N-[2-[(2-chloro-4,6-dinitrophenyl)azo]-5-(diethylamino)-4-methoxyphenyl]acetamide

Genetic toxicity: in vivo

Administrative data

Endpoint:

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

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

The test was conducted by means of Read Across approach. Further information was attached at section 13

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

see below

Principles of method if other than guideline:

Two doses only of the test compound were applied: Only two test compound concentrations were required for scoring as the test substance dosed at a limit concentration is not cytotoxic in the liver. A third dose was therefore considered unnecessary.

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Test material

Test material information

Constituent 1

Reference substance name:

Disperse Blue 291 - Similar Substance 05

IUPAC Name:

Disperse Blue 291 - Similar Substance 05

Test material form:

solid

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: ICI Barriered Animal Breeding Uni (BABU), Alderley Park, Macclesfield
- Age at study initiation:
- Weight at study initiation: 180-280 g
- Assigned to test groups randomly: yes - according to the order in which they are removed from the stock cage
- Fasting period before study:
- Housing: - pre dosing: up to 5 per cage
- for dosing: up to three per cage in a fume cupboard for a total period not exceeding 24 hours
- Diet (e.g. ad libitum): Porton Combined Diet, Special Diets Services Ltd, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: Since the animals were eventually moved in a fume cupboard, acclimatisation was not considered appropriate


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 21
- Humidity (%): 40 - 60
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: test substance in insoluble in water
- Concentration of test material in vehicle: 125 and 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg body weight

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Dosing suspensions of the test substance were prepared in corn oil.

Duration of treatment / exposure:

single dose

Frequency of treatment:

once

Post exposure period:

4 and 12 h after treatment

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 250 mg/kg bw/day (nominal)

Remarks:

in corn oil

Doses / concentrations 2

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

in corn oil

No. of animals per sex per dose:

5 males/dose/time point in treatment group
4 males/group/time point in vehicle and positive control group - only 1 animal/group/time point scored

Control animals:

yes, concurrent vehicle

Positive control(s):

2-acetylaminofluorene [2AAF] at twelve hours and N-nitrosodimethylamine [NDMA] or 6-p-dimethylaminophenylazobenzthiazole [6BT] at four hours

Examinations

Tissues and cell types examined:

Hepatocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on acute oral toxicity study in rats, in which the acute MTD for the test substance was >5000 mg/kg bw and a range finding study in 5 males at 2000 mg/kg bw

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Single oral dose by oral gavage. Slides from the animals were subsequently analysed. Of the two positive and two vehicle control animals in each experiment, only one of each was scored for induction of UDS

DETAILS OF SLIDE PREPARATION: Preparation of hepatocytes were made 4 or 12 h after dosage. Hepatocytes were prepared from treated animals by a two stage collagenase perfusion technique. Hepatocyte cultures were prepared by allowing cells to attach to plastic cover slips. Medium was removed from the dishes and replaced with fresh medium containing [3H] thymidine. After 4 h incubation at 37 °C within a 5% CO2/95% air (v/v) atmosphere, the medium was removed, the cells washed three times with medium containing unlabelled thymidine and the cultures incubated overnight with the same medium. Cultures were fixed and coverslips mounted onto microscope slides. Slides were coated with photographic emulsion and left for 14 d at 4 °C in the dark. The emulsion was developed, fixed and the cell nuclei and cytoplasm stained with Meyers haemalum and eosin Y phloxine.
Slides were examined microscopically for signs of undue cytotoxicity to enable selection of those to be examined for UDS.


METHOD OF ANALYSIS: Prior to microscopic assessment, all slides were furnished with code numbers, so that the counting was blind. The following counts were made:
- at least 25 but normally 50 cells/slide of two slides for each animal. The third slide was not normaly read, unles a total of 10 cells could not be obtained from the
first two slides examined.
- The nuclear count (the number of silver grains over the nucleus) and the cytoplasmic count (the number of grains in an adjacent, nuclear sized, most heavily labelled area of cytoplasm) were measured using an automated image analyser and the data captured directly into a computer.
- The mean net grain count (nuclear count - cytoplasmic count), the mean nuclear count and cytoplasmic counts and the percent of cells in repair (net grain count of 5 or greater) to be calculated.

Evaluation criteria:

EVALUATION CRITERIA
Criteria for a positive response:
- The criteria outlined in the ASTM Guideline are adopted, based on the mean net group grain count and the percentage of cells in repair. A treated group showing mean net nuclear grain counts of 5 or greater and 20% or greater of the cells in repair is considered to be a positive response. A compound can only be assigned as an unequivocal genotoxin in this assay if such a response is reproduced.

Criteria for a negative response:
- A negative response is obtained where the mean net nuclear grain count of the treated cultures is less than 0, and the percentage of cells in repair is also less than 20.


VALIDATION CRITERIA
Negative Controls
Cytoplasmic counts of less than 40 are considered acceptable. Mean net nuclear grain counts for the negative controls should be less than zero.

Positive Controls
Mean net grain counts for the positive control in each experiment shall be sufficient to be regarded as an unequivocal positive response; mean net nuclear grain value of 5 or greater, with at least 20% of the cells examined in repair (ie with 5 or more net grains).

Statistics:

difference between treated and control cultures

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

No significant adverse reactions to treatment

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: No adverse effects at 5000 mg/kg bw in acute oral or 2000 mg/kg body weight for four days in range finding study

RESULTS OF DEFINITIVE STUDY
- Animal Toxicity: No significant signs of acute toxic effects were observed.

Others:
Test substance caused no significant increases over the vehicle control in mean net nuclear grain count, nor in percentage of cells in repair, at either dose level (1250 or 2000 mg/kg bw) or time point (4 or 14 h) investigated. Hepatocytes from test substance treated animals had mean net nuclear grain values of less than zero. These data therefore provided no evidence for induction of UDS by test substance.

Applicant's summary and conclusion

Conclusions:

Under the test conditions, the test substance did not induce DNA repair in rat liver in vivo up to a limit dose of 2000 mg/kg bw.

Executive summary:

The test substance was tested for the ability to induce unscheduled DNA synthesis (UDS) in an in vivo rat hepatocyte assay. Male Fischer 344 rats were treated with a single oral doseby gavage at 1250, or 2000 mg/kg body weight. The highest test dose, 2000 mg/kg was the limit test dose for a non-toxic test agent in this assay. Animals were killed and hepatocytes prepared four hours and twelve hours following administration of the chemical. Two independent experiments were carried out for each time point.

Hepatocytes from treated rats were exposed to [³H]-thymidine and the amount of radioactivity incorporated into the nucleus [N] and an equal area of cytoplasm [C] determined by autoradiography. The cytoplasmic grain count was subtracted from that of the nucleus. The value obtained, the mean net nuclear grain count [N-C], is an index of UDS activity. In the respective testing laboratory, no negative control animal has shown a mean net nuclear grain count greater than zero. An [N-C] of more than zero in a treated animal is therefore considered indicative of a UDS response.

Each experiment was validated by concurrent control treatments of rats with corn oil, the solvent for the test substance and with the carcinogens 2-acetylaminofluorene [2AAF] at twelve hours and N-nitrosodimethylamine [NDMA] or 6-p-dimethylaminophenylazobenzthiazole [6BT] at four hours. Solvent treated rats gave rise to mean net grain counts of less than zero, whilst hepatocytes from 2AAF, 6BT or NDMA treated animals had mean net nuclear grain counts of greater than +5. These data showed that background levels of UDS were normal and that the tester animals were responsive to known carcinogens requiring metabolic activation for genotoxic activity.

Hepatocytes from treated animals were assessed for UDS at two dose levels of 1250 and 2000 mg/kg body weight. Treatments with the test substance in no case resulted in a mean net grain count greater than zero, at either time point.

It is concluded that, when tested up to a limit dose of 2000 mg/kg body weight, the test sample did not induce DNA repair (as measured by unscheduled DNA Synthesis) in hepatocytes from rats treated in vivo.