Reaction mass of 1,5-diamino-4,8-dihydroxy(4-hydroxyphenyl)anthraquinone and 1,5-diamino-4,8-dihydroxy-2-(4-methoxyphenyl)anthraquinone

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:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

from November 19, 1996 to March 4, 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The complete read across justification is detailed in section 13. Source study has reliability 1.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

(1983)

Deviations:

yes

Remarks:

lower relative humidity, solubility in water not indicated. However, these had no influence on the study results

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

(1992)

Deviations:

yes

Remarks:

lower relative humidity, solubility in water not indicated. However, these had no influence on the study results

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 03

IUPAC Name:

Similar Substance 03

Test material form:

solid

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: BRL; CH-4414 Füllinsdorf- Age at study initiation: 8 -12 weeks- Weight at study initiation: males mean value 30.8 g (SD ±2.9 g); Females mean value 22.5 g (SD ±1.5 g)- Assigned to test groups randomly: yes- Housing: single - Diet: pelleted standard diet, ad libitum (ALTROMIN 1324, D-32791 Lage/Lippe)- Water: tap water, ad libitum, (Gemeindewerke, D-64380 Roßdorf)- Acclimation period: 5 dENVIRONMENTAL CONDITIONS- Temperature: 21 ± 5 °C- Humidity: 20-70 %- Artificial light: 6.00 a.m-6.00 p.m

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: polyethylene glycol (PEG 400)- Justification for choice of solvent/vehicle: the vehicle was chosen to its relative non-toxicity for the animals. - Volume administered: 10 ml/kg bw

Details on exposure:

On the day of the experiment, the test substance was formulated in PEG 400. The vehicle was chosen to its relative non-toxicity for the animals. All animals received a single standard volume of 10 ml/kg bw orally.

Duration of treatment / exposure:

Single administration.

Frequency of treatment:

Once.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

200 mg/kg bw/day

Remarks:

24 h preparation interval

Doses / concentrations 2

Dose / conc.:

670 mg/kg bw/day

Remarks:

24 h preparation interval

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

24 h preparation interval

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

48 h preparation interval

No. of animals per sex per dose:

5/sex/group.

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive control: cyclophosphamideDissolved in: deionized waterDosing: 20 mg/kg bwRoute and frequency of administration: orally, onceVolume administered: 10 ml/kg bwSolution prepared on day of administration. The stability of positive control at room temperature is sufficient. At 25°C only 3.5 % of its potency is lost after 24 h.

Examinations

Tissues and cell types examined:

The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald/Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The highest dose (2000 mg/kg bw) was estimated by a pre-experiment to be suitable.TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 24 h and 48 h after a single administration of the test substance the bone marrow cells were collected for micronuclei analysis.METHOD OF ANALYSIS: Evaluation of the slides was performed using NIKON microscopes with 100× oil immersion objectives. At least 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and total erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 1000 erythrocytes. The analysis was performed with coded slides.

Evaluation criteria:

A test substance is classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant positive response for at least one of the test points. If no positive responses are seen, the substance is considered non-mutagenic in this system. This can be confirmed by means of the nonparametric Mann-Whitney test. However, both biological and statistical significance should be considered together.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

(only slight reduction of spontaneous activity which resolve within 48 h)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

As estimated by a pre-experiment 2000 mg/kg bw (highest guideline-recommended dose) of the test substance were administered as maximum dose and tolerated by the animals. The animals expressed slight toxic reactions.The mean number of normochromatic erythrocytes was not increased after treatment with the test substance as compared to the mean values of NCEs of the corresponding vehicle controls, indicating that the test substance had no cytotoxic properties in the bone marrow. In comparison to the corresponding vehicle controls there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test substance. The mean values of micronuclei observed after treatment were in the range of the vehicle control group.20 mg/kg bw cyclophosphamide used as positive control showed a statistically significant increase of induced micronucleus frequency.

Any other information on results incl. tables

Pre-experiment for toxicity: in a pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 2000 mg/kg bw/d test substance formulated in PEG 400. The volume administered was 10 ml/kg bw. Treated animals expressed toxic reactions as shown in table:

Toxic reactions	Hours post-treatment (male/female)
	1 h	6 h	24 h	48 h
Reduction of spontaneous activity	2/2	2/2	1/1	0/0

6 h after treatment the urine was red. On the basis of these data 2000 mg/kg bw were estimated to be suitable.

Deviations to the protocol:

One female did not reach the lower limit of the body weight range given in the protocol (19.3 g instead of 20 g). In deviation to the protocol the relative humidity was sometimes lower than 30 % (lowest value 20 %) and the temperature was lower than 21 ± 3 ⁰C (lowest temperature 16 ⁰C). As requested by the sponsor the stability of the test substance in PEG 400 which was used as vehicle was indicated (instead of the stability in water). Due to technical reasons 20 mg/kg bw instead of 40 mg/kg bw cyclophosphamide were applied as positive control. As the induced micronucleus frequency was lower than expected for 40 mg/kg bw CPA a second additional scoring of the slides of the positive control was performed. Therefore the number of evaluated polychromatic erythrocytes in this experimental group is 2000. These deviations had no influence on the integrity and validity of the study.

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance did not induce micronuclei in bone marrow cells of the mouse. Therefore, test substance is considered to be non-mutagenic in this micronucleus assay.

Executive summary:

A study was conducted to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse according to OECD guideline 474 and EU method B.12 in compliance with GLP.

The test substance was formulated in polyethylene glycol 400 (PEG 400). 24 and 48 h after a single administration, bone marrow cells were collected for micronuclei analysis. Ten animals (5/sex) per test group were evaluated. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test substance the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE. The following dose levels of the test substance were investigated:

- 24 h preparation interval: 200, 670 and 2000 mg/kg bw

- 48 h preparation interval: 2000 mg/kg bw

The highest dose (2000 mg/kg bw) was estimated by a pre-experiment to be suitable. The animals expressed slight toxic reactions. After treatment with test substance, the number of NCEs was not increased, indicating that test substance had no cytotoxic effects in the bone marrow. Further, there was no substantial enhancement in the frequency of micronuclei at any preparation interval and with any dose level used.

Under the study conditions, the test substance did not induce micronuclei in bone marrow cells of the mouse. Therefore, test substance is considered to be non-mutagenic in this micronucleus assay.