Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Disperse Blue 077 was found to have mutagenic effects in the bacterial reverse mutation assays. However, compounds containing nitro groups are known to exhibit false positive outcome due to the activity of nitro-reductases. Hence, the positive effect was considered bacteria specific. In order to investigate the role of nitroreductases further in the positive outcome of the bacterial reverse mutation, a modified bacterial reverse mutation assay that also investigates nitroreductase deficient strains i.e. TA98NR and TA100NR has been commissioned and results are expected in September 2020.

The source substance, Disperse Violet 057, did not lead mutations in mammalian cell gene mutation assay. Hence, taking into consideration the above information, Disperse Blue 077 was considered to be not mutagenic.

Further, Disperse Violet 057 did not exhibit clastogenic potential in a chromosomal aberration assay in vitro. Hence, using the principles of read across, Disperse Blue 077 was also considered to be not clastogenic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
None
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name: FAT 92'504/A
Terasil blau GRN Isomerengemisch roh feucht
- Batch-No.: 1/95
Aggregate State at RT: solid
Colour: dark blue
Analysis: cf. data in the sponsor's files
Purity: 46 % (active ingredient)
Stability: Pure: stable until April, 1999
Storage: room temperature
Expiration Date: April 1999

On the day of experiment, the test article was dissolved in DMSO. The solvent was chosen because of its solubility properties and its relative non toxicity to the bacteria.
The test article did precipitate in the overlay agar at concentrations of 2500 µg/plate and above.
Target gene:
Histidine auxotrophs
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Microsomal Fraction S9 Mix from rats
Test concentrations with justification for top dose:
33.3; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 µg/plate (active ingredient)
The highest dose of 5000 µg/plate was chosen on the basis of a pre-toxicity experiment, where no toxic effects were observed.
Vehicle / solvent:
- distilled water for TA 1535 and TA 100 without metabolic activation,
- DMSO for TA 1537, TA 98 without metabolic activation,
- DMSO for all strains with metabolic activation.
Untreated negative controls:
yes
Remarks:
Concurrent untreated and solvent controls were performed
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: Without S9: For TA 1535 and TA 100: Sodium azide and for TA 1535 and TA 98:4-nitro-o-phenylene-diamine, With S9: 2-aminoanthracene for all the strains
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation);
Evaluation criteria:
The generally accepted conditions for the evaluation of the results are:
- corresponding background growth on both negative control and test plates
- normal range of spontaneous reversion rates.

A test article is considered positive if either a dose related and reproducible increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.
A test article producing neither a dose related and reproducible increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
A significant response is described as follows:
A test article is considered mutagenic if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537 and TA 98 at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
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:
No toxic effects, evident as a reduction in the number of revertants, occurred in any of the test groups with and without metabolic activation up to the highest concentration.
The plates incubated with the test article showed normal background growth at all concentrations with and without metabolic activation.

Substantial, dose dependent increases in revertant colony numbers were observed in strains TA 98 and TA 1537 following treatment with FAT 92504/A; Terasil blau GRN Isomerengemisch roh feucht in the absence and presence of metabolic activation (S9 mix) in both experiments. TA 100 showed minor increases of colony numbers in the first experiment with and without metabolic activation but this effect could not be reproduced in the second experiment.

The enhancement factor of three was exceeded at concentrations of 333 µg/plate and above in the first and 1000 µg/plate and above in the second experiment in the absence of metabolic activation using strain TA 98. With metabolic activation the threshold of three was exceeded at a concentration of 1000 µg/plate and above in both experiments. In strain TA 1537 this threshold was reached or exceeded at 333 µg/plate and above in the presence or absence of metabolic activation in the first experiment and at 333 µg/plate or higher in the absence of metabolic activation and at 33 µg/plate and above with metabolic activation in experiment II.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

None

Conclusions:
FAT 92504/A was found to induce gene mutations by frameshift with the strains Salmonella typhimurium TA 98 and TA 1537.
Executive summary:

This study was performed to investigate the potential of FAT 92504/A (mixture of Disperse Blue 054 and Disperse Blue 077) to induce gene mutations according to the plate incorporation test (experiments I and II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100. The assay was performed in two independent experiments, both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 33.3; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 µg/plate (active ingredient). No toxic effects, evident as a reduction in the number of revertants, occurred in any of the test groups with and without metabolic activation up to the highest concentration. The plates incubated with the test article showed normal background growth at all concentrations with and without metabolic activation. Substantial, dose dependent increases in revertant colony numbers were observed in strains TA 98 and TA 1537 following treatment with FAT 92'504/A; Terasil blau GRN Isomerengemisch roh feucht in the absence and presence of metabolic activation (S9 mix) in both experiments. TA 100 showed minor increases of colony numbers in the first experiment with and without metabolic activation but this effect could not be reproduced in the second experiment. The enhancement factor of three was exceeded at concentrations of 333 µg/plate and above in the first and 1000 µg/plate and above in the second experiment in the absence of metabolic activation using strain TA 98. With metabolic activation the threshold of three wasexceeded at a concentration of 1000 µg/plate and above in both experiments. In strain TA 1537 this threshold was reached or exceeded at 333 µg/plate and above in the presence or absence of metabolic activation in the first experiment and at 333 µg/plate or higher in the absence of metabolic activation and at 33 µg/plate and above with metabolic activation in experiment II. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article (FAT 92504/A) did induce gene mutations by frameshifts in the genome of the strains TA 98 and TA 1537.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
Refer chapter 13 for detailed read across justification
Reason / purpose for cross-reference:
read-across source
Species / strain:
Chinese hamster lung fibroblasts (V79)
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
Conclusions:
Based on the read across data generated from the in-vitro mammalian chromosomal abberation study with FAT 36038/J, Disperse Blue 077 was considered to be not clastogenic in mammalian cells in vitro.
Executive summary:

Source substance, FAT 36038/J TE, was tested in the chromosome aberration assay using Chinese hamster ovary (CHO) cells in both the absence and presence of an Aroclor-induced rat liver S9 metabolic activation system according to OECD Guideline 473.

Based on the results of a preliminary toxicity test, the doses chosen for the chromosome aberration assay ranged from 10 to 250 μg/mL for the non-activated 4 and 20-hour exposure groups, and from 0.25 to 10 μg/mL for the S9-activated 4-hour exposure group.

In the initial chromosome aberration assay, 55 ± 5% cytotoxicity (reduction in cell growth index relative to the vehicle control) was not observed at any dose level in the non-activated 4-hour exposure group. Cytotoxicity was observed at dose levels ≥ 3 μg/mL in the S9-activated 4-hour exposure group and at dose levels 100, 175, 200 and 250 μg/mL in the non-activated 20-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at dose levels ≥ 50 μg/mL in the non-activated 4 and 20-hour exposure groups. The dose levels selected for microscopic analysis were 10, 25, and 50 μg/mL for the non-activated 4 and 20-hour exposure groups; and 0.25, 0.5, 1, and 3 μg/mL for the S9-activated 4-hour exposure group. No significant or dose-dependent increases in structural aberrations were observed in the non-activated 4 and 20-hour exposure groups (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

In the S9-activated 4-hour exposure group, a statistically significant increase (6.3%) in structural aberrations was observed at 3 μg/mL (p ≤ 0.05; Fisher’s Exact test). In order to confirm dose-responsiveness, an additional dose level of 1 μg/mL was included in the microscopic evaluation. However, the Cochran-Armitage test was negative for a dose-response (p > 0.05). In addition, the statistically significant increase was within the historical control range of 0.0% to 9.5%; but outside the 95% historical control limit.

No significant or dose-dependent increases in numerical (polyploid or endoreduplicated cells) aberrations were observed in any of the test substance treated groups (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). In order to confirm the positive response observed, the chromosome aberration assay was repeated in the S9-activated 4-hour exposure group at doses ranging from 0.1 to 10 μg/mL. Due to insufficient cell growth during baseline counts in the vehicle and untreated controls, the assay was repeated again in the S9-activated 4-hour exposure group at doses ranging from 0.1 to 10 μg/mL.

In the second repeat assay, 55 ± 5% cytotoxicity was observed at dose levels ≥ 5 μg/mL in the S9-activated 4-hour exposure group. The dose levels selected for microscopic analysis were 1, 2.5, and 5 μg/mL. No significant or dose-dependent increases in structural or numerical aberrations were observed in the S9-activated 4-hour exposure group (p > 0.05; Fisher’s Exact and Cochran-Armitage tests). These results indicated that the statistically significant increase observed in the initial assay at the cytotoxic dose was an isolated event which was not reproducible. Therefore, the test substance was considered to be negative for the induction of structural aberrations in all three exposure groups. All vehicle control values were within historical ranges, and the positive controls induced significant increases in the percent of aberrant metaphases (p ≤ 0.01). Thus, all criteria for a valid study were met.

Based on these findings, FAT 36038/J TE was concluded to be negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using CHO cells.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Refer chapter 13 for detailed read across justification
Reason / purpose for cross-reference:
read-across source
Species / strain:
Chinese hamster lung fibroblasts (V79)
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
Conclusions:
Based on the read across data generated from the in-vitro Mammalian Cell Forward Gene Mutation (CHO/HPRT) Assay with FAT 36038/J, the target substance FAT 40278 is also considered to be negative in the In Vitro Mammalian Cell Forward Gene Mutation (CHO/HPRT) Assay with Duplicate Cultures.
Executive summary:

No data on the mutagenic potential of Disperse Blue 077 in mammalian cells is available. To fill the data gaps, read across approach is adapted using similar substance FAT 36038/J. Read-across is claimed basis of structural relationship of the target and the source chemicals.

The source substance, FAT 36038/J TE, was evaluated according to OECD 476 guideline for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system (S9), as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr).

FAT 36038/J TE was prepared in DMSO and evaluated in a preliminary toxicity assay at concentrations of 2.93, 5.86, 11.7, 23.4, 46.9, 93.8, 188, 375, 750 and 1500 μg/mL with and without S9. The maximum dose evaluated was based on the solubility limitations of the test substance in the vehicle. Visible precipitate was observed at concentrations ≥11.7 μg/mL at the beginning and end of treatment. The pH of the cultures was adjusted at a concentration of 1500 μg/mL to maintain neutral pH, and the test substance had no adverse impact on the osmolality of the cultures. Adjusted relative survival was 27.70 and 11.61% at a concentration of 1500 μg/mL with and without S9, respectively.

Based on these results, FAT 36038/J TE was evaluated in the definitive mutagenicity assay at concentrations of 1.50, 3.00, 6.00, 7.00, 8.00, 9.00, 10.0, 11.0 and 11.7 μg/mL with S9 and 1.25, 2.50, 5.00, 8.00 and 11.7 μg/mL without S9. No visible precipitate was observed at the beginning or end of treatment, and the test substance had no adverse impact on the pH of the cultures. The average adjusted relative survival was 7.58 and 96.44% at a concentration of 11.7 μg/mL with and without S9, respectively. However, the limit dose was not achieved due to a shift in precipitate profile, and the entire assay was retested with an adjustment in dose levels.

In the mutagenicity assay retest, FAT 36038/J TE was evaluated at concentrations of 1.00, 3.00, 4.50, 6.00 and 12.0 μg/mL with S9 and 3.00, 6.00, 9.00, 10.0, 12.0, 25.0, 50.0, 100, 500 and 1500 μg/mL without S9. Visible precipitate was observed at concentrations ≥10.0 μg/mL at the beginning and end of treatment, and the pH of the cultures was adjusted at a concentration of 1500 μg/mL to maintain neutral pH. The average adjusted relative survival was 25.39 and 24.56% at concentrations of 6.00 μg/mL with S9 and 1500 μg/mL without S9, respectively. Cultures treated at concentrations of 1.00, 3.00, 4.50 and 6.00 μg/mL with S9 and 9.00, 12.0, 100, 500 and 1500 μg/mL without S9 were chosen for mutant selection (cultures treated at a concentration of 6.00 μg/mL without S9 were excluded from evaluation of mutagenicity because a sufficient number of higher concentrations was available; cultures treated at other concentrations were discarded prior to selection because a sufficient number of higher concentrations was available, or due to excessive toxicity). No significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated without S9 (p> 0.05). In contrast, the positive controls induced a significant increase in mutant frequency (p<0.01)

These results indicate FAT 36038/J was negative in the in vitro Mammlian Cell Forward Gene Mutation (CHO/HPRT) Assay with Duplicate cultures, under the conditons and according to the criteria of the test protocol.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Disperse Blue 077 was considered to be not clastogenic using the data available with a structurally similar substance.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 01, 1996 to January 13, 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
None
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: mammalian cell clastogenicity assay
Specific details on test material used for the study:
None
Species:
mouse
Strain:
NMRI
Details on species / strain selection:
Recommended by the guideline
Sex:
male/female
Details on test animals or test system and environmental conditions:
Source: BRL, CH-4414 Füllinsdorf
Number of Animals: 72 (36 males/36 females)
Initial Age at Start of Acclimatization: 8-12 weeks
Acclimatization: minimum 5 days
Initial Body Weight at Start of Treatment: males mean value 31.2 g (SD ± 3.0 g)
females mean value 22.9 g (SD ± 1.5 g)
According to the suppliers assurance the animals were in healthy condition. The animals were under quarantine in the animal house of C C R for a minimum of five days after their arrival. During this period the animals did not show any signs of illness or altered behaviour. The animals were distributed into the test groups at random and identified by cage number.

Husbandry
The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
Housing: single
Cage Type: Makrolon Type I, with wire mesh top (EHRET GmbH, D-79302 Emmendingen)
Bedding: granulated soft wood bedding (ALTROMIN, D-32791 Lage/Lippe)
Feed: pelleted standard diet, ad libitum (ALTROMIN 1324, D-32791 Lage/Lippe)
Water: tap water, ad libitum, (Gemeindewerke, D-64380 Roßdorf)
Environment: temperature 21 ±3°C
relative humidity 30-70%
artificial light 6.00 a.m. - 6.00 p.m.
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: PEG 400 was used as vehicle control.
- Justification for choice of solvent/vehicle: Widely accepted.
- Catalogue No.: 9726 (gas chromatography grade)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test article was formulated in PEG 400. The vehicle was chosen to its relative non-toxicity for the animals. All animals received a single standard volume of 10 ml/kg body weight orally.
Duration of treatment / exposure:
once
Post exposure period:
24 h preparation interval: 200, 670 and 2000 mg/kg b.w..
48 h preparation interval: 2000 mg/kg b.w..
Dose / conc.:
200 mg/kg bw/day
Remarks:
24 h preparation interval
Dose / conc.:
670 mg/kg bw/day
Remarks:
24 h preparation interval
Dose / conc.:
2 000 mg/kg bw/day
Remarks:
24 as well as 48h preparation interval
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide;
- Justification for choice of positive control(s): recommended by the guideline
- Route of administration: orally, once
- Doses / concentrations: 40 mg/kg b.w.
Tissues and cell types examined:
polychromatic erythrocytes (PCE)
Details of tissue and slide preparation:
Slide preparation:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifiiged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (MERCK, D-64293 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg). At least one slide was made from each bone marrow sample.

Analysis of cells:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 1000 the PCEs. The analysis was performed with coded slides.
Five animals per sex and group were evaluated as described. The remaining 6th animal of each test group was evaluated in case an animal had died in its test group.
Evaluation criteria:
A test article is classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant positive response for at least one of the test points.
A test article producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant positive response at any of the test points is considered non-mutagenic in this system.
However, both biological and statistical significance should be considered together.
Statistics:
Statistical significance was evaluated by means of the nonparametric Mann-Whitney test.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
After treatment with the test article the number of NCEs was not substantially increased as compared to the corresponding vehicle controls thus indicating that FAT 92'504/A had no cytotoxic effectiveness in the bone marrow.
In comparison to the corresponding vehicle controls there was no enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test article and with any dose level used.

40 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a statistically significant increase of induced micronucleus frequency.
Conclusions:
FAT 92504/A can be considered to be not-clastogenic in vivo.
Executive summary:

The test article FAT 92504/A (mixture of DISPERSE BLUE 054 and DISPERSE BLUE 077) was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test article was formulated in PEG 400. PEG 400 was used as vehicle control. The volume administered orally was 10 ml/kg b.w.. Twenty four and 48 h after the single administration of the test article the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal (exception animal no. 49 and 52 = 2000 PCEs) were scored for micronuclei.

The following dose levels of the test article were investigated:

24 h preparation interval: 200, 670, and 2000 mg/kg b.w.

48 h preparation interval: 2000 mg/kg b.w.

The mean number of normochromatic erythrocytes (NCEs) was not substantially increased after treatment with the test article as compared to the mean values of NCEs of the corresponding vehicle controls, indicating that FAT 92504/A had no cytotoxic properties in the bone marrow. In comparison to the corresponding vehicle controls there was no enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test article. The mean values of micronuclei observed after treatment with FAT 92'504/A were in the range of the vehicle control group. 40 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a statistically significant increase of induced micronucleus frequency. In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

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

Additional information

The genetic toxicity database of Disperse Blue 077 consists of a bacterial reverse mutation assay and a micronucleus assay. Further abstracts of a bacterial reverse mutation assay and a mammalian chromosomal aberration assay in vitro with Disperse Blue 077 commissioned by METI Japan (Ministry of Economy,Trade and Industry) are available. A positive mutagenic response was observed in the bacterial reverse mutation assays, hence mutagenic potential of the substance could not be ruled out. Hence to complete the genetic toxicity assessment of Disperse Blue 077, a study investigating mutagenic potential in mammalian cells is required. The genetic toxicity database of source substance Disperse Violet 057 consists of a couple bacterial reverse mutation assays, an in vitro mammalian cell gene mutation assay and an in vitro chromosomal aberration assay. Hence the studies with Disperse Violet 057 were used to complete the genetic toxicity assessment of Disperse Blue 077.

 

Bacterial reverse mutation assay with Disperse Blue 077

In a bacterial reverse mutation assay performed to investigate the potential of FAT 92504/A to induce gene mutations using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100 at concentrations 33.3; 100; 333.3; 1000; 2500; and 5000 μg/plate, with and without metabolic activation, substantial, dose dependent increases in revertant colony numbers were observed in strains TA 98 and TA 1537. Hence, it was concluded that during the described mutagenicity test and under the experimental conditions reported, the test article did induce gene mutations by frameshifts in the genome of the strains TA 98 and TA 1537.

In order to evaluate the mutagenic potential of Disperse Blue 77, METI (Ministry of economy, trade and industry), Japan commissioned a bacterial reverse mutation test in 2011, which was performed using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and E. coli WP2uvrA. The test was conducted using pre-incubation method with and without metabolic activation. A 2-fold or greater and dose-dependent increase in revertant colonies was observed in TA98 and TA1537 without metabolic activation (-S9 mix) andin TA100, WP2uvrA, TA98, and TA1537 with metabolic activation (+S9 mix). Hence, Disperse Blue 77 is considered to have produced mutagenic effect in this bacterial reverse mutation assay.

 

Assessment of the outcome of the reverse mutation assay:

It is well-known for aromatic nitro compounds to be positive in the Ames assay resulting from metabolism by the bacteria-specific enzyme nitro-reductase [Tweats et al. 2012]. However, it has been demonstrated in various publications that this is a bacteria-specific effect and that these Ames positive substances are not mutagenic in mammalian assays. Type II nitroreductases transfer a single electron to the nitro group, forming a nitro anion radical, which in the presence of oxygen generates the superoxide anion in a futile redox cycle, regenerating the nitro group [de Oliveira et al. 2010].

The nitroreductase family comprises a group of flavin mononucleotide (FMN)- or flavin adenine dinucleotide (FAD) -dependent enzymes that are able to metabolize nitroaromatic and nitroheterocyclic derivatives (nitrosubstituted compounds) using the reducing power of nicotinamide adenine dinucleotide (NAD(P)H). These enzymes can be found in bacterial species and, to a lesser extent, in eukaryotes. The nitroreductase proteins play a central role in the activation of nitrocompounds [de Oliveira et al. 2010].

That the reduction of these nitro-compounds to mutagenic metabolites is a bacteria-specific effect is demonstrated in the following by means of the two compounds AMP397 and fexinidazole.

AMP397 is a drug candidate developed for the oral treatment of epilepsy. The molecule contains an aromatic nitro group, which obviously is a structural alert for mutagenicity. The chemical was mutagenic in Salmonella strains TA97a, TA98 and TA100, all without S9, but negative in the nitroreductase-deficient strains TA98NR and TA100NR. Accordingly, the ICH standard battery mouse lymphoma tk and mouse bone marrow micronucleus tests were negative, although a weak high toxicity-associated genotoxic activity was seen in a micronucleus test in V79 cells [Suter et al. 2002]. The amino derivative of AMP397 was not mutagenic in wild type TA98 and TA100. To exclude that a potentially mutagenic metabolite is released by intestinal bacteria, a MutaTM Mouse study was done in colon and liver with five daily treatments at the MTD, and sampling of 3, 7 and 21 days post-treatment. No evidence of a mutagenic potential was found in colon and liver. Likewise, a comet assay did not detect any genotoxic activity in jejunum and liver of rats, after single treatment with a roughly six times higher dose than the transgenic study, which reflects the higher exposure observed in mice. In addition, a radioactive DNA binding assay in the liver of mice and rats did not find any evidence for DNA binding. Based on these results, it was concluded that AMP397 has no genotoxic potential in vivo. It was hypothesized that the positive Ames test was due to activation by bacterial nitro-reductase, as practically all mammalian assays including four in vivo assays were negative, and no evidence for activation by mammalian nitro-reductase or other enzymes were seen. Furthermore, no evidence for excretion of metabolites mutagenic for intestinal cells by intestinal bacteria was found.

Fexinidazole was in pre-clinical development as a broad-spectrum antiprotozoal drug by the Hoechst AG in the 1970s-1980s, but its clinical development was not pursued. Fexinidazole was rediscovered by the Drugs for Neglected Diseases initiative (DNDi) as drug candidate to cure the parasitic disease human African trypanomiasis (HAT), also known as sleeping sickness. The genotoxicity profile of fexinidazole, a 2-substituted 5-nitroimidazole, and its two active metabolites, the sulfoxide and sulfone derivatives were investigated [Tweats et al. 2012]. All the three compounds are mutagenic in the Salmonella/Ames test; however, mutagenicity is either attenuated or lost in Ames Salmonella strains that lack one or more nitroreductase(s). It is known that these enzymes can nitroreduce compounds with low redox potentials, whereas their mammalian cell counterparts cannot, under normal conditions. Fexinidazole and its metabolites have low redox potentials and all mammalian cell assays to detect genetic toxicity, conducted for this study either in vitro (micronucleus test in human lymphocytes) or in vivo (ex vivo unscheduled DNA synthesis in rats; bone marrow micronucleus test in mice), were negative.

Conclusion

Based on these data and the common mechanism between the reduction of these nitro-compounds, which is widely explored in literature [de Oliveira et al. 2010], it is concluded, that the mutagenic effects observed in the Ames tests with Disperse Blue 077 is a bacteria specific effect and not relevant to mammalians.

In order to investigate the role of nitroreductases further in the positive outcome of the bacterial reverse mutation, a modified bacterial reverse mutation assay that also investigates nitroreductase deficient strains i.e. TA98NR and TA100NR has been commissioned and results are expected in September 2020.

 

In vitro mammalian cell gene mutation assay with Disperse Violet 057 (read across)

In a study performed to evaluate the potential of Disperse Violet 057 to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system (S9), as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr), no significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated without S9 (p >0.05). In contrast, the positive controls induced a significant increase in mutant frequency (p <0.01). These results indicate Disperse Violet 057 was negative in thein vitromammalian cell forward gene mutation (CHO/HPRT) assay with duplicate cultures, under the conditions and according to the criteria of the test protocol.

Mammalian chromosomal aberration assay in vitro with Disperse Blue 077 (METI, Japan)

A chromosomal aberration test was performed using cultured mammalian cells (CHL/IU cells) with and without metabolic activation in order to evaluate whether Disperse Blue 077 induces chromosomal aberrations. No increase  in chromosomal  aberrations was observed  with or without  metabolic activation in short-term treatment, or without metabolic activation in continuous treatment for 24 hours. Hence, Disperse Blue 077 was considered to be not clastogenic to mammalian cells in vitro.

 

Micronucleus assay with Disperse Blue 077

Disperse Blue 077 was evaluated for clastogenic potential in a GLP-compliant micronucleus assay, conducted according to OECD Guideline 474. In this assay, there was no enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test article in comparison to the corresponding vehicle controls. Hence, it was concluded that the test article did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

 

Additionally, source substance, Disperse Violet 057 was also evaluated in two bacterial reverse mutation assays and an in vitro chromosomal aberration assay. While both reverse mutation assays conducted gave a positive response, the chromosomal aberration test conducted according to OECD 473 was concluded to be negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9-activated CHO cells.

 

As discussed above, both substances, Disperse Blue 077 as well as Disperse Violet 057, were found to induce mutagenic response in bacterial reverse mutation assays. However, the positive response can be considered bacteria specific and not relevant to mammalian cells. Disperse Blue 077 was not clastogenic in the mammalian chromosomal aberration assay in vitro as well as in the micronucleus assay. Disperse Violet 057 neither lead to mutations in the mammalian cell gene mutation assay in vitro, nor caused any clastogenic effects in an in vitro chromosomal aberration assay. Hence, based on the negative results in the in vitro mammalian cell gene mutation assay with Disperse Violet 057, and the in vitro chromosomal aberration assays with Disperse Blue 077 as well as Disperse Violet 057 and micronucleus assay with Disperse Blue 077, the target substance is considered to be neither mutagenic nor clastogenic, and concluded to be not genotoxic.

Justification for classification or non-classification

Based on the above discussion, Disperse Blue 077 was considered to be not genotoxic, and hence no classification is warranted as per CLP (Regulation EC No. 1272/2008) criteria.