Sodium 4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino]toluene-3-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

Study period:

From 31 November 1993 to 4 January 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Adopted 21st July 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

other: in vivo micronucleus test, limit dose test

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino]toluene-3-sulphonate

EC Number:

224-618-7

EC Name:

Sodium 4-[(9,10-dihydro-4-hydroxy-9,10-dioxo-1-anthryl)amino]toluene-3-sulphonate

Cas Number:

4430-18-6

Molecular formula:

C21H15NO6S.Na

IUPAC Name:

sodium 2-[(4-hydroxy-9,10-dioxo-9,10-dihydroanthracen-1-yl)amino]-5-methylbenzenesulfonate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by l'Oreal, batch No. 2060208
- Expiration date of the lot/batch: not specified
- Purity test date: 13 January 1993

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored in smoked glass flask at room temperature
- Stability under test conditions: no information
- Solubility and stability of the test substance in the solvent/vehicle: no information
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no information

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test substance was suspended in the vehicle at a concentration of 100 mg/ml and homogenized using a magnetic stirrer before and during administration. The preparations were made immediately before use.

Test animals

Species:

mouse

Strain:

Swiss

Remarks:

OF1/ICO: OF1 (IOPS Caw)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Iffa Crédo (France)
- Age at study initiation: 6 weeks
- Weight at study initiation: male : 32 to 37g ; female 23 to 31 g
- Assigned to test groups randomly: yes
- Fasting period before study: not specified
- Housing: groups of 5 mice were housed (same sex and group) in polycarbonate cages (33.5 x 18.7 x 13.0 cm) and each cage contained autoclaved sawdust
- Diet (e.g. ad libitum): A04C pelleted diet (U.A.R, France) ad libitum
- Water (e.g. ad libitum): filter tap water (0.22 micron filter, Millipore, France) contained in bottle, ad libitum
- Acclimation period: 5-day acclimatization

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2 deg Celsius
- Humidity (%): 50±20% relative humidity
- Air changes (per hr): filtered and non recycled air, no more details
- Photoperiod (hrs dark / hrs light): 12h/12h

IN-LIFE DATES: From: 2 December 1993 To: 9 December 1993

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: distilled water (C.I.T., millipore)
- Justification for choice of solvent/vehicle: no information
- Concentration of test material in vehicle: 100 mg/ml
- Amount of vehicle (if gavage or dermal): 20 ml/kg
- Type and concentration of dispersant aid (if powder): magnetic stirrer
- Lot/batch no. (if required): not specified
- Purity:not specified

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was suspended in the vehicle at a concentration of 100 mg/ml and homogenized using a magnetic stirrer before and during administration. The preparations were made immediately before use.

Duration of treatment / exposure:

24 and 48 hours

Frequency of treatment:

Each animal was given the test substance once

Post exposure period:

24 and 48 hours

Doses / concentrations

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

3 animals per sex per dose were used for preliminary study (total of 6) and 5 animals per dose per group were used for main test

Control animals:

yes

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): no justification
- Route of administration: oral gavage
- Doses / concentrations: 2.5 mg/ml dissolved in distilled water

Examinations

Tissues and cell types examined:

bone marrow in femur,erythrocytes examined

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: During the preliminary test , the administration of 2000 mg/kg of test substance induced no clinical signs nor mortality. Consequently, the dose of 2000 mg/kg, being the maximum dose requested by international guidelines, was selected for the cytogenetic study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):The test substance was administered by the oral route using a dose volume of 20 ml/kg. Each animal was dosed once. At the end of 24 hours or 48 hours of treatment, all the animals were sacrified by CO2 inhalation in excess. The femurs of the mice were removed and the bone marrow eluted out using fetal calf serum. After centrifugation, the supernatant was removed and the cells in sediment were suspended by shaking.

DETAILS OF SLIDE PREPARATION: After centrifugation, a drop of the resuspended cells was placed and spread on a slide. The slides were air-dried and stained with May-Grünwald-Giemsa. Two slides per animal were prepared, but only one was used for scoring. All the slides were coded for scoring

METHOD OF ANALYSIS: For each animal, the micronuclei were counted in 2000 polychromatic erythrocytes; the polychromatic erythrocytes (PE) and normochromatic (NE) erythrocytes ratio was established by scoring a total of 1000 erythrocytes (PE+NE)

Evaluation criteria:

The following criteria were used as an aid for determining a positive response:
-a statistically significant increase in the number of MPE for at least one of the sampling times when compared to the vehicle group
-this increase should be double the number of MPE of historical data

Statistics:

At each sampling time, the mean number of micronucleated polychromatic erythrocytes (MPE) and the PE/NE ratio from the treated groups were compared simultaneous vehicle groups. The intergroup comparison was performed using : for MPE the Chi-2 test, for the PE/NE ratio, the Student's "t" test, in which p=0.05 was used as the lowest level of significance.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

effect observed at 48 hours

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In all group treated with the registered item Acid Violet 43, the mean values of micronucleated polychromatic erythrocytes were similar to those of their respective vehicle groups at each sampling time, and no, statistically significant differences were observed. Moreover, the PE/NE ratio decreased significantly (p<0.05) from that respective vehicle control group at the 48 hour sampling time showing the toxic effect of this substance to bone marrow cells.

Any other information on results incl. tables

Table 1 Datasummary

Group	Doses	MPE/PE		PE/NE ratio		Time of sacrifice
	(mg/kg)	Mean	standard deviation	Mean	standard deviation	hours
Vehicle	/	1.1	1	0.9	0.1	24
Test substance	2000	1.8	0.8	0.8	0.2	24
CPA	50	34.3***	8.4	0.9	0.1	24
Vehicle	/	1.7	0.8	1.1	0.3	48
Test substance	2000	0.9	0.9	0.8*	0.2	48

 

*p<0.05

***p<0.001

CPA cyclophosphamide

Applicant's summary and conclusion

Conclusions:

Under experimental conditions of this study, the test substance Acid Violet 43 did not induce cytogenetic damage to bone marrow cells of mice when treated by oral route at 2000 mg/kg in the micronucleus test.

Executive summary:

This GLP-compliant study aims the potential aneuploidy effect of the registered substance Acid Violet 43 in mice, precisely potential genetic impact on bone marrow cell when treated orally in OECD 474 followed method for in vivo micronucleus test.

Swiss mice received a single oral dose of 2000 mg/kg bw Jarocol Violet 43. This dose level was not associated with any signs of toxicity. An additional positive control group of 5 mice/sex was given a single oral dose of cyclophosphamide (CPA) at 50 mg/kg. Animals from test item or vehicle control groups were killed either 24 or 48 hours after dosing, whereas CPA-treated animals were killed 24 hours after dosing. For each animal, smears were prepared from femur bone marrow and were scored blindly for the incidence of micronucleated polychromatic erythrocytes and for the polychromatic/normochromatic erythrocyte (PCE/NCE) ratio.

In all groups treated, the mean values of micronucleated polychromatic erythrocytes were similar to those of their respective vehicle group at each sampling time and no statistically significant differences were observed. At the 48-h sampling time, the PCE/NCE ratio was lower than in controls but this difference was mainly due to a high control value and does not clearly indicate bone marrow toxicity of the test substance.

Under experimental conditions of this study, the test substance Acid Violet 43 did not induce cytogenetic damage to bone marrow cells of mice when treated by oral route at 2000 mg/kg in the micronucleus test.