[No public or meaningful name is available]

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

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 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Version / remarks:

21. July 1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.39 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo)

Version / remarks:

19. May 2000

GLP compliance:

yes (incl. QA statement)

Type of assay:

unscheduled DNA synthesis

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

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat has been used for many years as suitable experimental animal in genotoxicity investigations. There are many data available from such investigations which may be helpful in the interpretation of results from the UDS test.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS: Wistar HanIbm: WIST (SPF)
- Source: RCC Ltd, Animal Breeding Services, CH-4414 Füllinsdorf
- Weight at study initiation: 183.7 ± 15.5 (SD) g (males)
- Number of animals: 32 (males)
- Assigned to test groups randomly: yes
- Housing: single housing
- Diet (e.g. ad libitum): pelleted standard diet (Altromin) ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12; artificial light 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.5% w/v sodium carboxymethylcellulose (CMC)
- Concentration of test material in vehicle: 0, 1000, 2000 mg/mL
- Amount of vehicle (if gavage or dermal): 20 mL/kg bw (10 mL/kg bw for positive control substances).

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment the test item was formulated in 0.5% CMC (Fluka, Cat. no: 21902). The vehicle was chosen to its non-toxicity for the animals. The animals received a single standard volume of 20 ml/kg body weight orally (the positive control groups were treated with 10 mL/kg body weight).

Duration of treatment / exposure:

The test item was administered once for 2 h or or 16 h.

Frequency of treatment:

single treatment

Post exposure period:

2 h and 16 h after dosing.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

4 males per dose group (main experiment).
2 female and 2 male per dose (pre-test).

Control animals:

yes, concurrent vehicle

Positive control(s):

- 4 male rats administered N,N'-dimethylhydrazinedihydrochloride (sym.) (DMH) (40 mg/kg bw) in 0.9 % NaCl solution (for 2 hours preparation interval).

- 4 male rats administered 2-acetylaminofluorene (2-AAF) (100 mg/kg bw) in dimethylsulfoxide/polyethylene glycol 400 (1 + 9) (for 16 hours preparation interval).

Examinations

Tissues and cell types examined:

hepatocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Preliminary study at max. dose of 2000 mg/kg body weight in 2 male and female rats

TREATMENT
The animals were starved overnight (2 hours treatment) or approximately 6 hours (16 hours treatment) before receiving the test item, the positive or vehicle control substance. Water was available ad libitum. Animals were weighed and the volume to be administered was adjusted to the body weight of each animal. The animals received the test item once. Four animals (males) were treated per group.

ISOLATION OF PRIMARY HEPATOCYTES
After anesthesizing the rats with Na-thiopental (Trapanal, Byk Gulden) the liver was perfused through the vena portae with Hanks' balanced salt solution (HBSS, Gibco/BRL) supplemented with 0.05 % w/v collagenase (Boehringer Mannheim) adjusted to pH 7.4 and maintained at 37 °C.

The isolated hepatocytes were washed twice with HBSS. The crude cell suspension was filtered through a stainless steel mesh (94 µm) to yield a single cell suspension. Cell viability was determined using trypan blue dye exclusion. In addition, the number of cells was determined.

CULTURE CONDITIONS
The washed cells were centrifuged and transferred into Williams medium E (WME, Gibco/BRL) supplemented with Hepes (2.38 mg/mL), Penicillin (100 units/mL), Streptomycin (0.10 mg/mL), L-Glutamine (0.29 mg/mL), Insulin (0.5 µg/mL) and Fetal calf serum (FCS, 100 µl/mL). This complete medium was adjusted to pH 7.6.

At least three cultures were established from each animal. Aliquots of 2.5 mL with freshly isolated hepatocytes in complete culture medium (2.0 x 10^5 viable cells/mL) were added to 35 mm six-well dishes (Greiner) containing one 25 mm round plastic coverslip (Thermanox) per well coated with gelatine.

After 1.5 h in a 95 % air / 5 % CO2 humidified incubator at 37 °C the culture medium was discarded. Then, the cell layer was rinsed once with PBS to remove non-adherent cells. Subsequently, 3HTdR (5 µCi/mL, specific activity 20 Ci/mmol; New England Nuclear) in 2.0 mL culture medium (WME, 1 % v/v FCS) was added to the cultures. After a labelling for 4 h, cells were washed twice with WME, 1 % v/v FCS and 0.25 mM unlabelled thymidine and incubated overnight. To prepare for autoradiography the medium was replaced by a hypotonic solution of 1 % (w/v) sodium citrate for 10 minutes to swell the nuclei for better grain detection. The cells on the coverslips were then fixed by three changes of methanol:acetic acid (3 + 1 v/v) for 20 min each, rinsed with 96 % (v/v) ethanol, and air-dried.

AUTORADIOGRAPHIC PROCESSING
Coverslips were mounted the side carrying the cells up on glass slides and coated with KODAK NTB2 (Tecnomara) photographic emulsion in the dark. The coated slides were stored in light-proof boxes in the presence of a drying agent for 14 days at 4° C. The photographic emulsion was then developped with Ilford Phenisol (Ilford Imaging) at room temperature, fixed with Rapid Fixer (Ilford Imaging) and stained with hematoxylin/eosin.

QUANTIFICATION OF UDS
Evaluation was performed microscopically on coded slides using NIKON microscopes with oil immersion objectives. Cells for scoring were randomly selected according to a fixed scheme. The number of silver grains in the nuclear area was counted automatically using the Sorcerer UDS device version 2.0 DT3152 (Perceptive Instruments). In addition, the number of grains of the most heavily labeled nuclear-sized cytoplasm area adjacent to the nucleus was counted. At least two slides per animal and 50 cells per slide were evaluated. Heavily radiolabelled cells undergoing replicative DNA synthesis were excluded from counting.

Three animals per group were evaluated as described above. The remaining animal per test group would have been evaluated if an animal died spontaneously or in case of technical problems concerning the isolation of hepatocytes. This was not the case in the present study.

METHOD OF ANALYSIS: light microscopy

Evaluation criteria:

Nuclear and net grain counts are estimated together. Increased net grains should be based on enhanced nuclear grain counts rather than on decreased cytoplasmic grain counts.

A test item is classified as positive if the mean number of net grains is higher than five per nucleus at one of the test points.

A group average between 0 and 5 net grains is considered as a marginal response. A dose-related increase in nuclear and net grains and/or a substantial shift of the
percentage distribution of the nuclear grain counts to higher values provide additional information to confirm a positive response with less than 5 net grains.

Statistical significance may give further evidence for a positive evaluation. Statistical significance can be evaluated by means of the non-parametric Mann-Whitney test. A test item producing net grains not greater than 0 at anyone of the test points is considered non-effective in this system.

Statistics:

A statistical evaluation of the results was not necessary to perform as the number of net grain counts of the groups treated with the test item were in the range of the corresponding controls.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

The treated animals expressed toxic reactions such as a reduction of spontaneous activity (2/4), ruffled fur (4/4) and eyelid closure (0/1) 2 h after treatment with 1000 / 2000 mg/kg b.w. of T-71.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

see attached "Tables 10.4 Mean Nucleus, Cytoplasmic Area, and Net Grains" for details of results.