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

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:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

according to guideline

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 in the main study. This test dose was accurately determined in a dose range finding study in order to find the maximum dose producing distinct toxicity but no lethality.

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

2,5-dimethoxyaniline

EC Number:

203-040-9

EC Name:

2,5-dimethoxyaniline

Cas Number:

102-56-7

Molecular formula:

C8H11NO2

IUPAC Name:

2,5-dimethoxyaniline

Details on test material:

- Name of test material (as cited in study report): Aminohydrochinondimethylether

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: HOECHST AG breeding colony
- Age at study initiation: 7 weeks
- Weight at study initiation: males: 25 - 34 g, females: 21 - 27 g

- Fasting period before study: no
- Housing: in fully air-conditioned rooms in Macrolon cages, on softwood granulate in groups of 5 animals
- Diet (e.g. ad libitum): rat/mice diet Altromin 1324, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

sesame oil (Oleum Sesami Ph. Eur. III)

Details on exposure:

The test compound was suspended in sesame oil and dosed once orally at 400 mg/kg bw to male and female mice, based upon the results of the previously conducted dose range finding assay.

Oral administration of 500 mg test substance per kg bw has caused partial lethality in male and female mice. The highest sublethal dose of 400 mg/kg bw was selected for the main study.

400 mg/kg bw = 4.0 % (w/v) = 10 ml/kg bw
0 mg/kg bw = 0 % (w/v) = 10 ml/kg bw

The test substance dilutions were freshly prepared at the day of administration. 1000 mg test material were weight in a breaker, ground in mortar, mixed with sesame oil, washed out in a 25 ml flask and topped up to the calibration mark. A suspension was formed.

Duration of treatment / exposure:

The test compound was suspended in sesame oil and dosed once orally at 400 mg/kg bw to male and female mice, based upon the results of the previously conducted dose range finding assay. The animals were killed 24,48 and 72 h after aministration.

Frequency of treatment:

once orally

Post exposure period:

Test compound and negative control: The animals were killed 24, 48 and 72 h after administration.
Positive control: The animals were killed 24 h after administration.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
400 mg/kg bw
Basis:
other: nominal in sesame oil

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0 mg/kg bw (negative control)
Basis:
other: nominal in sesame oil

No. of animals per sex per dose:

5 male and 5 female per group and killing time

Control animals:

yes, concurrent vehicle

Positive control(s):

Endoxan® (Cyclophosphamide): 50 mg/kg body weight p.o.

Examinations

Tissues and cell types examined:

Both femora were removed and the bones freed of muscle tissue. Bone marrow cells were collected (polychromatic/normochromatic erythrocytes).

Details of tissue and slide preparation:

Extraction of the bone marrow:
Animals were killed by carbon dioxide asphyxiation 24, 48 and 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 1200 rpm and almost smeared on a cleaned slide, identified by project code and animal number and air-dried for about 24 hours.

Straining procedure:
5 minutes in methanol
3 minutes in May-Grünwalds solution
2 minutes in May-Grünwalds solution diluted 1:1 with distilled water
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 cellls with micronuclei was recorded. As a control measure 1000 mature erythrocytes were also counted and examined for micronuclei. In addition, the ratio of polychromatic to normochromatic erythrocytes was determined. All bone marrow smears for evaluation are coded to ensure that the group which they belonged to remains unknown to the investigator. The number of polychromatic erythrocytes with micronuclei occuring in the 1000 polychromatic erythrocytes counted, and the numbner of normocytes with micronuclei occuring in the 1000 normocytes counted, were evaluated statistically; comparison of the dose groups with the stimultaneous control group was performed according to Wilcoxon (paired, one-sided, increase).

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 poylchromatic to normochromatic erythrocytes was also evaluated statistically by the method of Wilcoxon (paired, two sided).

All statistical results are based on a 95 % level of significance. Actual data were also compared with historical controls.

Statistics:

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 bassed 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

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The incidence of micronucleated polychromatic and normochromatic erythrocytes in the dose groups of Aminohydrochinondimethylether was within the normal range of the negative control groups. No statistically significant increase of micronucleated polychromatic erythrocytes has been observed. The number of normochromatic erythrocytes with micronuclei did not differ from the values of the stimultaneous control animals for each of the three killing times investigated. The ratio of polychromatic erythrocytes to normocytes remained essentially unaffected by the test compound.

Cyclophosphamide (Endoxan R) induced a marked and statistically significant increase of the number of polychromatic erythrocytes with micronuclei in both males and females indicating the sensitivity of the test system.

Any other information on results incl. tables

All animals survived after application of 400 mg / kg bw. The following signs of toxicity were observed: reduced spontaneous activity, norrowed palpebral fissures, stilted gait, piloerection, lacrimation and urine brown coloured. 48 hours after application all animals were free of clinical signs of toxicity.

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

Aminohydrochinondimethylether was tested in the micronucleus test. The test compound was supended in sesame oil and dosed once orally at 400 mg per kg body weight to male and female mice, based upon the results of the previously conducted dose range finding assay. Animals were killed 24, 48 or 72 hours after administration.

Endoxan R was used as positive control substance and was administered orally at the dose of 50 mg/kg bw.

The number of polychromatic and normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic / normochromatic erythroxytes in both male and female animals remained unaffected by the treatment with test substance 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 results indicate that, under the conditions of the present study, Aminohydrochinondimethylether is not mutagenic in the micronucleus test.