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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

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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.

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:
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.

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.

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.

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

Additional information

Justification for classification or non-classification

No classification

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