Sodium 4-[[3-(acetylamino)phenyl]amino]-1-amino-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance was evaluated for genotoxic potential in the standard Ames/salmonella gene mutation test and in the in vitro mammalian cell gene mutation test (HPRT-locus) and the in vitro cytogenicity chromosome aberration study. The test substance was ambiguous in the Ames test but negative in the in vitro mammalian gene mutation test. In the in vitro mammalian chromosome aberration test with a structural analogue, the test substance showed no clastogenic or aneugenic effect.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000-09-26 - 2000-10-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

The test compound was suspended in deionized water and a stock solution of 50 mg/mL was prepared for the highest concentration, which provided a final concentration of 5000 µg/plate. Further dilutions of 1600, 500, 160 and 50 µg/plate were used in the first plate incorporation test.
In the second experiment with the strain TA 98 dilutions of 5000, 1600, 500, 160, 50, 16, 5 and 1.6 µg/plate were chosen.
Visible precipitation of the test compound on the plates was observed at 1600 µg/plate and above.
Because of heavy precipitation of the test compound the bacterial lawn could only be evaluated at the dose level of 1600 µg/plate and lower concentrations.
In the first plate incorporation test toxicity was observed at a concentration of 1600 µg/plate in the absence of metabolic activation with the tester strain TA 100.

Vehicle / solvent:

Solvent: deionised water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation)

Evaluation criteria:

Assay considered valid if:
-Solvent control data are within the laboratory´s normal control range for the spontaneous mutant frequency
-Positive controls induced increases in the mutation frequency which were both statistically significant and within the laboratory´s normal range

Test compound is classified as mutagenic if has either following effects:
-it produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
-it induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn.

If test substance does not achieve either of the above criteria, it is considered to show no evidence of mutagenic activity in this system

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: -
- Effects of osmolality: -
- Evaporation from medium: -
- Water solubility: > 10 g/L
- Precipitation: yes; from 1600 µg/plate onwards
- Definition of acceptable cells for analysis: -
- Other confounding effects:


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: yes
- Negative (solvent/vehicle) historical control data: yes

Remarks on result:

other: no dose-dependency observed

Conclusions:

In the presence of metabolic activation, treatment of the bacterial strain with Acid Blue 324 resulted in increases in the number of revertant colonies with the strain TA 98 without showing any dose-dependency; hence not meeting the criteria for a positive result.

Executive summary:

Acid Blue 324 was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 98 and TA 102 of Salmonella typhimurium. This mutagenicity study was conducted (plate incorporation test), in the absence and in the presence of a metabolizing system derived from a rat liver homogenate. Additionally a repeat of the plate incorporation test was performed with the strain TA 98 in the presence of S9-mix.

For both studies, the compound was suspended in deionized water, and each bacterial strain was exposed to 5 dose levels, in the repetition with the strain TA 98 to 8 dose levels.

Visible precipitation of the test compound on the plates was observed at 1600 µg/plate and above. The concentrations for the first plate incorporation test were 50, 160, 500, 1600 and 5000 µg/plate. Because of the increased mutation frequencies (2.1 to 5.2 -fold without dose-dependency) observed with the strain TA 98, dose levels from 1.6 to 5000 µg/plate were chosen for the second plate incorporation test with TA98 in the presence of the metabolizing system.

Control plates without mutagen showed that the number of spontaneous revertant colonies was within the laboratory's historical control range. All the positive control compounds showed the expected increase in the number of revertant colonies.

In the first plate incorporation test toxicity was observed at a concentration of 1600 µg/plate in the absence of metabolic activation with the tester strain TA 100.

In the presence of metabolic activation, treatment of the bacterial strain with Acid Blue 324 resulted in increases in the number of revertant colonies with the strain TA 98 without showing any dose-dependency; hence not meeting the criteria for a positive result.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Nov 2000 to Feb 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

OECD Guideline For Testing Of Chemicals, 476 "Genetic Toxicology: In vitro Mammalian Cell Gene Mutation Test". Adopted: July 21st, 1997

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

U.S. Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.5300, In Vitro Mammalian Cell Gene Mutation Test, August 1998

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

EEC-Direktive 2000/32/EC, L136, Annex 4E, B.17: "Mutagenicity — In Vitro Mammalian Cell Gene Mutation Test". Adopted: May 19th, 2000

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: cell bank of "Genetic Toxicology", Aventis Pharma Deutschland GmbH, ProTox
Large stocks of the mycoplasma-free V79 cell line are stored in liquid nitrogen in the cell bank of "Genetic Toxicology", thus permitting repeated use of the same cell culture batch for numerous experiments. The identical characteristics of the cells ensure comparability of the experimental parameters.
Thawed stock cultures were kept at approx. 37 °C and approx. 4 % CO2 in 175 cm² plastic flasks. About 5 x 105 to 1 x 106 cells were seeded into each flask in 30 ml of MEM-medium supplement with approx. 10 % (v/v) FCS (fetal calf serum) containing approx. 2 mM L-glutamine and approx. 0.1 % (w/v) neomycinsulfate. The cells were subcultured twice a week.
For the selection of mutants the medium was supplemented with approx. 11 µg/ml thioguanine.


MEDIA USED
- Type and identity of media including CO2 concentration if applicable: MEM (minimal essential medium) with Hanks salts and 25 mM Hepes-buffer; approx. 37 °C and approx. 4 % CO2 in plastic flasks
- Properly maintained: [yes]
- Periodically checked for Mycoplasma contamination: [yes]
- Periodically 'cleansed' against high spontaneous background: [yes]

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

Concentration range in the main test (with and without metabolic activation): 25 to 4730 µg/mL

In a preliminary experiment for solubility Acid Blue 324 was studied with respect to its solubility in MEM Hank's cell culture medium. The concentrations at which visible precipitation was observed, was found at 250 to 4730 µg/mL. This experiment showed that 4730 µg/mL was the highest practicable concentration and corresponds to 10 mM, which is the highest dose level tolerated by the test system.
Accordingly, the preliminary toxicity study was carried out using a maximum concentration of 4730 µg/mL and a range of lower dose levels down to 10 µg/mL.
Following treatment in the absence of S9 metabolic activation, moderate toxicity was observed. Survival declined in a dose-related manner reaching 41.5% of the solvent control value at the highest dose level.
In the presence of S9 metabolic activation at a concentration of 4730 µg/mL survival was reduced to 64.4% of the solvent control value.
Based on these results 4730 µg/mL was selected as the maximum dose level for the main mutation experiments in both the absence and in the presence of S9-mix. Seven lower concentrations down to 25 µg/mL were also included.

Vehicle / solvent:

culture medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Exposure period (with metabolic activation): 4 hours
Exposure period (without metabolic activation): 4 hours

Expression time:
7 Tage

Selection time:
7 Tage

Evaluation criteria:

The assay is considered valid if the following criteria are met:
- The solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency
- The positive controls induced increases in the mutation frequency which were both statistically significant and within the laboratory's normal range
- The plating efficacy for the solvent control was greater than 50 %

The test compound is classified as mutagenic if:
- It reproducibly induces with one of the test compound concentrations a mutation frequency that is three times higher than the spontaneous mutant frequency in this experiment.
- There is a reproducible concentration-related increase in the mutation frequency. Such an evaluation may be considered independently from the enhancement factor for induced mutants.
- Survival of the responding dose group is at least 30 %.

However, in a case by case evaluation both decisions depend on the level of the corresponding negative control data.

Statistics:

The biometry of the results for the test compound is performed off-line with the MANN-WHITNEY-U-TEST

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Before treatment, the pH values and osmolality of the treatment media were determined. The addition of test compound solutions did not have any substantial effect on these parameters.

Only in the first independent experiment astatisticallysignificant enhancement of the mutation rate over the range of the solvent controls was found without metabolic activation at the concentrations of 100 µg/mL and 500 µg/mL of the test compound. This effect was not reproducible, not dose-dependent and not three fold higher than the solvent control values and therefore considered to be without biological relevance.

The criteria for a positive response were not fulfilled.

Conclusions:

Acid Blue 342 does not induce gene mutations in the HPRT-test with V79 Chinese hamster cells, both in the presence as well as in the absence of a metabolic activation system, under the experimental conditions described. Hence, Acid Blue 342 is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The study was performed to investigate the potential of Acid Blue 342 to induce gene mutations at the HPRT locus in V 79 cells of the Chinese hamster in vitro.

Two independent experiments were conducted both with and without an exogenous rat liver microsomal activation system (S9-mix).

The compound was suspended in MEM Hank's cell culture medium and tested at the following concentrations:

withoutS9:     mix:     25, 50, 100, 250, 500, 1000, 2500 and 4730* µg/mL

withS9-mix:    25, 50, 100, 250, 500, 1000, 2500 and 4730* µg/mL

* = 10mM

The concentration ranges were based on the results of preliminary tests for solubility and toxicity. The highest concentration showed toxic effects with and without metabolic activation.

Higher concentrations were not used because of the 10mMlimitation (OECD guideline).

Precipitation of the test compound was observed in a dose range of 250 to 4730 µg/mL.

Up to the highest investigated dose no relevant increase in mutant colony numbers was obtained in two independent experiments,

Only in the first independent experiment a statistically significant enhancement of the mutation rate over the range of the solvent controls was found without metabolic activation at the concentrations of 100 µg/mL and 500 µg/mL of the test compound. This effect was not reproducible, not dose-dependent and not three fold higher than the solvent control values and therefore considered to be without biological relevance.

Appropriate reference mutagens used as positive controls showed a distinct increase in induced mutant colonies, thus indicating the sensitivity of the assay and the efficacy of the S9-mix.

In conclusion, Acid Blue 342 does not induce gene mutations in the HPRT-test with V79 Chinese hamster cells, both in the presence as well as in the absence of a metabolic activation system, under the experimental conditions described.

Acid Blue 342 is therefore considered to be non-mutagenic in this HPRT assay.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

92/69/EWG, B.10 (Säuger zytogenetischer in vitro-Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from rat liver

Test concentrations with justification for top dose:

Preparation time 18 h:
with S9: 500, 1000, 2000 µg/mL
without S9: 125, 250, 500 µg/mL


Preparation time 6 and 28 h:
with S9: 2000 µg/mL
without S9: 500 µg/mL

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Analysis of metaphases
After the slides had been coded, 100 metaphases per experimental group were examined. The set of chromosomes was examined for completeness and the various chromosomal aberrations were assessed. Only metaphases with 22+/- 1 chromosomes are included in the analysis. The metaphases were examined for the following aberrations: gap (G), break (8), fragment (F), minute (M), deletion (D), exchanges including intrachanges (Ex), dicentrics (Di), chromosome disintegration (CD) and ring formation (RI). In addition, metaphases with 5 and
more aberrations were classified separately as multiple aberrations (rnA).
After the metaphases had been evaluated, the code was lifted. The values for the control group were compared with the results from the dose group and the positive control at each preparation time.

Evaluation criteria:

The evaluation of the results was performed as follows:
- The test substance is classified as mutagenic if it induces a significantly increased aberration rate as compared with the negative controls with one
of the concentrations tested . The significance is obvious either by an enhancement of the rate clearly exceeding the control range or it is proven
by adequate biometry (Binomial statistic with Fisher's exact test).
- The test substance is classified as mutagenic if there is a reproducible concentration related increase in the aberration rate.
- The test substance is classified as not mutagenic when it tests negatively both with and without metabolic activation.

Statistics:

Fisher - Exact test

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Solubility and toxicity
In a preliminary experiment the test substance was assayed with respect to its solubility in cell culture medium. The highest concentration at which no visible precipitation was observed, was found to be 2000 µg/ml.
The cytotoxicity experiment proved that the test substance was toxic to the V79 cells in the absence of metabolic activation (S9-mix) from 500 µg/ml up to the limit of solubility of 2000 µg/ml. And in the presence of metabolic activation (S9-mix) no indication of toxicity was observed up to the limit of solubility.
On the basis of these results the preparation of chromosomes was done after 2 h treatment with 500 µg/ml at 6, 18 and 28 hand 18 hours after treatment with 125 and 250 µg/ml without S9-mix; as also with 2000 µg/ml at 6, 18 and 28 hours and18 hours after treatment with 500 and 1000 µg/ml with S9-mix.
A cytotoxic effect was observed as a decrease of the mitotic index in all dose groups at the preparation times 6 and 18 hours after treatment. No decrease of the mitotic index was observed at the preparation time 28 hours.

Mutagenicity
The test substance was assessed for its mutagenic potential in vitro in the chromosome-aberration-test with two independent cell cultures without metabolic activation and two independent cell cultures with metabolic activation.
No relevant reproducible enhancement of the chromosome aberration rate over the range of the negative control was found with any of the concentrations used, neither with, nor without metabolic activation by S9-mix. The sensitivity of the test system was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control substances.
The results lead to the conclusion that the test compound is not mutagenic in the chromosome aberration.

Conclusions:

The test substance is considered to be non-mutagenic in this chromosome aberration assay

Executive summary:

The test substance was examined for mutagenic activity in V79 Chinese hamster cells. The induction of chromosome aberrations after in vitro treatment was investigated in the presence and absence of a fraction of liver homogenate for metabolic activation (S9-mix).

A preliminary cytotoxicity experiment was performed in order to select appropriate dose levels for the mutagenicity study. The test substance produced a significant cytotoxic effect without metabolic activation from 500 µg/ml up to the limit of solubility (2000 µg/ml). No cytotoxic effect was observed with metabolic activation up to the limit of solubility. For mutagenicity testing two independent

cell cultures with and without metabolic activation (S9-mix) up to the limit of solubility (2000 µg/ml) were used.

For main experiment dose levels of 125, 250 and 500 µg/ml were used in the absence and 500, 1000 and 2000 µg/ml in the presence of 59-mix metabolic activation.

The test compound did not induce a significant increase in the number of chromosome aberrations at any preparation time and dose level of the test substance. But a cytotoxic effect of the compound was observed in the rnain experiments. Marked increases in the rate of chromosome aberrations were obtained with the positive control substances indicating the sensitivity of the assay.

In conclusion, the test substance does not induce chromosome mutations (= aberrations) in V79 Chinese hamster cells, neither in the presence nor in the absence of a metabolic activation system, under the experimental conditions described.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In the in vivo mouse micronucleus test with a structural analogue, the test substance showed no clastogenic or aneugenic effect.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species: NMRI mouse
Strain: Hoe: NMRKf (SPF71)
Origin: HOECHST AG, Kastengrund, SPF breeding colony
Initial age at test: 7 weeks
Number of animals: 70 (35 males / 35 females)
Bodyweight at start of study: males mean= 30.5 g (26 - 35 g); females: mean= 25.6 g (23 - 31 g)
Acclimatization: at least 5 days
Food / water: mice diet Altromin 1324 (Altromin-GmbH, Lage/Lippe), ad libitum; tap water in plastic bottles, ad libitum
Housing: 5/cage
Room temperature: 22 +/- 2°C
Relative humidity: 55 +/- 10%
Lighting time: 12 h/12 h light/dark cyclus

Route of administration:

oral: gavage

Vehicle:

deionised water

Details on exposure:

The dose levels for micronucleus testing were selected on the basis of a preliminary study to determine the acute toxicity and the maximal applicable dose. Oral administration of 5000 mg/kg bodyweight did not lead to a partial lethality in male and female mice. It is considered the maximal applicable dose and was selected as dose level for the main study.

The 5000 mg/kg bw dose was prepared as a 25% (w/v) solution and administerd at a volume of 20 mL/kg bw devided into two equal parts of 10 mL/kg bw administered within 2 hours

The test compound dilutions were prepared fresh each day. 6250 mg test substance were weight in a beaker, mixed with deionisized water, washed out in a 25 ml flask and topped up to the calibration mark. A suspension was formed.

Duration of treatment / exposure:

animals were killed after 24, 48 or 72 hours after administration of the test compound

Frequency of treatment:

The test compound was given in two equal parts within two hours

Dose / conc.:

5 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 for each killing time

Control animals:

yes, concurrent vehicle

Positive control(s):

endoxane, 50 mg/kg bw

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

Extraction of the bone marrow
In conformity with the test procedure the animals were killed by carbon dioxide asphyxiation 24, 48 or 72 hours after application. For each animal, about 3 ml foetal bovine serum was poured into a centrifuge tube. Both femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. The mixture was then centrifuged for 5 minutes at 1200 rpm and almost all the supernatant discarded. One drop of the thoroughly mixed sediment was smeared on a cleaned slide, identified by project code and animal number and air-dried for about 24 hours.
Staining procedure
- 5 minutes in methanol
- 3 minutes in May-Grünwalds solution
- 2 minutes in May-Grünwalds solution diluted 1:1 with distilled water
- brief rinsing twice in distilled water
- 10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2 (Weise)
- rinsing in distilled water
- drying
- coating with Entellan

Evaluation criteria:

1000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. As a control measure 1000 mature erythrocytes were also counted and examined for micronuclei. In addition, the ratio of polychromatic to normochromatic erythrocytes was determined. All bone marrow smears for evaluation are coded to ensure that the group to which they belonged remains unknown to the investigator. The number of polychromatic erythrocytes with micronuclei occurring in the 1000 polychromatic erythrocytes counted, and the number of normocytes with micronuclei occurring in the 1000 normocytes counted, were evaluated statistically; comparison
of dose groups with the simultaneous control group was performed according to Wilcoxon (paired, one-sided, increase).
The results of the treatment groups (test substance) in the micronucleus test at each dose and killing time were compared with corresponding control values. The ratio of polychromatic to normochromatic erythrocytes was also evaluated statistically by the method of Wilcoxon (paired, two sided).

Statistics:

The number of polychromatic erythrocytes with micronuclei occurring in the 1000 polychromatic erythrocytes counted, and the number of normocytes with micronuclei occurring in the 1000 normocytes counted, were evaluated statistically; comparison of dose groups with the simultaneous control group was performed according to Wilcoxon (paired, one-sided, increase).
The results of the treatment groups (test substance) in the micronucleus test at each dose and killing time were compared with corresponding control values. The ratio of polychromatic to normochromatic erythrocytes was also evaluated statistically by the method of Wilcoxon (paired, two sided). The statistical evaluations were performed using the I1Diamant l1 computer program Version 2.0, supplied by the Department of Information and Communication Hoechst AG. All statistical results are based on a 95 %level of significance. Actual data were also compared with historical controls.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

All animals survived after application of 5000 mg/kg bodyweight. The following signs of toxicity were observed: faeces dark blue coloured and diarrhea. 24 hours p.a. in the cages no. 3, 4, 9 and 14 the soft-wood granulate was red coloured.
48 hours after application all animals were free of clinical signs of toxicity.
The dissection of the animals revealed the following macroscopic findings: At the killing time 24 hours p.a. at the dosage 5000 mg Remazol-Brillantblau R FWTRG per kg bw in some male and females the content of appendix and colon was blue coloured.

The incidence of micronucleated polychromatic erythrocytes in the treated groups was within the normal range of the negative control groups. No statistically significant increase of micronucleated polychromatic erythrocytes has been observed. The number of normochromatic erythrocytes with micronuclei did not differ significantly from the values of the simultaneous control animals for each of the three killing times investigated. The ratio of polychromatic erythrocytes to normocytes remained essentially unaffected by the test compound.

Conclusions:

No clastogenic or aneugenic effects were noted

Executive summary:

The test substance was tested in the micronucleus test. The test compound was administered orally by gavage to male and female mice. The following doses were tested: 0 and 5000 mg per kg bodyweight.

The 5000 mg per kg bodyweight dose level was chosen since a preliminary study had shown it to be the maximum applicable dose.

The animals were treated once with the test compound and according to the test procedure the animals were killed 24, 48 or 72 hours after administration of the test compound.

The test compound was given in two equal parts within two hours and according to the test procedure the animals were killed 24, 48 or 72 hours after administration of the test compound.

EndoxanR was used as positiv control substance and was administered orally at a dose of 50 mg per kg bodyweight.

The incidence of micronucleated polychromatic erythrocytes of the animals treated with the test substance was within the normal range of the negative control. The number of normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment with the test substance and was statistically not different from the control values.

Endoxan(R) induced in both males and females a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the system. The ratio of polychromatic erythrocytes to normocytes was not changed to a significant extend.

The results indicate that, under the conditions of the present study, the test substance is not mutagenic in the micronucleus test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Acid Blue 324 was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 98 and TA 102 of Salmonella typhimurium. This mutagenicity study was conducted (plate incorporation test), in the absence and in the presence of a metabolizing system derived from a rat liver homogenate. Additionally a repeat of the plate incorporation test was performed with the strain TA 98 in the presence of S9-mix.

For both studies, the compound was suspended in deionized water, and each bacterial strain was exposed to 5 dose levels, in the repetition with the strain TA 98 to 8 dose levels.

Visible precipitation of the test compound on the plates was observed at 1600 µg/plate and above. The concentrations for the first plate incorporation test were 50, 160, 500, 1600 and 5000 µg/plate. Because of the increased mutation frequencies (2.1 to 5.2 -fold without dose-dependency) observed with the strain TA 98, dose levels from 1.6 to 5000 µg/plate were chosen for the second plate incorporation test with TA98 in the presence of the metabolizing system.

Control plates without mutagen showed that the number of spontaneous revertant colonies was within the laboratory's historical control range. All the positive control compounds showed the expected increase in the number of revertant colonies.

In the first plate incorporation test toxicity was observed at a concentration of 1600 µg/plate in the absence of metabolic activation with the tester strain TA 100.

In the presence of metabolic activation, treatment of the bacterial strain with Acid Blue 324 resulted in increases in the number of revertant colonies with the strain TA 98 without showing any dose-dependency.

Due to the increased mutation frequency in Salmonella typhimurium TA 98 in the presence of a metabolizing system, a study was performed to investigate the potential of Acid Blue 342 to induce gene mutations at the HPRT locus in V 79 cells of the Chinese hamster in vitroto be able to differentiate between a true mutagenic or a bacteria-specific effect. Two independent experiments were conducted both with and without an exogenous rat liver microsomal activation system (S9-mix).

The compound was suspended in MEM Hank's cell culture medium and tested at the following concentrations:

without S9-mix:     25, 50, 100, 250, 500, 1000, 2500 and 4730* µg/mL

with S9-mix:    25, 50, 100, 250, 500, 1000, 2500 and 4730* µg/mL

* = 10mM

The concentration ranges were based on the results of preliminary tests for solubility and toxicity. The highest concentration showed toxic effects with and without metabolic activation. Higher concentrations were not used because of the 10mM limitation (OECD guideline). Precipitation of the test compound was observed in a dose range of 250 to 4730 µg/mL. Up to the highest investigated dose no relevant increase in mutant colony numbers was obtained in two independent experiments. Only in the first independent experiment a statistically significant enhancement of the mutation rate over the range of the solvent controls was found without metabolic activation at the concentrations of 100 µg/mL and 500 µg/mL of the test compound. This effect was not reproducible, not dose-dependent and not three fold higher than the solvent control values and therefore considered to be without biological relevance. Appropriate reference mutagens used as positive controls showed a distinct increase in induced mutant colonies, thus indicating the sensitivity of the assay and the efficacy of the S9-mix.

In conclusion, Acid Blue 342 does not induce gene mutations in the HPRT-test with V79 Chinese hamster cells, both in the presence as well as in the absence of a metabolic activation system, under the experimental conditions described. Acid Blue 342 is therefore considered to be non-mutagenicin this HPRT assay.

Consequently, the non-dose-dependent higher mutation frequency in TA 98 in the presence of S9 is considered either an incidental or a bacteria-specific effect and hence not relevant for humans.

The structural analogue was examined for mutagenic activity in V79 Chinese hamster cells. The induction of chromosome aberrations after in vitro treatment was investigated in the presence and absence of a fraction of liver homogenate for metabolic activation (S9-mix).

A preliminary cytotoxicity experiment was performed in order to select appropriate dose levels for the mutagenicity study. The test substance produced a significant cytotoxic effect without metabolic activation from 500 µg/ml up to the limit of solubility (2000 µg/ml). No cytotoxic effect was observed with metabolic activation up to the limit of solubility. For mutagenicity testing two independent

cell cultures with and without metabolic activation (S9-mix) up to the limit of solubility (2000 µg/ml) were used.

For main experiment dose levels of 125, 250 and 500 µg/ml were used in the absence and 500, 1000 and 2000 µg/ml in the presence of 59-mix metabolic activation.

The test compound did not induce a significant increase in the number of chromosome aberrations at any preparation time and dose level of the test substance. But a cytotoxic effect of the compound was observed in the rnain experiments. Marked increases in the rate of chromosome aberrations were obtained with the positive control substances indicating the sensitivity of the assay.

In conclusion, the test substance does not induce chromosome mutations (= aberrations) in V79 Chinese hamster cells, neither in the presence nor in the absence of a metabolic activation system, under the experimental conditions described.

Furthermore, the structural analogue was tested in the micronucleus test. The test compound was administered orally by gavage to male and female mice. The following doses were tested: 0 and 5000 mg per kg bodyweight.

The 5000 mg per kg bodyweight dose level was chosen since a preliminary study had shown it to be the maximum applicable dose.

The animals were treated once with the test compound and according to the test procedure the animals were killed 24, 48 or 72 hours after administration of the test compound.

The test compound was given in two equal parts within two hours and according to the test procedure the animals were killed 24, 48 or 72 hours after administration of the test compound.

EndoxanR was used as positiv control substance and was administered orally at a dose of 50 mg per kg bodyweight.

The incidence of micronucleated polychromatic erythrocytes of the animals treated with the test substance was within the normal range of the negative control. The number of normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment with the test substance and was statistically not different from the control values.

Endoxan(R) induced in both males and females a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the system. The ratio of polychromatic erythrocytes to normocytes was not changed to a significant extend.

The results indicate that, under the conditions of the present study, the test substance is not mutagenic in the micronucleus test.

Justification for classification or non-classification

Based on the above stated assessment the substance is deemed non-genotoxic and accordingly does not need to be classified according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) and according to CLP (Regulation (EC) No 1272/2008 Of The European Parliament And Of The Council) as implementation of UN-GHS in the EU.