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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Test item deemed to be non-cytotoxic and not mutagenic in the TK mutation system under the experimental conditions carried out.

Based on the results of the in vitro gene mutation study it was concluded that Disperse Blue 359 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

It is concluded that the from the in vitro micronucleus assay that the testing was valid and that Disperse Blue 359 is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described.

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Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6 weeks
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
yes
Remarks:
Cloning efficiency value (125%) outside the range and the value for mutation frequency was within 95% control limits of historical solvent control database, deviation had no effect on the results of the first mutation experiment in the absence of S9-mix.
GLP compliance:
yes
Type of assay:
in vitro mammalian cell transformation assay
Specific details on test material used for the study:
Batch 135214 of Disperse Blue 359 was a dark blue powder with a purity of 99%.
Target gene:
Mutations in L5178Y mouse lymphoma cells, specifically deficiencies in thymidine kinase (TK) levels due to the forward mutation of TK+/- to TK-/-
Species / strain / cell type:
mouse lymphoma L5178Y cells
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes (S9 homoegenate) prepared from male Sprague Dawley rats, dosed with phenobarbital (80mg/kg bodye weight) and ß-napyhoflavone (100mg/kg body weight)
Test concentrations with justification for top dose:
Based on the results of the dose-range finding test the following concentrations were selected for the first mutagenicity tests I the absence and presence of S9-mix: 0.20, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25 and 50 µg/ml of exposure medium. For the second mutagenicity test concentrations of 0.20, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5 and 25 µg/ml were used. These concentrations were chosen because the test item was deemed non-toxic and difficult to dissolve in aqueous solutions. The highest concentration was determined by solubility in the culture medium, the highest test item concentration showed slight precipitate in the exposure medium.
Vehicle / solvent:
Ethanol
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Key result
Species / strain:
mouse lymphoma L5178Y cells
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
Conclusions:
In conclusion, Disperse Blue 359 is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.
Executive summary:

The objective of this study was to evaluate the mutagenic potential of Disperse Blue 359 by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix). The TK mutational system detects base pair mutations, frame shift mutations and small deletions.

The test was performed in the absence of S9-mix with 3 and 24-hour treatment periods and in the presence of S9-mix with a 3 hour treatment period.

In the first experiment, Disperse Blue 359 was tested up to concentrations of 25 µg/ml in the absence and presence of S9-mix. The incubation time was 3 hours. In the second experiment, Disperse Blue 359 was again tested up to concentrations of 25 µg/ml in the absence of S9-mix. The incubation time was 24 hours. No toxicity was observed at this dose level in the absence and presence of S9-mix. Disperse Blue 359 precipitated in the culture medium at test item concentrations of 12.5 and 25 µg/ml after the three hour treatment and at 25 µg/ml and after the 24 hour treatment.

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, Disperse Blue 359 did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment.

In the presence of S9-mix, Disperse Blue 359 did not induce a significant increase in the mutation frequency.

In conclusion, Disperse Blue 359 is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 months
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
Batch 135214 of Disperse Blue 359 was a dark blue powder with a purity of 99%.
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
Cultured peripheral human lymphocytes were used as test system. Peripheral human lymphocytes are recommended in the international OECD guideline.
Blood was collected from healthy adult, non-smoking volunteers (aged 18 to 35 years).
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
cytokinesis-block proliferation index
Metabolic activation:
with and without
Metabolic activation system:
Rat S9 homogenate was obtained from Trinova Biochem GmbH, Giessen, Germany and is prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg).
Test concentrations with justification for top dose:
In the first cytogenetic assay, Disperse Blue 359 was tested up to 31.3 μg/ml for a 3 hours exposure time with a 27 hours harvest time in the absence and presence of S9-fraction. The test item precipitated in the culture medium at this dose level. In the second cytogenetic assay, Disperse Blue 359 was again tested up to 31.3 μg/ml for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. The test item precipitated in the culture medium at this dose level.
Vehicle / solvent:
Ethanol
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Colchicine was used in the -S9 mixture, Hanks' Balanced Salt Solution (HBSS) was used as the solvent for positive controls
Key result
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Disperse Blue 359 was not cytotoxic and difficult to dissolve in aqueous solutions, the highest concentration analysed was determined by the solubility in the culture medium.
Remarks on result:
other: In vitro gene mutation study in bacteria found test item not to be genotoxic

At a concentration of 31.3 μg/ml the test item precipitated in the culture medium. At the 3 h exposure time, blood cultures were treated in duplicate with 7.8, 15.6 and 31.3 μg test item/ml culture medium with and without S9 -mix (first cytogenetic assay). At the 24 hour exposure time single blood cultures were treated with 2.0, 3.9, 7.8, 15.6, 31.3 and 62.5 μg Disperse Blue 359/ml culture medium without S9-mix (dose range finding test).

All dose levels were selected for scoring of micronuclei. In the absence of S9-mix, Disperse Blue 359 did not induce a statistically significant increase in the number of mono- and binucleated with micronuclei at the highest concentration tested. In the presence of S9 -mix, Disperse Blue 359 induced a statistically significant increase in the number of binucleated cells with micronuclei at the highest concentration tested. In addition, a statistically significant Cochran Armitage trend test was obtained. However, since all individual number of binucleated cells with micronuclei were are inside the 95% control limits of the negative historical control data range, it was concluded that the increase was not biologically relevant.

To obtain more information about the possible clastogenicity and aneugenicity of Disperse Blue 359, a second cytogenetic assay was performed in which human lymphocytes were exposed for 24 hours in the absence of S9 -mix. The following dose levels were selected for the second cytogenetic assay:

Without S9-mix : 7.8, 15.6 and 31.3 μg/ml culture medium (3 hours exposure time, 27 hours harvest time).

The ability of Disperse Blue 359 to induce micronuclei in human peripheral lymphocytes was investigated in two independent experiments. The highest concentration analysed was selected based on the solubility of the test item in the culture medium.

The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition in the second experiment colchicine also showed a statistically significant increase in the number of binucleated cells with micronuclei. In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9 -mix) functioned properly.

Disperse Blue 359 did not induce a statistically significant and biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9 -mix, in either of the two experiments.

Conclusions:
It is concluded that the testing is valid and that Disperse Blue 359 is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.
Executive summary:

An in vitro micronucleus assay with Disperse Blue 359 in cultured peripheral human lymphocytes. This report describes the effect of Disperse Blue 359 on the number of micronuclei formed in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß naphthoflavone induced rat liver S9 -mix). The possibleclastogenicity and aneugenicity of Disperse Blue 359 was tested in two independent

experiments.

The study procedures described in this report are in compliance with the most recent OECDguideline. Batch 135214 of Disperse Blue 359 was a dark blue powder with a purity of 99%. The test item was suspended in ethanol. In the first cytogenetic assay, Disperse Blue 359 was tested up to 31.3 μg/ml for a 3 hours

exposure time with a 27 hours harvest time in the absence and presence of S9-fraction. The test item precipitated in the culture medium at this dose level. In the second cytogenetic assay, Disperse Blue 359 was again tested up to 31.3 μg/ml for a 24 hours exposure time with a 24 hours harvest time in the absence of S9 -mix. The test item precipitated in the culture medium at this dose level. The number of mono- and binucleated cells with micronuclei found in the solvent control

cultures was within the 95% control limits of the distribution of the historical negative control database. The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore

concluded that the test conditions were adequate and that the metabolic activation system (S9 -mix) functioned properly.

Disperse Blue 359 did not induce a statistically significant and biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9 -mix, in either of the two experiments.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 days
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Batch 135214 of Disperse Blue 359 was a dark blue powder with a purity of 99%.
Target gene:
Histidine requiring strains of Salmonella typhimurium and tryptophan requiring strains in Escherichia coli.

Each tester strain contained the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cellcoat)
gal : mutation in the galactose metabolism
chl : mutation in nitrate reductase
bio : defective biotin synthesis
uvrB : loss of the excision repair system (deletion of the ultraviolet-repair B gene)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes (S9 homogenate)
Test concentrations with justification for top dose:
Test item concentrations of 1.7 µg, 5.4 µg, 17 µg, 52 µg, 164 µg, 512 µg, 1600 µg and 5000 µg per plate were used for the dose range finding test. Test item was found to precipitate at doses equal to or higher than 164 µg for all strains barring TA98, whihc precipitated at 52 µg per plate.
Vehicle / solvent:
Dimethyl sulfoxide (DMSO)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 6-Chloro-9-[3-(2-chloroethylamino)propylamino]-2-methoxyacridine dihydrochloride
Details on test system and experimental conditions:
The characteristics of the different Salmonella typhimurium strains were as follows:
Strain Histidine mutation Mutation type
TA1537 hisC3076 Frameshift
TA98 hisD3052/R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions
*: R-factor = plasmid pKM101 (increases error-prone DNA repair)

Each tester strain contained the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cellcoat)
gal : mutation in the galactose metabolism
chl : mutation in nitrate reductase
bio : defective biotin synthesis
uvrB : loss of the excision repair system (deletion of the ultraviolet-repair B gene)
The Salmonella typhimurium strains are regularly checked to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.

The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilization of the strain using Tris-EDTA treatment. The strain is regularly checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants. Stock cultures of the five strains were stored in liquid nitrogen (-196°C).
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9 -metabolic activation. These results were confirmed in a follow-up experiment.
Based on the results of this study it is concluded that Disperse Blue 359 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reversemutation assay.
Executive summary:

The test item was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9 -mix (rat liver S9-mix induced by Aroclor 1254).

In the dose range finding test, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test item precipitated on the plates at dose levels of 164 μg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

Based on the results of the dose range finding test, the test item was tested in the first mutation assay at a concentration range of 5.4 to 512 μg/plate in the absence and presence of 5% (v/v) S9 -mix in the tester strains TA1535, TA1537 and TA98. The test item precipitated on the plates at dose levels of 164 μg/plate and upwards, except in tester strain TA98, where precipitation was already observed at 52 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In a follow-up experiment of the assay with additional parameters, the test item was tested at a concentration range of 15 to 154 μg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item was tested up to or beyond a precipitating dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9 -metabolic activation. These results were confirmed in a follow-up experiment.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification