1-hydroxy-4-(p-toluidino)anthraquinone

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Principles of method if other than guideline:

SOLVENT VIOLET 13 was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect on erythrocytes in bone marrow.
Four groups each comprising 5 males, received an intraperitoneal injection. Two groups were dosed with 2000 mg/kg body weight, one group was dosed with 1000 mg/ g body weight and one group was dosed with 500 mg/kg body weight. After dosing, the animals of the dose level of 2000 mg/kg body weight showed the following toxic signs: lethargy, rough coat and a hunched posture. The animals of the dose levels of 1000 and 500 mg/kg body weight showed no abnormalities after dosing, except two animals of the dose level of 500 mg/kg, which showed a rough coat.
A vehicle treated group served as negative control, a group treated with an intraperitoneal injection of cyclophosphamide (CP) at 50 mg/kg body weight served as positive control.
Bone marrow of the groups treated with SOLVENT VIOLET 13 was sampled 24 or 48 hours after dosing. Bone marrow from the negative control group was harvested at 24 hours after dosing only and bone marrow from the positive control group was harvested at 48 hours after dosing only.

GLP compliance:

yes

Type of assay:

mammalian germ cell cytogenetic assay

Test material

Test material information

Constituent 1

Reference substance name:

1-hydroxy-4-(p-toluidino)anthraquinone

EC Number:

201-353-5

EC Name:

1-hydroxy-4-(p-toluidino)anthraquinone

Cas Number:

81-48-1

Molecular formula:

C21H15NO3

IUPAC Name:

1-hydroxy-4-[(4-methylphenyl)amino]-9,10-anthraquinone

Specific details on test material used for the study:

Identification: SOLVENT VIOLET 13

Test animals

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

Test System: NMRI BR mice (SPF), Recommended test System in international guidelines
Source: Charles Ri er, Sulzfeld, Germany.
Number of Animais per Test: 5 male mice per sampling time in each treatment group.
Age at Start of Treatment: Young adult animals were selected (6 weeks old).

Sex:

male

Details on test animals or test system and environmental conditions:

Conditions
A controlled environment was maintained in the room with optimal conditions of approximately 15 air changes per hour, a temperature of 21± 3°C, a relative humidity of 30-70% and 12 hours artificial fluorescent light and 12 hours dark per day. Fluctuations from these optimal conditions were noted, but were considered not to have affected the integrity of the study.
Accommodation
Group housing of 5 animals per sex per cage in labelled polycarbonate cages containing purified sawdust as bedding material. Acclimatisation period was at least 5 days before statt of treatment under laboratory conditions.
Diet
Free access to Standard pelleted laboratory animal diet.
Water
Free access to tap-water.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

corn oil

Details on exposure:

The mice received an intraperitoneal injection of a maximum tolerated (high), an Intermediate and a low dose of SOLVENT VIOLET 13. The route of administration was selected taking into account the possible route of human exposure during manufacture, handling and use.
The dosing voiume was 10 ml/kg body weight.
The route and frequency of administration and the voiume administered of the negative and the positive control was the same as those of the test article.

Duration of treatment / exposure:

The animals were sacrificed by cervical dislocation 24 or 48 h after dosing SOLVENT VIOLET 13, 24 h after dosing of the vehicle and 48 h after dosing the positive contra!.

Frequency of treatment:

Single application

Post exposure period:

The animals were sacrificed by cervical dislocation 24 or 48 h after dosing SOLVENT VIOLET 13, 24 h after dosing of the vehicle and 48 h after dosing the positive contra!.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle

Doses / concentrations 2

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

Solvent Violet 13

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Solvent Violet 13

Doses / concentrations 4

Dose / conc.:

500 mg/kg bw/day

Remarks:

Solvent Violet 13

Doses / concentrations 5

Dose / conc.:

50 mg/kg bw/day

Remarks:

Cyclophosphamide

No. of animals per sex per dose:

5 male mice/dose

Control animals:

yes

Positive control(s):

The positive control used in the micronucleus test was cyclophosphamide (CP; CAS no. 50-18-0; Endoxan, Asta-Werke, F.R.G.) dissolved in physiological saline (Fresenius B.V., s-Hertogenbosch, The Netherlands) dosed at a single intraperitoneal injection of 50 mg salt/kg body weight.

Examinations

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

The animals were sacrificed by cervical dislocation 24 or 48 h after dosing SOLVENT VIOLET 13, 24 h after dosing of the vehicle and 48 h after dosing the positive contra!. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 2 ml of foetal calf serum. The cell Suspension was collected and centrifuged at 1000 rpm (approximately 100 g) for 5 min.

Evaluation criteria:

ACCEPTABILITY OF ASSAY
A micronucleus test is considered acceptable if it meets the following criteria:
a) The positive control substance induced a statistically significant (Wilcoxon Rank Sum Test, two-sided test at P < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes.
b) The incidence of micronucleated polychromatic erythrocytes in the control animals should reasonably be within the laboratory historical control data ränge (mean + three times the Standard deviation): Males: 0.70%o ± 2.67%o indicated are means for n=80.

DATA EVALUATION AND STATISTICAL PROCEDURES
Equivocal results should be clarified by further testing using modification of experimental conditions.
A test substance is considered positive in the micronucleus test if:
- It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test; two-sided test at P < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time).
A test substance is considered negative in the micronucleus test if:
- one of the tested concentrations or sampling times showed a statistically significant (P < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes.
The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Micronucleus Test
Since there were no substantial differences between the sexes in toxicity after dosing the animals with 2000 mg/kg body weight, the micronuclues test was performed with male animals only. Based on the results of the dose range finding study, dose levels of 2000, 1000 and 500 mg/kg body weight were selected as appropriate doses for the micronucleus test. Five male animals were used in each treatment group.

Mortality and systemic toxic signs
The animals of the vehicle control group and the animals of the positive control group showed no abnormalities.

Micronucleated polvchromatic erythrocytes
The mean number of micronucleated polychromatic erythrocytes scored in SOLVENT VIOLET 13 treated groups were compared with the corresponding solvent control group. No biologically significant increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of SOLVENT VIOLET 13 treated animals compared to the vehicle treated animals.
The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range.
Cyclophosphamide, the positive control substance, induced a statisticaily significant increase in the number of micronucleated polychromatic erythrocytes in both sexes. Hence, the acceptability criteria of the test were met.

Ratio polychromatic to normochromatic erythrocytes
The groups that were treated for 24 hours with SOLVENT VIOLET 13 showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle Controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The dose group of 2000 mg SOLVENT VIOLET 13/kg body weight (48 hours treatment) and the group that was treated with cyclophosphamide showed a decrease in the ratio of olychromatic to normochromatic erythrocytes compared to the vehicle control.

CONCLUSION
It is concluded that this test is val d and that SOLVENT VIOLET 13 is not mutagenic in the micronucleus test under the experimental conditions described in this report.

Applicant's summary and conclusion

Conclusions:

Negative (not mutagenic in the micronucleus test under the experimental conditions).

Executive summary:

SOLVENT VIOLET 13 was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect on erythrocytes in bone marrow.

Four groups each comprising 5 males, received an intraperitoneal injection. Two groups were dosed with 2000 mg/kg body weight, one group was dosed with 1000 mg/ g body weight and one group was dosed with 500 mg/kg body weight. After dosing, the animals of the dose level of 2000 mg/kg body weight showed the following toxic signs: lethargy, rough coat and a hunched posture. The animals of the dose levels of 1000 and 500 mg/kg body weight showed no abnormalities after dosing, except two animals of the dose level of 500 mg/kg, which showed a rough coat.

A vehicle treated group served as negative control, a group treated with an intraperitoneal injection of cyclophosphamide (CP) at 50 mg/kg body weight served as positive control.

Bone marrow of the groups treated with SOLVENT VIOLET 13 was sampled 24 or 48 hours after dosing. Bone marrow from the negative control group was harvested at 24 hours after dosing only and bone marrow from the positive control group was harvested at 48 hours after dosing only.

Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes.

No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with SOLVENT VIOLET 13.

The groups that were treated for 24 hours with SOLVENT VIOLET 13 showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The group that was treated for 48 hours with SOLVENT VIOLET 13 and the group that was treated with cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle control.

It is concluded that SOLVENT VIOLET 13 is not mutagenic (negative) in the micronucleus test under the experimental conditions described in this report.