Sodium 1-amino-9,10-dioxo-4-(2,4,6-trimethylanilino)anthracene-2-sulfonate, reaction products with acidified C10-14-tert-alkyl(linear and branched) amines

Genetic toxicity: in vivo

Administrative data

Endpoint:

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

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study (OECD test guideline 486)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

unscheduled DNA synthesis

Test material

Test material information

Constituent 1

Reference substance name:

2-methylundecan-2-aminium 1-amino-9,10-dioxo-4-[(2,4,6-trimethylphenyl)amino]-9,10-dihydroanthracene-2-sulfonate

EC Number:

700-958-6

Molecular formula:

UVCB Substance

IUPAC Name:

2-methylundecan-2-aminium 1-amino-9,10-dioxo-4-[(2,4,6-trimethylphenyl)amino]-9,10-dihydroanthracene-2-sulfonate

Details on test material:

- Description: blue powder
- Expiration date of the batch: 01-Apr-1997
-Stability under storage conditions: stable
- Stability in solvent: at least 24 hours in PEG 400
- Storage: at room temperature

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

Strain: Wistar/WU
Source: Charles River Wiga GmbH, 97633 Sulzfeld, Germany
Number of animals: 25 males
Acclimatisation: at least 5 days
Age of the animals: 6-8 weeks old
Initial body weight at start of treatment: 274-357 g

Housing: single
Cage type: Makrolon Type I, with wire mesh top (EHRET GmbH, D-79312 Emmendingen 14, F.R.G.)
Bedding: granulated soft wood bedding (ALTROMIN, D-65931 Lage/Lippe, F.R.G.)
Feed: pelleted standard diet (ALTROMIN 1324, D-65931 Lage/Lippe, F.R.G.)
Water: tap water, ad libitum (Gemeindewerke, D-64380 RoBdorf, F.R.G.)

Environment:
Temperature: 21 ± 3 °C
Relative humidity: 30-70 %
Artificial light: 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

On the day of the experiment (immediately before treatment), the test item was formulated in PEG 400. The vehicle was chosen according to its relative non-toxicity for the animals. All animals were challenged orally once. The volume administered was 10 mL/kg bw.

Details on exposure:

The test item was formulated in PEG 400. This suspending agent was used as vehicle control. The test item was tested at the following dose levels:
2 hour treatment period: 2000 mg/kg bw
16 hour treatment period: 200 and 2000 mg/kg bw

The animals were starved overnight (2 hours treatment) or approximately 6 hours (16 hours treatment) before receiving the test item, water was available continuously. The animals received the test article once. Five animals (males) were treated per dose group.
After a treatment period of 2 or 16 hours, the animals were narcotised and sacrificed by liver perfusion. Primary hepatocyte cultures were established and exposed for 4 hours to 3HTdR (methyl-3H-thymidine) which is incorporated if UDS occurs. At least three cultures were established for each animal.

Duration of treatment / exposure:

2 or 16 hours

Frequency of treatment:

single treatment

Post exposure period:

After a treatment period of 2 or 16 hours, the animals were narcotised and sacrificed.

Doses / concentrations

Remarks:

Doses / Concentrations:
2 hour treatment period: 2000 mg/kg bw ; 16 hour treatment period: 200 and 2000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

2-Acetylaminofluorene in dimethyl sulfoxide/polyethylene glycol 400 (1+9): 100 mg/kg bw

Examinations

Tissues and cell types examined:

Primary hepatocyte cultures were established and examined.

Details of tissue and slide preparation:

Isolation of the Primary Hepatocytes
The animals were sacrificed by liver perfusion. After anaesthetising the rats with Nembutal (Sanofi/Ceva, D-40472 Düsseldorf, F.R.G.) the liver was perfused through the vena portae with Hanks' balanced salt solution (HBSS, Gibco/BRL, D-76344 Eggenstein, F.R.G.) supplemented with collagenase (0.05 % w/v, Boehringer Mannheim, D-68305 Mannheim, F.R.G.) adjusted to pH 7.4 and maintained at 37 °C. The hepatocytes were isolated from the liver and washed twice with HBSS. The crude cell suspension was filtered through a 94 µm stainless steel mesh to yield a single cell suspension. The quality of the actual performed perfusion was determined by the trypan blue dye exclusion method. In addition, the number of the isolated cells was determined.

Culture Conditions
The washed hepatocytes were centrifuged and transferred into Williams medium E (WME, Gibco/BRL, D-76344 Eggenstein, F.R.G.) supplemented (1) with HEPES (2.38 mg/mL), glutamin (0.29 mg/mL), penicillin (100 units/mL), insulin 0.50 µg/mL, streptomycin (0.10 mg/mL), fetal calf serum (100 µL/mL). The medium without the cells was adjusted to pH 7.6. At least three cultures were established for each animal. Aliquots of 2.5 mL with freshly isolated hepatocytes in complete culture medium (1.0 x 10E5 living cells/mL) were added to 35 mm six-well cluster dishes (Greiner, D-72603-Nurtingen, F.R.G.) containing one gelatinised 25 mm round plastic coverslip (Thermanox, Flow Laboratories, D-53340 Meckenheim, F.R.G.) per well.

After an attachment period of approximately 1.5 hours in a 95 % air/5 % carbon dioxide 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, D-63033 Dreieich, F.R.G.) in 2.0 mL culture medium (WME, 1 % FCS) was added to the cultures. After a labelling time of 4 hous the cells were washed twice with WME supplemented with 1 % FCS and 0.25 mM unlabelled thymidine. Cultures were incubated overnight using the same medium. To prepare for autoradiography the medium was replaced by a hypotonic solution of 1 % sodium citrate for 10 minutes to swell the nuclei for better grain quantification. 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 % ethanol, and air dried.

Autoradiographic processing
The cover slips were mounted the side carrying the cells up on glass slides and coated with KODAK NTB2 (Technomara, D-35463 Fernwald, F.R.G.) photographic emulsion in the dark. The coated slides were stored in light-proof boxes in the presence of a drying agent for 12-14 days at 4 °C. The photographic emulsion was then developed with KODAK D-19 (Technomara, D-35463 Fernwald, F.R.G.) at room temperature, fixed in TETENAL (Tetenal, D-22844 Norderstedt, F.R.G.) and stained with hematoxylin/eosin.

Quantification of UDS
Evaluation was performed microscopically on coded slides using NIKON microscopes with oil immersion objectives. The cells for scoring were randomly selected according to a fixed scheme. The number of silver grains above the nucleus was counted automatically using the ARTEK 880 or 982 counter. In addition, the number of grain counts of one nuclear-sized cytoplasm adjacent to the nucleus was counted. At least two slides per animal and 50 cells per slide were evaluated. Heavily labelled S-phase cells were excluded from counting. Four animals per group were evaluated as described above.

Evaluation criteria:

Nuclear and net grain counts were estimated together. Increased net grains should be based on enhanced nuclear grain counts rather than on decreased cytoplasmic grain counts. A test item was classified as positive if the mean number of net grains was higher than 5 per nucleus at one of the test
points. A group average between 0 and 5 net grains was 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 provided additional information to confirm a positive response with less than 5 net grains. Statistical significance gave further evidence for a positive evaluation. Statistical significance was evaluated by means of the nonparametric Mann-Whitney test. A test item producing net grains not greater than 0 at anyone of the test points was considered non-effective in this system.

Statistics:

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

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

but tested up to 2000 mg/kg bw

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative