Trisodium 5-hydroxy-1-(4-sulphophenyl)-4-(4-sulphophenylazo)pyrazole-3-carboxylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There are many in vitro studies available on Acid Yellow 23 performed following the official guidelines:

OECD 471, Ames test

OECD 473, Chomosomal aberration

OECD 476, Mouse Lymphoma

OECD 480, Genetic Toxicology Saccharomyces cerevisiae

OECD 482, DNA repair assay

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:

2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles, acceptable for assessment. GLP compliant.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA102

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S-9 mix

Test concentrations with justification for top dose:

312.5; 625; 1250; 2500; and 5000.0 µg/plate

Vehicle / solvent:

- Vehicle/solvent used: water for injections (Batch EVBO3A).- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

1µg/plate: TA1535 and TA100 without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

50 µg/plate: TA1537 without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

0.5 µg/plate: TA98 without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

0.5 µg/plate: TA102 without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

2 µg/plate: Escerichia coli WP2 uvra without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: a-anthramine

Remarks:

2 µg/plate: TA1535, TA1537, TA98 and TA100 with S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-anthramine

Remarks:

10 µg/plate: TA102 and E.C. WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)NUMBER OF REPLICATIONS: The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.DETERMINATION OF CYTOTOXICITY: Toxicity of the test article may be evidenced by a reduction in the number of spontaneous revertants, a clearing of the bacterial background lawn, or by degree of survival of treated cultures.

Evaluation criteria:

The generally accepted conditions for the evaluation of the results are:- corresponding background growth on both negative control and test plates- normal range of spontaneous reversion rates.A test article is considered as positive if either a significant dose-related increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.A test article producing neither a significant dose-related increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.A test article is considered as mutagen if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537, TA 1538, and TA 98 it is at least three times higher as compared to the spontaneous reversion rate.Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98, TA 100

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

2.3.1 TreatmentThe test item was tested in a preliminary test and two mutagenicity experiments. The preliminary test, both experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate incorporation method.The second experiment with S9 mix was performed according to the preincubation method.The direct plate incorporation method was performed as follows: test item solution (0.1 mL), S9 mix when required or phosphate buffer pH 7.4 (0.5 mL) and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 45°C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.The preincubation method ^'^' ^^ was performed as follows: test item solution (0.1 mL), S9 mix (0.5 mL) and the bacterial suspension (0.1 mL) were incubated for 60 minutes at 37°C under shaking before adding the overlay agar and pouring onto the surface of a minimum agar plate.After 48 to 72 hours of incubation at 37°C, revertants were scored with an automatic counter (Cardinal counter. Perceptive Instruments, Suffolk CB9 7 BN, UK).2.3.2 Preliminary toxicity testTo assess the toxicity of the test item to the bacteria, six dose-levels (one plate/dose-level) were tested in the TA 98, TA 100, TA 102 and WP2 uvrA strains, with and witiiout S9 mix. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.2.3.3 Mutagenicity experimentsIn two independent experiments, using three plates/dose-level, each strain was tested, with and without S9 mix, with:- at least five dose-levels of the test item,- the vehicle control,- the appropriate positive control.The sterility of the S9 mix was checked before the beginning and at the end of each experiment and was found to be satisfactory.

Conclusions:

Not mutagenic

Executive summary:

Under the experimental conditions, the test item did not show mutagenicity activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia Coli. Acid Yellow 23 could be considered not mutagenic.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

S. typhimurium strains TA, TA94 and

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Test concentrations with justification for top dose:

At least four non-toxic concentrations of compound were tested with three plates per dose level.

Vehicle / solvent:

DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: DAB - dimtehylaminoazobenzene

Details on test system and experimental conditions:

All the 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-phos- phate, 4mM-NADPH, 4mM-NADH, 33mM-KC1, 8 mM-MgCI 2, 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).

Evaluation criteria:

The criteria for a posztive, i.e. mutagemc, effect m the test was that the test chemical should induce at least a doubling of the spontaneous reversion count, together w~th a clear dose-response, many tester strain in duplicate experiments.

Species / strain:

other: all strains

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:

valid

Conclusions:

Not mutagenic

Executive summary:

The tested substance does not show any mutagenic effects in all of the tested S.Typhimurium strains.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

other: mouse lymphoma TK+/- assay

Target gene:

L5178Y TK +/- mouse lymphoma cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Test concentrations with justification for top dose:

Cells at a concentration of 6 × 105/ml were exposed for 4 h to a range of concentrations from 0.0 to 10000 /g/ml or the limit of solubility.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

ethylmethanesulphonate

Details on test system and experimental conditions:

L5178Y TK +/- mouse lymphoma cells were originally obtained from Dr. Donald Clive, Burroughs Wellcome Company, Research Triangle Park, NC. The cells were grown in Fischer's medium for leukemic cells of mice (Gibco, Grand Island, NY) supplemented with 10% horse serum (Gibco, Grand Island, NY), antibiotics (50 U penicillin/mi and 50 /~g streptomycin/ml), and 0.02% Pluronic F-68 (BASF Wyandotte Corp., Wyandotte, MI). All serum lots were prescreened for their ability to support optimal growth. The cells were checked for the presence of mycoplasma by agar block isolation (Rodwell and Mitchell, 1979) and Hoechst staining (McGarrity et al., 1983) before and after cryopreservation.The toxicity of each chemical was first de- termined both with and without $9 prepared from Aroclor-1254-induced male Fischer 344 rats. $9 mix was prepared according to the procedure of Clive et al. (1979). The cells were then washed, resuspended in growth medium, and incubated at 37 °C for 48 h. The rate of cell growth was de- termined for each of the treated cultures and compared to the rate of growth of the solvent controls. The doses of chemical selected for testing were within the range yielding approximately 0-90% cytotoxicity. Cells in duplicate cultures were exposed to the test chemical, positive control, and solvent control for 4 h at 37 ° C; washed twice with growth medium; and maintained at 37 °C for 48 h in log phase growth to allow recovery and mutant expression. The cultures were adjusted to 0.3 × 106 cells/ml at 24-h intervals. They were then cloned in soft agar medium containing Fischer's medium, 20% horse serum, 2 mM sodium pyruvate, 0.02% Pluronic F-68 and 0.35% Noble agar. Resistance to trifluorothymidine (TFT) was determined by adding 3 #g/ml TFT to one set of plates. The 100 × stock solution of TFT in saline was stored at -70°C and thawed immediately before use. Plates were incubated at 37°C in 5% CO 2 in air for 12 days, and then counted with an automatic colony counter (Biotran II, New Brunswick Scien- tific Co., Edison, N J). Mutant frequencies were expressed as mutants per 104 surviving cells.

Evaluation criteria:

A response was considered positive if there was a dose-related increase in the mutant frequency above the spontaneous control frequen- cy, with a 2-fold increase at more than 1 dose and relative total growth greater than 10%.

Species / strain:

mouse lymphoma L5178Y cells

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:

valid

Conclusions:

Not mutagenic in the mouse lymphoma assay.

Executive summary:

The tested subctance show negative results in the mouse lymphoma assay in the absence of metabolic activation. No results are available in this assay with metabolic activation.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

yes

Type of assay:

other: Mouse Lymphoma

Target gene:

Mouse Lymphoma L51878Y cells (forward mutation at Thymidine Kinase (TK+/-) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Test concentrations with justification for top dose:

156.3; 312.5; 625; 1250; 250; 5000 μg/ml (± S9)Toxicity: in a preliminary experiment, the concentration range of the test item was 39 t0 5000 μg/ml without S9 (4h/24h) and with S9 (4h). No toxicity was observed at all doses.

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

other: MDNA

Evaluation criteria:

A response was considered positive if there was a dose-related increase in the mutant frequency above the spontaneous control frequen- cy, with a 2-fold increase at more than 1 dose and relative total growth greater than 10%.

Species / strain:

mouse lymphoma L5178Y cells

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:

valid

Conclusions:

Not mutagenic

Executive summary:

There was no significant increase in mutation frequencies under all condition doses.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

S. typhimurium strains TA, TA94 and

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium, other: TA92

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium, other: TA94

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Test concentrations with justification for top dose:

5 mg/plate

Vehicle / solvent:

Phospate buffer

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Details on test system and experimental conditions:

All the 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-phos- phate, 4mM-NADPH, 4mM-NADH, 33mM-KC1, 8 mM-MgCI 2, 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).

Species / strain:

other: all strains

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

Conclusions:

Not mutagenic

Executive summary:

The tested substance does not show any mutagenic effects in all of the tested S.Typhimurium strains.

Reference 6

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

other: DNA repair assays

Species / strain / cell type:

hepatocytes:

Details on mammalian cell type (if applicable):

Rat hepatocytes were isolated and cultured by the two-step in situ liver perfusion method described by Maslansky and Williams [1982]. Approximately 2 X lo5 viable hepatocytes were seeded into 25-mm wells and were allowed to attach to Thermanox plastic coversli s (Flow Labs, Rockville, MD) for 2 hr. The cells were then incubated as detailed in Kornbrust and Barfknecht [1984a,b]. The 4-hr incubation period was chosen because previous studies had shown that longer incubations do not improve the detection of DNA repair and may actually decrease the observed response for many chemicals owing to a large increase in the backround radioactivity (ie, cyto- plasmic grains, see below). After the [H]-thymidine incubation, cultures were washed and incubated overnight with unlabeled media.

Metabolic activation:

not specified

Test concentrations with justification for top dose:

1x10(-3), 1x10(-4), 1x10(-5) and 1x10(-6) Molar

Vehicle / solvent:

DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Details on test system and experimental conditions:

Animals: Male Sprague Dawley cesarean-derived (Crl: COBS@ CD@ [SD] BR) rats (Charles River Breeding Laboratories, Inc., Kingston, NY), weighing 200-300 g, were used for all in vivo/in vitro HPC/DR assays, and also served as the source of hepatocytes for the in vitro HPC/DR assays. Prior to use, the animals were housed in humidity- and temperature-controlled rooms with 12-hr light/dark cycles, and allowed access to food and water ad libitum. For the preliminary in vitro assay test, stock solutions of the dyes were prepared in dimethylsulfoxide (DMSO) or water and incubated with the primary hepatocyte cultures during the 4-hr [3H]-thymidine labeling period. The final concentration of DMSO was always 1 % . Control incubations were conducted with and without 1% DMSO. This concentration of DMSO had no effect on DNA repair.

Evaluation criteria:

DNA repair was quantified by the autoradiographic determination of incorpo- rated [3H]-thymidine, similar to the method of Williams [1977] and Bermudez et a1 [1979], as detailed in Kornbrust and Barfknecht [1984a,b]. Net nuclear grains (NNG) were determined by counting the number of grains in each nuclei and subtracting the average number of grains present in three equal-size adjacent cytoplasmic areas. A strict procedure for random selection of cells was used [Mirsalis and Butterworth, 19801. The average NNG for 60 cells (iSD) as well as the percentage of cells with 2 5 NNG was determined for each dye concentration (in vitro HPC/DR assay) or each dye administered (in vivo/in vitro HPC/DR assay).

Species / strain:

hepatocytes: rat

Metabolic activation:

not specified

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

Conclusions:

Not mutagenic

Executive summary:

The tested substance showed negative results in the in vitro HPC/DR assay.

Reference 7

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

S. typhimurium strains

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Test concentrations with justification for top dose:

Tested at 5 dose levels in triplicate. The range of concentrations for testing was based on pre- liminary toxicity tests in which the viability of the bacterial cells on complete medium was measured at concentrations up to 10 mg/plate or to the limit of solubility.When solubility and toxicity were not limiting factors, the maximum concentra- tion tested was 10 mg/plate.

Vehicle / solvent:

DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

Remarks:

without metabolic activation

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation

Details on test system and experimental conditions:

For the plate-incorporation assay, Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were used. These strains were kindly supplied by Dr. Bruce Ames, University of California at Berkeley. Strain checks were performed immediately after receipt of the cultures and on a routine basis thereafter.Each chemical was tested without metabolic activation and with $9 mix from Aroclor 1254-induced male Fischer 344 rats and Syrian golden hamsters. The $9 mix was prepared according to the procedure described by Ames et al. (1975) and contained 0.1 ml S9 per ml of $9 mix; an aliquot of 0.5 ml of S9 mix was added per plate.

Evaluation criteria:

a re- sponse was considered positive if there was a dose-related increase in the number of revertants above spontaneous solvent controls, with a 2-fold increase for strains TA1535, TA1538, TA98 and TA100, and a 3-fold increase for TA1537.

Species / strain:

other: all strains

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:

valid

Conclusions:

Not mutagenic

Executive summary:

The tested substance does not show any mutagenic effects in all of the tested S.Typhimurium strains.

Reference 8

Endpoint:

in vitro transformation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

other: in vitro mammalian cells transformation assay

Version / remarks:

the BHK21/CI3 cell transformation test of Stylesa--m a validation study commissioned by the Ecological and Toxicological Association of the Dyestuffs Manufacturing Industry (ETAD), involving control substances (dyestuffs and closely related chemicals), which have been adequately shown to be carcinogenic or non-carcmogenic mammal tests.

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell transformation assay

Species / strain / cell type:

other: BHK21/C13

Details on mammalian cell type (if applicable):

Baby Syrian hamster kidney fibroblasts (BHK21/C 13) were maintained m the laboratory m Oulbecco's modification of Eagle's minimal essential medmm supplemented wlth l 0 ~/o newborn calf serum From these stock cultures, 1 ml tester cultures were prepared as 10-6 cells ml-1 in medium without serum and treated in triplicate with test compound in the presence and absence of S9-mlx. All tests were carried out at least twice The cultures were shaken for 4h at 37°C and the cells washed and resuspended in growth medmm containing serum Cell survival and growth in seml-solid agar were determined to assess cytotoxicity and transforming abdtty, respectively, of the chemicals

Metabolic activation:

with and without

Test concentrations with justification for top dose:

not specified

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Species / strain:

other: Baby Syrian hamster kidney fibroblasts (BHK21/C 13)

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

Conclusions:

Not mutagenic

Executive summary:

The tested substance does not show any effect in the in vitro cell transformation.

Reference 9

Endpoint:

genetic toxicity in vitro, other

Remarks:

Mitotic gene conversion in Saccharomyces cerevisiae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1979

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 480 (Genetic Toxicology: Saccharomyces cerevisiae, Gene Mutation Assay)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

yeast cytogenetic assay

Target gene:

Mitotic gene conversionin diploid Saccharomyces cerevisiae

Species / strain / cell type:

Saccharomyces cerevisiae

Details on mammalian cell type (if applicable):

The diploid yeast BZ 34 was kindly donated by Dr. Robert Mortimer of the 310 Donner Laboratory, Berkeley.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

not specified

Test concentrations with justification for top dose:

5 mg/ml

Vehicle / solvent:

The stationary phase tests were carried out in potassium phosphate buffer (0.1 M, pH 7). In a typical treatment a cell suspension of about 10 s cells/ml was prepared from a culture with a low background conversion frequency. 1 ml of this was added to 9 ml of phosphate buffer which contained the colour at 5 mg/ml.

Details on test system and experimental conditions:

The treatment was carried out at 30°C in a shaker water-bath in the dark. After a lapse of 4 h, suitable dilutions were made and plated on yeast extract, peptone, dextrose (YEPD) and Arg- plates for scoring survival and gene conversion respectively.Treatment under growth conditions was done in YEPD broth. 10 ml of broth were inoculated with 1000 cells and placed on a shaker water-bath at 30°C. 311 After 24 h of growth a given food colour was added (5 mg/ml) and the treat- ment continued in the dark. At 72 h (i.e. 48 h after addition of the colour) the treatment was terminated. The pH at this stage was about 6. Cell counts were made with a haemocytometer to see whether the food colours inhibited cell division. Suitable dilutions were made and plated on YEPD as well as Arg- plates. After 3 days the colonies were counted and the number of convertants per million survivors was calculated. Control samples without the colours were treated identically. N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) served as a positive control. Treatments were repeated 2--3 times for each colour.

Species / strain:

Saccharomyces cerevisiae

Metabolic activation:

not specified

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:

That the absence of lethal and genetic effects may be due to the lack of penetration of the dye into the cell can be ruled out by the following observations. When the cells were plated after treatment without further dilution on Arg- plates, the colonies picked up the colour of the dye in ques- tion. This suggested that the dye could indeed enter the cells. This was further confirmed in some cases by spectrophotometric measurements. The absorbance of the supernatant after treatment was less than that before treatment. How- ever, the dye uptake was not enough to be seen on microscopic examination of invidual cells.

Conclusions:

Not mutagenic

Executive summary:

Under the test conditions neither significant cell killing nor inhibition of cell division was observed.

Reference 10

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Chromosomal aberration

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Stocks of the V79 cell line (1-mL portions) were maintained at -196°C in liquid nitrogen using 7% DMSO in culture medium as a cryoprotectant. Each batch used for the cytogenetic experiments was checked for - mycoplasma contamination. - karyotype stability. - plating efficiency (= colony forming ability) incl. vital staining.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Test concentrations with justification for top dose:

In the pretest for toxicity based on a BALB/c 3T3 Neutral Red Uptake Cytotoxicity Test 600 µg/mL (approx. 200 µg/mL active component) teh tested substancewas used as top concentration. The cells were prepared at a sampling time of 18 hours (about 1.5-fold cell cycle time) after 4 and 18 hours exposure time without S9 mix and after 4 hours exposure time with S9 mix.Test groups in the 1st Experiment: With and Without S9 mix (4-hour exposure 18 hours sampling time): Negative Control 150.0 µg/mL 300.0 µg/mL 600.0 µg/mL 1200.0 µg/mL 1800.0 µg/mL 2400.0 µg/mL Test groups in the 2nd Experiment: With and Without S9 mix (4-hour exposure 18 hours sampling time): Negative Control 468.8 µg/mL 937.5 µg/mL 1875 µg/mL 3750 µg/mL 7500 µg/mL 15000 µg/mL Test groups in the 3rd Experiment: With S9 mix (4-hour exposure 28 hours sampling time): Negative Control 468.8 µg/mL 937.5 µg/mL 1875 µg/mL 3750 µg/mL 7500 µg/mL 15000 µg/mL Test groups in the 3rd Experiment: With and without S9 mix (18-hour exposure 18 and 28 hours sampling time): Negative Control 117.2 µg/mL234.4 µg/mL468.8 µg/mL 937.5 µg/mL 1875 µg/mL 3750 µg/mL 7500 µg/mL 15000 µg/mL

Vehicle / solvent:

The test substance is an aqueous solution, therefore, culture medium (MEM: Minimal Essential Medium) was selected as most suitable vehicle.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Culture media MEM (minimal essential medium with Earle's salts) containing a L-glutamine source supplemented with − 10% (v/v) fetal calf serum (FCS) − 1% (v/v) penicillin/streptomycin (10 000 IU / 10 000 µg/mL) − 1% (v/v) amphotericin B (250 µg/mL) During exposure to the test substance (only 4-hour treatment), MEM medium was used without FCS supplementation. Cell culture Deep-frozen cell suspensions were thawed at 37°C in a water bath, and volumes of 0.5 mL were transferred into 25 cm2 plastic flasks containing about 5.0 mL MEM supplemented with 10% (v/v) FCS (fetal calf serum). Cells were grown with 5% (v/v) CO2 at 37°C and > 90% humidity and subcultured twice weekly. Cell monolayers were suspended in culture medium after dispersion with 0.25% (w/v) trypsin solution. Cell cycle time The cell cycle of the untreated V79 cells lasted for about 12 - 14 hours (last measurement based on the BrdU method of Speit et al. [ 12]: Nov 2007 & Feb 2009) under the selected culture conditions. Thus, the selected 1st sampling time of 18 hours (see item 3.7.1.) was within the guideline recommended interval of about 1.5-fold of the normal cell cycle time. The later sampling time of 28 hours fulfilled the recommendation of an interval with more than 1.5fold of the normal cell cycle time to cover a possible substance induced cell cycle delay. S9 mix: 24 hours after the last administration with phenobarbital, the rats were sacrificed and the livers were prepared using sterile solvents and glassware at a temperature of +4°C. The livers were weighed and washed in a weight-equivalent volume of a 150 mM KCl solution, then cut into small pieces and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9 000 x g for 10 minutes at +4°C, 5-mL portions of the supernatant (so-called S9 fraction) were stored at -70°C to -80°CThe S9 mix was prepared freshly prior to each experiment ( 4). For this purpose, a sufficient amount of S9 fraction was thawed at room temperature, and 1 part S9 fraction was mixed with 9 parts S9 supplement (cofactors). This preparation, the so-called S9 mix, was kept on ice until used.The concentrations of the cofactors in the S9 mix were: - MgCl2 8 mM - KCl 33 mM - glucose-6-phosphate 5 mM - NADP 4 mM- phosphate buffer (pH 7.4) 15 mM.

Statistics:

The statistical evaluation of the data was carried out using the MUCHAN program system. The proportion of metaphases with structural aberrations was calculated for each group. A comparison of each dose group with the negative control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test was BonferroniHolm corrected versus the dose groups separately for each time and was performed one-sided. If the results of this test are statistically significant compared with the respective negative control, labels (* p < 0.05, ** p < 0.01) are printed in the tables

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the 1st Experiment in the absence and presence of metabolic activation no cytotoxicity was observed up to the highest applied concentration (2 400 µg/mL; approx. 800 µg/mL active component).According to the results of the present in vitro cytogenetic study, the test substance did not lead to a biologically relevant increase in the number of structural chromosomal aberrations incl. and excl. gaps either without S9 mix or after the addition of a metabolizing system in three experiments performed independently of each other selecting different exposure times (4 and 18 hours) and sampling times (18 and 28 hours). The types and frequencies of structural chromosome aberrations were close to the concurrent negative control values at both sampling times and clearly within in the range of the historical negative control data. In this study, no relevant increase in the number of cells containing numerical chromosomal aberrations was observed in the absence and the presence of metabolic activation The structural chromosome aberration rates of the vehicle control groups were within our historical negative control data range and, thus, fulfilled the acceptance criteria of this study. The increase in the frequencies of structural chromosome aberrations induced by the positive control substances EMS and CPP clearly demonstrated the sensitivity of the test system and of the metabolic activity of the S9 mix employed. The values were within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study

Conclusions:

Not mutagenic

Executive summary:

Under the experimental conditions chosen here, the conclusion is drawn that the tested substance is not a chromosome-damaging (clastogenic) substance under in vitro conditions using V79 cells in the absence and the presence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Based on the results of the in vivo studies available on Acid Yellow 23, no mutagenic effetcs were observed.

The available tests were conducted following the guidelines:

OECD 487, Unscheduled DNA synthesys

NO OECD, rat gut micronucleus assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

other: HPC/DN repair assay

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

cesarean-derived

Sex:

male

Details on test animals or test system and environmental conditions:

Male Sprague Dawley cesarean-derived (Crl: COBS@ CD@ [SD] BR) rats (Charles River Breeding Laboratories, Inc., Kingston, NY), weighing 200-300 g, were used for all in vivo/in vitro HPC/DR assays, and also served as the source of hepatocytes for the in vitro HPC/DR assays. Prior to use, the animals were housed in humidity- and temperature-controlled rooms with 12-hr light/dark cycles, and allowed access to food and water ad libitum.

Route of administration:

oral: gavage

Vehicle:

water

Duration of treatment / exposure:

single application

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

not specified

Control animals:

other: Negative control: Acid Red 14 (Carmoisine)

Positive control(s):

Solvent Yellow 3 (0-aminoazotoluene)

Evaluation criteria:

Consistent with the criteria employed by other investigators [Williams, 1977; Bermudez et al, 1979; Probst et al, 19811, average net nuclear grain counts of 5 or greater were assumed to constitute a positive response, since these differed from the control net nuclear counts by greater than 2 SD. On a historical basis in this laboratory, control incubations have invariably yielded an average value for net nuclear grains of less than zero. In the present study, NNG counts ranged from -0.6 to -2.8 and from -0.9 to -2.1 for no-solvent and 1% DMSO control incubations, respectively. In addition, the proportion of cells with 2 5 NNG was 6 8% for all control incubations [4.1 f 2.6% (mean -t standard deviation), n = 17). Therefore, net nuclear grain counts below zero were considered negative responses. For those dyes that produced responses between zero and 5 average net nuclear grains, it was generally not possible to demonstrate a statistically significant difference from the control value within a given experiment. Therefore, these responses were judged to be equivocal, unless, in addition to an average net nuclear grain count between zero and 5, at least 25% of the cells examined contained 2 5 net nuclear grains, in which case the response was considered weakly positive. Concentrations of the dyes that produced approximately 90% or greater detachment of the hepatocytes from the coverslips (as assessed visually by comparing to control slides) were assumed to be toxic and were not counted.

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Not mutagenic

Executive summary:

The tested substance showed negative results in the in vivo HPC/DR assay.

Reference 2

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

rat gut micronucleus assay

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

other: rat gut micronucleus assay

Version / remarks:

This test is considered particularly suitable for the detection of clastogenic and aneugenic effects of compounds given orally by gavage and targeting the intestine or the colon (Vanhauwaert et al., 2001).

Deviations:

not specified

Principles of method if other than guideline:

The rat gut micronucleus assay is a good choice for alternative in vivo genetic toxicology testing strategies based on literatura data. The test assessed in the in vivo micronucleus test on gut for genotoxic effects (frequency of micronucleated cells in the colon) and toxicity (apoptotic and mitotic cells).

GLP compliance:

not specified

Remarks:

Not indicated in the report

Type of assay:

other: rat gut micronucleus assay

Species:

mouse

Strain:

other: ddY

Sex:

male

Details on test animals or test system and environmental conditions:

Supplier: Japan SLC Co., Shizuoka, JapanAge: 7 weeksRats were caged singly in plastic cage and housed in fully air-conditioned rooms with temperature =19-23°C and humidity continuosly recorded.Animals were subjected to 12 hours artificial light and 12 hours darkness in each 24 hours period.Feeding: ad libitum commercial pellet diet (A04, SAFE, Villemoisson, France)Drinking water: tap water ad libitumAcclimatization: one week

Route of administration:

oral: gavage

Vehicle:

physiological saline

Duration of treatment / exposure:

single application

Post exposure period:

24 hours

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 animals

Control animals:

other: water and oil vehicles

Positive control(s):

1,2-dimethylhydrazine (DMH)Dimethylaminoazobenzene (DAB), an azo dye compound which is considered a hepatocarcinogen in several animal species (IARC, 1975) but was also shown to damage colonic cell DNA in mice at low dose (Tsuda et al., 2001)

Tissues and cell types examined:

Colon tissue

Details of tissue and slide preparation:

After cervical dislocation, the colon was excised, flushed free of faecal debris with PBS, cut open longitudinally and rolled from anus to caecum (Moolenbeek and Ruitenberg, 1981). The ‘‘Swiss rolls” were fixed in 10% neutral formalin, embedded in paraffin (Paraplast, CML, France), sectioned through the roll (5 lm) and stained with Feulgen-fast green after hydrolysis 1 h at 60–65 °C in HCl N.

Evaluation criteria:

For each animal, over 1000 epithelial cells were scored manually using a whole number of crypts in which a single continuous row of epithelium cells could be discerned from the proximal end adjacent to the muscle layer to the distal end at the mucosal surface. Cells were scored as normal, micronucleated, apoptotic or mitotic cells. A micronucleated cell was defined as a cell with a normal nucleus and one or two micronuclei which neither exceeded one-third of the diameter of the main nucleus or overlapped with it. Cells with a fragmented nucleus were defined as apoptotic cells. Mitotic cells were identified as cells in which the chromatin was condensed and visible as chromatids. Generally, mitotic figures were horizontally displaced away from epithelial cells, towards the crypt lumen.

Statistics:

For the gut micronucleus test, data of treated groups were compared to respective control group using the Pearson’s v2 test on the pooled animal data of each treatmentgroup. P values <0.05 indicated that differences were statistically significant

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In negative control groups, (water and oil vehicles) the background incidence of micronucleated cells was very low. Treatments of mice with amaranth, sunset yellow, tartrazine or DAB did not increase the frequency of micronucleated colonic cells, at any dose level. Positive control (DMH, 30 mg/kg b.w.) showed a statistically significant increase in the frequency of micronucleated gut cells. Only the positive control DMH induced a statistically significant increase of apoptotic colonic cells. The number of nuclear anomalies (micronuclei + apoptotic bodies) per crypt was 1.36 ± 0.12 in the DMH group.DMH induced a statistically significant decrease in the frequency of mitotic figures while food dyes increased the mitotic cells at all dose levels when compared to water treated group. The increases were not related to the doses and reached the level of significance with the exception of the tartrazine 1000 mg/kg b.w. group. The incidence of mitotic cells was not modified following DAB exposure.

Conclusions:

Not mutagenic

Executive summary:

The acute oral exposure to the tested substance did not induce genotoxic effect in the gut micronucleus assay in mice.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Justification for selection of genetic toxicity endpoint
The experimental reports and the publications are complete and weel documented.
The tests were performed following official guidelines.

Justification for classification or non-classification

There are both in vitro and in vivo mutagenic tests performed on Acid Yellow 23 and for another form of this dye.

The chromosomal aberration test performed on a differet form of Acid Yellow 23 showed negative result.

The results of the other in vitro tests (Mouse Lymphoma, DNA repair assay and Genetic Toxicology Saccharomyces cerevisiae toxicity) gave negative results and no mutagenic effects were seen.

Based on the information present in the official ECHA guideline:

"Guidance on Informatio requirement and Chemical Safety Assessment - R.7a - Endpoint specific guidance"

the substance should be considered as not genotoxic considering the results of the in vivo genotoxicty tests.
The results of the in vivo micronucleous test does not show any effects and the absence of mutagenic effects was confirmed by the in vivo unscheduled DNA synthesis test results.

Based on the above considerations, no concern related to genetic toxicity is expected.

No classification for mutagenicity is warranted under Regulation (CE) 1272/2008.