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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Direct Blue 094 does not need to be classified for mutagenicity.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 09 March, 1994 to 22 March, 1994
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
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine auxotrophs
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 post mitochondrial supernatant (S9 fraction)
Test concentrations with justification for top dose:
Six concentrations of FAT 11127/C (Solophenyl Marine BL roh trocken) ranging from 20.6 to 5000.0 µg/plate were tested with strain Salmonella typhimurium TA 100 to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and without metabolic activation. Normal background growth was observed. The numbers of revertant colonies were not reduced. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000.0 µg/plate with and without metabolic activation.
Vehicle / solvent:
Dimethyl Sulfoxide (DMSO)
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
cyclophosphamide
mitomycin C
other: 2-Aminoanthracene: TA 100, TA102, TA 98 and TA 1537 with S9
Details on test system and experimental conditions:
Agar plate incorporation : 0.1 ml of the overnight cultures were mixed with 2 ml of top agar, either 0.5 ml of 100 mM sodium phosphate buffer (experiments without activation) or 0.5 ml of the activation mixture (experiments with activation) and 0.1 ml of a solution of the test substance, the positive control or the solvent as a negative control and poured on minimal agar in Petri dishes. Each Petri dish contained about 20.0 ml of minimal agar (1.5% agar supplemented with 2% salts of the Vogel-Bonner Medium E and 2% glucose). The top agar was composed of 0.6% agar and 0.6% NaCl and was supplemented with 10% of 0.5 mM L-histidine and 0.5 mM (+) biotin dissolved in water.

Incubation period : 48 hours at 37±1.5°C in darkness.
Evaluation criteria:
Assay accpetance criteria
A test is considered acceptable if the mean colony counts of the negative control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response. In either case the final decision is based on the scientific judgement of the Study Director.

Criteria for a positive response
The test substance is considered to be positive if the following condition is met:
• At least a reproducible meaningful increase of the mean number of revertant per plate above that of the negative control at any concentration for one or more of the strains tested. Generally, a concentration-related effect should be demonstrable.
Statistics:
A statistical analysis of the test data was not performed. At present the use of statistical methods concerning this particular test system is not generally recommended.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
other: TA 100, TA 1535, WP2 uvrA, TA 102, TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Range finding test :

Six concentrations of FAT 11127/C (Solophenyl Marine BL roh trocken) ranging from 20.6 to 5000.0 µg/plate were tested with strain Salmonella typhimurium TA 100 to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and without metabolic activation. Normal background growth was observed. The numbers of revertant colonies were not reduced. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000.0 µg/plate with and without metabolic activation.

Mutagenicity test, original experiment

In the experiments performed with and without metabolic activation, treatment of strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 with FAT 11127/C did not lead to an increase in the incidence of histidine-prototrophic mutants in comparison with the negative control.

Mutagenicity test, confirmatory experiment:

Since the purity of the test material is about 80%, in this experimental part the concentration range of 77.2 to 6250.0 µg/plate was used. In the experiments performed with and without metabolic activation for strains TA 100, TA 102, TA 1535 and TA 1537, no increase in the incidence of either histidine-prototrophic mutants was observed in comparison with the negative control. However, in the experiment with strain TA 98 without metabolic activation, a marginal increase in the number of back-mutants was observed at the concentration of 6250 µg/plate. No effect was seen in the corresponding experiment with microsomal activation.

Mutagenicity test, confirmatory experiment 2 :

To confirm the effect observed in the first confirmatory without metabolic activation on strain TA 98, this experiment was repeated. Again, a marginal increase in the number of back-mutants was registered at the highest concentration.

In the mutagenicity tests normal background growth was observed with all strains at all concentrations. The numbers of revertant colonies were not reduced. The test substance exerted no toxic effect on the growth of the bacteria.

There were no known circumstances or occurrences in this study that were considered to have affected the quality or integrity of the test data.

Conclusions:
FAT 11127/C exerted a marginal mutagenic effect in strain TA 98 without metabolic activation.
Executive summary:

FAT 11127/C was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimurium viz TA 98, TA 100, TA 102, TA 1535 and TA 1537 according to OECD Guideline 471. The test was performed with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. The compound was tested as a suspension in DMSO at five concentrations in the range of 312.5 to 5000.0 µg/plate in the presence and absence of a metabolic activation system.

 

In order to confirm the results, the experiments were repeated with and without metabolic activation at five concentrations in the range of 77.2 to 6250.0 µg/plate. Each strain was additionally tested in the presence and in the absence of a metabolic activation system with a suitable, known mutagen as positive control.

 

In both experiments, performed with and without metabolic activation, none of the tested concentrations of FAT 11127/C led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control with strains TA 100, TA 102, TA 1535 and TA 1537. With strain TA 98, however, a slight increase in the number of histidine-prototrophic mutants was observed in the experiments without metabolic activation at the concentration of 6250.0 µg/plate. No effect was seen in the corresponding experiment with metabolic activation. In order to confirm the effect observed in the first confirmatory without metabolic activation on strain TA 98, this experiment was repeated. Again a marginal increase in the number of back-mutants was registered at the highest concentration.

Hence it can be concluded that, based on the results of these experiments and on standard evaluation criteria, FAT 11127/C (Solophenyl Marine BL roh trocken) exerted a marginal mutagenic effect in strain TA 98 without metabolic activation.

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

Genetic toxicity in vivo

Description of key information

no data available

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

Additional information

Bacterial reverse mutation assay:

FAT 11127/C was tested for mutagenic effects in vitro in histidine-requiring strains of Salmonella typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537 according to OECD Guideline 471. The test was performed with and without the addition of rat-liver post mitochondrial supernatant (S9 fraction) as an extrinsic metabolic activation system. The compound was tested as a suspension in DMSO at five concentrations in the range of 312.5 to 5000.0 µg/plate in the presence and absence of a metabolic activation system.

 

In order to confirm the results, the experiments were repeated with and without metabolic activation at five concentrations in the range of 77.2 to 6250.0 µg/plate. Each strain was additionally tested in the presence and in the absence of a metabolic activation system with a suitable, known mutagen as positive control.

 

In both experiments, performed with and without metabolic activation, none of the tested concentrations of FAT 11127/C led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control with strains TA 100, TA 102, TA 1535 and TA 1537. With strain TA 98, however, a slight increase in the number of histidine-prototrophic mutants was observed in the experiments without metabolic activation at the concentration of 6250 µg/plate.No effect was seen in the corresponding experiment with metabolic activation. In order to confirm the effect observed in the first confirmatory without metabolic activation on strain TA 98, this experiment was repeated. Again a marginal increase in the number of back-mutants was registered at the highest concentration.

Hence, it can be concluded that, based on the results of these experiments and on standard evaluation criteria, FAT 11127/C (Solophenyl Marine BL roh trocken) exerted a marginal mutagenic effect in strain TA 98 without metabolic activation.

However, Direct Blue 094 was identified as a potential benzidine congener by the Australian National Industrial Chemicals Notification and Assessment Scheme (NICNAS) and assessed using the Inventory Multi-Tiered Assessment and Prioritization (IMAP) framework. Using these assessments and available data, an expert statement has been prepared to investigate the genotoxic concerns regarding the chemical. The expert statement concludes that the final dye when fully metallized/completely complexed with copper will not be able to produce the mutagenic action. The dye is supposed to be sold in the this fully metallized form. The current study available with Direct Blue 094, seems to have been conducted with a test material with some impurities (not fully complexed dye), hence these impurities have been considered to have been the reason behind the positive outcome of the study. The Expert Statement has been presented below (also attached in section 13):

Expert statement on the genotoxicity of Direct Blue 094

(EC: 278-174-4, CAS: 75284-35-4)

Preface:

Direct Blue 094 was identified as a potential benzidine congener by the Australian National Industrial Chemicals Notification and Assessment Scheme (NICNAS) and assessed using the Inventory Multi-Tiered Assessment and Prioritization (IMAP) framework. The substance was included in a group of chemicals representing all metallized 3,3-disubstituted benzidine-congener-based dyes having the potential to be metabolized to the benzidine congener 3,3’-dihydroxybenzidine (EC: 679-800-2, CAS: 2373-98-0). As the dye synthesis commences with the mutagenic 3,3’-dimethoxybenzidine, the finished dye or dye products may contain significant levels of impurities that may be reduced to the benzidine congener.

Listing in national inventories:

Currently, the substance is listed in the Australian Inventory NICNAS, as well as in the Canadian DSL, the US TSCA and the European C&L inventory of the CLP Regulation.

Human Health Information:

Currently only few data are available to assess the toxicological properties of the test substance. Acute oral toxicity studies conducted by Ciba-Geigy in 1974 and 1978 identified the test substance to be not toxic to rats. In addition, the test substance did not show any skin or eye irritating effects when to the skin and eye of rabbits. An in vivo test on Guinea pigs for skin sensitization (Buehler test) was also negative. Further data on toxicokinetics or higher TIER studies are currently not available.

Genotoxicity Information:

Currently only insufficient information is available to assess the genotoxic potential of the test substance. In 1994 a mutagenicity test in bacteria (Ames test) was conducted involving three experiments (one range finding test and two confirmatory experiments) on five different tester strains. While the range finding test was judged negative, the confirmatory tests have been judged weakly positive by the study director. Unfortunately, in both cases the effects with the highest concentration of the test material have been in the range of 2-times the historical control data, and in the second confirmatory test the negative and the positive control has been out of the range of historical control data. But looking at the isolated experiments taking the experimental blank control value as reference, both confirmatory results would need to be judged positive. Taking all these circumstances into account, the test result is evaluated ambiguous and further data are required to assess the genetic toxicity of the test substance.

QSAR models evaluated for this substance resulted in “no results” in case of the OECD Toolbox and in “potentially mutagenic” in case of a model developed for the REACH Dyes Consortium in cooperation with Prof. Dr. Roberto Todeschini, University of Milan, Italy. But this model uses data that are within the database of existing studies on comparable substances, in this case the results on Direct Blue 218 have been used as described also below. Therefore, a reliable prediction based on QSAR besides the known read-across candidate Direct Blue 218 is currently not available.

Available data of comparable substances:

Currently few data on comparable substances are available. Under REACH only Direct Blue 218 has been registered in the 1-10 tons volume band and data have been disseminated by ECHA. For genotoxicity assessment in vitro and in vivo data are available for that substance. The substance has been investigated in an Ames test on Salmonella typhimurium strain 1538 with and w/o metabolic activation only and was found to be negative [Reid TM, et al., Environmental Mutagenesis 6: 705-717 (1984)]. Other authors confirmed this result in Ames tests on various tester strains [Mortelmans K, et al., Environ. Mutagen. 8(Suppl. 7):1 -119, (1986)]; Prival MJ, et al., Mutation Research, 136: 33 -47(1984); Gregory AR et al., Journal of Applied Toxicology, Vol. 1, No. 6, 308-313 (1981); Prival MJ, et al., Environmental Mutagenesis Volume 4, Issue 3: 414 (1982)]. In eukaryotic cells an in vitro mammalian chromosome aberration test was performed to assess the mutagenic nature of the dye in CHO cells. Also, this test gave no indication of a mutagenic behaviour of the test item [National Toxicology Program, NTP TR 430, NIH Publication No. 94-3161, 1994]. Finally, the clastogenic behaviour was assessed in a chromosomal aberration test which was also negative [National Toxicology Program, NTP TR 430, NIH Publication No. 94-3161, 1994]. In vivo, Direct Blue 218 was investigated in a Drosophila SLRL assay on male and female animals. As a result, Direct Blue 218 did not induce a significant increase in the frequency of SLRL mutations when administered by feeding and injection to male Canton S Drosophila melanogaster flies and hence it is not likely to classify as a gene mutant in vivo [Woodruff RC, et al., Environmental Mutagenesis 7:677-702 (1985)]. For a second dye, Direct Blue 080, an Ames test conducted showed negative results. Further data on comparable dyes are currently not available.

Genotoxicity of metabolites and degradation products

Currently almost no data on toxicokinetics and potential degradation products of the dye are available. In general, reductive cleavage of the azo-bond may occur in the liver, the skin and intestine, mediated by NAHD-dependent microsomal cytochrome P450 enzymes in hepatocytes [Platzek T, et al., Human and experimental Toxicology 18pp. 552-559 (1999)]. In addition, bacteria in the intestine have been demonstrated to be able to cleave the azo bond as well playing an important role for the cleavage of azo bonds in mammals. As important metabolites formed by such processes 3,3’-Dihydroxybenzidine and Dimethoxybenzidine were identified to be produced by the mechanisms. This metabolite demonstrated to be positive in various strains of Salmonella typhimurium when tested according to the Ames protocol as well as in an SCE assay in mammalian cells in vitro, a chromosomal aberration test in vitro and in a DNA fragmentation test in rat hepatocytes and human bladder cells (summarized in IMAP Group Assessment Report, NICNAS March 2019).

Chemical reaction scheme of Direct Blue 094

The synthetic route for Direct Blue 094 consists of coupling of diazotated 3,3’-dimethoxybenzdine (CAS 119-90-4) (Figure 2, upper right) asymmetrically to 4,5-dihydroxy-2,7-naphthalenedisulfonic acid (chromotropic acid) followed by coupling to 7-(benzoylamino)-4-hydroxy-2-naphthalene sulfonic acid (benzoyl-J-acid). In the final step a “demethylating coppering” affords the copper complex [Morgan D, et al., Environmental Health Perspectives 102 (2) pp. 63-78 (1994)]. If the metallization is not completed, mutagenic benzidine congener may be still present and may cause mutagenic or even carcinogenic effects.

Summary

In summary, several data from various authors and assessments of national institutes like NICNAS or NTP have shown, that the complexed direct dyes do not exhibit mutagenic or even carcinogenic effects as such, but in most cases non-reacted or non-complexed residual material may cause mutagenic effects in bacteria or mammalian cells. The dye under assessment is considered to be fully formed copper complex and hence chances of it producing the mutagenic action are minimal. Under metabolic conditions on the skin, in the intestine or the liver involving cytochrome P450 enzymes, benzidine congeners may be produced to exhibit their toxic effects. However, the fully complexed/metallized structures are resistant to such metabolic changes and hence are considered to not undergo in vivo metabolism to produce such mutagenic benzidine congeners.

Justification for classification or non-classification

The bacterial reverse mutation assay available seems to have been conducted with a test material that was not fully complexed and contained impurities that may have lead to the positive outcome in the study. The dye is supposed to be sold as a fully complexed dye, hence it is considered to not have such a mutagenic action in the bacterial reverse mutation assay as proven from the studies with structurally similar substances. Hence Direct Blue 094 does not need to be classified for mutagenicity according to Regulation (EC) No. 1272/2008.

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