1,1,2,2,3,3,4-heptafluorocyclopentane

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 1997-09-03 to 1997-10-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

430-710-1

EC Name:

-

Cas Number:

15290-77-4

Molecular formula:

C5H3F7

IUPAC Name:

1,1,2,2,3,3,4-heptafluorocyclopentane

Specific details on test material used for the study:

Batch No.: 9706-1A
Purity: 98.65 %

Method

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Human lymphocytes
- Suitability of cells: The cells have been used for this type of study for a number of years. They were easy to culture in vitro but do not divide unless stimulated to do so. This is achieved by adding phytohaemagglutinin (PHA) to the culture which results in a high mitotic yield.
- Normal cell cycle time (negative control): 16 hours

For lymphocytes:
Human blood was collected aseptically from healthy male donors, pooled and diluted with RPMI 1640 tissue culture medium (Imperial) containing 10% foetal calf serum (Gibco). Aliquots (0.4 mL blood 4.5 mL medium : 0.1 mL phytohaemagglutinin (Gibco)) of the cell suspension were placed in sterile universal containers and incubated at 37°C in a slanted position for approximately 48 hours. The cultures were gently shaken once daily to resuspend the cells.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : Harlan Olac Ltd
- method of preparation of S9 mix: S9 mix contained: S9 fraction (25% v/v), MgCl2 (8mM), KCl (33 mM), sodium orthophosphate buffer pH 7.4 (100 mM), glucóse-6-phosphate (5 mM), NADP (2 mM) and NADPH (2 mM). All the cofactors were filter-sterilised before use.
- concentration or volume of S9 mix and S9 in the final culture medium: 1.25 mL

Test concentrations with justification for top dose:

FIRST TEST
Set 1: With S9 mix - 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 μg/mL
Set 2: Without S9 mix - First: 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 μg/mL; Repeated: 250, 500, 750, 1000, 1200, 1400, 1600, 1800 and 2000 μg/mL.
SECOND TEST
Set 3: Without S9 mix - 100, 200, 400, 600, 800, 1000 and 1200 μg/mL.
Set 4: Without S9 mix - 50, 100, 200, 400, 600, 800, 1000 and 1200 μg/mL.
Set 5: With S9 mix -31.3, 62.5, 125, 250, 375, 500, 750 and 1000 μg/mL.

On dosing at 1 % v/v into aqueous tissue culture medium, giving a final concentration of 4090 μg/mL small oily globules were observed. Concentrations with high ionic strength and osmolality may cause chromosomal aberrations. Therefore, concentrations greater than 5000 μg/mL or 10 mM are not used in this test. In this case, the highest final concentration used for subsequent testing was 2000 μg/mL (10 mM) as this is the concentration recommended for use according to Japanese guidelines.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulphoxide

- Justification for choice of solvent/vehicle: The test item was found to be soluble in dimethyl sulphoxide at 409 mg/mL..

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- FIRST TEST:
For set 1, aliquots of S9 mix (1.25 mL) were added to one set of duplicate cultures followed by 62.5 μL aliquots of test item.
For set 2, 50 μL aliquots of the various dilutions of test item, were added to one set of duplicate cultures.
- SECOND TEST:
Three sets of cultures were initiated and maintained as previously described.
Three hours after dosing, the cultures containing S9 mix were centrifuged and the cell pellets resuspended in fresh medium. They were then incubated for a further 21 hours. Cultures treated in the absence of S9 mix were incubated for 24 and 48 hours.


TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: FIRST TEST: 3 hours treatment and 21 hours recovery; SECOND TEST: continuous treatment of 24 and 48 hours
- Harvest time after the end of treatment: FIRST TEST: 21 hours recovery; SECOND TEST: At the end of treatment


FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Harvesting and fixation: Two hours before the end of the 24 hour incubation period, mitotic activity was arrested by the addition of Colcemid (Sigma) to each culture at a final concentration of 0.1 μg/mL. After two hours incubation, each cell suspension was transferred to a conical centrifuge tube and centrifuged for 5 minutes at 500 g. The cell pellets were treated with a hypotonic solution (0.075M KCI prewarmed at 37°C). After a 10 minute period of hypotonic incubation at 37°C, the suspensions were centrifuged at 500 g for 5 minutes and the cell pellets fixed by addition of freshly prepared cold fixative (3 parts methanol : 1 part glacial acetic acid). The fixative was replaced several times. The pellets were allowed to fix for at least two hours.
- Methods of slide preparation: The pellets were resuspended using a whirlimixer, then centrifuged at 200 g for 10 minutes and finally resuspended in a small volume of fresh fixative. Two or three drops of the cell suspensions were dropped onto pre-cleaned microscope slides which were then allowed to air-dry. The slides were then stained in 10% Giemsa, prepared in buffered water (pH 6.8). After rinsing in buffered water the slides were left to air-dry and then mounted in DPX.
- Microscopic Examination:
The prepared slides were examined by light microscopy at a magnification of x160. The proportion of mitotic cells per 1000 cells in each culture was recorded except for positive control treated cultures. From these results the dose level causing a decrease in mitotic index of approximately 50% of the solvent control value or, if there was no decrease, the maximum concentration, 10 mM, was used as the highest dose level for the metaphase analysis. Intermediate and low dose levels usually approximated to 50% and 25% of the highest dose level.
The concentration of each positive control compound selected for analysis was the lowest concentration dosed unless a preliminary scan of metaphase figures indicated an insufficient level of aberrant cells
The slides were then coded. Metaphase figures were identified, using a magnification of x160 and examined at a magnification of x1000 using an oil immersion objective. Approximately 100 metaphase figures were examined, where possible, from each culture. Only cells with 44 - 46 chromosomes were analysed for structural aberrations. The vernier readings of all aberrant metaphase figures were recorded. Additionally, the proportion of polyploid cells in 100 metaphase figures, where possible, was recorded.

Statistics:

The number of aberrant metaphase figures, or polyploid cells, in each treatment group was compared with the solvent control value using Fisher's test (Fisher 1973).

Results and discussion

Test results

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

STUDY RESULTS
- Positive control data : Both positive control compounds caused large statistically significant increases (P<0.001) in the proportion of aberrant cells.

Chromosome aberration test (CA) in mammalian cells:
FIRST TEST
- Toxicity data:
In set 1, test item caused a reduction in the mitotic index to 53% of the solvent control value at 500 μg/mL. This dose level, together with lower dose levels of 250 and 125 μg/mL, were selected for metaphase analysis. To clarify the results from the metaphase analysis an additional dose level, 1000 μg/mL, was subsequently analysed. This gave a mitotic index of 27% of the solvent control value.
In set 2, test item caused a reduction in the mitotic index to 75% of the solvent control value at 1000 μg/mL. However, because of a sudden decrease in the mitotic index to 7% of the solvent control value at 2000 μg/mL, the test was repeated. In the repeat test, test item reduced the mitotic index to 39% of the solvent control value at 1400 μg/mL. The higher concentrations (1600, 1800 and 2000 μg/mL) were excessively toxic. Therefore, 1400μg/mL was selected as the highest concentration for metaphase analysis. The lower dose levels selected were 1000 μg/mL, which reduced the relative mitotic index to 42%, and 750 μg/mL, which was the highest non-toxic dose level. To clarify the results from the metaphase analysis an additional dose level, 1200 μg/mL, was subsequently analyzed. This gave a mitotic index of 25% of the solvent control value.
- Metaphase analysis:
In set 1, test item caused a statistically significant increase (P<0.001) in the number of aberrant cells at the highest doss level analysed, 1000 μg/mL. This increase, to 6.0% with and without gaps, lies outside the upper 95% limit of the historical control range of (4.5%).
In set 2, test item caused statistically significant increases in the number of aberrant cells at 1000, 1200 and 1400 μg/mL. The increases seen at 1000 and 1200 μg/mL, to 5.0% and 4.6%, respectively, lie outside the upper 95% limit of the historical control range (4.0%). However, at all dose levels the damage was predominantly seen in one culture.
No statistically significant increases in the proportion of polyploid cells were observed in either treatment set.

SECOND TEST
- Toxicity data:
In set 3, test item caused a reduction in the mitotic index to 45% of the solvent control value at 800 μg/mL. This dose level, together with 400 and 200 μg/mL, were selected for metaphase analysis. To clarify the results from the metaphase analysis, additional dose levels were analyzed. These were 600 and 1000 μg/mL, with mitotic indices of 52% and 28%, respectively, of the solvent control value.
In set 4, test item reduced the mitotic index to 45% of the solvent control value at 1000 μg/mL. This dose level was the highest dose level selected for the metaphase analysis. The lower dose levels selected were 800 and 400 μg/mL, which were moderately toxic and non-toxic, respectively. To clarify the results from the metaphase analysis an additional dose level was analysed. This was 1200 μg/mL, with a mitotic index of 30% of the solvent control value.
In set 5, test item, reduced the mitotic index to 49% of the solvent control value at the highest dose level, 1000 μg/mL. This dose level, together with 500 and 250 μg/mL, were selected for metaphase analysis. To clarify the results from the metaphase analysis an additional dose level was analysed. This was 750 μg/mL, with a mitotic index of 54% of the solvent control value.
- Metaphase analysi:
In set 3, test item caused a statistically significant increase (P<0.001) in the number of aberrant cells at the highest dose level, 1000 μg/mL, to 6.0% and 7.0%, without and with gaps, respectively. These increases lie outside the upper 95% limit of the historical control range (4.0% and 4.25%, respectively). However, this dose level was excessively toxic in one of the replicate cultures and only 20 cells could be scored.
In set 4, test item caused a statistically significant increase (P<0.01) in the number of aberrant cells at 800 μg/mL, but only when gap damage was included. However, this increase, to 4.0%, lies just inside the upper 95% limit of the historical control range (4.25%).
In set 5, test item caused a statistically significant increase (P<0.01) in the number of aberrant cells at the highest dose level, 1000 μg/mL, but only excluding gap damage. This increase, to 3.0%, lies inside the upper 95% limit of the historical control range of (4.5%).
No statistically significant increases in the proportion of polyploid cells were observed in either treatment set.


HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
See “Appendix 1 Historical control data” in attached background material

Applicant's summary and conclusion

Conclusions:

The test item has shown evidence of clastogenic but not polyploidy-inducing activity in this in vitro cytogenetic test system.

Executive summary:

 The test item was tested to determine whether it would cause chromosomal aberrations or polyploidy in human lymphocytes cultured in vitro according to OECD 473. Two sets of tests were carried out. The first test used a three hour culture treatment, with and without S9 mix, with a harvest at 24 hours (sets 1 and 2, respectively). The second test used 24 and 48 hour continuous treatments without S9 mix, and the test with S9 mix was repeated (set 3, 4 and 5, respectively). Cultures were treated with a range of dose levels, the solvent control and positive control compounds.

Cultures in all treatment sets were analysed for structural chromosomal aberrations and for the incidence of polyploidy.

The test item caused statistically significant increases in the proportion of aberrant cells in all five sets. Some of these increases lay outside the upper 95% limit ofthe historical control range. No statistically significant increases in the proportion of polyploid cells were observed in either treatment set.

It is concluded that the test item has shown evidence of clastogenic but not polyploidy-inducing activity in this in vitro cytogenetic test system.