Trisodium 3-hydroxy-4-(4'-sulphonatonaphthylazo)naphthalene-2,7-disulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test compound amaranth [Food Red 2] was not mutagenic in the study conducted using Salmonella typhimurium TA97a, TA98 and TA100 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)

Rationale for reliability incl. deficiencies:

other: data from peer- reviewed journals

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Ames mutagenicity assay was performed to evaluate the mutagenic nature of the test compound amaranth.

GLP compliance:

not specified

Type of assay:

bacterial gene mutation assay

Species / strain / cell type:

S. typhimurium, other: Salmonella typhimurium TA97a, TA98 and TA100

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Metabolic activation system:

no metabolic activation system used

Test concentrations with justification for top dose:

10,100,250,500 and 1000 μg /plate

Vehicle / solvent:

Vehicle
Vehicle(s)/solvent(s) used: Sterile double distilled water
Justification for choice of solvent/vehicle: No data available

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

for TA97a and TA98, 20 μg/plate nitro phenylene diamine and for TA100, 1.5 μg/plate sodium azide

Positive control substance:

other: for TA97a and TA98, 20 μg/plate nitro phenylene diamine and for TA100, 1.5 μg/plate sodium azide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
Preincubation period: No data available
Exposure duration: 48⁰C
Expression time (cells in growth medium): 48⁰C
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 available

NUMBER OF REPLICATIONS: triplicate

Evaluation criteria:

Increase in the number of mutagenic revertants

Statistics:

ANOVA test was performed at 0.05 level

Species / strain:

S. typhimurium, other: Salmonella typhimurium TA97a, TA98 and TA100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Mutagenicity of food colours in tester strains of Salmonella typhimurium

 

Dose           μg/plate	Mean of the No. Revertant Colonies ± S.D.
	TA97a	TA98	TA100
10	120.33 ± 15.50	222.0 ± 49.79	103.7 ± 9.07
100	115.67 ±  17.78	205.7 ±  4.04	92.7 ± 16.17
250	92.33 ±  12.50	170.7 ± 68.72	92.0 ±  27.73
500	120.67 ± 22.94	178.3 ± 30.29	83.7 ±  7.095
1000	122.33± 24.82	195.7± 36.23	109.0 ± 10.19
10	134.00 ± 42.51	49.3 ± 13.7	98.0  ± 7.55
Solvent control	122.30 ± 9.61	21.3 ± 9.02	121.0  ±  3.61
NPD	827.70 ± 106.53	522.0 ± 50.48	-
SA	-	-	1624.67 ±  89.76

 

Where:

NPD= Nitrophynylenediamine

SA= Sodium azide

S.D.= Standard Deviation

Anova Value of TA97a (Amaranth —2.11, ns)

TA 98 (Amaranth –10.19*)

TA100 (Amaranth —2.196, ns)

P< 0.01 (5.06)

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation

The test compound amaranth [Food Red 2] was not mutagenic in the study conducted using Salmonella typhimurium TA97a, TA98 and TA100 without metabolic activation system.

Executive summary:

Ames mutagenicity assay was performed to evaluate the mutagenic nature of the test compound Amaranth in plate incorporation assay.

 

The test material was tested at a concentration of 10,100,250,500 and 1000 μg /plate. The plates were inverted within an hour and placed in a dark vented incubator at 37⁰C for 48 hours. Positive controls (for TA97a and TA98, 20 μg/plate nitro phenylene diamine and for TA100, 1.5 μg/plate sodium azide) and negative controls were maintained concurrently for all the experiments. Three plates were used for each set. After 48 hours of incubation, the revertant colonies were counted. ANOVA test was performed at 0.05 level.

 

Amaranth [Food Red 2] was determined to be non - mutagenic under the study conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Gene toxicity in vitro:

Peer reviewed articles were viewed to determine the mutagenic nature of the test compoundAmaranth(CAS no 915-67-3). The studies are summarized as below:

 

OECD Guideline 471 (Bacterial Reverse Mutation Assay) (Aparajita Das and Anita Mukherjee; 2004) to determine the mutagenic nature of the test compound Amaranth. Ames mutagenicity assay was performed to evaluate the mutagenic nature of the test compound Amaranth in plate incorporation assay. The test material was tested at a concentration of 10,100,250,500 and 1000 μg /plate. The plates were inverted within an hour and placed in a dark vented incubator at 37⁰C for 48 hours. Positive controls (for TA97a and TA98, 20 μg/plate nitro phenylene diamine and for TA100, 1.5 μg/plate sodium azide) and negative controls were maintained concurrently for all the experiments. Three plates were used for each set. After 48 hours of incubation, the revertant colonies were counted. ANOVA test was performed at 0.05 level.Amaranth [Food Red 2] was determined to be non - mutagenic under the study conditions.

 

This study presents data from primary screening by the Ames test (primary mutagenicity screening of food additives currently used in japan; 1984) on 200 food additives used in Japan. All the samples were supplied from the Japan Food Additives Association, Tokyo, at the request of the Ministry of Health and Welfare of Japan, where the purity and quality of each sample were checked. Reverse mutation assays usingS. typhimuriumstrains TA92, TA1535, TA100, TA1537, TA94 and TA98 were carried out according to the method of Ames, McCann & Yamasaki.Acetone was used as a solvent since test compound is insoluble in water.All samples were kept in a refrigerator before use. Reverse mutation assays usingS. typhimuriumstrains TA92, TA1535, TA100, TA1537, TA94 and TA98 were carried out according to the method of Ames, McCann & Yamasaki. All these test strains were originally provided by Dr B. N. Ames, University of California, Berkeley, USA. The liver microsome fraction (S-9) was prepared from the liver of Fischer rats (Charles River Japan Co.) pretreated 5 days before with polychlorinated biphenyls (500 mg/kg body weight of Kanechlor KC-400 in olive oil, ip). The reaction mixture (S-9 mix) contained 5 mM-glucose 6-phosphate, 4mM-NADPH, 4mM-NADH, 33mM-KCl, 8 mM-MgCI₂, 100 mM-phosphate buffer (pH 7.4) and 3.75 ml S-9 (129 mg protein) in a total volume of 12.5 ml. Cells cultured overnight were pre-incubated with both the test sample and the S-9 mix for 20 min at 37°C before plating. Duplicate plates were used for each of six different concentrations of the sample. The number of revertant (his +) colonies was scored after incubation at 37°C for 2 days. The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated). A negative result indicates that no significant increases in the number of revertant colonies were detected in anyS. typhimuriumstrains at the maximum dose. At the maximum dose of 5.0mg/plate, the test chemical was non mutagenic.

 

From the same study, Chromosomal aberration testsin vitrousing a Chinese hamster fibroblast cell line were carried out on 190 synthetic food additives and 52 food additives derived from natural sources, all of which are currently used in Japan. The maximum dose of each sample was selected by a preliminary test in which the dose needed for 50% cell-growth inhibition was estimated using a cell densitometer. The cell line was originally established from the lung of a newborn female at the Cancer Research Institute, Tokyoand was maintained by 4-day passages in Minimum Essential Medium (MEM; GIBCO) supplemented by 10% calf serum. The modal chromosome number is 25 and the doubling time was approximately 15 hr.Ethanol was used as solvent since it is insoluble in water. Chromosome preparations were made as follows: Colcemid (final concentration 0.2µg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution (1.5%, at pH 6.8) for 12-15 min. A hundred well-spread metaphases were observed under the microscope (x 600 with a no cover objective lens). The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0%. The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%. When no reasonable dose-response relationships were found, additional experiments were carried out at similar dose levels. For a quantitative evaluation of the clastogenic potential of the positive samples, the D20 was calculated, which is the dose (mg/ml) at which structural aberrations (including gaps) were detected in 20% of the metaphases observed. In addition, the TR value was calculated, which indicates the frequency of cells with exchange-type aberrations per unit dose (mg/ml). Since the incidence of aberrations for the test chemical was 12.0% at the maximum dose, it can be concluded that the test chemical is mutagenic.

 

Amaranth was subjected to liquid tests (both aerobic with microsomes and anaerobic) (joseph p. brown, gerald w. roehm and ronald j. brown; 1978) and to plate tests involving initial 16 h anaerobic incubations to facilitate microbial reduction of the azo bond to evaluate the mutagenic potential.Salmonella typhimuriumstrains TA1535, TA100, TA1537, TA1538, TA98 were obtained from Dr. Bruce N. Ames (University of California, Berkeley). Manipulation of these tester strains was carried out as recommemdedby Ames. Frozen permanents of each tester strain and the S-9 fractions were stored in sterile plastic vials under liquid nitrogen (Linde LR-30). Broth cultures of the tester strains were prepared by inoculating 10 ml nutrient broth -- 0.5% NaCI contained in each of five 25-ml Delong culture flasks from the respective frozen permanents, and incubating overnight (14--16 h) at 37°C in a New Brunswick G24 gyrotory incubator shaker at 350 r.p.m. The mammalian liver homogenate fraction "S-9" was prepared essentially as described by Ames . Female Sprague Dawley rats (Simonsen Laboratories, Gilroy, Calif.) were maintained on Simonsen white laboratory diet. Five days prior to sacrifice the animals were injected i.p. with about 0.5 ml of a corn oil solution of Aroclor 1254 (400 mg/ml) to give a dose of 500 mg/kg body weight. Removal of livers and subsequent preparation of the S-9 fraction and "S-9 mix" Amaranth was weighed into tared, numbered screwcap vials were dissolved in glass distilled deionized water or dimethylsulfoxide (DMSO) at a concentration of 10 mg/ml. The chemical reduction of water-soluble azo dyes was accomplished by dissolving an equal weight (molar excess) of sodium dithionite (Na2S204) in the test solution and swirling gently until the solution turned colorless. Reduction was carried out immediately prior to adding to the top agar and plating.

(a) Plate Tests

Red No. 2 was plated in the usual fashion at concentrations from 50 µg/plate to 500µg/plate and then was incubated anaerobically for 16 h before the usual 3-day aerobic incubation.

All results with the five tester strains with and without microsomal activation were negative.

(b) Liquid tests

Aerobic liquid tests were conducted with Red No. 2.Anaerobic liquid tests were carried out with, Red. No.2 at a concentration of 10 mg/ml medium. After 16 h anaerobic incubation, 25 to 100µl of the cell suspensions were added to top agar with or without microsomes and plated (250 - 1000µg dye/plate). The plates were then incubated aerobically for 3 days.

The results were wholly negative in both anerobic and aerobic liquid tests.

The criteria adopted for scoring a mutagenic response in routine plate tests is that the observed number of revertants exceed twice the background value for that given assay and exceed the 99.9% confidence limit based on the historical controls.

Amaranth was non- mutagenic in both the tests conducted.

 

A chromosomal aberration test (Mutation Research, 195 (1988)) on 951 test substances using mammalian cells in culture was performed. Chinese hamster fibroblast cell lines were used for the test. 250 micrograms/ml of the test chemical was tested using CHL cell line. The cell line was exposed to test chemical for 48 hours without metabolic activation. Mutagenic or clastogenic activity was observed for the test chemical

Therefore, genotoxicity of Amaranth was confirmed by the Chromosomal Aberration assay using CHL cell line

 

Food dyes were tested in the Salmonella-mammalian microsome mutagenicity Assay (Mutagenicity Testing of Some Commonly Used Dyes; 1981) using Plate incorporation assay. Amaranth dye was purchased fromFisher Scientific Co., Fair Lawn, N.J.

The Salmonella typhimurium tester strains TA1535, TA1537, TA1538, TA98, and TA100 were grown in nutrient broth shaken for 14 h at 37°C. Ames test was performed using Plate - incorporation method as described by Ames et al. with the modifications of Andrews et al. The revertant colonies were counted by using a hand-held tally. Dimethyl sulfoxide was the solvent control.Liver homogenates (S9) were prepared from male Sprague-Dawley rats stimulated with Aroclor 1254 (500 mg/kg intraperitoneally 5 days before sacrifice). The S9 mix, added in samples of 0.5 ml per plate, contained 3 mg of protein.A compound was considered mutagenic when the number of revertants above background was at least twice the value of the historical control mean or twice the value of the current control mean, whichever was greater, and a dose-response curve could be demonstrated.The positive control chemicals sodium azide, 9 -aminoacridine, 2- nitrofluorene, and 2-aminoanthracene were used with the tester strains.

The historical mean values and standard deviations for the normal background reversion to prototrophy of the three tester strains used throughout these studies were reported. Amaranth was found to be non – mutagenic in the presence and absence of metabolic activation in all tester strains in the plate- incorporation assay.

 

Bacterial mutagenicity was determined by the method of Ames (angela e. auletta, janice m. kuzava and amar s. parmar, 1977) withS. typhimuriumstrains TA1535, TA100, TA1538, TA98 and TA1537.One-tenth ml of the appropriate food coloring in distilled water and 0.1 ml of bacterial culture were added to a soft agar overlay supplemented with 0.5 mM biotin and 0.5 mM histidine. The agar was mixed and poured over a base plate of Spizzizen's minimal medium [10]. Revertant colonies were scored after 48 h at 37°C. The livers of male Fischer rats (F344/f Mai) pretreated with Aroclor 1254 for the non-specific induction of enzyme activity served as the sources of enzymes for metabolic activation. In the assay, 0.5 ml S-9 Mix, consisting of liver post-mitochondrial supernatant and cofactors, was added to the soft agar overlay along with test substance and bacteria. Plates were poured and incubated as described above. The appearance of colored colonies on the agar plates was taken as an indication that the coloring had penetrated the bacteria and therefore was in close proximity to the DNA. MNNG , BaP and 4-NQO were used as positive controls. BaP with S-9was used at 10µg/plate for strain TA98. Because of the increased sensitivity of TA100 to the action of this agent, it was used at 5µg/plate with this strain. An examination of the background lawn failed to show any overt indication of toxicity. Each determination is the mean of 3 plates and is shown with the standard deviation around the mean. Known positive mutagens used as control compounds resulted in an increase in the number of revertants per plate. All strains gave negative results with Amaranth dye. The test compound amaranth [Food Red 2] was non – mutagenic toSalmonella typhimuriumTA1535, TA100, TA1538, TA98 and TA1537 strains tested with and without metabolic activation.

 

The mutagenic activity of Amaranth was evaluated with the Salmonella/microsome assay, (S.D. Varella, G.L. Pozetti, W. Vilegas, E.A. Varanda; 2005) using Salmonella typhimurium strains TA98, TA100 and YG1024. S. typhimurium strains TA98 and TA100 were kindly provided by Dr. B. Ames, University of California, Berkeley, CA, USA and YG1024 by Dr. T. Nohmi and M. Watanabe, National Institute of Hygienic Sciences, Tokyo, Japan. The concentrations were selected based on toxicity, in a preliminary test. In all subsequent assays, the upper limit of the dose range tested was either the highest non-toxic dose or the lowest toxic dose determined in this preliminary assay. Toxicity was apparent either as a reduction in the number of his+ revertants, or as an alteration in the auxotrophic background (i.e. background lawn). The Salmonella mutagenicity test was performed using the pre-incubation method for 20–30min with S. typhimurium strains TA100, TA98 and YG1024 (derivative of TA98, that have higher levels of O acetyltransferase), with and without metabolic activation. The S9-mix was freshly prepared before each test using an Aroclor-1254-induced rat liver fraction purchased (lyophilized) from Moltox—Molecular Toxicology Inc. The positive controls used sodium azide for TA100 (-S9), 4-nitro-o-phenylenediamine for TA98 and YG1024 (-S9) and 2-anthramine in assays with metabolic activation. Amaranth was dissolved in DMSO. The concentrations tested were 0.46, 0.92, 1.83, 2.75 and 3.65mg/plate. The mutagenic index (MI) was also calculated for each dose; that is the average number of revertants per plate divided by the average number of revertants per plate from the negative (solvent) control. A sample was considered positive when the mutagenic index was equal to or greater than 2 for at least one of the tested doses and if it had a reproducible dose–response curve. The statistical analysis was performed with the Salanal computer program, adopting the Bernstein model. Amaranth, independent of metabolic activation, was non-mutagenic in TA100. When strain TA98 was exposed to amaranth with metabolic activation, there was mutagenic activity. Amaranth was mutagenic with and without metabolic activation to YG1024 strain.

 

Food dyes were tested (king-thom chung, george e. Fulk, and a. W. Andrews; 1981) in the Salmonella-mammalian microsome mutagenicity Assay using Liquid Pre –incubation assay. Amaranth dye was purchased from Fisher Scientific Co., Fair Lawn, N.J. The Salmonella typhimurium tester strains TA1535, TA1537, TA1538, TA98, and TA100 were grown in nutrient broth shaken for 14 h at 37°C.The liquid pre - incubation assays were timed for 30 min at 37°C in a Dri-block. The revertant colonies were counted by using a hand-held tally. Dimethyl sulfoxide was the solvent control.Liver homogenates (S9) were prepared from male Sprague-Dawley rats stimulated with Aroclor 1254 (500 mg/kg intraperitoneally 5 days before sacrifice). The S9 mix, added in samples of 0.5 ml per plate, contained 3 mg of protein.A compound was considered mutagenic when the number of revertants above background was at least twice the value of the historical control mean or twice the value of the current control mean, whichever was greater, and a dose-response curve could be demonstrated. The positive control chemicals sodium azide, 9-aminoacridine, 2- nitrofluorene, and 2-aminoanthracene were used with the tester strains. The historical mean values and standard deviations for the normal background reversion to prototrophy of the three tester strains used throughout these studies were reported. Amaranth was found to be non – mutagenic in the presence and absence of metabolic activation in all tester strains in the liquid- preincubation assay.

 

 

Food dyes were tested in the Rec – assay. Amaranth dye was obtained (Yasuhide TonoGAI, Yoshio Ito, Masahiro IWAIDA, Masatomo TATI, Youki Ose and Takahiko Sato; 1979) from National Institute of Hygienic Sciences, Japan. Bacillus subtilis H 17 (Rec+) and M45 (Rec- ) was donated from the National Institute of Genetics. They were cultured in Difco nutrient broth (1% meat extract, 1% polypeptone,0.5% chloride) and diluted with the same broth on use. Preparation of plates: Agar was added to nutrient broth at the concentration of 1.5%, heated for 10min. at 1kg/cm2, then 20ml of the melted medium was added to sterile Petri dish (diameter : 9.0 cm). Agar plates were streaked with inoculum of two strains in the same manner as was reported by Kada et al and paper disk, 8mm in diameter, immersed with either 10µM of dye or 0.5 - 5.0mM of halogen in DMSO solution, was placed on the surface so as to cover the beginning of the bacterial streaks. After incubation for 24 hrs at 37°C, the length of growth inhibition of the bacterial streak was measured. Amaranth was found to be non – mutagenic absence of metabolic activation in the Rec – assay.

 

Based on the majority of studies reviewed, the test material Amaranth (915-67-3) is not mutagenic in vitro.

Justification for selection of genetic toxicity endpoint
Data is from peer- reviewed journals.

Justification for classification or non-classification

Gene toxicity in vitro:

Based on the key study used and its relative supporting data, the test materialAmaranth(915-67-3) is not mutagenic in vitro.