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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 26 April 1993 to 10 May 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

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 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

Dated : 23 May 1983

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus 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: in smoked glass flask stored 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 dissolved in the solvent at concentrations from 28 to 280 mg/ml, depending on the highest concentration selected. The preparations were made immediately before use.

Method

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: donated by 2 healthy donors (one man and one woman)
- Suitability of cells: Human lymphocytes are chosen for cytogenetic assays because aberrations can be easily scored, their karyotype is stable (46 chromosomes) and the assays are reproducible.
- Cell cycle length, doubling time or proliferation index: cell cycle time of 12-14 hours
- Sex, age and number of blood donors if applicable: one man and one women, no more details
- Whether whole blood or separated lymphocytes were used if applicable:whole blood was used
- Methods for maintenance in cell culture if applicable: RPMI 1640 medium containing 20% fetal calf serum, L Glutamine, penicillin, streptomycin and fungison, PHA (phytohaemaglutinin was used as mitogen activator
- Modal number of chromosomes: 46
- Normal (negative control) cell cycle time: 12-14 hours

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 medium containing 20% fetal calf serum, L Glutamine, penicillin, streptomycin and fungison, PHA. Tubes were placed at 37 deg Celsius, in a humidified atmosphere of 5% CO2/95% air
- Properly maintained: not specified
- Periodically checked for Mycoplasma contamination: not specified
- Periodically checked for karyotype stability: not specified
- Periodically 'cleansed' against high spontaneous background: not specified

Cytokinesis block (if used):

colcemid solution 0.2 µg/mL

Metabolic activation:

with and without

Metabolic activation system:

S9 mix prepared from a liver microsomal fraction (S9) of rats induced with Aroclor 1254

Test concentrations with justification for top dose:

The concentrations of the test item Acid Violet 43 for chromosomal aberrations scoring were 500, 250 and 125 µg/mL without S9 and 625, 312.5 and 156.25 µg/mL with S9 mix in the first test. 500, 375 and 125 µg/mL were used without S9 mix and 750, 500 and 125 µg/mL with S9 mix were used in the repeat test. The highest concentration being the concentration which, in each case, showed moderate to marked toxicity.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water
- Justification for choice of solvent/vehicle: no information

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 2 hours in presence of S9 mix (first and repeat test), 24 hours without S9 mix (first test), 24 and 48 hours without S9 mix (first and repeat tests)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours (first experiment), 24 and 48 hours (repeat test)

SPINDLE INHIBITOR (cytogenetic assays): colcemid solution 0.2 µg/mL

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 cultures per dose group

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:The cells were centrifugated and the medium removed. After hypotonic treatment (KCl 0.075M), the cells were fixed in a methanol/acetic acid mixture (3/1 ; v/v), spread on glass slides and stained with Giemsa. At least 2 slides per culture were prepared. All slides were coded for scoring.

NUMBER OF CELLS EVALUATED: The number of cells in mitosis was evaluated on a total of 1000 cells.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 200 metaphases/concentration were analysed whenever possible; for the positive controls, only 100 metaphases/concentration were scord and only for the first harvest time.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
- Any supplementary information relevant to cytotoxicity: Mitotic Index = (number of cells in mitosis/number of cells examined)

OTHER EXAMINATIONS:
- Determination of polyploidy: The numerical aberrations (polyploidy) were recorded only for the later harvest time.

Evaluation criteria:

The following critera were used as an aid for determining a positive response:
-a reproducible and statistically significant increase in the aberrant cells frequency for at least one of the tested concentration.
-A test substance was considered as non-clastogenic in this test system if there is no significant increase in aberrant cells frequency at any dose above concurrent control frequencies and in both of the two harvest tomes. Both biological and statistical significance was considered together in the evaluation.

Statistics:

For each test and for each harvest time, the aberrant cells frequency (excluding gaps) in treated cultures was compared to that of the negatives cultures. The comparison was performed using the Chi-2 test in which p=0.05 was used as the lowest level of significance.

Results and discussion

Test results

Test results 1

Species / strain:

lymphocytes: human

Remarks:

first test and repeat test, at 24 hours harvest time

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

lymphocytes: human

Remarks:

48 hours harvest time (Repeat test)

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

lymphocytes: human

Remarks:

48 hours harvest time (repeat study)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: In the first experiment, concentrations used were 5000, 2500, 1250, 625, 312.5 and 156.25 µg/mL. without and with S9 mix. At 5000, 2500, 1250, the mitotic index was nul, hence the high concentrations used were 625 µg/mL (with S9 mix) and 500 µg/mL (reducing 70% of control mitotic index)

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: not specified

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: not reported
- Negative (solvent/vehicle) historical control data: not reported

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: mitotic index used (number of cells in mitosis / number of cells observed)
- Other observations when applicable:metaphases counting

Any other information on results incl. tables

Table 1 :Summaryof theresults

		numberof aberrations		% aberrantcells
	Concentration µg/mL	+ Gap	- Gap	+ Gap	- Gap	Mitotic Index
First experiment	0	0	0	0	0	3.7
With metaboli cactivation	156.25	2	0	1	0	3.2
	312.5#	0	0	0	0	1.1
	625#	8	4	4.7	2	1.8
	CPA	39	35	24	24***	1.8
First experiment	0	3	3	1.5	1.5	2.6
With metabolic activation	125	1	1	0.5	0.5	3.9
	250#	8	5	4	2.9	2.7
	250 #	3	2	1.7	1.1	1.9
	500	2	2	1.7	1.7	0.7
	MMC	50	49	29	29***	2.5
Repeatexperiment	0	8	0	3.5	0	2.7
Withoutmetabolicactivation	125	4	0	1.5	0	1.9
24hoursharvestingtime	375	5	3	3.4	2	1.1
	500	5	2	1.8	1.2	0.9
	MMC	46	39	37.2	34.9***	1.3
Repeat experiment	0	7	1	3.5	0.5	3.7
With metabolic activation	125	1	1	0.5	0.5	2.5
24hours harvesting time	500	7	3	3.5	1.5	1.9
	750	7	3	3.7	1.8	1.4
	CPA	59	46	37.3	32.5	2.1
Repeat experiment	0	3	2	0.5	0.5	2.6
Without metabolic activation	125	4	0	2	0	1.3
48 hours of harvesting time	375	12	54	5.5	2.5	0.8
	500	20	12	7.3	5.1***	1.4
Repeat experiment	0	4	1	2	0.5	3.1
With metabolic activation	125	3	0	1.5	0	3.3
48hours of harvesting time	500 #	1	1	2	1	0.4
	750	12	8	5.5	4.0*	0.7

 

*** p<0.001

# 180 metaphases counted

Applicant's summary and conclusion

Conclusions:

Under experimental conditions of this study, the test item Acid Violet 43 induced moderate clastogenic effect on human lymphocytes in 48 hours period of treatment at the highest concentration with and without metabolic activation. The treatment period was for 24 and 48 hours on the repeat test.

Executive summary:

This GLP-compliant study was performed to assess the potential clastogenicity of the registered substance Acid Violet 43 in human lymphocytes according in vitro cytogenicity test and OECD 473 method.

Acid Violet 43 was investigated in two independent experiments in the absence and presence of metabolic activation system. Duplicate cultures were treated with each concentration of Acid Violet 43 or with known clastogens in the presence (cyclophosphamide) or absence of S9 (mitomycin C). The concentrations of the test item Acid Violet 43 for chromosomal aberrations scoring were 500, 250 and 125 µg/mL without S9 and 625, 312.5 and 156.25 µg/mL with S9 mix in the first test. 500, 375 and 125 µg/mL were used without S9 mix and 750, 500 and 125 µg/mL with S9 mix were used in the repeat test. The highest concentration being the concentration which, in each case, showed moderate to marked toxicity. In both experiments, continuous treatment (until harvesting) was performed in the absence of S9 mix, whereas pulse (2-hour) treatment was performed in the presence of S9 mix. Cells were harvested 24 hours after the beginning of treatment in both experiments and additionally at 48 hours in the second experiment. Chromosome preparations were stained and examined microscopically for mitotic index and for aberrations when selected.

For the 24-h harvest time the test substance did not induce any significant increase in the aberrant cells frequency, with and without metabolic activation in both experiments. For the 48-h harvest time performed during the repeat test, a significant increase in the aberrant cells frequency was recorded in the 2-h treatment group at the highest concentration (750 μg/ml) with metabolic activation system as well as in the 48-h treatment group at the highest concentration (500 μg/ml) without metabolic activation system. Aberrations consisted almost of chromatid deletions, and the proportion of cells bearing numerical aberrations was also slightly increased. These changes were observed together with a reduction in mitotic index to 46% and 52 % of controls.

Under experimental conditions of this study, the test item Acid Violet 43 induced moderate clastogenic effect on human lymphocytes in 48 hours period of treatment at the highest concentration with and without metabolic activation system.