Methyl N-[3-(acetylamino)-4-[(2,4-dinitrophenyl)azo]phenyl]-N-(3-methoxy-3-oxopropyl)-β-alaninate

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:

From 20. June to 28. Oct 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

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)

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Test material

Test material information

Constituent 1

Reference substance name:

Methyl N-[3-(acetylamino)-4-[(2,4-dinitrophenyl)azo]phenyl]-N-(3-methoxy-3-oxopropyl)-β-alaninate

EC Number:

274-828-8

EC Name:

Methyl N-[3-(acetylamino)-4-[(2,4-dinitrophenyl)azo]phenyl]-N-(3-methoxy-3-oxopropyl)-β-alaninate

Cas Number:

70729-65-6

Molecular formula:

C22H24N6O9

IUPAC Name:

methyl N-[3-(acetylamino)-4-[(2,4-dinitrophenyl)azo]phenyl]-N-(3-methoxy-3-oxopropyl)-β-alaninate

Test material form:

solid: particulate/powder

Details on test material:

Disperse Red 311

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Zeneca Barriered Animal Breeding Unit (BABU), Alderley Park, Macclesfield
- Diet (e.g. ad libitum): Porton Combined Diet , ad libitum
- Water (e.g. ad libitum): Filtered tap water, ad libitum

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.5% (w/v) hydroxy propyl methyl cellulose in 0.1% aqueous polysorbate 80
- Justification for choice of solvent/vehicle: Forms good suspension
- Concentration of test material in vehicle: 125 and 200 mg/mL
- Amount of vehicle: 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Dosing suspensions of the test substance were prepared in 0.5% (w/v) hydroxy propyl methyl cellulose in 0.1% aqueous polysorbate 80
A solution of dimethylhydrazine dihydrochloride was prepared in deionised water.

Duration of treatment / exposure:

16 h

Frequency of treatment:

Single dose

Post exposure period:

2 and 16 h after treatment

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 250 mg/kg bw (total dose)

Doses / concentrations 2

Dose / conc.:

2 000 mg/kg bw (total dose)

No. of animals per sex per dose:

5 males/dose in treatment groups
2 males/experiment in vehicle and positive control group

Control animals:

yes, concurrent vehicle

Positive control(s):

Dimethylhydrazine dihydrochloride
- Route of administration: oral, gavage
- Doses / concentrations: 30 mg/kg bw (3 mg/mL)

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 (recently conducted study in same lab), in which the acute MTD for the test substance was >2000 mg/kg

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 2 or 16 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:
- 30 morphologically normal cells/slide and a total of 60 cells/animal.
- 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 (AMS 40-10) 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:

Criteria for a positive response:
- A mean animal net nuclear grain count [N-C] value of greater than zero represents a biologically significant departure from normal.
- The radiolabelling of the nucleus exceeds that of the cytoplasm, indicating of a real net synthesis of nuclear DNA
Criteria for a negative response:
- The mean net nuclear grain count of all test substance treated animals is less than 0.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

Colouration of the urine was observed in animals dosed with Disperse Red 311

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: >2000 mg/kg in acute oral study

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

Others:
Treatments with Disperse Red 311 in no case resulted in a mean net nuclear grain count greater than zero, at either time point investigated.
Colouration of the urine was observed in animals dosed with Disperse Red 311, which clearly indicates that the test material was absorbed following administration via the oral route.

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:

Disperse Red 311 was tested for the ability to induce unscheduled DNA synthesis (UDS) in an in vivo rat hepatocyte assay. Male Alpk:APfSD rats were treated with a single oral dose of Disperse Red 311 by gavage at dose levels of 1250 or 2000 mg/kg bodyweight. The latter dose level is the limit dose level for this assay. Animals were killed and hepatocytes isolated and prepared two and sixteen hours after dosing. 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 and an equal area of cytoplasm 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 this laboratory no negative control animal has shown a mean net nuclear grain count of greater than zero. An [N-C] value of greater than zero is therefore considered indicative of a UDS response.

Each experiment was validated by concurrent control treatments of rats with 0.5% (w/v) hydroxy propyl methyl cellulose in 0.1% aqueous polysorbate 80, the vehicle for Disperse Red 311 and with the carcinogen dimethylhydrazine dihydrochloride [DMH.2HC1]. Vehicle-treated rats gave rise to mean net nuclear grain counts of less than zero, whilst hepatocytes from DMH.2HC1-treated animals had mean net nuclear grain counts of greater than +5. These data show that the Background levels of UDS in this study were normal and that the test animals were responsive to a known carcinogen requiring metabolic activation for the demonstration of genotoxic activity.

Hepatocytes from Disperse Red 311 treated animals were assessed for UDS at both dose levels tested. Treatments with Disperse Red 311 in no case resulted in a mean net nuclear grain count greater than zero, at either time point investigated.

Colouration of the urine was observed in animals dosed with Disperse Red 311, which clearly indicates that the test material was absorbed following administration via the oral route.

It is concluded that, when tested up to a limit dose level of 2000 mg/kg, the test sample of Disperse Red 311 did not induce DNA repair (as measured by unscheduled DNA synthesis) in rat liver.