[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

1998

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

458-880-0

EC Name:

-

Cas Number:

85209-93-4

Molecular formula:

Hill formula: C29 H42 O4 P Li

IUPAC Name:

2,4,8,10-tetra(tert-butyl)-6-hydroxy-12H-dibenzo- [d,g][1,3,2]dioxaphosphocin 6-oxide, lithium salt

Test material form:

solid: particulate/powder

Method

Species / strain

Species / strain / cell type:

lymphocytes: human, cultured in vitro in whole blood culture

Details on mammalian cell type (if applicable):

CELLS USED
- Source of lymphocytes: Human blood collected aseptically from two healthy, non-smoking male donors and pooled.
- Normal cell cycle time (negative control): cells do not divide unless stimulated with phytohaemagglutinin. In this laboratory the cell cycle time is approximately 13 - 14 hours.

For lymphocytes:
- Sex, age and number of blood donors: two male, non-smoking donors
- Whether whole blood or separated lymphocytes were used: whole blood was used
- Whether blood from different donors were pooled or not: pooled blood was used
- Mitogen used for lymphocytes: phytohaemagglutinin

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: RPMI 1640 supplemented with 10% foetal calf serum, 1 I.U./ml sodium heparin, 20 I.U./ml penicillin / 20 µg/ml streptomycin and 2.0 mM glutamine. Aliquots (0.4 ml blood : 4.5 ml medium : 0.1 ml phytohaemagglutinin) of cell suspension were placed in sterile universal containers and incubated at 37°C for approximately 48 h. The cultures were gently shaken daily to resuspend the cells.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Colcemid

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from Sprague-Dawley rats (Aroclor 1254 induced rat liver fraction)

Test concentrations with justification for top dose:

EXPERIMENT 1:
Concentrations prepared without and with metabolic activation (S9): 0, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 μg/mL.

Due to a steep toxic response observed in both the absence and presence of S9 mix at concentrations above 78.13 μg/mL, a repeat test was performed using the following concentrations of T-71: 0, 6.25, 12.5, 25, 50, 75, 100 and 125 μg/mL.

EXPERIMENT 2:
Concentrations of T-71 were as follows:
Without S9 mix: 0, 3.13, 6.25, 12.5, 25, 50 and 75 μg/mL.
With S9 mix: 0, 6.25, 12.5, 25, 50 and 75 μg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: cell culture medium
- Justification for choice of solvent/vehicle:
Information provided by the sponsor indicated that dimethyl sulphoxide, purified water and acetone were not suitable solvents. T-71 was also found to be insoluble in ethanol, however a dosable suspension was formed in culture medium at 10 mg/mL. On dosing at 50% v/v into aqueous tissue culture medium, giving a final concentration of 5000 µg/mL, heavy precipitate was observed. In this case, the highest concentration used for subsequent testing was 5000 µg/mL.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Cyclophosphamide tested at 10 μg/mL, vehicle sterile purified water: with metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Mitomycin C tested at 0.2 μg/mL (3 hour treatment) and 0.1 μg/mL (continuous treatment), vehicle sterile purified water: without metabolic activation

Details on test system and experimental conditions:

CELL DIVISION STIMULANT:
Phytohaemagglutinin

METHOD OF APPLICATION:
in cell culture medium

DURATION
- Exposure duration:
3 hours in Experiment 1 (without and with metabolic activation) and in Experiment 2 (with metabolic activation).
20 hours in Experiment 2 (without metabolic activation)
[After the 3 h treatment the cells were cultivated with fresh media for 17 h].
- Final concentration of S9 mix:
Experiment 1: cultures contained 1 ml S9 mix and 2.5 mL cell culture medium
Experiment 2: cultures contained 1 ml S9 mix and 2.5 mL cell culture medium
- Fixation time (start of exposure up to fixation or harvest of cells):
20 hours in each of both experiments

SPINDLE INHIBITOR (cytogenetic assays):
Colcemid® was added to the cultures (0.1 µg/mL culture medium) 18 hours after treatment start.
2 h later, the cells were treated with hypotonic solution (0.075 M KCl) for 10 min at 37 °C. After incubation in the hypotonic solution, the cells were fixed with 3 + 1 methanol + glacial acetic acid.

STAIN (for cytogenetic assays):
After fixation the cells were stained with 10% Giemsa.

NUMBER OF REPLICATIONS:
Duplicate cultures were treated at each concentration.

NUMBER OF CELLS EVALUATED:
100 metaphases per culture, amounting to a total of 200 metaphases per dose concentration, were scored for structural chromosomal aberrations.
This number was reduced In cultures showing a high level of aberrant cells, where 10 cells in 100 metaphases with structural aberrations (excluding gaps) were observed.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (% cells in mitosis) determined by counting the number of mitotic cells in 1000 cells;

Microscopic examination of the metaphases included the recording of the following parameters:
- Aberrant cells (including and excluding gaps),
- Number of gaps,
- Types of aberrations
Chromatid break, Chromosome break, Chromatid gap, Chromatid exchange, Chromosome exchange, Chromosome gap,
Others: Cells with greater than eight aberrations, pulverised cells and pulverised chromosomes

Determination of polyploidy:
- Polyploid and endoreduplicated cells were noted when seen.

Evaluation criteria:

An assay is considered to be acceptable if the vehicle and positive control values lie within the current historical control range.

The test substance is considered to cause a positive response if the following conditions are met:
-Statistically significant increases (P<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) are observed at one or more test concentration.
-The increases exceed the negative control range of this laboratory, taken at the 99% confidence limit.
-The increases are reproducible between replicate cultures.
-The increases are not associated with large changes in pH, osmolality of the treatment medium or extreme toxicity.
-Evidence of a dose-relationship is considered to support the conclusion.

A negative response is claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed, at any dose level.

Statistics:

One-tailed Fisher exact test (Fisher 1973) for comparison of the number of aberrant metaphase cells in each test substance group with the solvent control value.

FISHER, R.A. (1973) The Exact Treatment of 2 x 2 Table in: Statistical Methods for Research Workers. Hafner Publishing Company, New York.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

in both experiments following 3 h treatment

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

With metabolic acitviation and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

See table on results 'Table CA NA-70.pdf' attached as background material.

Applicant's summary and conclusion

Conclusions:

It is concluded that the test substance T-71 has shown no evidence of clastogenic activity in this in vitro cytogenetic test system, under the experimental conditions described.