Reaction mass of Chromate(2-), [3-hydroxy-4-[(2-hydroxy-1-naphthalenyl)azo]-7-nitro-1-naphthalenesulfonato(3-)] [N-[7-hydroxy-8-[(2-hydroxy-5-nitrophenyl)azo]-1-naphthalenyl]]-, lithium sodium and Chromate(2-), [3-hydroxy-4-[(2-hydroxy-1-naphthalenyl)azo]-7-nitro-1-naphthalenesulfonato(3-)][N-[7-hydroxy-8-[(2-hydroxy-5-nitrophenyl)azo]-1-naphthalenyl]acetamidato(2-)]-, lithium sodium

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Only bacteria-specific effects were noted in the bacteria reverse mutation assay, whereas the mutagenicity study in mammalian cells was negative. In addition, the test substance did not show clastogenic or aneugenic effects at test concentration not exibiting a high cytotoxicity.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from rat liver

Test concentrations with justification for top dose:

with metabolic activation: 10, 50, 100, 500, 1000 and 5000 µg/plate
without metabolic activation: 10, 50, 100, 500, 1000 and 5000 µg/plate

Vehicle / solvent:

water

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 2-Aminoanthracene; 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, 4-nitro-o-phenylenediamine

Details on test system and experimental conditions:

Bacterial strains
Salmonella typhimuriurn TA100, TA98, TA1535, TA1537 and TA1538 were obtained from Professor B.N. Ames, University of California, U.S.A.
Escherichia coli WP2 uvrA was provided by Dr. T. Kada, National Institute of Genetics, Mishima, Japan.

Cultures of each strain were prepared by incubating an aliquot of parent stock culture in nutrient broth at 37°C for 18 hours.

After mixing 0.1 ml of test material solution, 0.5 ml of phosphate buffer or S-9 mix, 0.1 ml of culture of tester strain, and 2 ml of top agar in a test tube, the mixture was poured on minimal agar plate. The plates were incubated at 37°C for 2 days and scored, for revertants.

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Under the study conditions, the test substance was found to be mutagenic in most Salmonella strains in bacterial reverse mutation assay.

Executive summary:

A bacterial reverse mutation assay (Ames) was carried out, to evaluate the test substance for the mutagenic potential in a microbial assay with and without the addition of mammalian metabolic activation preparation. The test was carried out in concentrations of 10, 50, 100, 500, 1000 and 5000 µg/plate in strains of Salmonella typhimurium TA100, TA98, TA1535, TA1537 and TA1538 and Escherichia coli WP2 uvrA. Cultures of each strain were prepared by incubating an aliquot of parent stock culture in nutrient broth at 37°C for 18 hours. After mixing 0.1 ml of test material solution, 0.5 ml of phosphate buffer or S-9 mix, 0.1 ml of culture of tester strain, and 2 ml of top agar in a test tube, the mixture was poured on minimal agar plate. The plates were incubated at 37°C for 2 days and scored, for revertants.

Significant differences were observed in the number of mutant colonies between the test compound dosages and the control with Salmonella typhimurium TA100, TA98, TA1535, TA1537 and TA1538. No increases in mutation frequencies were observed in Escherichia coli WP2 uvrA.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from rat liver

Test concentrations with justification for top dose:

with metabolic activation: 10, 50, 100, 500, 1000 and 5000 µg/plate
without metabolic activation: 10, 50, 100, 500, 1000 and 5000 µg/plate

Vehicle / solvent:

water

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 2-Aminoanthracene; 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, 4-nitro-o-phenylenediamine

Details on test system and experimental conditions:

Bacterial strains
Salmonella typhimuriurn TA100, TA98, TA1535, TA1537 and TA1538 were obtained from Professor B.N. Ames, University of California, U.S.A.
Escherichia coli WP2 uvrA was provided by Dr. T. Kada, National Institute of Genetics, Mishima, Japan.

Cultures of each strain were prepared by incubating an aliquot of parent stock culture in nutrient broth at 37°C for 18 hours.

After mixing 0.1 ml of test material solution, 0.5 ml of phosphate buffer or S-9 mix, 0.1 ml of culture of tester strain, and 2 ml of top agar in a test tube, the mixture was poured on minimal agar plate. The plates were incubated at 37°C for 2 days and scored, for revertants.

For each concentrations 2 plates were prepared and scored.

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Under the study conditions, the test substance was found to be mutagenic in some Salmonella strains in the bacterial reverse mutation assay.

Executive summary:

A bacterial reverse mutation assay (Ames) was carried out, to evaluate the test substance for the mutagenic potential in a microbial assay with and without the addition of mammalian metabolic activation preparation. The test was carried out in concentrations of 10, 50, 100, 500, 1000 and 5000 µg/plate in strains of Salmonella typhimuriurn TA100, TA98, TA1535, TA1537 and TA1538 and Escherichia coli WP2 uvrA. Cultures of each strain were prepared by incubating an aliquot of parent stock culture in nutrient broth at 37°C for 18 hours. After mixing 0.1 ml of test material solution, 0.5 ml of phosphate buffer or S-9 mix, 0.1 ml of culture of tester strain, and 2 ml of top agar in a test tube, the mixture was poured on minimal agar plate. The plates were incubated at 37°C for 2 days and scored, for revertants.

The test substance was tested positive without metabolic activation in Salmonella typhimurium TA 98 and TA 1538. The test result was ambigous in TA 100 and TA 1538 with metabolic activation and in TA 1537 without metabolic activation. Negative results were obtained in E. coli and Salmonella typhimurium TA 1535 with and without metabolic activation and in TA 100 without and TA 1537 and TA 98 with metabolic activation.

Species/strain	Metabolic activation	Genotoxicity	Cytotoxicity
E. coli WP2 uvr A	with and without	negative	no, but tested up to limit concentrations
S. typhimurium TA 1535	with and without	negative	yes
S. typhimurium TA 100	without	negative	yes
S. typhimurium TA 100	with	ambiguous	no, but tested up to limit concentrations
S. typhimurium TA 98	without	positive	yes
S. typhimurium TA 98	with	negative	no, but tested up to limit concentrations
S. typhimurium TA 1537	without	ambiguous	yes
S. typhimurium TA 1537	with	negative	no, but tested up to limit concentrations
S. typhimurium TA 1538	without	positive	Yes
S. typhimurium TA 1538	with	ambiguous	no, but tested up to limit concentrations

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 03-Feb-2003 to 27-Feb-2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: Draft OECD Guideline (proposal): In Vitro Mammalian Micronucleus Test

Version / remarks:

June 2002 (3rd International Workshop on genotoxicity Testing IWGT, Plymouth, UK)

Qualifier:

according to guideline

Guideline:

other: Global Drug Safety Evaluation International study protocol guidelines, Aventis Pharma (DI&A/LO/DSE). In vitro micronucleus test

Version / remarks:

December 2000, 23

Principles of method if other than guideline:

In this study performed for screening purposes without GLP inspection one experiment with metabolic activation (3/21 h treatment/recovery time) and one experiment without metabolic activation (24/24 h treatment/recovery time) was conducted.

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from rat liver

Test concentrations with justification for top dose:

with metabolic activation: 300, 350, 400, 450, 475 and 500 µg/mL
without metabolic activation: 25, 50, 80, 100, 120 and 140 µg/mL

Vehicle / solvent:

cell culture medium

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

(with metabolic activation)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

(without metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

Solvent controls, positive controls and 12 concentrations of the test compound were tested on microtiter plates. Due to toxicity the highest concentration used was 500 µg/ml. For each group two cell cultures were treated. Cytotoxicity was demonstrated by the cell survival rate. The survival rate was determined by cell counting using a cell counter (Casy® counter, Scharfe System). Relative cell growth was expressed as a percentage of the solvent control.
On the basis of cytotoxicity at least five test groups were selected for evaluation. In case of soluble non-toxic compounds, the highest concentration groups were evaluated. Otherwise a maximum concentration resulting in approximately 50 % or less cell survival was selected for evaluation.
In case of inappropriate toxicity a repeat experiment with modified concentrations was performed. Cells were harvested in 200 µL KCl-solution, centrifuged for 5 minutes at approx. 300 g and 100 µL from the supernatant was replaced by fixative (ethanol/ glacial acetic acid).
After a second centrifugation step, the supernatant was discarded and stored for approximately 30 minutes at 4 °C after adding 200 µL fixative. After a further centrifugation, the supernatant was discarded and the sediment was suspended with 30 — 45 µL fixative. Approximately 20 µL of the suspension was transferred onto a slide and air-dried for about 24 hours. Subsequently, the slides were stained as follows:
- 8 min in May-Gruenwald's solution (1 : 1 in phosphate buffer solution pH 7.2)
- 30 min in Giemsa solution (1 : 9 in phosphate buffer solution pH 7.2)
- rinsing in distilled water
- drying
1000 cells of each test and control group of both cultures were counted and the mean is calculated. The number of cells with micronuclei was recorded, not the number of individual micronuclei. Subsequently the micronuclei ratio in relation to the solvent control was determined. Statistical analysis was performed with the Chi square-test. If mean micronucleus values in the concentration groups are below the mean micronucleus number of the solvent control no statistical analysis has to be performed.

Evaluation criteria:

Criteria for a valid assay
The study is considered valid if solvent and positive control values correspond to the historical control data range and positive control values show a statistically significant increase (P < 0.05) in micronuclei formation.


Criteria for clastogenicity
Both biological and statistical significances are considered together for evaluation purposes. The compound is considered positive (clastogenic) if a dose-dependent or statistically significant increase (P < 0.05; at least one concentration) in micronuclei formation outside of the historical control data range of the solvent control is observed.

Statistics:

The biometry of the results was performed with a one-sided Fisher's exact test.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

the two highest concentrations causing clastogenicity were highly cytotoxic (survival rate between 28 and 42%)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No increase in micronucleus formation was observed in the presence of metabolic activation in all concentration groups.
In the absence of metabolic activation a significant increase in micronucleus formation was observed at the highest concentrations (120 and 140 µg/mL). However, this clastogenic effect correlated with heavy cytotoxicity (survival rate 41.7% and 28.0%,respectively) and is therefore considered to have only limited biological relevance. All other dose groups showed no increase in micronucleus formation.

Conclusions:

Under the study conditions, the test substance induced structural chromosome aberrations in V79 Chinese hamster cells at highly cytotoxic concentrations in the absence of a metabolic activation system.
In the presence of a metabolic activation system and at weakly cytotoxic concentrations in the absence of a metabolic activation system, the test substance did not induce clstogenic effects.

Executive summary:

The purpose of this study was to investigate the potential of Acid Black 220 to induce micronucleus formation in L5178Y cells with and without metabolic activation by liver homogenate from rats pretreated with Aroclor 1254 (S9-mix). L5178Y cells were treated in 96-well microplates for 3 hours with and 24 hours without metabolic activation following a recovery time of 21 and 24 hours,respectively. Cells were treated with the test compound diluted in the cell culture medium and evaluated for micronucleus formation up to concentrations producing distinct cytotoxicity (less than 50 % cell survival). Maximum concentrations evaluated were 500 µg/mLin the presence of S9-mix and 140 µg/mLin the absence of S9-mix.

Visible precipitation of the test compound in cell culture medium was not observed.

No increase in micronucleus formation was observed in the presence of metabolic activation in all concentration groups.

In the absence of metabolic activation a significant increase in micronucleus formation was observed at the highest concentrations (120 and 140 µg/mL). However, this clastogenic effect correlated with heavy cytotoxicity (survival rate 41.7% and 28.0%,respectively) and is therefore considered to have only limited biological relevance. All other dose groups showed no increase in micronucleus formation.

The solvent and positive controls showed the expected increase in micronucleated cells indicating the validity of the study.

Under the experimental conditions of this in-vitro micronucleus test in mouse lymphoma cells, Acid Black 220 caused clastogenic effects in the absence of metabolic activation correlated with heavy cytotoxicity, indicating limited biological relevance.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study following official guidelines, GLP compliant, performed on a mixture containing the test substance and one analogue substance as pure sodium salts

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

clone 65/3Origin: Dr. D. Wild, Freiburg, Germany

Metabolic activation:

with

Metabolic activation system:

Post mitochondrial supernatant (S9 fraction) from Aroclor 1254 induced rat liver

Test concentrations with justification for top dose:

Cytotoxicity testRange with metabolic activation: 0.24 to 500.0 ug/mlRange without metabolic activation: 0.24 to 500.0 ug/mlMutagenicity testOriginal experiment:Range with metabolic activation: 18.52 to 500.0 ug/mlRange without metabolic activation: 14.81 to 400.0 ug/mlConfirmatory experiment:Range with metabolic activation:18.52 to 500.0 ug/mlRange without metabolic activation: 12.96 to 350.0 ug/ml

Vehicle / solvent:

dimethylsulfoxide (suspension)Since the test item was insoluble in all common solvents, it had to be prepared as a suspension in DMSO. The highest suitable concentration of test item in DMSO was determined in a preliminary solubilisation test to be 50.0 mg/ml. Lower concentrations of the test substance were obtainedby appropriate dilution of the stock suspension with DMSO. The respective suspensions were added 1:100 to the cell culture medium. The final concentration of DMSO in the culture medium was 1%. The highest concentration caused a homogenious turbidity after 100-fold dilution with culture medium as determined in the preliminary solubility test by microscopic evaluation. Due to the dense black staining by the test compound, observation of the cultures with the naked eye did not allow todecide whether the compound precipitated or not. It was therefore not determined which concentrations caused the formation of precipitates in the toxicity and mutagenicity tests. The test substance suspensions were prepared immediately before the start of the test.

Untreated negative controls:

yes

Remarks:

dimethylsulfoxide

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

ethylmethanesulphonate

Remarks:

N-dimethylnitrosamine with metabolic activation, ethylmetanesulphonate without metabolic activation

Evaluation criteria:

All mutant frequencies are normalized to a virtual cloning efficiency of 100% at the end of the expression period. If the cloning efficiency of the viability cultures is lower than 15%, the corresponding mutant frequency is usually not calculated, owing to the high statistical insignificance of theresult. For every concentration a mean mutant factor, which is defined as the ratio of the mean mutant frequencies of the treated cultures with the mean mutant frequencies of the solvent control cultures, will be calculated.Criteria for a positive responseThe test substance will be considered to be mutagenic if:• The assay is valid (see assay acceptance criteria)• The mutant frequency at one or more concentrations is significantly greater than that of the negative control and the number of normalized mutant clones in the treated and untreated cultures differs by more than 20.• There is a significant dose-relationship as indicated by the linear trend analysis.• The effects described above are reproducible.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines [9]

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

The test item was tested for in vitro gene mutation in mammalian cells following OECD 476. Under the experimental conditions the test item did not show any mutagenic potential.

Executive summary:

The test item was tested for in vitro gene mutation in mammalian cells following OECD 476. Chinese Hamster lung fibroblasts V79 were exposed to the test item with and without metabolic activation at dose ranging up to 500 µg/l and 400 µg/l respectively. After the expression period, the mutant frequency was determined by 6-thioguanine screening and the substance resulted not having mutagenic potential.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The target substance is the sodium/lithium salt of the source substance, which is the sodium salt.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source: Acid Black 107 + Acid Black 164 + Acid Black 220 Na
Target; Acid Black 220

3. ANALOGUE APPROACH JUSTIFICATION
Source and target contain the same structure. The source is a sodium salt, whereas the target is a sodium/lithium salt.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

The test item was tested for in vitro gene mutation in mammalian cells following OECD 476. Under the experimental conditions the test item did not show any mutagenic potential.

Executive summary:

The test item was tested for in vitro gene mutation in mammalian cells following OECD 476. Chinese Hamster lung fibroblasts V79 were exposed to the test item with and without metabolic activation at dose ranging up to 500 µg/L and 400 µg/L, respectively. After the expression period, the mutant frequency was determined by 6-thioguanine screening and the substance resulted not having mutagenic potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No micronuclei formation was onserved in the micronucleus test in rats.

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:

From 28-Nov-2002 to 23-Jan-2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Gartenstrasse 27, 33178 Borchen
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: Male animals-mean = 181.4 g (= 100 %) (min = 173 g, max = 191 g); female animals- mean = 146.5 g (=100 %) (min = 140 g, max = 154 g)
- Assigned to test groups randomly: Yes
- Housing: Five animals per cage in transparent macrolon cages (type IV) on soft wood granulate in an air conditioned room.
- Diet (e.g. ad libitum): Rat/mice diet ssniff R/M-H (V 1534), ad libitum, ssniff® GmbH, Postbox 2039, 59480 Soest
- Water (e.g. ad libitum): Tap water in plastic bottles, ad libitum
- Acclimation period: 5 d under study conditions
- Animal identification: Fur marking with KMnO4 and cage numbering

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C (except short lasting deviations due to disturbances of air condition)
- Humidity (%): 50% (except short lasting deviations due to disturbances of air condition)
- Photoperiod (hrs dark / hrs light): 12 h light/dark cycle

IN-LIFE DATES: From December 03, 2002 to December 05, 2002

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: deionised water
- Concentration of test material in vehicle: 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SUSPENSION: On the day of first administration the test substance was dissolved in deionized water at an appropriate concentration.
A magnetic stirrer was used to keep the preparation homogeneous until dosing had been completed.

Stability and homogeneity in the vehicle : guaranteed for 96 hours in deionized water by the sponsor (archived with the raw data)

Duration of treatment / exposure:

2 d

Frequency of treatment:

twice at an interval of 24 h

Dose / conc.:

2 000 mg/kg bw/day

No. of animals per sex per dose:

5/sex/group.

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive control: Cyclophosphamide
Dissolved in: distilled water
Dose: 40 mg/kg bw
Route and frequency of administration: Oral (gavage), once
Volume Administered: 10 mL/kg bw

Tissues and cell types examined:

- 2,000 polychromatic erythrocytes were counted for each animal.
- The number of cells with micronuclei was recorded, not the number of individual micronuclei.
- The ratio of polychromatic erythrocytes to 200 normochromatic erythrocytes was determined.
- Main parameter for the statistical analysis, i.e. validity assessment of the study and mutagenicity of the test substance, was the proportion of polychromatic erythrocytes with micronuclei out of the 2000 counted erythrocytes.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: No preliminary experiments were performed, as corresponding toxicological information was available. Since 5000 mg per kg body weight resulted in no lethality in acute oral toxicity testing a limit test with 2000 mg per kg body weight was performed.
.

-Extraction of the bone marrow: Animals were killed by carbon dioxide asphyxiation 24 h after dosing. One femora was removed and the bone freed of muscle tissue. The proximal end of the femora was opened, the bone marrow flushed into a centrifuge tube containing about 3 mL of fetal bovine serum and a suspension was prepared. The mixture was then centrifuged for 5 minutes at approximately 1200 rpm, after which almost all the supernatant was discarded. One drop of the thoroughly mixed sediment was smeared onto a cleaned slide, identified by project code and animal number and air-dried for about 12 h.

-Staining procedure: The slides were stained as follows:-
-5 minutes in methanol
-5 minutes in May-Grunwald's solution
-brief rinsing twice in distilled water
-10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2 (Weise)
-rinsing in distilled water
-drying
-coating with Entellan

Evaluation criteria:

Both biological and statistical significances were considered together for evaluation purposes. A test substance is considered as positive if there is a significant dose- related increase in the number of micronucleated polychromatic erythrocytes compared with the concurrent negative control group. A test substance producing no significant dose-related increase in the number of micronucleated polychromatic erythrocytes is considered non-clastogenic in this system.

Statistics:

Assuming the study is valid based on a monotone-dose-relationship, one-sided Wilcoxon tests were performed initially comparing control values with those of the highest dose group. A significance level of 5% is adopted for all tests.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

Exposure was assured by the presence of light grey coloured tissues at necropsy

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

All animals survived after treatment. No signs of toxicity were detected. All dosed animals showed black discoloured faeces and Body surface showed grey discolorations which were noted on hairless areas 24 hours after the first administration up to the end of study.
The dissection of the animals revealed a nearly black coloured content of the gastro-intestinal tract and light grey coloured tissues.
The bone marrow smears were examined for the occurrence of micronuclei in red blood cells. The incidence of micronucleated polychromatic erythrocytes in the test tem-treated groups was within the normal range of the negative control groups (mean of micronucleated polychromatic erythrocytes per 2000 cells: 1.7 — 4.9). No statistically significant increase in micronucleated polychromatic erythrocytes was observed. The ratio of polychromatic erythrocytes to total erythrocytes remained essentially unaffected by the test compound and differed less than 20% from the control values.
Cyclophosphamide (Endoxan®) induced a marked and statistically significant increase in the number of polychromatic erythrocytes with micronuclei, thus indicating the sensitivity of the test system.

Conclusions:

Under the study conditions, the test substance did not induce micronuclei in bone marrow cells of the rat. Therefore, the test substance was not considered to be clastogenic in this micronucleus assay.

Executive summary:

A study was conducted to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the rat according to OECD Guideline 474, EPA OPPTS 870.5395 and EU Method B.12, in compliance with GLP.

The test compound was dissolved in deionized water and was given twice at an interval of 24 hours as oral doses of 2000 mg per kg body weight to male and female rats (Hsd:Sprague Dawley), based on available acute oral toxicity study data (see 5.6). At study start the animals were 6 weeks of age and had mean body weights of 181.4 g (M) and 146.5 g (F). According to the test procedure the animals were killed 24 hours after the last administration.

Endoxan^®was used as positive control substance and was administered once orally at a dose of 40 mg per kg body weight.

The number of polychromatic erythrocytes containing micronuclei was not significantly increased compared with the control. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment with the test substance and differed less than 20% from the control value.

Endoxan^®induced a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromatic erythrocytes to total erythrocytes was not changed to a significant extent.

Under the conditions of the present study the results indicate that the test substance is not clastogenic in the micronucleus testin vivo.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The both main constituents of Acid Black 220 were tested separately in the bacterial reverse mutation assay (Ames test). Both constituents (Component A and Component B) were tested in concentrations of 10, 50, 100, 500, 1000 and 5000 µg/plate in strains of Salmonella typhimurium TA100, TA98, TA1535, TA1537 and TA1538 and Escherichia coli WP2 uvrA with and without the addition of mammalian metabolic activation preparation. Cultures of each strain were prepared by incubating an aliquot of parent stock culture in nutrient broth at 37°C for 18 hours. After mixing 0.1 ml of test material solution, 0.5 ml of phosphate buffer or S-9 mix, 0.1 ml of culture of tester strain, and 2 ml of top agar in a test tube, the mixture was poured on minimal agar plate. The plates were incubated at 37°C for 2 days and scored, for revertants.

In Component A was tested positive with and without metabolic activation in Salmonella typhimurium TA100, TA1537 and TA1538. Positive results were also seen in Salmonella typhimurium TA 98 without metabolic activation. The test result was ambiguous in TA 98 and in TA 1535 with metabolic activation. Negative results were obtained in E. coli with and without metabolic activation and in TA 1535 without metabolic activation.

Species/strain	Metabolic activation	Genotoxicity	Cytotoxicity
E. coli WP2 uvr A	with and without	negative	no, but tested up to limit concentrations
S. typhimurium TA 100	with and without	positive	no, but tested up to limit concentrations
S. typhimurium TA 1537	with and without	positive	no, but tested up to limit concentrations
S. typhimurium TA 1538	with and without	positive	no, but tested up to limit concentrations
S. typhimurium TA 1535	without	negative	yes
S. typhimurium TA 1535	with	ambiguous	yes
S. typhimurium TA 98	without	positive	no, but tested up to limit concentrations
S. typhimurium TA 98	with	ambiguous	no, but tested up to limit concentrations

 

Component B was tested positive without metabolic activation in Salmonella typhimurium TA 98 and TA 1538. The test result was ambiguous in TA 100 and TA 1538 with metabolic activation and in TA 1537 without metabolic activation. Negative results were obtained in E. coli and Salmonella typhimurium TA 1535 with and without metabolic activation and in TA 100 without and TA 1537 and TA 98 with metabolic activation.

Species/strain	Metabolic activation	Genotoxicity	Cytotoxicity
E. coli WP2 uvr A	with and without	negative	no, but tested up to limit concentrations
S. typhimurium TA 1535	with and without	negative	yes
S. typhimurium TA 100	without	negative	yes
S. typhimurium TA 100	with	ambiguous	no, but tested up to limit concentrations
S. typhimurium TA 98	without	positive	yes
S. typhimurium TA 98	with	negative	no, but tested up to limit concentrations
S. typhimurium TA 1537	without	ambiguous	yes
S. typhimurium TA 1537	with	negative	no, but tested up to limit concentrations
S. typhimurium TA 1538	without	positive	yes
S. typhimurium TA 1538	with	ambiguous	no, but tested up to limit concentrations

 

The test substance was tested for mutagenic effects on V79 Chinese hamster cells in vitro suspended in DMSO. The cells were treated in the experiments with metabolic activation for 5 hours and in the experiments without metabolic activation for 21 hours. The results of each experiment were confirmed in a second and independent experiment (confirmatory experiment). Based on a preliminary range finding test to assess cytotoxicity, a maximum test substance concentration of 500 µg/mL with and 400 µg/mL without metabolic activation was chosen as highest concentrations for the first mutagenicity assay.

The first mutagenicity test with metabolic activationwas performed at the concentrations of 18.52, 55.56, 166.67 and 500 µg/mL. The mean growth inhibiting values found at the highest concentration after treatment and expression were 36.56% and less than 3.06%, respectively. In the confirmatory experiment the concentrations applied were 18.52, 55.56, 166.67 and 500 µg/mL. The highest concentration revealed a mean acute growth inhibition of 10.75%. The mean growth inhibitory effect after the expression period was 6.44%. N-Nitrosodimethylamine (DMN, 1.0 µL/mL) was used as positive control.

In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-Thioguanine (6-TG).

The first mutagenicity test without metabolic activationwas performed at concentrations of 14.81, 44.44, 133.33 and 400 µg/mL. The mean growth inhibition value found at the highest concentration after treatment was 92.65%. No toxicity was noted after expression. In the confirmatory experiment the concentrations applied were 12.96, 38.89, 116.67 and 350 µg/mL. The highest concentration revealed a mean acute, growth inhibitory effect of 76.50%. The mean growth inhibition after the expression period was 16.93%. Ethylmethansulfonate (EMS, 0.3 µL/mL) was used as positive control.

In both experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-TG.

Based on the results of two independently performed experiments and under the given experimental conditions, it is concluded that the test substance and its metabolites did not show any mutagenic activity in this forward mutation system.

 

In a screening study to assess the potential of Acid Black 220 to induce micronucleus formation in L5178Y cells with and without metabolic activation by liver homogenate from rats pretreated with Aroclor 1254 (S9-mix). L5178Y cells were treated in 96-well microplates for 3 hours with and 24 hours without metabolic activation following a recovery time of 21 and 24 hours, respectively. Cells were treated with the test compound diluted in the cell culture medium and evaluated for micronucleus formation up to concentrations producing distinct cytotoxicity (less than 50 % cell survival). Maximum concentrations evaluated were 500 mg/mL in the presence of S9-mix and 140 µg/mL in the absence of S9-mix.

Visible precipitation of the test compound in cell culture medium was not observed.

No increase in micronucleus formation was observed in the presence of metabolic activation in all concentration groups.

In the absence of metabolic activation a significant increase in micronucleus formation was observed at the highest concentrations (120 and 140 µg/mL). However, this clastogenic effect correlated with heavy cytotoxicity (survivalrate 41.7% and 28.0%, respectively) and is therefore considered to have only limited biological relevance. All other dose groups showed no increase in micronucleus formation.

The solvent and positive controls showed the expected increase in micronucleated cells indicating the validity of the study.

Under the experimental conditions of this in-vitro micronucleus test in mouse lymphoma cells, Acid Black 220 caused clastogenic effects in the absence of metabolic activation correlated with heavy cytotoxicity, indicating limited biological relevance.

 

Furthermore, a study was conducted to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the rat according to OECD Guideline 474, EPA OPPTS 870.5395 and EU Method B.12, in compliance with GLP.

The test compound was dissolved in deionized water and was given twice at an interval of 24 hours as oral doses of 2000 mg per kg body weight to male and female rats (Hsd:Sprague Dawley), based on available acute oral toxicity study data (see 5.6). At study start the animals were 6 weeks of age and had mean body weights of 181.4 g (M) and 146.5 g (F). According to the test procedure the animals were killed 24 hours after the last administration.

Endoxan® was used as positive control substance and was administered once orally at a dose of 40 mg per kg body weight.

The number of polychromatic erythrocytes containing micronuclei was not significantly increased compared with the control. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment with the test substance and differed less than 20% from the control value.

Endoxan® induced a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromatic erythrocytes to total erythrocytes was not changed to a significant extent.

Under the conditions of the present study the results indicate that the test substance is not clastogenic in the micronucleus test in vivo.

 

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.  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 has been widely demonstrated in the two compounds AMP397 and fexinidazole (see attachment).

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 test with Acid Black 220 is a bacteria specific effect and not relevant to mammalians.

In summary,only bacteria-specific effects were noted in the bacteria reverse mutation assay, whereas the mutagenicity study in mammalian cells was negative. In addition, the test substance did not show clastogenic or aneugenic effects.

 

Justification for classification or non-classification

Based on the above reported test results and read across considerations Acid Black 220 is not classified for genetic toxicity according to the CLP Regulation (EC n. 1272/2008).