Tetrasodium 1-acetamido-2-hydroxy-3-(4-((4-sulphonatophenylazo)-7-sulphonato-1-naphthylazo))naphthalene-4,6-disulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The mutagenic potency ofC.I Brilliant black BN( food black 1) was tested by the plate incorporation method usingSalmonella typhimuriumstrainTA98, TA100, TA1535, TA1537, and TA1538. When the test bacterial strain is exposed with the test chemical for 48hrs, no mutagenic response was seen in any of the strains of Salmonella typhimurium(with and without metabolic activation system).

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Qualifier:

according to guideline

Guideline:

other: as mentioned below

Principles of method if other than guideline:

Bacterial gene mutation assay was carried out using S. typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538.

GLP compliance:

not specified

Type of assay:

bacterial gene mutation assay

Target gene:

Plate incorporation method

Species / strain / cell type:

S. typhimurium, other: TA98, TA100, TA1535, TA1537, and TA1538.

Details on mammalian cell type (if applicable):

No data available

Additional strain / cell type characteristics:

other: Salmonella typhimurium is a histidine auxotroph strain.

Metabolic activation:

with and without

Test concentrations with justification for top dose:

Test concentration: Dose (µg /plate): conc. in the range of 10-10000 µg /plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Appropriate solvent was used for preparing the stock solution of the test chemical. Name of the solvent is not specified in the study.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

yes

Positive controls:

yes

Positive control substance:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporationDURATION- Preincubation period: 48-h- Exposure duration: 37 ± 2ᵒC- Expression time (cells in growth medium): 48 hr- Selection time (if incubation with a selection agent): No data available - Fixation time (start of exposure up to fixation or harvest of cells): No data availableSELECTION AGENT (mutation assays): No data availableSPINDLE INHIBITOR (cytogenetic assays): No data availableSTAIN (for cytogenetic assays): No data availableNUMBER OF REPLICATIONS: TriplicatesNUMBER OF CELLS EVALUATED: 1-2 × 109 cells/mlDETERMINATION OF CYTOTOXICITY- Method: No data available- Other: Cytotoxicity was observed in a preliminary dose range-finding study using strain TA100.OTHER EXAMINATIONS:- Determination of polyploidy: No data available- Determination of endoreplication: No data available- Other: : No data availableOTHER: The test materials were stored at -20ᵒC, 4ᵒC, or room temperature as required to maintain compound stability. The test chemical was analyzed for purity by Midwest Research Institute (MRI).Five doses of test chemical, together with the appropriate concurrent solvent and positive controls, were tested on each tester strain without metabolic activation and also with activation by induced rat and hamster liver S9 preparations. The test bacterial cultures were grown overnight in Oxoid nutrient broth no. 2 and were removed from incubation when they reached a density of (1-2) × 109 cells/ml.On the day of use, all tester strain cultures were checked for genetic integrity.

Evaluation criteria:

The result was considered positive if at least a doubling in the mean number of revertants/plate of at least one tester strain was found.

Statistics:

No data available

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537, and TA1538.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: No data available- Effects of osmolality: No data available- Evaporation from medium: No data available- Water solubility: No data available- Precipitation: No data available- Other confounding effects: No data availableRANGE-FINDING/SCREENING STUDIES: Preliminary dose range-finding study was performed using Salmonella typhimurium strain TA100.COMPARISON WITH HISTORICAL CONTROL DATA: No data availableADDITIONAL INFORMATION ON CYTOTOXICITY: No data available

Remarks on result:

other: other: Bacterial tester strain

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):negative Negative (with and without)The test substance C.I Brilliant black BN ( food black 1) had shown non-mutagenic response in the test bacterial strain TA98, TA100, TA1535, TA1537, and TA1538. (with and without metabolic activation system).

Executive summary:

The mutagenic potency ofC.I Brilliant black BN( food black 1) was tested by the plate incorporation method usingSalmonella typhimuriumstrainTA98, TA100, TA1535, TA1537, and TA1538. When the test bacterial strain is exposed with the test chemical for 48hrs, no mutagenic response was seen in any of the strains of Salmonella typhimurium(with and without metabolic activation system).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Genetic toxicity in vitro:

In study conducted by H. E. Seifried, et. al. (2006),The mutagenic potency of C.I Brilliant black BN( food black 1) was tested by the plate incorporation method usingSalmonella typhimuriumstrainTA98, TA100, TA1535, TA1537, and TA1538. When the test bacterial strain is exposed with the test chemical for 48hrs, no mutagenic response was seen in any of the strains of Salmonella typhimurium(with and without metabolic activation system).

 

 Also from same study,the mutagenic potency of the C.I Brilliant black BN( food black 1) was tested on theL5178Y TK^+/-3.7.C mouse lymphoma cells. The test compound was found to be non-mutagenic in absence of activation system and positive in presence of activation system, when the mammalian cell was exposed for 4 hrs along with the total expression time of 48 hrs.

 C.I. Briliant black BNwas found to have no mutagenic inducing potency on the test mammalian cell in absence of metabolic activation system and showed positive mutagenic effect in presence of metabolic activation system at lowest dose 470µg/ml The size of mutant mouse lymphoma colonies was determined using an Artek 982 colony counter/sizer or the ProtoCol colony counter and the mutant frequencies were expressed as mutants per 10⁶surviving cells.

 

The mutagenic potency of the C.I Brilliant black BN ( food black 1) (H. E. Seifried,et. al.2006) was tested on theL5178Y TK^+/-3.7.C mouse lymphoma cells. The test compound was found to be non-mutagenic in absence of activation system and positive in presence of activation system, when the mammalian cell was exposed for 4 hrs along with the total expression time of 48 hrs. C.I. Briliant black BN was found to have no mutagenic inducing potency on the test mammalian cell in absence of metabolic activation system and showed positive mutagenic effect in presence of metabolic activation system at lowest dose 470µg/ml. The size of mutant mouse lymphoma colonies was determined using an Artek 982 colony counter/sizer or the Protocol colony counter and the mutant frequencies were expressed as mutants per 10⁶surviving cells.

 

 

In ain- vitrogene mutation test (Screening of food dyes for Genotoxic activity 1979), WP2 uvr AE. coli were treated withBlack PN by using Microcosmical fluctuation test. Significant effects were observed on survival of treated WP2 uvr AE. Colistrain with and without (10 mg/ml) metabolic activation. Therefore, Black PN was considered to be negative (with and without) metabolic activation on WP2 uvr AE. coli whentestedin vitro by usingMicrocosmical fluctuation test .

 

From the same study (Screening of food dyes for Genotoxic activity 1979), In ain- vitrogene mutation test, WP100E. coli were treated withBlack PN by using microsomal rec assay. No effects were observed on survival of treated WP100E. colistrain without metabolic activation. Therefore, Green S was considered to be Negative without metabolic activation(10 mg/ml) on WP100E. coli whentestedin vitro by usingmicrotonal rec assay.

 

In ain- vitrogene mutation test (r. b. haveland-smith and r. d. combes, 1979), TA 1538Salmonella typhimurium were treated withBlack PN by using Microcosmical fluctuation test. no effect were observed with and without (10 mg/ml) metabolic activation on survival of bacterial gene. Therefore, Black PN was considered to be Negative (with and without) metabolic activation on TA 1538Salmonella typhimurium whentestedin vitro by usingMicrocosmical fluctuation test.

 

In ain- vitrogene mutation test(r. b. haveland-smith and r. d. combes, 1979), WP100E. coli were treated withBlack PN by using microsomal rec assay. No effects were observed on survival of treated WP100E. colistrain without metabolic activation. Therefore, Green S was considered to be Negative without metabolic activation(10 mg/ml) on WP100E. coli whentestedin vitro by usingmicrotonal rec assay.

 

The genetic toxicity test (r. b. haveland-smith and r. d. combes, 1979), was performed in human peripheral blood lymphocytes by Cytokinesis-block micronucleus assay (CBMN).Heparinized peripheral venous blood samples were collected from healthy, nonsmoking donors and processed for the micronucleus assay. Whole blood samples were incubated in mediumat 37°C.Lymphocytes were treated with the tested chemicals for 24 h; Cytochalasine B (6 μg ml-1) was added 44 h after LF-7 stimulation. After incubation the cultures were harvested and treated with hypotonic solution. Lymphocytes were spread onto microscopic slides and stained with acridine orange (2 μg ml-1) or Giemsa dye. A nuclear division index (NDI) was calculated according to Eastmond and Tucker [14] as follows: NDI = (M1 + 2 x M2 +3 x M3 + 4 x M4)/N, where M1-M4 respectively represent the number of cells with one to four nuclei and N is the total number of cells scored. The frequency of MN induction was evaluated by scoring 4000 nucleated lymphocytes (1000 cells per slide) per sample. It was found that a significantly (p<0.001) higher incidence of MN was found after treatment with all the concentrations of brilliant black 1compared to the control cells. Therefore, genetic toxicity test for brilliant black 1(2519-30-4) in Human peripheral blood lymphocytes was found to be positive.

 

The genetic toxicity test was performed in human peripheral blood lymphocytes by Comet assay. Heparinized peripheral venous blood samples were collected from healthy, nonsmoking donors and processed for the Comet assay. Comet assay was performed under alkaline conditions and in dim light. Lymphocytes were isolated in Histopaque-1077 gradient and treated for 1 h with the control solution and tested chemicals at 37°C.50 μl of lymphocytes suspension and 150 μl of 0.75% LMP agarose were placed onto a microscopic slide coated with 1% NMP agarose solution. The slides were subjected for 1 h to alkaline (pH 10) lysis prior to electrophoresis. Electrophoresis was performed for 20 min at 25 V and 300 mA. After electrophoresis, the slides were neutralized for 15 min and kept in a refrigerator until analysis. For microscopic analyses, the slides were immersed in distilled water for 20 min, stained with 100 μl of DAPI, and incubated for 12-16 h in a humid chamber, at 4°C. Comet cells were sorted into 4 classes (0-III) according to the degree of DNA damage. The nuclei isolated from the control lymphocytes were round-shaped and did not form comets. After treatment with BB, nuclei with variable shapes and comets of different lengths appeared and the percentage of class II and III nuclei rose gradually with increased concentrations of BB. No intact nuclei representing class I was found after BB treatment at the highest concentration. Therefore, the genetic toxicity test (Comet assay) for brilliant black BN (2519-30-4) in human peripheral blood lymphocytes was found to be positive.

 

From read across data Red 2G (CAS no. 3734-67-6),Genotoxicity study (C. N. Edwards and R. D. Combes 1984) was observed in Male Sprague-Dawley rats given Red 2G orally at the concentration 800 mg/kg body weight were investigated by fluctuation test. Test chemical present in urine and faecal extracts were evaluated for mutagenicity by using fluctuation test. Salmonella typhimurium Strain- TA98 and TA100 were exposed to the urine and faecal extracts of Male Sprague-Dawley rats. .Therefore it was considered that Red 2G did not induce any mutation in the presence and absence of metabolic activator.

 

Genotoxicity study for Red 2G (R.B. Haveland-Smith, 1979) was observed in Escherichia coli strain WP2, WP67 and WP100 by REC and POL Assays. Red 2G is a base-substitution mutagen for bacteria when tested in the presence of a microsomal activation system. The effects were observed only at very high concentrations of 10 mg/ml .No effect was detectable at 1 mg/ml or below, suggesting that its mutagenic potency is relatively low for bacteria.Thereforeit was considered thatRed 2G does not induce mutation with and without metabolic activation at low concentration as 1 mg/ml. so the result was found to be negative.

 

Genotoxicity study for Red 2G (R.B. Haveland-Smith, 1979) was observed in Escherichia coli strain WP2 WP2uvrA at the concentration of 10, 5 and 1 mg /ml in presence and absence of metabolic activation by Fluctuation tests. The tubes were scored for turbidity. When dyes such as Red 2G was used in this system, it may be impossible to detect the turbidity in the tubes by eye or to use a growth indicator such as bromothymol blue, due to masking by the colour. In this case, the presence of viable prototrophic revert ants was verified by streaking lapful’s from each tube onto supplemented agar. The results indicate that Red 2G was found to be Negative (without) metabolic activation Escherichia coli strain WP2uvrA at the concentration of 10mg /ml. While the result was found to be positive (with) metabolic activation Escherichia coli strain WP2uvrA at the concentration of 10 and 5mg /ml but not significant at 1 mg /ml.

 

Genotoxicity study for Red 2G (R.B. Haveland-Smith, 1979) was observed in Salmonella typhimurium strain TA1538 at the concentration of 10mg /ml in presence and absence of metabolic activation by Fluctuation tests. The tubes were scored for turbidity. When dyes such as Red 2G was used in this system, it may be impossible to detect the turbidity in the tubes by eye or to use a growth indicator such as bromothymol blue, due to masking by the colour. In this case, the presence of viable prototrophic revertants was verified by streaking loopfuls from each tube onto unsupplemented agar. No significant change was observed in the tubes. Therefore the result was found to be Negative (with and without) metabolic activator for Red 2G in Salmonella typhimurium strain TA1538.

 

Genotoxicity study for Red 2G (C. N. Edwards and R. D. Combes 1984) was observed in Male Sprague-Dawley rats given Red 2G orally at the concentration 800 mg/kg body weight were investigated by plate incorporation assays. Test chemical present in urine and faecal extracts were evaluated for mutagenicity by using plate incorporation assays. Salmonella typhimurium Strain- TA98 and TA100 were exposed to the urine and faecal extracts of Male Sprague-Dawley rats. .Therefore it was considered that Red 2G did not induce any mutation in the presence and absence of metabolic activator.

 

From the above target and read across data indicates positive and negative results. While considering the majority of the data available for target and read across chemicals the target chemical C.I Brilliant black BN ( food black 1) is classified as "Not Mutagenic".

Justification for selection of genetic toxicity endpoint
The test substance C.I Brilliant black BN ( food black 1) had shown non-mutagenic response in the test bacterial strain TA98, TA100, TA1535, TA1537, and TA1538. (with and without metabolic activation system).

Justification for classification or non-classification

The chemical C.I Brilliant black BN ( food black 1) gives negative results for gene toxicity in vitro studies conducted in bacteria as well as animal cell lines. Thus, the chemical C.I Brilliant black BN ( food black 1) is classified as "Not Mutagenic".