Disodium 6-hydroxy-5-[(2-methoxy-4-sulphonato-m-tolyl)azo]naphthalene-2-sulphonate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from publication

Data source

Reference

Reference Type:

publication

Title:

Micronucleus assay study of the test chemical

Author:

Masamitsu Honma

Year:

2015

Bibliographic source:

Food and Chemical Toxicology 84 (2015)

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

Determination of genotoxicity of the test chemical by Mammalian Erythrocyte Micronucleus test.

GLP compliance:

no

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 6-hydroxy-5-[(2-methoxy-4-sulphonato-m-tolyl)azo]naphthalene-2-sulphonate

EC Number:

247-368-0

EC Name:

Disodium 6-hydroxy-5-[(2-methoxy-4-sulphonato-m-tolyl)azo]naphthalene-2-sulphonate

Cas Number:

25956-17-6

Molecular formula:

C18H16N2O8S2.2Na

IUPAC Name:

disodium 6-hydroxy-5-[(2-methoxy-3-methyl-4-sulfonatophenyl)diazenyl]naphthalene-2-sulfonate

Test material form:

solid: particulate/powder

Details on test material:

IUPAC name: Disodium 6-hydroxy-5-[(2-methoxy-4-sulphonato-m-tolyl)azo]naphthalene-2-sulphonate
Mol. formula: C18H14N2Na2O8S2
Molecular Weight: 496.4266 g/mol
InChI: 1S/C18H16N2O8S2.2Na/c1-10-7-14(16(28-2)9-17(10)30(25,26)27)19-20-18-13-5-4-12(29(22,23)24)8-11(13)3-6-15(18)21;;/h3-9,21H,1-2H3,(H,22,23,24)(H,25,26,27);;/q;2*+1/p-2/b20-19+;;
Smiles: c12c(cc(cc2)S(=O)(=O)[O-])ccc(c1/N=N/c1c(cc(c(c1)C)S(=O)(=O)[O-])OC)O.[Na+].[Na+]
Physical state: Solid particulate/powder

Test animals

Species:

mouse

Strain:

other: CD1 mice (Crlj:CD1)

Details on species / strain selection:

No data

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Japan
- Age at study initiation: 6 weeks
- Weight at study initiation: No data
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study: No data
- Housing:No data
- Diet (e.g. ad libitum): CRF-1 pellet feed, Oriental Yeast
- Water (e.g. ad libitum): ad libitum
- Acclimation period:7 days before treatement

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data
- Humidity (%): No data
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): No data

IN-LIFE DATES: From: To:No data

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Vehicle(s)/solvent(s) used: 0.5% carboxymethylcellulose sodium
- Justification for choice of solvent/vehicle: The test chemical was soluble in 0.5% carboxymethylcellulose sodium

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:The test chemical was dissolved in 0.5% carboxymethylcellulose sodium solution prior to administration.

DIET PREPARATION
- Rate of preparation of diet (frequency):No data
- Mixing appropriate amounts with (Type of food): No data
- Storage temperature of food:No data

Duration of treatment / exposure:

2 days

Frequency of treatment:

Daily with a 24 hr interval

Post exposure period:

No data

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

30 mice
6 mice per group

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin C

Examinations

Tissues and cell types examined:

Bone marrow cells

Details of tissue and slide preparation:

Tissue preparation:
Mice were sacrificed by cervical dislocation at 23-24 h after the final treatment.Femurs was removed and bone marrow cells were flushed from femurs into centrifuge tubes using calf serum. Cells were then resuspended in Dulbecco's phosphate-buffered saline and were centrifuged, and supernatants were then removed. These procedures were performed twice, and then cells were fixed in 10% neutral-buffered formalin solution. After exchanging the fixative twice by centrifugation, cells were re-suspended in formalin solution, and the cell suspension was filtered through a cell strainer.

Slide preparation:
Fixed cell suspensions were dropped onto cover slips and were immediately placed on acridine orange coated slides. Two thousand polychromatic erythrocytes (PCEs) per animal were analyzed using a fluorescent microscope with a 1000 lens, equipped with a blue excitation filter and a barrier filter, and numbers of micronucleated polychromatic erythrocytes (MNPCEs) were counted. To investigate the influence of the test substance on bone marrow cell proliferation, numbers of PCEs in a total of 500 erythrocytes were counted.

Evaluation criteria:

No data

Statistics:

Frequencies of MNPCEs in treatment and positive control groups were compared with those in the negative control group using conditional binomial tests.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Remarks:

0.5% carboxymethylcellulose sodium

Negative controls validity:

valid

Remarks:

0.5% CMC

Positive controls validity:

valid

Remarks:

Mitomycin C

Remarks on result:

other: No mortality observed

Additional information on results:

In micronucleus tests, no significant differences in MNPCEs were found between test chemical treatment groups and the negative control group. The frequency of PCEs, which offers an index of the influence of the test substance on bone marrow cells, did not differ between any of the treatment groups and the negative control group.The frequency of MNPCEs in the positive control group was increased (p ≤ 0.01) in comparison with that in the negative control group. No detahs, clinical signs and body weight changes were observed in control and tratment groups.

Any other information on results incl. tables

Table: The results of micronucleus test in bone marrow of CD1 mice after test chemical treatment.

Compound	Dose(mg/kg)	No. of animal	% MNPCE^a	% PCE
Control	0	5	0.14±0.07	63.3±8.1
Test chemical	500	5	0.20±0.04	61.8±5.1
	1000	5	0.14±0.07	61.4±9.1
	2000	5	0.17±0.14	62.9±5.0
MMC	1	5	3.66±0.94**	56.0±8.1

**:p < 0.01, significant difference from control (Kastenbaum and Bowman method, upper-trailed).

Control: negative control (0.5% Carboxymethylcellulose sodium, 10 ml/kg).

MMC: positive control (Mitomycin C, dose once a day, for 2 days, i.p., 1 days after administration).

^aPolychromatic erythrocytes possessing one or more than one micronuclei (MNPCEs) were counted.

Applicant's summary and conclusion

Conclusions:

In a Mammalian Erythrocyte Micronucleus Test, the frequency of MNPCEs in the positive control group was increased (p ≤ 0.01) in comparison with that in the negative control group. Thus, the test chemical was considered to be not mutagenic in nature.

Executive summary:

In a Mammalian Erythrocyte Micronucleus Test, the mutagenic nature of test chemical was evaluated in male CD1 mice (Crlj: CD1) mouse. The mice were dosed orally by gavage at a concentration of 500, 1000 and 2000 mg/kg bw. 0.5% carboxymethylcellulose sodium was used a vehicle and Mitomycin C was used as a positive control substance. 30 mice (6 mice /group) were dosed daily at an interval of 24 hrs for 2 days. Bone marrow cells were examined. The target tissues were treated to achieve cells, slide preparation of the cells was done and later the slide was used for electrophoresis. Frequencies of micro nucleated polychromatic erythrocytes (MNPCEs) in treatment and positive control groups were compared with those in the negative control group using conditional binomial tests.In micronucleus tests, no significant differences in MNPCEs were found between test chemical treatment groups and the negative control group. The frequency of PCEs, which offers an index of the influence of the test substance on bone marrow cells, did not differ between any of the treatment groups and the negative control group. The frequency of MNPCEs in the positive control group was increased (p ≤ 0.01) in comparison with that in the negative control group. No deaths, clinical signs and body weight changes were observed in control and treatment groups. Thus the test chemical was considered to be not mutagenic in nature.