2,2'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[3-oxo-N-phenylbutyramide]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenicity in bacterial reverse mutation assays (Ames test) with the Prival modification for azo-dyes have been performed for Diarylide Yellow Pigments Pigment Yellow 12 and its structural analogues. 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 Pigment Yellow 12 and the 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 Pigment Yellow 12 which gave negative results with and without metabolic activation.

Two tests were performed with Pigment Yellow 13, a structural analogue to Pigment Yellow 12, 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

Reference

Reference 1

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

year of publication: 1987

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 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat or hamster liver S9 mix

Test concentrations with justification for top dose:

100, 333, 1000, 3333, 10000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (TA 1535, TA 100), 9-aminoacridine (TA 1537), 4-nitro-o-phenylenediamine (TA98)

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes 37°C without shaking
- Exposure duration: 2 days

NUMBER OF REPLICATIONS: 3 plates per strain and dose level, including the controls; experiments were repeated

Evaluation criteria:

An individual trial was judged as:
- mutagenic: if dose-related increase over the corresponding solvent control was seen
- weakly mutagenic: if low-level dose response
- questionable: if dose-related increase was judged to be insufficiently high to be called weakly mutagenic or only a single dose was elevated or a non -dose-related increase was seen

A chemical was judged mutagenic, if it produced a reproducible, dose-related increase in revertants over the corresponding solvent control in replicate trials.

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:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: was observed at all tested concentrations, the revertant colonies were counted manually

Remarks on result:

other: all strains/cell types tested

The test item showed no mutagenic activity in the preincubation test performed with modifications similar to Prival with (induced rat and hamster liver S9) and without metabolic activation.

Conclusions:

Interpretation of results:
negative

The test item did not exert mutagenic activity in the reverse bacterial mutation assay (preincubation assay, also with Prival modification) with and without metabolic activation.

Executive summary:

Mutagenic activity of the test item was investigated in Salmonella typhimurium strains TA 1535, TA 1537, TA98 and TA100 with (Aroclor 1254-induced rat liver S9 mix or with Aroclor 1254-induced hamster liver S9 mix; i.e. modified Prival test) and without metabolic activation at concentrations of 100, 333, 1000, 3333 and 10000 µg/plate using the preincubation method.

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

Reference 3

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:

no data

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 476 (In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

Mouse Lymphoma Study

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 from the livers of either Aroclor 1254-induced or non-induced male Fischer 344 rats

Test concentrations with justification for top dose:

0.0312, 0.0625, 0.125, 0.25, 0.5 µg/ml (Nonactivation Trial 1)
0.1, 0.2, 0.3, 0.4, 0.5 (Nonactivation Trial 2; Induced S9 Trial 1, 2, 3)

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:

other: methylmethanesulfonate, ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): 10-12 days


SELECTION AGENT (mutation assays): trifluorothymidine


NUMBER OF REPLICATIONS: all treatment levels within an experiment were performed in duplicate; experiments were performed twice (nonactivated) or in triplicate (S9)


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Statistics:

statistical analysis for trend and peak responses

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

- results without metabolic activation were negative in two replicates, the tested concentrations were non-toxic

Trial 1 (mean mutant frequency): 36, 40, 44, 40 and 46 at 0.03, 0.06, 0.125, 0.25 and 0.5 µg/ml; DMSO control: 31

Trial 2 (mean mutant frequency): 30, 30, 45, 38 and 37 at 0.1, 0.2, 0.3, 0.4 and 0.5 µg/ml; DMSO control: 30

- with metabolic activation one trial revealed increased mutant frequencies at concentrations >/= 0.2 µg/ml in comparison to the vehicle control, but without dose response relationship; in two further trials negative responses were observed

Trial 1 (mean mutant frequency): 25, 61, 68, 69 and 62 at 0.1, 0.2, 0.3, 0.4 and 0.5 µg/ml; DMSO control: 30

Trial 2 (mean mutant frequency): 48, 57, 62, 61 and 60 at 0.1, 0.2, 0.3, 0.4 and 0.5 µg/ml; DMSO control: 54

Trial 3 (mean mutant frequency): 79, 68, 76, 94 and 88 at 0.1, 0.2, 0.3, 0.4 and 0.5 µg/ml; DMSO control: 71

- solvent and positve controls were within the normal range

Conclusions:

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

The test item did not induce mammalian cell gene mutations under the conditions tested.

Executive summary:

Induction of mammalian cell gene mutations in vitro has been investigated in mouse lymphoma L5178Y cells in the presence (rat liver S9) and absence of metabolic activation. The test item did not induce gene mutations in concentrations up to 0.5 µg/ml under the tested conditions.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Reason / purpose for cross-reference:

read-across source

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

not specified

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Conclusions:

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

The toxicity potential of registration substance is assessed using analogue approach.
The test item did not induce mammalian cell gene mutations under the conditions tested.

Executive summary:

The toxicity potential of registration substance is assessed using analogue approach.

Induction of mammalian cell gene mutations in vitro has been investigated in mouse lymphoma L5178Y cells in the presence (rat liver S9) and absence of metabolic activation. The test item did not induce gene mutations in concentrations up to 0.5 µg/ml under the tested conditions.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Reason / purpose for cross-reference:

read-across source

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

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:

not examined

Positive controls validity:

valid

Conclusions:

The toxicity potential of registration substance is assessed using analogue approach.
Interpretation of results:
negative

The test item did not exert mutagenic activity in the reverse bacterial mutation assay (preincubation assay, also with Prival modification) with and without metabolic activation.

Executive summary:

The toxicity potential of registration substance is assessed using analogue approach.

Mutagenic activity of the test item was investigated in Salmonella typhimurium strains TA 1535, TA 1537, TA98 and TA100 with (Aroclor 1254-induced rat liver S9 mix or with Aroclor 1254-induced hamster liver S9 mix; i.e. modified Prival test) and without metabolic activation at concentrations of 100, 333, 1000, 3333 and 10000 µg/plate using the preincubation method.

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

Reference 6

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

key study

Reason / purpose for cross-reference:

read-across source

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Additional strain / cell type characteristics:

not specified

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

Conclusions:

The toxicity potential of registration substance is assessed using analogue approach.
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:

The toxicity potential of registration substance is assessed using analogue approach.

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 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 structural analogue 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

Reference 1

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

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

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. QA statement)

Remarks:

in accordance with Directive 88/320/EEC

Type of assay:

micronucleus assay

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system 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.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

key study

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

The toxicity potential of registration substance is assessed using analogue approach.
Interpretation of results: negative
The test item was considered to be non-genotoxic under the conditions of this in vivo micronuclei assay.

Executive summary:

The toxicity potential of registration substance is assessed using analogue approach.

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 12 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 12 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 Pigment Yellow 12 has not to be classified as germ cell mutagens according to Regulation (EC) No 1272/2008.