Diammonio(ethyl)[4-[[4-[ethyl(3-sulphonatobenzyl)amino]phenyl](2-sulphonatophenyl)methylene]cyclohexa-2,5-dien-1-ylidene](3-sulphonatobenzyl)ammonium

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

The study is conducted on a read across test material. The complete read across justification is attached in section 13. The reliability of the original study is 2.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1988

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

1000 instead of 2000 cells scored per animal

GLP compliance:

not specified

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 1

IUPAC Name:

Similar Substance 1

Specific details on test material used for the study:

purity not specified
Test item name: Acid Blue 9
supplied by the Japan Food Additives Association, Tokyo

Test animals

Species:

mouse

Strain:

other: ddY

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Shizuaka Agnicultural Cooperative Association for Laboratory Animals, Shizuoka
- Age at study initiation: Eight-weeks
- Diet ad libitum
- Water ad libitum

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

physiological saline

Duration of treatment / exposure:

single injection

Frequency of treatment:

First experiment: one treatment
Second experiment: one treatment per day for four consecutive days

Post exposure period:

26h after single dosing
24h after last of the four doses

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

single treatment

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

single treatment

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

single treatment

Doses / concentrations 4

Dose / conc.:

1 010 mg/kg bw/day (actual dose received)

Remarks:

four treatments in 24h intervals

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

MMC as positive control
physiological saline as vehicle control

Examinations

Tissues and cell types examined:

Femoral marrow cells

Details of tissue and slide preparation:

Femoral marrow cells were flushed out with foetal bovine serum and smeared on clean glass slides. Cells were fixed with methanol for 5 min, and stained with Acridine Orange for the pilot experimnent and with Giemsa for the main study.
1000 cells per animal were scored

The number of micronucleated polychromatic erythrocytes (MNPCE-s) was recorded.
The proportion of polychromnatic erthrocytes (PCEs) among the total erythrocytes was also evaluated by observing 1000 erythrocytes on the same slide.

Evaluation criteria:

A positive response was defined as an increase in MNPCE which was statistically different from the control.

Statistics:

In the first step of the procedure, the frequencN of MNPCEs in each treatment group was compared with the binomial distribution specified by histonical control data from the laboratory (1 or 2) where the test was performed (Hayashi ei al. 1985). In the second step. the dose-response relationship was tested by the Cochran-Armitage trend test (Armitage, 1955, Cochran, 1954; Margolin & Risko, 1988).

Results and discussion

Test results

Test results 1

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Mortality at 2000 mg/kg bw

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: single dosing up to 2000 mg/kgbw

Test results 2

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

One animal died after the ip application of 2000 mg/kg bw.

Any other information on results incl. tables

4x 1010 mg/kg bw:

Micronucleated PCE: 0.18 +/0.15 %      PCE 43.6 +/-13.8 %       no mortality

2000 mg/kg bw

Micronucleated PCE: 0.34 +/- 0.15%       PCE 51.4 +/-4.1 %       1/6 animals died

1000 mg/kg bw

Micronucleated PCE: 0.08 +/-0.08%       PCE 49.0 +/- 4 %       no mortality

500 mg/kg bw

Micronucleated PCE: 0.22 +/- 0.15%       PCE 52.7 +/- 4%       no mortality

vehicle control

Micronucleated PCE: 0.33 +/- 0.08%       PCE 48.8 +/- 4.8%       no mortality

Applicant's summary and conclusion

Conclusions:

Acid Blue 9 was not genotoxic in the micronucleus study in mice after intraperitoneal injection.