150 vehicle (water) was used as negative control.
Cell Culture and Treatment: Blood sample (vein) was taken from healthy adult male into a heparinized tube. Whole blood was cultured in RPMI 1640 medium containing 15% fetal bovine serum (FBS), antibiotics, L-glutamine and 1% PHA-M (phytohemagglutinin M) for 2 days before treatment with AF0150 or control compounds. For the initial assays, the culture were treated for 3 hours in the presence or absence metabolic activation (S9), washed and then incubated in fresh culture medium until harvested at 22 hours alter inititation of treatment. For confirmatory assay without S9, the culture was treated for 19.3 hours, washed and then incubated in fresh culture medium until harvested at 22 hours of initiation of treatment. For confirmatory assays with S9, the culture was treated for 3 hours in the medium without FBS, washed and then incubated in fresh complete culture medium. In all the assays, Colcemid (0.1 uWm1) was added to the cell culture at 2 hours prior to cell harvesting for a metaphase-arresting.
Cell Harvest, Staining and Analysis: The cells were centrifuged, treated with hypotonic KC1 (75 mM) and fixed with fixative solution (methanol:acetic acid), followed by slide preparation and Giemsa staining. Cells with 46 centromeres were analyzed with microscopy for different types of chromosomal aberrations. Percentages of mitotic cells (mitotic index) and polyploid and endoreduplication were also measured.

Evaluation criteria:

Cells with 46 centromeres were analyzed with microscopy for different types of chromosomal aberrations. Percentages of mitotic cells (mitotic index) and polyploid and endoreduplication were also measured.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

In both initial and confirmatory assays, AF0150 at the doses of 2-5 mg/ml decreased in some degree the mitotic index as compared to negative control group (Table 1). At the 5 mg/ml group in the initial assay without S9 activation the mitotic index decreased by 47%, suggesting the dose selection was appropriate.
Human blood lymphocytes treated with AF0150 at the doses of 2, 3, 4, 5 mg/m1 had no significant increases in chromosomal aberrations, polypoidy, or endoreduplication in the two independent assays with or without metabolic activation. Positive control compounds, mitomycin (without metabolic activation) and cyclophosphamide (metabolic activation) induced statistically significant increases in cells with chromosomal aberrations.

Table 1.Mitotic Index (MI, %) and Chromosomal Aberration in PCE (CA-PCE, %)
of AF0150-Treated Human LvmnhocvteIn vitro

Treatment	Initial Assay				Confirmatory Assay
	-S9		+S9		-S9		+S9
	MI	CA-PCE	MI	CA-PCE	MI	CA-PCE	MI	CA-PCE
2 mg/mi *	5.4	0.5	4.1	0	2.6	0.5	5.4	0.5
3 mg/m1	4.5	1.0	3.9	0	2.8	0	2.9	1.0
4 mg/mi	6.5	0	4.8	0.5	2.6	0.5	4.2	1.0
5 mg/m1	3.4	0	3.3	1.0	2.9	0	4.1	0
CPt			1.6	32			3.6	12
MMCt	0.8	62			1.8	34
H2O	6.4	1.5	4.5	0	3.5	0	4.2	0

 

* AF0150 concentration in cultured human lymphocytes.

tCP (cyclophosphamide, with S9 activation) and MMC (mitomycin C, without S9 activation) were positive controls.

Conclusions:

AF0150, which active ingredient is tetradecafluorohexane, is not mutagenic

Executive summary:

In both initial and confirmatory assays, AF0150 at the doses of 2-5 mg/ml decreased in some degree the mitotic index as compared to negative control group (Table 1). At the 5 mg/ml group in the initial assay without S9 activation the mitotic index decreased by 47%, suggesting the dose selection was appropriate.

Human blood lymphocytes treated with AF0150 at the doses of 2, 3, 4, 5 mg/m1 had no significant increases in chromosomal aberrations, polypoidy, or endoreduplication in the two independent assays with or without metabolic activation. Positive control compounds, mitomycin (without metabolic activation) and cyclophosphamide (metabolic activation) induced statistically significant increases in cells with chromosomal aberrations.

In conclusion, AF0150 treatmentin vitrodid not induce chromosomal aberrations in cultured human whole blood lymphocytes.

Reference 3

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 4-September 17, 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

AF0150 lot number: ZZ16054 (400 mg/vial)

Target gene:

Thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

other: heterogenous at the TK locus

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

The final concentrations were 1, 2, 3, 4, 5 mg/ml for AF0150; 5 and 10 ng/ml for MMS; and 2 and 4 ug/ml for MCA in the presence or absence of metabolic activation (S9).

Vehicle / solvent:

water

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

methylmethanesulfonate

Details on test system and experimental conditions:

Cell Culture: The mouse lymphoma L5178Y cell line, heterozygous at the TK locus, was used for this assay. The cells were cultured in growth medium (RPMI 1640 medium containing 10% heat-inactivated horse serum, Pluronic F68, L-glutamine, sodium pyruvate, and antibiotics) in a shaking incubator at 37°C. Treatment medium was Fisher's medium containing above supplements with 5% horse serum. Cloning medium was Fisher's medium containing 20% horse serum and 0.24% agar but no Pluronic F68. Selection medium was the cloning medium supplied with 3 ug/ml TFT.

AF0150 and Positive Control Preparations: AF0150 (400 mg fill) was reconstituted with 10 ml SWFI to a final concentration of 40 mg/m1 and used within 30 minutes alter reconstitution. Methyl methanesulfonate (MMS, without S9 activation) and methylcholanthrene (MCA, with S9 activation) were used as positive controls. AF0150 vehicle, SWFI, was used as negative control.

Treatment: The tested compounds were added to 6x106 cells in 10 ml treatment medium per tube. The final concentrations were 1, 2, 3, 4, 5 mg/ml for AF0150; 5 and 10 ng/ml for MMS; and 2 and 4 ug/ml for MCA in the presence or absence of metabolic activation (S9). After treatment for 4 hours, cells were washed and incubated in 20 ml of growth medium for a 2-day expression period.

Cloning and Mutant Selection: A total of 3x106 cells were then suspended in the cloning medium or selection medium and a certain number of cells were seeded in 100-mm dishes followed by incubation at 37°C for 10-14 days for cloning and mutant analysis. The mutant frequency was calculated as the ratio of the total number of mutant colonies found in each selection dish to the total number of cells seeded, adjusted by the absolute selection cloning efficiency (cultures from cells in cloning medium).

Evaluation criteria:

The mutant frequency was calculated as the ratio of the total number of mutant colonies found in each selection dish to the total number of cells seeded, adjusted by the absolute selection cloning efficiency (cultures from cells in cloning medium).

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Results/Discussion

AF0150 at 5 mg/ml in the assays without activation and 4 and 5 mg/ml in the assays with activation induced moderate cytotoxicity in terms of decreases in cell growth by more than 50%, suggesting the dose selection was appropriate. The average cloning efficiencies for the vehicle controls was 93.0-101.7% without activation and 91.4-110.0% with S9 metabolic activation. The positive controls, MMS (nonactivation) and MCA (activation), induced significant increases in mutant frequency by more than 5-fold as compared to vehicle control groups.

Incubation of cells with AF0150 at concentrations from 1-5 mg/ml did not increase mutant frequency in the presence and absence of metabolic activation from two separate assays. However, as seen in Tables 1 and 2, the relative growth rate in all AF0150 dose groups tended to decrease as compared to vehicle control groups, corresponding to lower mutant frequency than in vehicle control group. This suggests that the AF0150-induced inhibition of cell growth could result in underestimating mutation potential, although positive controls showed more significant inhibition of cell growth.

Conclusions:

AF0150, which active ingredient is tetradecafluorohexane, is not mutagenic

Executive summary:

AF0150 at 5 mg/ml in the assays without activation and 4 and 5 mg/ml in the assays with activation induced moderate cytotoxicity in terms of decreases in cell growth by more than 50%, suggesting the dose selection was appropriate. The average cloning efficiencies for the vehicle controls was 93.0-101.7% without activation and 91.4-110.0% with S9 metabolic activation. The positive controls, MMS (nonactivation) and MCA (activation), induced significant increases in mutant frequency by more than 5-fold as compared to vehicle control groups.

Incubation of cells with AF0150 at concentrations from 1-5 mg/ml did not increase mutant frequency in the presence and absence of metabolic activation from two separate assays. However, as seen in Tables 1 and 2, the relative growth rate in all AF0150 dose groups tended to decrease as compared to vehicle control groups, corresponding to lower mutant frequency than in vehicle control group. This suggests that the AF0150-induced inhibition of cell growth could result in underestimating mutation potential, although positive controls showed more significant inhibition of cell growth.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 5-20, 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
- Expiration date of the lot/batch:
- Purity test date:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity:
- Specific activity:
- Locations of the label:
- Expiration date of radiochemical substance:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

young adult male and female mice, Crl:CD-1 (ICR) BR strain. Standard procedures were followed for housing, handling, feeding and care of the animals. Alter acclimation for at least 7 days, animals were randomly assigned into 5 groups (6 animals/sex/group) and received designed treatments.

Sex:

male/female

Details on test animals or test system and environmental conditions:

The animals were 8 weeks old with body weights of 29.2-34.1 (males) and 22.2-28.6 (females) at initiation of treatment.

Route of administration:

intravenous

Vehicle:

Water

Details on exposure:

AF0150 and Positive Control Preparations: AF0150 (400 mg fill) was reconstituted with 10 ml SWFI to a final concentration of 40 mg/ml and was used within 30 minutes alter reconstitution.

Animals received IV injection of a single dose of AF0150 or saline (negative control), and oral administration of 80 mg/kg cyclophosphamide (positive control).
Table 1. Micronucleus Studv Desisn in Mice


Treatment Dosing Vol. Route
(mUkg) of Dosing Harvest Time, 24 hr Harvest Time, 48 hr

Male Female Male Female
200 mg/kg AF0150 5.0 IV 6 6 - -
400 mg/kg AF0150 10 IV 6 6 6 6
800 mg/kg AF0150 20 IV 6 6 6 6
Saline 20 IV 6 6 6 - -
Cyclophosphamide 10 PO 6 6 - -

Duration of treatment / exposure:

Animals received IV injection of a single dose of AF0150 or saline (negative control), and oral administration of 80 mg/kg cyclophosphamide (positive control).

Frequency of treatment:

unique administration

Post exposure period:

24-48h

Dose / conc.:

0 mg/kg bw (total dose)

Remarks:

Control

Dose / conc.:

200 mg/kg bw (total dose)

Dose / conc.:

400 mg/kg bw (total dose)

Dose / conc.:

800 mg/kg bw (total dose)

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

80 mg/kg oral cyclophosphamide

Tissues and cell types examined:

Toxic signs were observed at least daily for all animals throughout study

Details of tissue and slide preparation:

Bone marrow was collected from the hind limb and transferred to centrifuge tubes containing 3-5 ml bovine serum. The cells were centrifuged, prepared for slides, fixed in methanol and stained with May-Grunwald and Giemsa.

Evaluation criteria:

The slides were scored for polychromatic erythrocyte (PCE), normochromatic erythrocyte (NCE) and micronucleated PCE. The micronucleus frequency, expressed as percentage of micronuleated cells, was determined by analyzing the number of micronucleated PCEs from 2000 PCEs per animal.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Treatments with AF0150, saline or CP had no significant toxic signs in all animals. As seen in Table 2, AF0150 at all doses was no cytotoxic to bone marrow in ternis of PCE:NCE ratio. There was no significant difference in micronucleated PCEs between AF0150- and saline-treated mice. The positive control, cyclophosphamide, induced statistically significant increases in micronucleated PCEs in bath sexes as compared to saline control group.

Table 2.Micronucleus Assay Summary

 

TREATMENT	DOSE	HARVEST TIME (HA)	% MICRONUCLEATED PCEs                                                                            RATIO PCE:NCE MEAN OF 2000 PER ANIMAL * S.E.                                               MEAN s S.E. MALES               FEMALES           TOTAL             MALES                FEMALES
controls
VEHICLE	0.0% saline	24 hr	0 08 4 0.03	0.04 s 0.03	0.06 ± 0.02	0.59 s 0.04	0.79 ± 0.05
		48 tu	0.08 ± 0.03	0.04 s 0.03	0.06 .± 0.02	0.46 s 0.02	0.52 ± 0.06
POSTIVE	CP 80.0 mg/kg	24 hr	3.52 ± 0 19»	2.28 * 0.29*	2.90 ± 0.26•	0.71 4 0.07	0.68 s 0.09
TEST ARTICLE	200 mg/ka	24 hr	0-05 ± 0.03	0.07 4 0.01	0.06 s 0.02	0.74 * 0.09	0.76 * 0.05
	400 mg/kg	24 hr	0.15 ± 0 04	0.08 4. 0.04	0,12 ± 0.03	0,69 * 0.04	0.52 4 0.04
	800meg	24 hr	0.05 ± 0.02	0.10 4 0.04	0.08 s 0.02	0.64 * 0.06	0.72 ± 0.11
		48 hr	0.03 * 0.01	0.04 ± 0.03	0.04 s 0.02	0 50 * 0.08	0.43 ± 0.02

 

· Significantly greater than the corresponding vehicle control, p<0.01.

CP = Cvclophosphamide

PCE =Polychromatic erythrocyte NCE = Normochromatic erythrocyte

Conclusions:

AF0150, which active ingredient is tetradecafluorohexane, is not toxic to bone marrow in mice.

Executive summary:

Treatments with AF0150, saline or CP had no significant toxic signs in all animals. As seen in Table 2, AF0150 at all doses was no cytotoxic to bone marrow in ternis of PCE:NCE ratio. There was no significant difference in micronucleated PCEs between AF0150- and saline-treated mice. The positive control, cyclophosphamide, induced statistically significant increases in micronucleated PCEs in bath sexes as compared to saline control group.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

No study was available assessing toxicity to reproduction of tetradecafluorohexane by oral route.

However, extensive data was available for the drug AF0150, which new drug application (NDA) was successfully assessed by US-FDA. AF0150 is tetadecafluorohexane in phospholipids, administered by intraveinous route. As discussed in toxicokinetics statement, formulation and route of administration of AF0150 is assumed to dramatically increase bioavalability of tetradecafluorohexane, without modifying further ADME since tetradecafluorohexane is not metabolized and rapidly eliminated in expired air. In cell models, tetradecafluorohexane is expected to be released rapidly, considering the lack of stabilisation effect of blood pressure.

AF1050 data are therefore considered relevant to assess genetic toxicity of tetradecafluorohexane.

The standard genotoxicity battery was used to test the potential genotoxicity of AF0150, including a bacterial reverse mutation test, a mammalian cell DNA damage (chromosomal aberration assay with human lymphocytes and forward mutation with mouse lymphoma tk cells), and anin vivomouse micronucleus assay. The AF0150 dose selection and treatments (in sealed culture tubes and flasks) were appropriate based on cytotoxicity or maximal solubility of the product. Appropriate negative and positive controls were included in allin vitroandin vivostudies, and the expected results were produced from the positive control compounds. All studies were conducted in the presence and absence of metabolic activation (using induced rat liver S9).

AF0150 at doses up to 20 mg/plate had no significant increase in bacteriai reverse mutation in all bacteriai strains detecting C-G and A-T point mutation. At doses up to 5 mg/ml, AF150 did not induce significant chromosomal aberrations and forward mutation as compared to vehicle control group. Single IV injection of AF0150 at the dose up to 800 mg/kg did not increase micronucleated polychromatic erythrocytes in mouse bone marrow.

AF0150, and therefore tetradecafluorohexane, are concluded to be negative in the following genotoxicity assays:

1.     In vitrobacterial reverse mutation assay

2.     In vitrochromosomal aberration assay in culture human lymphocytes

3.     In vitromouse lymphoma tk forward mutation assay

4.     In vivomicronucleus assay in mice

Overall, tetradecafluorohexane is considered having no genetic toxicity properties.