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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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.

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.

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.

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:

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 2

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 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 bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Please refer to attached read across justification document (Chapter 13).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Please refer to attached read across document (Chapter 13).

3. ANALOGUE APPROACH JUSTIFICATION
Please refer to attached read across justification document (Chapter 13).

4. DATA MATRIX
Please refer to attached read across justification document (Chapter 13).

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 5

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

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Please refer to attached read across justification document (Chapter 13).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Please refer to attached read across document (Chapter 13).

3. ANALOGUE APPROACH JUSTIFICATION
Please refer to attached read across justification document (Chapter 13).

4. DATA MATRIX
Please refer to attached read across justification document (Chapter 13).

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.

Reference 6

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

Justification for type of information:

Please refer to attached read across justification document (Chapter 13).

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

No classification

No adverse effects were detected in vitro in bacterial or mammalian cells.