Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: well performed GLP and OECD guideline study following a previous guideline version

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Principles of method if other than guideline:
Study was performed according to a previous guideline version. Following this protocol only a single dose was tested. This test dose was accurately determined in a dose range finding study in order to find the highest administrable non lethal dose level.
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2,5-Dianilino Terephthalic Acid

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain specifics: Hoe: NMRKf (SPF71)
- Source: Hoechst AG, breeding colony
- Age at study initiation: 9 weeks
- Weight at study initiation: males: 27-37 g, females: 24-31 g
- Housing: in groups of five in Macrolon(R) cages on softwood granulate, in fully air-conditioned rooms
- Diet (e.g. ad libitum): rat/mouse diet Altromin 1324, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3 °C
- Humidity (%): 50 ± 20 %
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: sesame oil
- Concentration of test material in vehicle: 30 % (w/v)
- Amount of vehicle (if gavage or dermal): 10 ml/kg body weight
- Purity: quality according to Ph. Eur. III
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
30 % was the highest concentration achievable in sesame oil
Duration of treatment / exposure:
single oral administration
Frequency of treatment:
single oral administration
Post exposure period:
killing at 24 hours (dose group, negative control and positive control), 48 hours (dose group and negative control) or 72 hours (dose group and negative control) after administration
Doses / concentrations
Remarks:
Doses / Concentrations:
3000
Basis:
nominal conc.
mg/kg body weight
No. of animals per sex per dose:
5 males and 5 females (10 animals) per dose group and killing time
Control animals:
yes, concurrent vehicle
Positive control(s):
50 mg/kg body weight cyclophosphamide (endoxan, 5 males and 5 females)

Examinations

Tissues and cell types examined:
1000 polychromatic erythrocytes from the femoral bone marrow of each animal
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES:
Animals were killed 24, 48 or 72 h after application

DETAILS OF SLIDE PREPARATION:
Femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. The mixture was centrifuged for 5 minutes at 1200 rpm and the supernatant discarded. One drop of the thoroughly mixed sediment was
smeared on a slide.

Staining procedure:
- 5 minutes in methanol
- 5 minutes in May-Grünwalds solution
- brief rinsing twice in distilled water
- 10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2
- rinsing in distilled water
- drying
- coating with Entellan
Evaluation criteria:
A substance is considered as positive if there is a significant increase in the number of micronucleated polychromatic erythrocytes at least at one point of time. A test substance producing no significant increase in the number of micronucleated polychromatic erythrocytes at any counting time is considered non-mutagenic in this system.
Statistics:
The number of polychromatic erythrocytes with micronuclei occurring in 1000 polychromatic erythrocytes, and the number of normocytes with micronuclei occurring in 1000 normocytes, were evaluated statistically; comparison of dose groups with the simultaneous control group was performed according to Wilcoxon (paired, one-sided, increase)

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
A RANGE-FINDING STUDY was conducted to determine the highest administrable non lethal dose level.

RESULTS OF RANGE-FINDING STUDY

- Dose range: 3000 mg/kg body weight
- Clinical signs of toxicity in test animals: intensive yellow coloured urine
- Lethality: 0 of 3 males and 0 of 3 females


RESULTS OF DEFINITIVE STUDY

- All animals survived after application of 3000 mg per kg bodyweight.
- Clinical signs: urine intensive yellow coloured and faeces violet coloured (males only). 48 hours after application all animals were free of clinical signs.
- Macroscopic findings: stomach and intestinum contents were violet coloured in some animals
- Induction of micronuclei (for Micronucleus assay): no statistically significant increase of micronucleated polychromatic erythrocytes in dosed animals
- Ratio of PCE/NCE (for Micronucleus assay): unaffected by the test compound

Any other information on results incl. tables

Mean mutation indices in polychromatic erythrocytes:

Sex

Sampling after Dosing

Number of Animals

Vehicle control group

Dose group

Positive control group

Male

24 h

5

1.0

2.2

31.2

Female

24 h

5

1.0

2.0

32.4

Male

48 h

5

1.0

0.2

-

Female

48 h

5

1.0

0.5

-

Male

72 h

5

1.0

0.3

-

Female

72 h

5

1.0

0.6

-

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The results indicate that, under the conditions of the present study, the test item is not mutagenic in the micronucleus test.
Executive summary:

The test item was tested in the micronucleus test according to OECD guideline 474. The test compound was suspended in sesame oil and dosed once orally at 3000 mg per kg body weight to male and female mice, based on the results of the previously conducted dose range finding assay. Animals were killed 24, 48 or 72 hours after administration.

EndoxanR was used as positive control substance and was administered orally at a dose of 50 mg per kg body weight.

The number of polychromatic and normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment with the test item and was statistically not different from the control values.

EndoxanR induced in both males and females a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity af the system.

The results indicate that, under the conditions of the present study, the test item is not mutagenic in the micronucleus test.