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The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenic properties of test substance was investigated in a bacterial reverse mutation assay (Ames test; test strains used: S. typhimurium TA1535, TA1537, TA98, TA100 and E,coli WP2P and WP2P uvrA), in an in vitro gene mutation study. Negative results were obtained with and without metabolic activation.
A reliable study on the induction of chromosome aberration in mammalian cells in vitro is available for test substance, which gave negative results with and without metabolic activation.

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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:
supporting study
Reliability:
2 (reliable with restrictions)
Qualifier:
according to guideline
Guideline:
other: Annex V - method B.14
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
Species / strain / cell type:
E. coli, other: WP2P and WP2P uvrA
Metabolic activation:
with and without
Metabolic activation system:
Aroclor - induced rat liver S9
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 100 ... 5000 µg/plateConcentration range in the main test (without metabolic activation): 100 ... 5000 µg/plate
Vehicle / solvent:
Solvent: Dimethylsulphoxide
Details on test system and experimental conditions:
Concentration of the test substance resulting in precipitation: 500 µg/plate
Species / strain:
other: as specified above
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Additional information on results:
Observations:The substance did not induce any significant, reproducibleincreases in the observed number of revertent colonies inany strain either in the precense or absence of metabolicactivation
Remarks on result:
other: other: Main test
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):negative with metabolic activationnegative without metabolic activationUnder the conditions of this assay, the substance gave a negative, ie non-mutagenic, response with S.typhimutium strains TA1535, TA1537, TA98 and TA100 and with E.Coli strains WP2P and WP2P uvrA in presence and absence of an auxiliry metabolising system (S9)
Executive summary:

The substance has been evaluated in bacterial mutagenicity assay using four strains of salmonella typhimurium and two strains of escherichia coli following protocols complying with OECD guideline number 471 and 472 and with the UK department of Health guidelines.

In two separate experiments, the compound did not induce any significant, reproducible increases in the observed numbers af revertant colonies in any of the tester strains used, either in the presence or absence of an auxiliary metabolising system (S9)

In each experiment, the positive controles responded as expected, indicating that the assay was performing satisfactorily.

Under the conditions of this assay, the substance gave a negative, ie non-mutagenic, response with S.typhimutium strains TA1535, TA1537, TA98 and TA100 and with E.Coli strains WP2P and WP2P uvrA in presence and absence of an auxiliry metabolising system S9.

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
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 473 (1997) ICH Tripartite Harmonised Guideline on Genotoxicity: Specific Aspect of Regulatory Tests (1995) EPA Health Effects Test Guidelines Oppts 870.5375 (1998)
GLP compliance:
yes
Type of assay:
other: in vitro mammalian cytogenicity (B10)
Species / strain / cell type:
mammalian cell line, other: Chinese hamster ovary (CHO) cells
Metabolic activation system:
The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was prepared from male Sprague Dawley rats induced with Aroclor 1254 and obtained from Molecular Toxicology Incorporated, USA.
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 52.77 ... 1200 µg/mlConcentration range in the main test (with metabolic activation): 35.18 ... 800 µg/mlConcentration range in the main test (without metabolic activation): 52.77 ... 1200 µg/mlConcentration range in the main test (without metabolic activation): 8.798 ... 250 µg/ml
Vehicle / solvent:
McCoy's 5A
Details on test system and experimental conditions:
Exposure period (with metabolic activation): 3 hoursExposure period (without metabolic activation): 3 hoursExpression time:Two types of experiments was conducted.Cultures received pulse treatments (both in the absence andpresence of S-9) for 3 hours only. They were then washed andincubated for further 17 hours before harvesting.Or cultures treated for 20 hours before harvesting.Selection time:Approximately 2 hours prior to harvest, colchicine was addedto give a final concentration of approximately 1 µg/ml toarrest dividing cells in metaphase.Fixation time:Cells was keept in fixative in the refrigerator beforeslides were prepared but slides were not made on the day ofthe harvest to ensure cells were adequately fixed.
Key result
Species / strain:
other: as specified above
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Additional information on results:
Observations:It is concluded that Hypersol Synergist L 4722 did not induce chromosome aberrations in cultured Chinese hamsterovary (CHO) cells when tested to it's limit of cytotoxicityin both absence and presence of metabolic activation (S-9).
Remarks on result:
other: other: main test
Remarks:
Migrated from field 'Test system'.
Conclusions:
It is concluded that Hypersol Synergist L 4722 did not induce chromosome aberrations in cultured Chinese hamster ovary (CHO) cells when tested to it's limit of cytotoxicity in both absence and presence of metabolic activation (S-9).
Executive summary:

Hypersol Synergist L4722 was tested in an in vitro cytogenetics assay using duplicate cultures of chinese hamster ovary (CHO) cells in two independent experiments. Treatments covering a broard range of doses, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S9). The test article was formulated as a suspension in McCoy's 5A culture medium (McCoy's 5A) and the highest dose level used, 1200 µg/ml, was determined following a preliminary toxicity (slide quality assessment) trial.

In experiment 1, treatment in absence and presence of S-9 was for 3 hours followed by a 17 -hour recovery period prior to harvest (3 -17). the S-9 used was prepared from a rat liver post-mitochondrial fraction (S-9) from Aroclor 1254 induced animals. The test article dose levels for chromosome analysis were selected by evaluating the effect of t Hypersol Synergist L4722 on relative cell number. Chromosome aberrations were analysed at three dose levels. The highest concentration chosen for analysis 959.9 µg/ml in absence of S-9 and 491.5 µg/ml in the presence of S-9, induced approximately 47% and 49% reduction in cell number in the absence and presence of S-9 respectively.

In experiment 2, treatment in the absence of S-9 was continuous for 20 hours. Treatment in the prescence of S-9 was for 3 hours only followed by 17 hour recovery period prior to harvest (3 -17). Chromosome aberrations were analysed at three dose levels. The highest concentration chosen for analysis, 52.44 µg/ml in absence of S-9 and 409.6 µg/ml in the presence of S-9, induced approximately 51% and 53% reduction in cell number in the absence ond presence of S-9 respectively.

Appropriate negative (solvent) control cultures were included in the test system in both experiments under each treatment condition. The proportion of cells with structural aberrations in these cultures fell within historical solvent control ranges. 4 -nitroquinoline-1 -oxide and cyclophosphamide were employed as positive control chemicals in the absence and presence of liver S-9 respectively. Cells receiving these were sampled in each experiment, 20 hours after the start of treatment; both compounds induced statistically significant increases in the proportion of cells with structural aberrations.

Treatment of cultures with t Hypersol Synergist L4722 in abcence and the presence of S-9 (both experiments) resulted in frequencies of cells with aberrations witch were similar to those seen in concurrent vehicle control cultures. The aberrant cell frequency of all Hypersol Synergist L4722 treated cultures fell within historical negative control (normal) ranges.

It is concluded that Hypersol Synergist L 4722 did not induce chromosome aberrations in cultured Chinese hamster ovary (CHO) cells when tested to it's limit of cytotoxicity in both absence and presence of metabolic activation (S-9).

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:
other: Acceptable, sufficiently documented publication which meets basic scientific principles
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
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
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
Remarks:
Migrated from field 'Test system'.

- 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 (migrated information):
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:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
year of publication: 1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented publication, which meets basic scientific principles
Principles of method if other than guideline:
Bacterial reverse mutation assay (Ames test)
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.
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
Remarks:
Migrated from field 'Test system'.

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 (migrated information):
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 gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From 14 JUL 2006 to 25 JUL 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD TG 471), with Prival modification for azo-dyes
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
in accordance with German Chemikaliengesetz and Directive 88/320/EEC
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:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 (experiment I); hamster liver S9 (experiment II)
Test concentrations with justification for top dose:
Experiment I: 3, 10, 33, 100, 333, 1000, 2500, 5000 µg/plate
Experiment II: 33, 100, 333, 1000, 2500, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility properties of the solvent and its relative non-toxicity to the bacteria
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide (TA 1535 and TA 100), 4-Nitro-o-phenylene-diamine (TA 1537 and TA 98), methyl methane sulfonate (WP2 uvrA)
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 1535, TA 100, TA 1537, WP2 uvrA), congo red (TA 98)
Remarks:
with metabolic activation (hamster liver S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Experiment I: plate incorporation assay with induced rat liver S9 mix (induction with phenobarbital/ß-naphthoflavone)
Experiment II: preincubation assay with non-induced hamster liver S9 mix

DURATION
- Preincubation period: Experiment II: 30° C for 30 up to 35 (strains TA 98, TA 100, WP2 uvrA) minutes
- Exposure duration: at least 48 hours

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

Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, WP2 uvrA) or thrice (strains TA 1535, TA 1537) the colony count of the corresponding solvent colony is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the thresshold is regarded as an indication of mutagenic potential if reproduced in an independent second experiment. however, whenever the colony counts remain within the historical range of negative annd solvent controls such as an increase is not considered biologically relevant.
Statistics:
Arithmetic means and standard deviation of the counted colonies were calculated.
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:
E. coli WP2 uvr A
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: of the test item occured at concentrations of 1000 µg/plate and more. Nevertheless the colonies could be counted manually.

COMPARISON WITH HISTORICAL CONTROL DATA: there were no biologically relevant deviations
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:
Interpretation of results (migrated information):
negative

The test item did not exert mutagenic activity in the reverse bacterial mutation assay (plate incorporation assay and pre-incubation 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 and TA100 as well as Escherichia coli strain WP2 uvrA with (induced rat liver S9 mix) and without metabolic activation at concentrations of 3, 10, 33, 100, 333, 1000, 2500 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 33, 100, 333, 1000, 2500 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 conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Possible mutagenic potential of test substance has been investigated in several reliable tests in vitro (bacterial reverse mutation assays (tests) and chromosome aberration tests in mammalian cells. All tests consistently yielded negative results indicating that the test substance has no mutagenic potential. Besides these negative findings on mutagenicity the test substance is unlikely to be bioavailable after oral, dermal and inhalation exposure and therefore has not to be classified as germ cell mutagen.

Justification for classification or non-classification

Due to the negative findings with test substance in the mutagenicity assays in vitro and in consideration that it is unlikely to be bioavailable after oral, dermal and inhalation exposure the test substance has not to be classified as germ cell mutagen accordingto Regulation (EC) No 1272/2008 and Council Directive 67/548/EEC.