Reaction products of 4-amino-3-hydroxy-7-nitronaphthalene-1-sulfonic acid and 2′,4′-dihydroxyacetophenone chelated with iron, sodium salts

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

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

Adequacy of study:

key study

Study period:

1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Source study has reliability 2. Details on the read across are attached in section 13.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

not specified

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 01

IUPAC Name:

Similar Substance 01

Test material form:

solid: particulate/powder

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River GmbH, WIGA, D-8741 Sulzfeld, FRG
- Weight at study initiation: mean 26.7 g
- Assigned to test groups randomly: yes
- Housing: individually in Makrolon cages, type MI
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: about one week

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24°C
- Humidity: 30 - 70%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle/solvent used: CMC (carboxymethyl cellulose)
- Concentration of test material in vehicle: 8.5, 17, 34 g/100 ml
- Amount of vehicle: 20 ml/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
All test substance formulations were prepared immediately before administration. The amount of substance or volume to be administered was related to the specific weight of the individual animals on the day of the experiment.

Duration of treatment / exposure:

16, 24, 48 h for the highest dose of test material; 24 h for all other dose groups and controls

Frequency of treatment:

Single application

Post exposure period:

16, 24, 48 h for the highest dose of test material; 24 h for all other dose groups and controls

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 700 mg/kg bw/day (nominal)

Remarks:

#1

Doses / concentrations 2

Dose / conc.:

3 400 mg/kg bw/day (nominal)

Remarks:

#2

Doses / concentrations 3

Dose / conc.:

6 800 mg/kg bw/day (nominal)

Remarks:

#3

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

- Substance: cyclophosphamide
- Route of administration: oral by gavage
- Doses / concentrations: 40 mg/kg bw

Examinations

Tissues and cell types examined:

Bone marrow of the two femora

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION
In the determination of the acute oral toxicity all animals survived the high dose of 6800 mg/kg body weight without any clinical signs or symptoms. A volume as high as 20 ml/kg body weight had to be selected in order to be able do administer this amount. Higher doses suspended in an 0.5 % aqueous CMC formulation led to a viscous mass which could no longer be administered.

DETAILS OF SLIDE PREPARATION
Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained. Staining in eosin and methylene blue solution for 5 minutes. Rinsed in aqua dest., then placed in fresh aqua dest. for 2 or 3 minutes. Staining in Giemsa solution for 12 minutes. After being rinsed twice in aqua dest. and clarified in xylene, the preparations were embedded in Entellan.

METHOD OF ANALYSIS
As a rule, 1000 polychromatic erythrocytes from each of the male and female animals of every test group are evaluated and investigated for micronuclei. The normochromatic erythrocytes (= normocytes), which occur, are also scored.

Evaluation criteria:

The increase in the micronucleus rate in polychromatic erythrocytes of treated animals as compared with the solvent control group provides an index of a chromosome breaking (clastogenic) effect or of a spindle activity of the substance tested.

Statistics:

Two statistical tests were used to answer the questions of whether there are significant differences between control group and dose group or between the individual dose groups concerning the rate of micronuclei in polychromatic erythrocytes: first, the exact test according to FISHER, which was applied to register significant differences between the relative frequencies of a characteristic of two groups, and, second, the asymptotic U test according to MANN-WHITNEY (rank test modified according to WILCOXON). The relative frequencies of cells with micronuclei per animal were use das a criterion of the rank determination for the U test.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals: a single oral administration in doses of 6800 mg/kg, 3400 mg/kg or 1700 mg/kg body weight was tolerated by all animals without any signs of toxicity.
- Necropsy: gross-pathological examination of the animals sacrificed at the end of the study did not reveal any changes of the internal organs which could the attributed to the test substance administered.

Any other information on results incl. tables

Results:

Substance	Dose (mg/kg bw)	Interval: 16 hours				Interval: 24 hours				Interval: 48 hours
		Polychromatic erythrocytes investigated	Normocytes / 10000 polychromatic erythrocytes	Cells with micronuclei		Polychromatic erythrocytes investigated	Normocytes / 10000 polychromatic erythrocytes	Cells with micronuclei		Polychromatic erythrocytes investigated	Normocytes / 10000 polychromatic erythrocytes	Cells with micronuclei
				per 1000 polychromatic erythrocytes	per 1000 normochromatic erythrocytes			per 1000 polychromatic erythrocytes	per 1000 normochromatic erythrocytes			per 1000 polychromatic erythrocytes	per 1000 normochromatic erythrocytes
vehicle control, 0.5 % CMC		-				10000	3485	1.8	0
test substance	6800	10000	2988	1.4	1.34	10000	4349	1.4	1.61	10000	4359	1.3	1.61
test substance	3400	-				10000	3506	1.4	0.86
test substance	1700	-				10000	4307	1.9	0.46
cyclophosphamide	40	-				10000	5623	23.4	1.78

Applicant's summary and conclusion

Conclusions:

No induction of chromosome breaking (clastogenic) effect or a spindle activity in polychromatic erythrocytes from bone marrow of femora mice.

Executive summary:

Method:

The substance was tested for chromosome aberration potential following OECD guideline 474, by oral administration.

Results:

Under experimental conditions, test substance did not induce chromosome breaking (clastogenic) effect or a spindle activity in polychromatic erythrocytes from bone marrow of femora mice.