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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenicity in bacterial reverse mutation assays (Ames test) has been tested for a series of Diarylide Yellow Pigments. But tests performed with the Prival modification for azo-dyes have only been performed for Diarylide Yellow Pigments Pigment Yellow 83 and its structural analogues, Pigment Yellow 12, Pigment Yellow 13, and Pigment Yellow 81. Ames tests without Prival modification were performed for Pigment Yellow 14, Pigment Yellow 17, Pigment Yellow 55, Pigment Yellow 126, Pigment Yellow 127, Pigment Yellow 174 and Pigment Yellow 176. All available tests revealed negative results with and without metabolic activation.

In vitro gene mutation in mammalian cells (Mouse Lymphoma Assay) has been studied for the structural analogues, Pigment Yellow 12 and Pigment Yellow 170. Both tests were negative in the presence and absence of metabolic activation.

A reliable study on the induction of chromosome aberration in mammalian cells in vitro is available for the strcutural analogue, Pigment Yellow 12, which gave negative results with and without metabolic activation. This study is supported by another test performed with another structural analogue, Pigment Yellow 14, which also gave negative results in the presence and absence of metabolic acitivation. But reliability of the study performed with Pigment Yellow 14 could not be assigned (reliability 4) due to the short documentation. Incubation of rat hepatocytes in vitro with Pigment Yellow 12 resulted in an increased induction of the tail moment. The relevance of these data is doubtful, because a) the Comet assay is mainly used to monitor tissue exposure to a toxic agent. In vitro sinlge cell exposure followed by single cell electorphoresis is not properly validated at the moment; b) a known carcinogen was used as positive control substance. At the moment it cannot be excluded that the effects observed in the presence of the test item are due to mechanical disturbances. However no appropriate positive control substance (e.g. an inert material expected to cause physical effects on the cells) was investigated. Due to these shortcomings the test was considered not to be reliable.

Two tests were performed with Pigment Yellow 13, a strucutral analogue to Pigment Yellow 83, to investigate the induction of unscheduled DNA synthesis in human fibroblasts and rat hepatocytes. Both tests were performed without metabolic activation and gave negative results.

For further information please refer to read across document, IUCLID Chapter 13

Link to relevant study records

Referenceopen allclose all

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:
From 11 JUN 2002 to 27 JUN 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
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 102
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9; hamster liver S9
Test concentrations with justification for top dose:
50, 160, 500, 1600, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility properties of the solvent
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide (TA 100, TA1535), 9-aminoacridine (TA 1537), 2-nitrofluorene (TA 98), mitomycin C (TA 102)
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation (rat liver S9 mix)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (TA 100, TA 1535, TA 1537, TA 102), congo red (TA 98)
Remarks:
with metabolic activation (hamster liver S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- plate incorporation assay with induced rat liver S9 mix (10% (v/v); induction with Aroclor 1254)
- preincubation assay with uninduced hamster liver S9 mix (30% (v/v))

DURATION
- Preincubation period: 20 to 30 minutes at 30 °C
- Exposure duration: at least 48 hours

NUMBER OF REPLICATIONS: 3 plates per strain and dose level, including controls

Evaluation criteria:
A test compound is classified as mutagenic if it has either of the following effects:
- it produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
- it induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn
Statistics:
Arithmetic means and standard deviation of the counted colonies were calculated, as well as the respective dose/control ratio.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
visible precipitation of the test item:
- plate incorporation test: at 50 µg/plate and above
- preincubation test: with S9-mix at 500 µg/plate and above and without S9-mix at 160 µg/plate and above

COMPARISON WITH HISTORICAL CONTROL DATA: there were no deviations observed

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

The test item showed no mutagenic activity in both experiments (plate incorporation assay, preincubation assay) each with and without metabolic activation.

Conclusions:
negative

The test item did not exert mutagenic activity in the reverse bacterial mutation assay (plate incorporation assay and preincubation assay according to Prival) with and without metabolic activation.
Executive summary:

Mutagenic activity of the test item was investigated in Salmonella typhimurium strains TA 1535, TA 1537, TA98, TA100 and TA 102 with (induced rat liver S9 mix) and without metabolic activation at concentrations of 50, 160, 500, 1600, and 5000 µg/plate using the plate incorporation assay. Due to the test items characteristic as an azo-dye the test was also conducted using the Prival modification, i.e. testing the above mentioned bacterial strains in the preincubation assay with uninduced hamster liver S9 mix for metabolic activation. This test was performed using the concentrations 50, 160, 500, 1600, and 5000 µg/plate.

The test item did not reveal any mutagenic activity under the conditions tested. The appropriate reference mutagenes showed distinct positive mutagenic effects.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
year of publication: 1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Principles of method if other than guideline:
in vitro mammalian chromosome aberration test: NTP-Chinese hamster Ovary Cell Cytogenetics
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from Aroclor 1254-induced male Sprague Dawley rats
Test concentrations with justification for top dose:
1.6, 5, 16, 50, 160 µg/ml without metabolic activation (highest concentration not evaluated)
0.5, 1.6, 5, 16, 50 µg/ml with metabolic activation (highest concentration not evaluated)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethylsulfoxide (DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 8-12 h without metabolic activation; 2 h with metabolic activation
- Expression time (cells in growth medium): 10 h (with metabolic activation)
- Selection time (if incubation with a selection agent): 2 h
- Fixation time (start of exposure up to fixation or harvest of cells): up to 12 h


SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: no data


NUMBER OF CELLS EVALUATED: 100 first-division metaphase cells; only 50 first-division metaphase cells for the second dose of the positive control Mitomycin C


OTHER EXAMINATIONS:
- "simple" aberrations: breaks and terminal deletions
- complex" aberrations: rearrangements and translocations
- "other" aberrations: pulverized cells, despiralized chromosomes, cells containing 10 or more aberrations

Evaluation criteria:
For a positive response the presence of a dose-response and the significance of the individual dose points compared to the vehicle control were mandatory.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Frequency of effects:

Without metabolic activation:

1, 1, 2, 2% cells with aberrations at 1.6, 5.0, 16 and 50 µg/ml, respectively; DMSO control: 1%

With metabolic activation:

2, 1, 4, 0% cells with aberrations at 0,5, 1.6, 5.0 and 16 µg/ml, respectively; DMSO control: 1%

Conclusions:
Interpretation of results:
negative with metabolic activation
negative without metabolic activation

The test item Diarylanilide yellow did not induce chromosome aberrations under the conditions tested.
Executive summary:

Induction of chromosome aberrations by the test item has been investigated in Chinese hamster ovary cells in vitro in the presence (induced rat liver S9) and absence of metabolic activation. The test item did not induce chromosome aberrations in tests concentrations up to 16 µg/ml (with metabolic activation) or 50 µg/ml (without metabolic activation) under these test conditions.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 21 MAR 1994 to 14 JUN 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
in accordance with Directive 88/320/EEC
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium supplemented with 10% donor horse serum and 20 mM HEPES buffer (R10)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix from Aroclor 1254 pretreated rats
Test concentrations with justification for top dose:
62.5, 125, 250, 500, 1000 µg/l
Vehicle / solvent:
- dimethyl sulphoxide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Details on test system and experimental conditions:

DURATION
- Exposure duration: 3 hrs with manual shaking at approximately 0.5 hour intervals
- Expression time (cells in growth medium): 24 hrs
- Selection time (if incubation with a selection agent): 10-14 days


SELECTION AGENT (mutation assays): trifluorothymidine (4 µg/ml)

NUMBER OF REPLICATIONS: 2 experiments, treatments performed in duplicate, both with and without metabolic activation

DETERMINATION OF CYTOTOXICITY
- Method: colony formation

Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: a precipitate of the test item was noted at all dose levels

RANGE-FINDING/SCREENING STUDIES:
- the maximum dose level of 1000 µg/ml was selected on the basis that it was the maximum practical dose level that could be achieved

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

- no marked reductions in viability of cells treated with the test item in the presence or absence of metabolic activation

- vehicle controls were within the normal range (10 - 125 x 10E-6)

- positive controls gave marked increases in mutant frequency, demonstrating that the test system was operating satisfactorily

- the test item did not induce any statistically significant or dose related increase in mutant frequency

- experiment 1 and 2 gave concordant results

Conclusions:
negative without metabolic activation
negative with metabolic activation

The test substance did not increase the mutant frequency at the TK+/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.
Executive summary:

Mouse Lymphoma cells (L5178Y TK+/-) were treated with the test substance at five concentrations in the range of 62.5 to 1000 µg/ml. A heavy precipitate of the test substance was noted at all dose levels. The solvent control gave mutant frequencies within the normal range. The positive controls gave marked increases in the mutant frequency, both in the presence or absence of metabolic activation, indicating the satisfactory performance of the test system. The test substance demonstrated no statistically significant or dose related increases in mutant frequency at any dose level, either with or without metabolic activation, thus indicating that the test item is not mutagenic under the conditions tested.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Induction of micronuclei in vivo was investigated in a OECD guideline tests with mice which were intraperitoneally treated with Pigment Yellow 170 in concentrations up to 5000 mg/kg bw, the maximum recommended dose. No induction of micronuclei was observed at any dose group.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 26 APR 1994 to 06 JUN 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
only 1000 instead of 2000 immature erythrocytes were evaluated per animal
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
only 1000 instead of 2000 immature erythrocytes were evaluated per animal
GLP compliance:
yes (incl. certificate)
Remarks:
in accordance with Directive 88/320/EEC
Type of assay:
micronucleus assay
Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan U.K. Ltd., blackthorn, Bicester, Oxon, U.K.
- Age at study initiation: 5-8 weeks
- Weight at study initiation: males: 21-30 g; females: 20-25 g
- Assigned to test groups randomly: yes
- Housing: in groups up to five by sex
- Diet: Rat and Mouse Expanded Diet No. 1 (Special Diet Services Ltd. Witham, Essex, U.K.), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 44-50
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12


Route of administration:
intraperitoneal
Vehicle:
- Vehicle used: arachis oil
- Concentration of test material in vehicle: 125, 250 or 500 mg/ml
- dose volume: 10 ml/kg

Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- test material was freshly prepared as a suspension at the appropriate concentration in arachis oil
- analysis of concentration, homogeneity and stability was not performed

APPLICATION:
- single intraperitoneal application
Duration of treatment / exposure:
1250 and 2500 mg/kg group: 24 hrs
5000 mg/kg group: 24, 48 and 72 hrs
Frequency of treatment:
single
Post exposure period:
none
Remarks:
Doses / Concentrations:
1250, 2500, 5000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5 males and 5 females per dose and exposure duration
Control animals:
yes, concurrent vehicle
Positive control(s):
- cyclophosphamide
- Route of administration: intraperitoneal
- Doses / concentrations: 50 mg/kg
- Exposure duration: 24 hrs
Tissues and cell types examined:
bone marrow cells from the femur
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
- maximum recommended dose level and two lower dose levels

DETAILS OF SLIDE PREPARATION:
- femur was dissected from each animal, aspirated with foetal calf serum and bone marrow smears were prepared following centrifugation and re-suspension. Smears were air-dried, fixed in absolute methanol and stained with May-Grünwald/Giemsa

METHOD OF ANALYSIS:
- blind examination with light microscopy
- scoring of micronucleated cells per 1000 polychromatic erythrocytes per animal
- normochromatic erythrocytes associated with 1000 erythrocytes were counted ans scored for incidence of micronuclei
- calculation of polychromatic to normochromatic erythrocytes

Evaluation criteria:
- a statistically significant increase in the number of micronucleated polychromatic erythrocytes in the treatment groups in comparison to the control group is evaluated as positive mutagenic response
- a positive response for bone marrow toxicity is demonstrated when the dose group mean polychromatic to normochromatic ratio is shown to be statistically significant from the concurrent vehicle control group
Statistics:
- Student's t-test
- one way analysis of variance
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 500-5000 mg/kg bw, i.p; 500-2000 mg/kg bw, oral
- Clinical signs of toxicity in test animals: hunched posture and fur stained orange; one premature death at 2000 mg/kg bw i.p., which was considered to be due to poor dosing technique rather than to toxicity of the test material



RESULTS OF DEFINITIVE STUDY
- Ratio of PCE/NCE (for Micronucleus assay): group means for control groups: 1.46 - 1.83; treatment groups did not deviate significantly

- no premature deaths in any dose group

- fur stained with test material in all animals treated with the test item

- hunched posture in some animals at 5000 mg/kg bw

- one animal with lethargy at 5000 mg/kg bw

There was no statistically significant increase in the frequency of micronucleated PCE's and in the PCE/NCE ratio in any dose group when compared to the concurrent vehicle control groups.

The positive control goup showed a marked increase in the incidence of micronucleated PCE.

Conclusions:
Interpretation of results: negative
The test item was considered to be non-genotoxic under the conditions of this in vivo micronuclei assay.
Executive summary:

Genetic toxicity of the test item has been investigated in an in vivo micronucleus assay in male and female ICR mice (5 per sex and group). The test item was administered at the maximum recommended dose level of 5000 mg/kg bw, with 2500 and 1250 mg/kg bw as the two lower dose levels. Animals were killed 24, 48 or 72 hours later. Evaluation of polychromatic (PCE) or normochromatic (NCE) erythrocytes did not reveal any evidence of an increase in the incidence of micronucleated cells. No significant change in the PCE/NCE ratio was observed after dosing with the test item, however, clinical signs were observed in animals dosed with the test item and as such are evidence of systemic exposure. The positive control material produced a marked increase in the frequency of micronucleated PCE. The test material was considered to be non-genotoxic under the conditions of the test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

There is no evidence for species specific effects of the substance. Therefore, the results of the in vitro/in vivo data are regarded as relevant for humans.

Additional information

Possible mutagenic potential of Pigment Yellow 83 and its structural analogues, the Diarylide Yellow Pigments, has been investigated in several reliable tests in vitro (bacterial reverse mutation assays (Ames tests), gene mutation and chromosome aberration tests in mammalian cells, test for unscheduled DNA synthesis and sister chromatide exchanges) as well as in vivo (micronucleus assay in mice). All these tests consistently yielded negative results indicating that Diarylide Yellow Pigments and consequently Pigment Yellow 83 have no mutagenic potential. Besides these negative findings on mutagenicity Diarylide Yellow Pigments are unlikely to be bioavailable after oral, dermal and inhalation exposure and therefore have not to be classified as germ cell mutagens.

For details please refer to read across justification, IUCLID Chapter 13

Justification for classification or non-classification

Due to the negative findings with Diarylide Yellow Pigments in the mutagenicity assays in vitro and in vivo and in consideration that they are unlikely to be bioavailable after oral, dermal and inhalation exposure Diarylide Yellow Pigments and consequently Pigment Yellow 83 have not to be classified as germ cell mutagens according to Regulation (EC) No 1272/2008.