2-(3-(4-amino-9,10-dihydro-3-sulpho-9,10-dioxoanthracen-4-yl)aminobenzenesulphonyl)vinyl) disodium sulphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No genotoxic effects were seen in either mutagenicity studies in bacteria and mammal cells or cytogenicity studies in mammal cells.

Link to relevant study records

Reference

Reference 1

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)

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.

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

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)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media:MEM (minimal essential medium) with Hank's salts and 25 mM Hepes-buffer
- Properly maintained: ye
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no data

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix rat

Test concentrations with justification for top dose:

Test groups
a: without metabolic activation: 100, 200, 300, 400 and 500 µg/ml
b: with metabolic activation: 500, 1000, 1500 and 2000 µg/ml

Control groups
negative controls:
a: untreated control
b: cultures treated with the solvent

positive controls:
a: without metabolic activation:EMS (Ethyl methane sulfonate)
b: with metabolic activation: DMBA (9,10-dimethyl-1,2-benzanthracene)

Vehicle / solvent:

DMSO

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:

The cultures are prepared and treated with the test compound the same way as for the mutagenicity experiment. Subsequently the cultures are kept in the incubator for 20 hours. The following day they are trypsinated t counted t diluted and 500 cells per 25 cm2 flask are plated. After incubation for 7 days the colonies are stained with methylene blue and counted.

For toxic substances a percentage survival rate relative to the solvent control is calculated for each treatment. The Dose level which results in a predicted survival of about 30 % is estimated from the results obtained. This dose is chosen as the highest dose level. The lowest doses are chosen of the level from the negative controls. If the test substance is not sufficiently toxic to reduce survival up to the 30 % level (the maximum of 5 mg/ml or the lowest concentration at which visible precipitation is observed) will be selected as maximal dose.

Two days old exponentially growing cultures more than 50 %confluent are trypsinated and a single cell suspension is prepared. The trypsin concentration is 0.2 % in Ca-Mg-free salt solution. The Ca-Mg-free salt solution is prepared as follows (per litre): NaCl 8000 mg; KCl 400 mg; glucose 1000 mg; NaHC03 350 mg; Trypsin 2000 mg.
Subsequently the cells are replated for mutagenicity testing and for determination of plating efficiency.

The treatment schedule of the mutagenicity test is done as follows:
Day 1: Subculturing of a exponentially growing culture
a) About 500 cells in 25 cm2 flasks with 5 ml medium for determination of the plating efficiency; in duplicate for each experimental point.
b) 6 x 105 - 1 x 106 cells in 175 cm2 flasks with 30 ml medium for the mutagenicity test, one flask per experimental point.
Day 2: Treatment of a) and b) with the test substance in the presence and absence of S9-mix (final protein concentration: 0.3 mg/ml) for 4 hours.
Day 5: Subculturing of b) in 175 cm2 flasks
Day 8: Fixation and staining of the colonies in a)-flasks for the determination of the plating efficiency.
Day 9: Subculturing of b) in five 80 cm2 flasks with culture medium containing 6-thioguanine: Mutant selection (about 400000 cells/flask); subculturing of b) in two 25 cm2 flasks for plating efficiency (about 500 cells per flask)
Day 16: Fixation and staining of colonies of b) - from subcultures seeded on day 9.

All incubations were done at 37°C and 5 %CO2.
Staining was done with 10 %methylene blue in 0.01 nKOH solution.
Only colonies with more than 50 cells were counted.

Evaluation criteria:

Criteria for a valid assay
- 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%

Criteria for a positive response
- 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:

MANN-WHITNEY-U-TEST

Species / strain:

Chinese hamster lung fibroblasts (V79)

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

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

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:

In a preliminary experiment Remazol-Brillantblau R FWTRG was assayed with respect to its solubility in cell culture medium. The lowest concentration at which visible precipitation was observed, was found to be 2000 µg/ml.
The cytotoxicity experiment proved Remazol-Brillantblau R FWTRG was not toxic to the V79 cells in the presence of metabolic activation (S9-mix). In the absence of metabolic activation (S9-mix) cytotoxic effects were observed from 750 µg/ml up to the limit of solubility. Further experiments on toxicity revealed a slight cytotoxic effect without metabolic activation. In the presence of metabolic activation a dose dependent increase of cytotoxicity was observed.

On the basis of the preliminary experiment 500 µg/ml was determined in the absence and 2000 µg/ml in the presence of S9-mix as maximal dose level for mutagenicity testing in experiment I and II.

The test substance Remazol-Brillantblau R FWTRG was assessed for 1ts mutagenic potential in vitro in the HGPRT-test in two independent experiments without metabolic activation and two independent experiments with metabolic activation.
No relevant reproducible enhancement of the mutant colonies or mutant frequency over the range of the negative control was found with any of the concentrations used, either with or without metabol ic 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 permit to draw the conclusion that the test compound is not mutagenic in the HGPRT-test with cells of the V79 Chinese-hamster cell line.

Conclusions:

Reactive Blue 19 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. It is therefore considered to be non-mutagenic in this HPRT assay.

Executive summary:

The test substance was examined for mutagenic activity in V79 Chinese Hamster cells with two independent experiments. The induction of 6-thioguanine resistant mutants 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. In this experiment the test substance did not produce any significant cytotoxic effect up to the limit of sol ubil ity (2000 µg/ml) with metabolic act ivat ion (S9-mix). Without metabolic activation (S9-mix) the test substance produce cytotoxic effects at the dose level 750 µg/ml up to the limit of solubility (2000 µg/ml).

In the absence of S9 metabolic activation dose levels of 100,200,300,400,500 µg/ml and in the presence of S9 metabolic activation dose levels of 500, 1000, 1500, 2000 µg/ml were used.

The test compound did not induce a significant increase in the number of mutant colonies or in the mutation frequency at any dose level of the test substance.

A slight not relevant cytotoxic effect of the test compound was observed in the main experiments in the absence of metabolic activation and a dose depedent increase of cytotoxicity in the presence of metabolic activation system. Marked increases in mutation frequency were obtained with the positive control substances indicating the sensitivity of the assay.

In conclusion, the test substancevdoes not induce gene mutations in the HGPRT-test with V79 Chinese hamster cells, either in the presence or in the absence of a metabolic activation system, under the experimental conditions described.

Reference 3

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:

November 1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat S9-mix induced with Aroclor 1254

Test concentrations with justification for top dose:

4 to 10000 µg/plate

Vehicle / solvent:

aqua bidest

Untreated negative controls:

yes

Remarks:

sterility check

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-aminoanthracene

Remarks:

all strains with S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 100, TA1535 w/o S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537 w/o S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA 98 w/o S9

Details on test system and experimental conditions:

Two independent mutagenicity tests were formed. In both cases top agar is prepared for the Salmonella strains by mixing 100 ml agar (0.6% agar, 0.5 % NaCl) with 10 ml of a 0.5 mM histidine-biotin solution. The following ingredients are added (in order) to 2 ml of molten top ager at 45°C:
0.1 ml of an overnight nutrient broth culture of the bacterial tester strain
0.1 ml test compound solution
0.5 ml S-9 Mix (if required) or buffer
After mixing, the liquid is poured into a petridish with minimal agar (1.5 % agar, Vogel-Bonner E medium with 2 % glucose). After incubation for 48 to 72 hours at 37°C in the dark, colonies (his+ revertants) are counted.

Evaluation criteria:

Cytotoxicity: reduced growth rate or thinning of bacterial lawn
Positive: - dose-related and reproducible increase in number of revertant colonies
- doubling of spontaneous mutation rate in at least one tester strains either with or without S9

Statistics:

NA

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

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

True negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

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

True negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

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

True 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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

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

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test compound did not cause a significant increase in the number of revertant colonies with any of the tester strains neither in the absence nor presence of S-9 Mix. No dose dependent effect was obtained.
It is concluded that the test substance is not mutagenic in these bacterial test systems neither in the absence nor in the presence of an exogenous metabolizing system.
This test was performed according to the methods described. No unforeseen circumstances were observed which have affected the quality and integrity of this study.
This study was conducted in compliance with the principles of good laboratory practice.

Conclusions:

Reactive blue 19 is not mutagenic in this bacterial test system either with or without exogenous metabolic activation at the dose levels investigated.

Executive summary:

Remazol-Brillantblau R was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 1538, TA 98 of Salmonella typhimurium. The mutagenicity studies were conducted in the absence and in the presence of a metabolizing system deri ved from rat li ver homogenate. A dose range of 6 different doses from 4 µg/plate to 10 000 µg/plate was used. Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies.

Toxicity: The test compound proved to be not toxi c to the bacterial strains. 10 000 µg/plate was chosen as top dose level for the mutagenicity study.

Mutagenicity: In the absence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of a metabolic activation system, treatment of the cells with Remazol-Brillantblau R did not result in relevant increases in the number of revertant colonies. Summarizing, it can be stated that Remazol-Brillantblau R is not mutagenic in these bacterial test systems neither with nor without exogenous metabolic activation at the dose levels investigated.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1988

Justification for type of information:

see Read Across Justification in section 13

Reason / purpose for cross-reference:

read-across source

Metabolic activation system:

S9-Mix from rat liver

Vehicle / solvent:

water

Details on test system and experimental conditions:

The first experiment was performed with all tester strains using three plates per dose to get information on mutagenicity and toxicity for calculation of an appropriate dose range. A reduced rate of spontaneously occuring colonies as well as visible thinning of the bacterial lawn were used as indicator for toxicity.
Thinning of the bacterial lawn was controlled microscopically.
In combination with the second experiment, toxicity testing was performed as follows: 0.1 ml of the different dilutions of the test compound were thoroughly mixed with 0.1 ml of 10-6 dilution of the overnight culture of TA 100 and plate with histidine and biotin rich top agar (3 plates per dose). The solvent control is compared with the number of colonies per plate in the presence of the test compound. Results are given as a ratio of these values (= surviving fraction).

Top agar is prepared for the Salmonella strains by mlxlng 100 ml agar (0.6 % agar, 0.5 %NaCl) with 10 ml of a 0.5 mM histidine-biotin solution. With E. coli histidine is replaced by tryptophan (2.5 ml, 0.5 mM). The following ingredients are added (in order) to 2 ml of molten top agar at 45°C:
0.1 ml of an overnight nutrient broth culture of the bacterial tester strain
0.1 ml test compound solution
0.5 ml S9 Mix (if required) or buffer
After mixing, the liquid is poured into a petridish with minimal agar (1.5 % agar, Vogel-Bonner E medium with 2 % glucose). After incubation for 48 to 72 hour at 37°C in the dark, colonies (his+ revertants) are counted.

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

up to 10000 µg/plate

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:

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

Remarks:

up to 10000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

up to 10000 µg/plate

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 nor precipitates, but tested up to recommended limit concentrations

Remarks:

up to 10000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

up to 10000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

up to 10000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test compound did not cause a significant increase in the number of revertant colonies with any of the tester strains either in the absence or presence of S-9 Mix. No dose dependent effect was obtained.
A slight increase of revertant colonies was observed with the Salmonella strain TA 1537 eiter in the absence or in the presence of exogenous metabolic activation.
In a third independent experiment these results have been confirmed.
It is concluded that the test substance is not mutagenic in these bacterial test systems either in the absence or in the presence of an exogenous metabolizing system.
This test was performed according to the methods described. No unforeseen circumstances were observed which have affected the quality and integrity of this study.
This study was conducted in compliance with the principles of good laboratory practice.

Conclusions:

The test substance is not mutagenic in these bacterial test systems either with or without exogenous metabolic activation in either strain at the dose levels investigated.

Executive summary:

The test substance was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 1538, TA 98 of Salmonella typhimurium and Escherichia coli WP2uvrA.

The mutagenicity studies were conducted in the absence and in the presence of a metabolizing system derived from rat liver homogenate. A dose range of 6 different doses from 4 or 20 microgram/plate to 5000 or 10000 microgram/plate was

used.

Control plates without mutagen showed that the number of spontaneous revertant colonies was similiar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies.

Toxicity: The test compound proved to be not toxic to the bacterial strains. 10000 microgram/plate was chosen as top dose level for the mutagenicity study.

Mutagenicity: In the absence of the metabol ic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains, except the salmonella strain TA 1537. Also in the presence of a metabolic activation system, treatment of the cells with Reaktiv-Blau F-64357 FW did not result in relevant increases in the number of revertant colonies. Slight increase of revertant colonies was observed with TA 1537.

Summarizing, it can be stated that the test substance is not mutagenic in these bacterial test systems either with or without exogenous metabolic activation at the dose levels investigated.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No genotoxic effects were seen in the mouse micronucleus test

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 Remazol-Brillantblau R FWTRG per 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 Remazol-Brillantblau R FWTRG 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 (total dose)

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

Positive controls validity:

valid

Additional information on results:

All animals survived after application of 5000 mg/kg body weight of the test substance. 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/kg bw in some male and females the content of appendix and colon was blue coloured.

The incidence of micronucleated polychromatic erythrocytes in the substance 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:

Under the conditions of the present study, the test substance is not cytogenic in the micronucleus test.

Executive summary:

Reactive Blue 19 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/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. Endoxan^R 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 cytogenic in the micronucleus test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Reactive Blue 19 was tested for mutagenicity with the bacterial strains TA100, TA1535, TA1537, TA1538, TA98 of Salmonella typhimurium and Escherichia coli WP2uvrA and in an HPRT assay in mammalian cells (V79). The test substance is not mutagenic in these test systems either with or without exogenous metabolic activation at the dose levels investigated.

 

Reactive Blue 19 was tested for clastogenicity and aneugenicity in the in vitro chromosome aberration assay (V79 cells) and the in vivo micronucleus test in the mouse. The results indicated that, under the conditions of the present studies, the test substance is not genotoxic in these test systems.