4-chloro-2,5-dimethoxyaniline

Genetic toxicity: in vivo

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

Study period:

1992-07-27 to 1993-03-31

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well conducted GLP-study, following previous OECD-TG. One dose instead of three doses tested (rationale given).

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 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1983

Deviations:

no

GLP compliance:

yes

Remarks:

GLP compliance statement attached to full study report.

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

4-chloro-2,5-dimethoxyaniline

EC Number:

228-782-0

EC Name:

4-chloro-2,5-dimethoxyaniline

Cas Number:

6358-64-1

Molecular formula:

C8H10ClNO2

IUPAC Name:

4-chloro-2,5-dimethoxyaniline

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Strain: Hoe: NMRKf (SPF71)
- Source: Hoechst AG, Kastengrund, SPF breeding colony
- Initial age at test: 9 weeks
- Bodyweight at start of study:
males: mean = 36.8 g (28 - 39 g)
females: mean = 26.6 g (23 - 31 g)
- Acclimatization: at least 5 days
- Diet: rat/mice diet Altromin 1324, ad libitum
- Water: tap water in plastic bottles, ad libitum
- Housing: in fully air-conditioned rooms in groups of five animals
- Romm temperature: 20 +/- 3 °C
- Rel. humidity: 50 +/- 20 %
- Lighting: 12 hours daily
- Identification: fur-marking with KMnO4 and cage numbering

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle: sesame oil

Details on exposure:

- Preparation of diet: The test compound dilutions were freshly prepared at the day of administration. 3000 mg 4-chloro-2,5-dimethoxyanilin TF were weight in a beaker, filled in a mortar, suspended with sesame oil, washed out in a 25 ml flask and topped up to the calibaration mark. A suspension was formed.

Duration of treatment / exposure:

24, 48, 72 hours after administration

Frequency of treatment:

1 each

Post exposure period:

24, 48, 72 hours

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
1200 mg/kg bw (12.0 % (w/v)) (24 hrs)
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
50 mg/kg bw (0.5 % (w/v)) (positive control; 24 hrs)
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1200 mg/kg bw (12.0 % (w/v)) (48 hrs)
Basis:
actual ingested

Doses / concentrations 4

Remarks:

Doses / Concentrations:
1200 mg/kg bw (12.0 % (w/v)) (72 hrs)
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

other: Yes, positive control substance

Positive control(s):

- Positive control substance: Cyclophosphamide (Endoxam (R))
- Dose/Exposure: single dose of 50 mg/kg bw, 24 hour group

Examinations

Tissues and cell types examined:

Study of the induction of micronuclei in none marrow cells.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Oral administration of 1200 mg 4-chloro-2,5-dimethoxyanilin TF per kg bodyweight did not lead to a partial lethality but caused strong clinical signs of toxicity in male and female mice, therefore it is considered the maximum applicable dose and was selected as dose level for the main study.

DETAILS OF SLIDE PREPARATION:
In conformity with the test procedure the animals were killed by carbon dioxide asphyxiation 24, 48 or 72 hours after application. for each animal, about 3 ml foetal bovine serum was poured into a centrifuge tube. Both 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. A suspension was formed. The mixture was then centrifuged for 5 minutes at approx. 1000 rpm and almost all the supernatant discarded. One drop of the thoroughly mixed sediment was smeared on a cleaned slide, identified by project code and animal number and air-dried for about 24 hours.
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 (Weise)
- rinsing in distilled water
- drying
- coating with Entellan

Evaluation criteria:

1000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. As a control measure 1000 mature erythrocytes were also counted and examined for micronuclei. In addition, the ratio of polychromatic to normochromatic reythrocytes was determined.
Both biological and statistical significances are considered together in evaluation.
A substance is considered as positive if there is a significant increase in the number of micronucleated polychromatic reythrocytes for at least one of the time points. A test substance producing no significant increase in the number of micronucleated polychromatic reythrocytes at any one of the time points is considered non-mutagenic in this system.

Statistics:

The results of the treatment groups (test substance) in the micronucleus test at each dose and killing time were compared with corresponding control values. The ratio of polychromatic to normochromatic reythrocytes was also evaluated statistically by the method of Wilcoxon (paired, two sided) [1]. The statistical evaluations were performed using the "Diamant" computer program Version 2.0, supplied by the Department of Information and Communication Hoechst AG. All statistical results are based on a 95% level of significance. Actual data were also compared with historical controls.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Oral administration of 1200 mg of test substance resulted in death of one male out of 30 animals treated. This animal was replaced and survived after treatment.

Vehicle controls validity:

valid

Negative controls validity:

other: negative control = vehicle control in this study = valid

Positive controls validity:

valid

Additional information on results:

- Oral administration of 1200mg of the test item per kg bw resulted in death of one male out of 30 animals trated
- This animal was replaced and survived after treatment
- Signs of toxicity observed in test animals: reduced spontaneous activity, uncoordinated gait, ataxic gait, abdominal position, irregular breathing, panting, righting, reflex reduced, righting reflex negative, forward movement in crawling posture, stupor, narrowed palpebral fissures, closed palpebral fissures, piloerection, hindlimbs in the abduction position, increased tonus of the abdominal position, urine brown-orange coloured.
- 48 hours after application all animals were free of clinical signs of toxicity.
- The dissection of some animals revealed the following macroscopic findings: small intestine filled with conspicuous compact content, large intestine absolute empty and bladder filled with brown liquid.
- Bone marrow smears were examined for the occurence of micronuclei in red blood cells
- Incidence of micronucleated polychromatic and normochromatic erythrocytes in the treated groups were within the normal range of the negative control groups
- No statistically significant increase of micronucleated polychromatic erythrocytes has been observed
- Number of normochromatic erythrocytes with micronuclei did not differ from the values of the simultaneous control animals for each of the three killing times investigated
- Ratio of polychromatic erythrocytes to normocytes remained essentially unaffected by the test compound

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The administration of 4-chloro-2,5-dimethoxyanilin TF did not lead to a substantial increase of micronucleated polychromatic erythrocytes and is not mutagenic in the micronucleus test.

Executive summary:

4-Chloro-2,5-dimethoxyaniline TF was tested in the micronucleus test. The test compound was suspended in sesam oil and dosed once oral at 1200 mg per kg bodyweight to male and female mice, upon the results of the previously conducted dose range finding assay (see appendix p. 24). According to the test procedure the animals were killed 24, 48 or 72 hours after administration.

Endoxan(R) was used as positive control substance and was administered orally at a dose of 50 mg per kg bodyweight.

The number of polychromatic and normochromatic reythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic reythrocytes in both male and female animals remained unaffected by the treatment with 4-chloro-2,5-dimethoxyaniline TF and was statistically not different from the control values.

Endoxan(R) induced in both males and females a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the system. The ratio of polychromatic erythrocytes to normocytes was not changed to a significant extent.

The results indicated that, under the conditions of the present study, 4-chloro-2,5-dimethoxyaniline TF is not mutagenic in the micronucleus test.