Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
genetic toxicity in vivo, other
Remarks:
single cell gell / Comet assay
Type of information:
experimental study planned
Study period:
not yet defined
Justification for type of information:
TESTING PROPOSAL ON VERTEBRATE ANIMALS


NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out : Acid Brown 126

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION [please address all points below]:
- Available GLP studies : assessment on genotoxic potential of test substance was initially based on th following tests:
IN VITRO GENE MUTATION STUDY IN BACTERIA [Bacterial reverse mutation assay (OECD 471)]
- Available non-GLP studies : Not Available
- Historical human data : Not Available
- (Q)SAR : Not Available
- In vitro methods : assessment on genotoxic potential of test substance was initially based on th following tests:
IN VITRO GENE MUTATION STUDY IN BACTERIA [Bacterial reverse mutation assay (OECD 471)]
- Weight of evidence: available results from in vitro study (GLP) were taken into account, but no definitive conclusion could be drawn
- Grouping and read-across: no read across data available

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- According to the Annex VIII, Column 2 of the REACH Regulation, appropriate in vivo studies shall be considered in case of positive result in any of the genotoxicity studies in Annex VII or VIII. Specific adaptation possibilities apply in case of in vivo studies already available.
Based on the positive result in the Ames test, on the expected negative result in the in vitro mammalian cells assays and on the lack of results from in vivo studies, an appropriate in vivo genotoxicity study is proposed.

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed: single cell gel / Comet assay.
Since it is acknowledged that many azo dyes can be converted into mutagens by the intestinal microflora, appropriate in vivo follow up studies to evaluate genotoxic activity in tissues/organs, including the gastrointestinal tract, after oral administration would be transgenic rodent mutation assay or a rodent Comet assay. As reported in literature, from the analysis of 91 chemicals with published data from both tests, the comet assay appears to yield similar results to the TGR assay in liver and gastrointestinal tract; hence the comet assay in an acceptable choice when the intention is to confirm in vitro gene mutation activity in terms of genotoxicity in general
Given that the substance showed positive results in the Ames test, it should be necessary to consider performing an in vivo mammalian Alkaine comet assay (OECD 489) in rats following recommendations stated in ECHA's 2015 REACH progress report.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
other: reference to other assay considered in the justification for the testing proposal
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From July 2nd to July 13th, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial gene mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
Stocks of Salmonella tester strains (TA 1535, TA 1537, TA 98, TA 100) were obtained from Dr. B.N.Ames, University of California. Stocks of E. coli tester strains (WP2 uvrA) was obtained from Life Science Research, Occold, Suffolk, UK.
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system: S9
Test concentrations with justification for top dose:
-Toxicity test: 5000, 1580, 500, 158 and 50.0 μg/plate
- Main test I: 5000, 2500, 1250, 625 and 313 μg/plate.
- Main test II: 5000, 2500, 1250, 625 and 313 μg/plate.
- Main test III: 1100, 846, 651, 501 and 385 μg/plate.


The overlay mixture was composed as follows:
Overlay agar (held at 45°C) 2.0mL
Test or control item solution 0.1mL
S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.5mL
Bacterial suspension 0.1mL
Main Assays II and III were performed using a pre-incubation method. The components were added in turn to an empty test-tube:
Bacterial suspension 0.1mL
Test or control item solution 0.1mL
(2-Aminoanthracene solution) (0.05 mL)
Modified S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.5mL
The incubate was vortexed and placed at 37°C for 30 minutes. Two mL of overlay agar was then added and the mixture vortexed again and poured onto the surface of a minimal medium agar plate and allowed to solidify.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:
Sterile water for injection
Dimethylsulfoxide (DMSO)

- Justification for choice of solvent/vehicle:
selected since it is compatible with the survival of the bacteria and the S9 metabolic activity
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene; Trypan blue
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate

FORMULATION PROCEDURE
Solutions of the test item, as received, were prepared immediately before use in sterile water for injection. Solutions were prepared on a weight/volume basis without correction for the displacement due to the volume of the test item. Concentrations were expressed in terms of material as received. All test item solutions were used within 1 hour and 58 minutes from the initial preparation. All dose levels in this report are expressed to three significant figures.

METHOD OF APPLICATION: in agar (plate incorporation)
- Nutrient Broth: Oxoid Nutrient Broth No. 2 was prepared at a concentration of 2.5% in distilled water and autoclaved prior to use. This was used for the preparation of liquid cultures of the tester strains.
- Nutrient Agar: Oxoid Nutrient Broth No. 2 (25 g) and Difco Bacto-agar (15 g) were added to distilled water (1 litre) and autoclaved. The solutions were then poured into 9 cm plastic Petri dishes and allowed to solidify and dry before use. These plates were used for the non-selective growth of the tester strains.
- Minimal Agar: Minimal medium agar was prepared as 1.5% Difco Bacto-agar in Vogel-BonnerMedium E, with 2% Glucose, autoclaved and poured into 9 cm plastic Petri dishes.
- Top Agar: "Top Agar" (overlay agar) was prepared as 0.6% Difco Bacto-agar + 0.5% NaCl in distilled water and autoclaved. Prior to use, 10mL of a sterile solution of 0.5mMBiotin + 0.5mM Histidine (or 0.5mMtryptophan) was added to the top agar (100 mL).

DETERMINATION OF CYTOTOXICITY
A preliminary toxicity test was undertaken in order to select the concentrations of the test item to be used in theMain Assays. In this test a wide range of dose levels of the test item, set at half-log intervals, were used. Treatments were performed both in the absence and presence of S9 metabolism using the plate incorporation method; a single plate was used at each test point and positive controls were not included. Toxicity was assessed on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.

SOLUBILITY
Solubility of the test item was evaluated in a preliminary trial using sterile water for injection. This solvent was selected since it is compatible with the survival of the bacteria and the S9 metabolic activity. The test item was found to be soluble at 50.0mg/mL. This result permitted a maximum concentration of 5000 μg/plate to be used in the toxicity test.

INCUBATION AND SCORING
The prepared plates were inverted and incubated for approximately 72 hours at 37°C. After this period of incubation, plates were held at 4°C for approximately 24 hours before scoring.
Evaluation criteria:
For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.
Statistics:
The regression analysis fits a regression line to the data by the least squares method, after square root transformation of the plate counts to satisfy normal distribution and homoscedasticity assumptions. The regression equation is expressed as:
y = a + b x
where:
y = transformed revertant numbers
a = intercept
b = slope value
x = dose level (in the units given).
Regression lines are calculated using a minimum of the three lowest dose levels, and then including the further dose levels in turn. The correlation co-efficient (r), the value of students "t" statistic, and the p-value for the regression lines are also given.
Species / strain:
other: Salmonella typhimurium (TA1535, TA1537, TA98 and TA100)
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
other: Salmonella typhimurium (TA1535, TA1537, TA98 and TA100)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid

SOLUBILITY

The test item was found to be soluble at 50.0 mg/mL. This result permitted a maximum concentration of 5000 μg/plate to be used in the toxicity test.

TOXICITY TEST

Neither precipitation of the test item, nor toxicity was observed at the end of the incubation period, with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism. Plates treated with the test item showed a dose dependent brown colour of the agar, which did not interfere with the scoring of revertant colonies and evaluation of the background lawn. No relevant increases were observed with the remaining tester strains..

MAIN TEST

Neither toxicity, nor precipitation of the test item was observed at the end of the incubation period with any tester strain, at any dose level, in the absence or presence of S9 metabolic activation.

As no relevant increase in revertant numbers was observed at any concentration tested, Main Assay II was carried out. Based on the chemical structure of the test item (azo-dye), the experiment was performed using the pre-incubation method in the presence of a reductive metabolic system (hamster S9 supplemented with flavin mononucleotide cofactor). The test item was assayed at the same concentrations used in Main Assay I. Neither precipitation of the test item, nor toxicity was observed at the end of the incubation period, with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism.

In the presence of S9 metabolism, a relevant increase in revertant numbers (2.3-fold) was seen with TA98 tester strain at an intermediate dose level (625 μg/plate). No relevant increases were observed with the remaining tester strains.In order to confirmthis result,Main Assay III was performed using the Prival modification method. The dose range used was slightly modified to investigate more closely those doses of the test item most likely to exhibit a mutagenic response. The following dose levels were assayed: 1100, 846, 651, 501 and 385 μg/plate.

Relevant increases in revertant numbers were observed at 651, 501, 385 μg/plate in the presence of the reductive metabolic system. These increases were greater than twice the control value and so can be considered as clear evidence of mutation induction.

Plates with the test item showed a dose dependent brown colour of the agar (brown-yellow in the presence of the reductive metabolic system), which did not interfere with the scoring of colonies and evaluation of the background lawn. The sterility of the S9 mix and of the test item solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions. Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly.

 

VALIDITY CRITERIA:

The assay was considered valid if the following criteria were met:

1. Mean plate counts for untreated and positive control plates should fall within 2 standard deviations of the current historical mean values.

2. The estimated numbers of viable bacteria/plate should fall in the range of 100 – 500 millions for each strain.

3. No more than 5% of the plates should be lost through contamination or other unforeseen event.

Criteria for outcome of the assays:

For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

EVALUATION

Results show that mean plate counts for untreated and positive control plates fell within the normal range based on historical control data. The estimated numbers of viable bacteria/plate (titre) fell in the range of 100 - 500 million for each strain. No plates were lost through contamination or cracking. The study was accepted as valid.The test item induced reproducible, more than two-fold increases in the number of revertant

colonies with TA98 tester strain in the presence of a reductive metabolic activation system. A statistically significant dose-effect relationship was indicated. On the basis of the stated criteria, it must be concluded that the test item is mutagenic in bacteria under the reported experimental conditions.

Conclusions:
Mutagenic in Salmonella typhimurium in the presence of a reductive metabolic activation system.
Executive summary:

The test item was examined according to the OECD Guideline 471 for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbital and 5,6-benzoflavone (standard metabolic activation) in the toxicity test and in Main Assay I, and liver S9 fraction from uninduced hamsters (reductive metabolic activation system using the Prival modification method) in Main Assays II and III. The test item was used as a solution in sterile water for injection.

Toxicity test

The test item Acid Brown 126 was assayed in the toxicity test at a maximum concentration of 5000 μg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 μg/plate. Neither precipitation of the test item, nor toxicity was observed at the end of the incubation period, with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism. No relevant increases were observed with the remaining tester strains.

Main Assays

On the basis of toxicity test results, inMain Assay I, using the plate incorporation method and the standard S9 metabolic activation system, the test item was assayed at the following dose levels: 5000, 2500, 1250, 625 and 313 μg/plate. Neither precipitation of the test item, nor toxicity was observed at the end of the incubation period, with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism.

As no relevant increase in revertant numbers was observed at any concentration tested, Main Assay II was carried out. Based on the chemical structure of the test item (azo-dye), the experiment was performed using the pre-incubation method in the presence of a reductive metabolic system (hamster S9 supplemented with flavin mononucleotide cofactor). The test item was assayed at the same concentrations used in Main Assay I. Neither precipitation of the test item, nor toxicity was observed at the end of the incubation period, with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism.

In the presence of S9 metabolism, an increase in revertant numbers (2.3-fold) was seen with TA98 tester strain at an intermediate dose level (625 μg/plate). No relevant increases were observed with the remaining tester strains. In order to confirmthis result, Main Assay III was performed using the Prival modification method. The dose range used was slightly modified to investigate more closely those doses of Acid Brown 126 most likely to exibit a mutagenic response. The following concentrations were assayed: 1100, 846, 651, 501 and 385μg/plate.

Once again, relevant increases in revertant numberswere observed at 651, 501, 385μg/plate. These increases were greater than twice the control values and so can be considered as clear evidence of mutation induction.

 Conclusion

It is concluded that the test item Acid Brown 126 induces reverse mutation inSalmonella typhimuriumin the presence of a reductive metabolic activation system, under the reported experimental conditions.

Reason / purpose for cross-reference:
other: reference to other assay considered in the justification for the testing proposal
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
other: assessment
Adequacy of study:
key study
Study period:
2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Principles of method if other than guideline:
Data from substance review and handbook with collection of data.
GLP compliance:
not specified
Remarks:
not applicable
Type of assay:
other: Follow-up of In-vitro positive results
Genotoxicity:
positive
Remarks:
based on assessment
Remarks on result:
not measured/tested
Remarks:
assessment derived from substance review and handbook with collection of data.
Conclusions:
The substance is expected to be negative in in vitro mammalian cell assays
Executive summary:

As expected from the chemical structure the positive results observed in the Bacterial Mutation Assay are attributable to the reduction of the azo-bonds, which produce the relese of aromatic amines, most od which are under suspicion for genotoxicity and carcinogenicity.

The azo dyes would clearly not be expected to give positive results in mammalian cells, where reductive metabolic conditions are not applied.

Since the specific metabolic pathways (reduction of the azo linkage and metabolic oxidation of the using mammalian cells in vitro, an in vivo test is considered to be necessary to effectively evaluate the mutagenicity potential of the test chemical. Given that the test item showed positive results in the Ames test, it should be necessary to consider performing an in vivo mammalian alkaline comet assay (OECD 489) in rats following recommendations stated in ECHA's 2015 REACH progress report.

Data source

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
GLP compliance:
yes
Type of assay:
mammalian comet assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction products of diazotised 3-amino-2-hydroxy-5-nitrobenzenesulphonic acid, coupled with 1,3-diaminobenzene and diazotised sodium 4-aminobenzenesulfonate, metallised with Basic Chromium (III) Sulphate
EC Number:
947-395-4
Molecular formula:
not applicable
IUPAC Name:
Reaction products of diazotised 3-amino-2-hydroxy-5-nitrobenzenesulphonic acid, coupled with 1,3-diaminobenzene and diazotised sodium 4-aminobenzenesulfonate, metallised with Basic Chromium (III) Sulphate
Test material form:
solid: particulate/powder

Results and discussion

Applicant's summary and conclusion