Dialuminium tris[2-(2,4,5,7-tetrabromo-6-oxido-3-oxoxanthen-9-yl)benzoate]

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Data source

Reference

Reference Type:

publication

Title:

Micronucleus Tests in Mice on 39 Food Additives and Eight Miscellaneous Chemicals

Author:

M. Hayashi, M. Kishi, T. Sofuni And M. Ishidate, Jr

Year:

1988

Bibliographic source:

Food Chem Toxicol., 26(6), 487-500

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

Micronucleus test of Phloxine (Acid Red 92) in mice

GLP compliance:

not specified

Type of assay:

other: In vitro mammalian micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Dipotassium 3,6-dichloro-2-(2,4,5,7-tetrabromo-6-oxido-3-oxoxanthen-9-yl)benzoate

Cas Number:

6441-77-6

Molecular formula:

C20H6Br4Cl2O5.2K

IUPAC Name:

Dipotassium 3,6-dichloro-2-(2,4,5,7-tetrabromo-6-oxido-3-oxoxanthen-9-yl)benzoate

Test material form:

solid: particulate/powder

Details on test material:

- Name of the test material: Food Red No.104 (phloxine)
- IUPAC name: Dipotassium 3,6-dichloro-2-(2,4,5,7-tetrabromo-6-oxido-3-oxoxanthen-9-yl)benzoate
- Molecular weight: 792.9646 g/mol
- Molceular Formula: C20H6Br4Cl2O5.2K
- Substance type: Organic
- Purity: No data

Specific details on test material used for the study:

- Name of the test material: CI Food Red 92
- IUPAC name: Dipotassium 3,6-dichloro-2-(2,4,5,7-tetrabromo-6-oxido-3-oxoxanthen-9-yl)benzoate
- Molecular weight: 792.9646 g/mol
- Molceular Formula: C20H6Br4Cl2O5.2K
- Substance type: Organic
- Purity: No data

Test animals

Species:

mouse

Strain:

other: ddY

Details on species / strain selection:

No data

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Shizuoka Agriculture Cooperative Association for Laboratory Animals, Shizuoka
- Age at study initiation: 8 weeks
- Weight at study initiation: No data available
- Fasting period before study: No data available
- Housing: No data available
- Diet (e.g. ad libitum): CE-2 food pellets, ad libitum
- Water (e.g. ad libitum): Water, ad libitum
- Acclimatization period: No data available

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

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: Saline
- Justification for choice of solvent/vehicle: The test chemical was soluble in saline
- Concentration of test material in vehicle: 0, 30, 60 or 120 mg/Kg
- Amount of vehicle (if gavage or dermal): No data
- Type and concentration of dispersant aid (if powder): No data
- Lot/batch no. (if required): No data
- Purity: No data

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Prior to treatment, the test compound was dissloved in saline at dose levels of 0, 30, 60 or 120 mg/Kg

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

VEHICLE
- Justification for use and choice of vehicle (if other than water): Saline
- Concentration in vehicle: 0, 30, 60 or 120 mg/kg
- Amount of vehicle (if gavage): No data available
- Lot/batch no. (if required): No data available
- Purity: No data available

Duration of treatment / exposure:

24 hours (when exposed to only one injection) or 96 hours (when injected 4 times with 24 hrs apart)

Frequency of treatment:

Once ((when exposed to only one injection) or 4 times (with 24 hrs apart each injection)

Post exposure period:

No data available

Doses / concentrations

Remarks:

Doses/Concentrations: 0, 30, 60 or 120 mg/kg (when injected only once) or 60 mg/kg (when injected 4 times 24 hrs apart)

No. of animals per sex per dose:

Total: 30 mice
Control: 6 males
30 mg/kg: 6 males
60 mg/kg: 12 males
120 mg/kg: 6 males

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin C
- Justification for choice of positive control(s): No data
- Route of administration: Intraperitoneal
- Doses / concentrations: 2.0 mg/Kg

Examinations

Tissues and cell types examined:

Femoral cells were observed for the presence of micronucleated polychromatic erythrocytes (MNPCEs)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The maximum doses of the test compounds were determined by pilot experiments using the multisampling at multi-dose levels method. For some chemicals, which had not been subjected to the pilot experiments, the maximum dose levels were set at the supposed maximum tolerated dose referring to the LDs0

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 24 hrs or 96 hrs

DETAILS OF 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 experiment and with Giemsa for the full-scale test.

METHOD OF ANALYSIS: The preparations were coded and analysed without any knowledge of the treatment. One thousand polychromatic erythrocytes per mouse were scored using a light microscope, with a high power objective (x 100), and the number of micronucleated polychromatic erythrocytes (MNPCEs) was recorded. The proportion of polychromatic erythrocytes (PCEs) among the total erythrocytes was also evaluated by observing 1000 erythrocytes on the same slide.

OTHER: No data

Evaluation criteria:

One thousand polychromatic erythrocytes per mouse were scored using a light microscope, and the number of micronucleated polychromatic erythrocytes (MNPCEs) was recorded. The proportion of polychromatic erythrocytes (PCEs) among the total erythrocytes was also evaluated by observing 1000 erythrocytes on the same slide.

Statistics:

A two-stage statistical procedure was used. In the first step of the procedure, the frequency of MNPCEs in each treatment group was compared with the binomial distribution specified by historical control data from the laboratory where the test was performed. In the second step, the dose-response relationship was tested by the Cochran-Armitage trend test. A positive result was recorded only when one or more treatment group(s) showed a statistically significant difference from the spontaneous level of MNPCEs and the trend test indicated a positive dose response. This procedure was not applied to tests with multiple treatments which, in most cases, contained only one dose group. Such data were evaluated statistically using the first step only.

A Monte-Carlo simulation study showed that the probability of a type I error (the probability of a false positive), in this two-step procedure was, in general, closer to the nominal significance level than that of the usual conditional binomial testwhich has been widely used to evaluate micronucleus test data. Similarly, this method was a more powerful method of analysis than the conditional binomial test.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No mutagenic potential

Additional information on results:

No data

Any other information on results incl. tables

Table: Results of the micronucleus test using mouse bone marrow cells

Chemical	Vehicle	Route	No. of doses	Time between doses (hr)	Sampllng time (hr)	Dose level (mg/Kg)	MNPCE (%)	PCE (%)	Mortality	Trend test
CI Food red 92	Saline	ip	1		24	0	0.20±0.13	56.9 ± 5.6	0/6	NS
						30	0,22 ± 0.18	55.3 ± 5.2	0/6
						60	0.23 ± 0.14	40.9 ± 16.5	0/6
						120	0.08±0.11	37.7 ± 7.0	0/6
						MMC 2.0	6.48 ± 1.87*	37.8 ± 6.1	0/6
		ip	4	24	24	60	0.25 ± 0.12	67.0 ± 5.5	0/6

Applicant's summary and conclusion

Conclusions:

Food Red 92 (Phloxine) is considered to be non-mutagenic in male ddY mice since no mutagenic effects were seen in a micronucleus test.

Executive summary:

In the in vivo micronucleus test performed Acid Red 92 (Phloxine) was investigated in male ddY mice for mutagenicity. The test chemical was administered by intraperitoneal injection once (at doses 0, 30, 60 or 120 mg/kg) or 4 times 24 hours apart (at a dose of 60 mg/kg/injection). 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 experiment and with Giemsa for the full-scale test. One thousand polychromatic erythrocytes per mouse were scored using a light microscope. After treatment, the number of micronucleated polychromatic erythrocytes (MNPCEs) was recorded and the proportion of polychromatic erythrocytes (PCEs) among the total erythrocytes was evaluated. Based on the results, no mutagenic effects could be detected. Therefore, Acid Red 92 is considered to be non-mutagenic when male ddY mice were exposed to the test chemical.