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EC number: - | CAS number: -
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Ames test:
The mutagenicity of the test item was tested in four strains of Salmonella typhimurium (TAl00, TA1535, TA98, and TA1537) and one strain of Escherichia coli (WP2uvrA). The results showed that the test item did not increase the number of revertant colonies more than 2-fold compared to the negative control, in the presence or absence of metabolic activation, at all concentrations tested and for all strains used. The test item was concluded to be non-mutagenic for the five bacterial strains listed.
Chromosomal Aberration:
A study was performed to assess the ability of Dye-2019 to cause structural chromosome aberrations in human lymphocytes cultured in vitro.
Human lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin (PHA), and exposed to the test item both in the absence and presence of exogenous metabolic activation (S9 mix). Vehicle and positive control cultures were also included where appropriate. Two hours before the end of the incubation period, cell division was arrested using Colcemid®, the cells harvested and slides prepared, so that metaphase cells
could be examined for chromosomal damage.
The study consisted of a preliminary toxicity test and a main test. In both types of tests the cells were treated for 3 and 21 hours in the absence of S9 mix and for 3 hours in the presence of S9 mix. The mitotic index was assessed for all cultures to determine cytotoxicity. Based on the data from the preliminary toxicity test, test item concentrations were selected for the main test.
In the main test, justification for the highest analyzed concentration was based on the limit final concentration (2000 μg/mL) for this test system, where relatively no cytotoxicity was observed.
The following test item concentrations were selected for metaphase analysis:
In the absence of S9 mix, 3-hour treatment: 500, 1000 and 2000 μg/mL.
In the presence of S9 mix, 3-hour treatment: 500, 1000 and 2000 μg/mL.
In the absence of S9 mix, 21-hour treatment: 500, 1000 and 2000 μg/mL.
Under the conditions described above, Dye-2019 caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations (excluding gaps), at any analyzed concentration, when compared with the vehicle control. There was no evidence of a linear dose-concentration relationship. The mean proportion of cells with chromosomal aberrations (excluding gaps) for the vehicle and test item treated cultures were within the laboratory historical 95% confidence limits.
No statistically significant increases in the proportion of polyploid or endoreduplicated metaphase cells were observed during metaphase analysis, under any treatment condition, when compared with the vehicle control.
Both positive control compounds caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.
It was concluded that Dye-2019 has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.
Link to relevant study records
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- October 20 2020 to November 27 2020
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Specific details on test material used for the study:
- Not specified
- Target gene:
- Human lymphocytes.
- Species / strain / cell type:
- lymphocytes: Human
- Details on mammalian cell type (if applicable):
- Human lymphocytes have been used in this type of study for a number of years (Evans and
O'Riordan, 1975, Scott et al., 1990). They are cultured in vitro but do not divide unless
stimulated to do so. This is achieved by adding phytohaemagglutinin (PHA) to the culture
which results in a high mitotic yield (Nowell, 1960).
In this study, human blood with tissue culture medium was incubated in the presence of PHA
before being treated with the test item. Following treatment the cells were arrested at
metaphase using the mitotic inhibitor, Colcemid®. Chromosomes in these metaphase cells
were then examined for the presence of chromosome aberrations. The best estimate of the
aberration frequency is at the first cell division after initiation of treatment since certain types
of damage may be lost during subsequent cell divisions. The cultures were harvested after a
period of time which is approximately 1.5 times the cell cycle after initiation of treatment,
21 hours (Henderson et al., 1997). - Metabolic activation:
- with and without
- Metabolic activation system:
- Preparation of S9 Fraction
S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and
5,6-benzoflavone to stimulate mixed-function oxidases in the liver, was stored at -90
to -70°C.
Preparation of S9 Mix
S9 mix contained: S9 fraction (10% v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate
buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADP (4 mM). All the cofactors
were filter sterilized before use. - Test concentrations with justification for top dose:
- Preliminary toxicity test: 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL
Main test: 500, 1000, 2000 µg/mL
The osmolality and pH of Dye-2019 in medium were measured by analysing samples of
HML media, dosed at 10% (v/v), with either the vehicle (water) or a Dye-2019 formulation at
20 mg/mL (to give a final concentration of 2000 μg/mL). For medium dosed with Dye-2019
at 2000 μg/mL; no fluctuations in osmolality of the medium of more than 50 mOsmol/kg and
no fluctuations in pH of more than 1.0 unit were observed compared with the vehicle control.
The maximum final concentration tested in the preliminary toxicity test was 2000 μg/mL as
this is the standard limit concentration within this test system as recommended in the current
OECD Guideline 473 (2016). - Vehicle / solvent:
- Water
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- Culture of Lymphocytes
Human blood was collected from two healthy, non-smoking, adult (between 26-35 years of
age) donors, pooled (in equal volumes from each donor) and diluted with HML media. As
lymphocytes do not normally undergo cell division, they were stimulated to do so by the
addition of phytohaemagglutinin (PHA), a naturally occurring mitogen. Cultures were
established from the pooled sample and dispensed as 5 mL aliquots (in sterile universal
containers) so that each contained blood (0.4 mL), HML media (4.5 mL) and PHA solution
(0.1 mL). All cultures were then incubated at 34 to 39°C, and the cells re-suspended (twice
daily) by gentle inversion.
Media
The following media, obtained from a suitable supplier, was used:
HML media RPMI 1640, supplemented with 10% fetal calf serum, 0.2 IU/mL sodium
heparin, 20 IU/mL penicillin / 20 μg/mL streptomycin and 2.0 mM
L-glutamine.
Preliminary Toxicity Test
In all exposure conditions the highest concentration tested was 2000 μg/mL and no
precipitate was observed by eye at the end of treatment at 2000 μg/mL as assessed in
concurrently treated HML media-only cultures.
In the absence of S9 mix following 3-hour treatment, Dye-2019 caused no biologically
significant reduction in the mitotic index at 2000 μg/mL, the highest tested concentration,
when compared with the vehicle control.
In the presence of S9 mix following 3-hour treatment, Dye-2019 caused a reduction in the
mitotic index to 64% at 2000 μg/mL, the highest tested concentration, when compared with
the vehicle control.
In the absence of S9 mix following 21-hour treatment, Dye-2019 caused no biologically
significant reduction in the mitotic index at 2000 μg/mL, the highest tested concentration,
when compared with the vehicle control.
The concentrations used in the main test were based upon these data.
Main Test
In all treatment conditions the highest concentration tested was 2000 μg/mL since
precipitation did not limit the top concentration tested.
3-Hour Treatment in the Absence of S9 Mix
Toxicity Data
Dye-2019 caused no reduction in the mitotic index at 2000 μg/mL, compared with the mean
vehicle control value. The concentrations selected for metaphase analysis were 500, 1000
and 2000 μg/mL.
Metaphase Analysis
Dye-2019 caused no statistically significant increases in the proportion of cells with
chromosomal aberrations (excluding gaps) at any analyzed concentration, when compared
with the vehicle control. There was no evidence of a linear dose-concentration relationship.
All mean values (excluding gaps) for the vehicle control (water), and all Dye-2019 treatment
concentrations were within the laboratory historical 95% confidence limits.
The positive control compound, Mitomycin C, caused statistically significant increases
(p<0.001) in the proportion of aberrant cells and was within the laboratory historical 95%
confidence limits. This demonstrated the sensitivity of the test system.
3-Hour Treatment in the Presence of S9 Mix
Toxicity Data
Dye-2019 caused no reduction in the mitotic index at 2000 μg/mL, compared with the mean
vehicle control value. The concentrations selected for metaphase analysis were 500, 1000
and 2000 μg/mL.
Metaphase Analysis
Dye-2019 caused no statistically significant increases in the proportion of cells with
chromosomal aberrations (excluding gaps) at any analyzed concentration, when compared
with the vehicle control. There was no evidence of a linear dose-concentration relationship.
All mean values (excluding gaps) for the vehicle control (water), and all Dye-2019 treatment
concentrations were within the laboratory historical 95% confidence limits.
The positive control compound, Cyclophosphamide, caused statistically significant increases
(p<0.001) in the proportion of aberrant cells and was within the laboratory historical 95%
confidence limits. This demonstrated the efficacy of the S9 mix and the sensitivity of the test
system.
21-Hour Treatment in the Absence of S9 Mix
Toxicity Data
Dye-2019 caused no biologically significant reduction in the mitotic index at 2000 μg/mL,
compared with the mean vehicle control value. The concentrations selected for metaphase
analysis were 500, 1000 and 2000 μg/mL.
Metaphase Analysis
Dye-2019 caused no statistically significant increases in the proportion of cells with
chromosomal aberrations (excluding gaps) at any analyzed concentration, when compared
with the vehicle control. There was no evidence of a linear dose-concentration relationship.
All mean values (excluding gaps) for the vehicle control (water), and all Dye-2019 treatment
concentrations were within the laboratory historical 95% confidence limits.
The positive control compound, Mitomycin C, caused statistically significant increases
(p<0.001) in the proportion of aberrant cells and was within the laboratory historical 95%
confidence limits. This demonstrated the sensitivity of the test system.
Slide Preparation
The fixed pellets were re-suspended, then centrifuged at 500g for 5 minutes and re-suspended
in a small volume of fixative. A few drops of the cell suspensions were dropped onto precleaned
microscope slides and allowed to air dry. One slide was prepared per culture. The
slides were then stained in 10% Giemsa, prepared in buffered water (pH 6.8). After rinsing
in buffered water the slides were left to air-dry and coverslips mounted using DPX. The
remainder of the cell suspensions in fixative were stored at 2 to 8C until slide analysis was
completed.
Microscopic Examination
The prepared slides were examined by light microscopy and the incidence of mitotic cells per
1000 cells assessed. Slides were assessed for mitotic index (except when clear evidence of
overt toxicity was observed, or in cultures where there were no signs of cytotoxicity).
Main Test Procedure
The procedure for the main tests was the same as that for the preliminary tests, with the
following exceptions; positive control cultures were included for all tests, duplicate cultures
were prepared for each treatment level and two slides were prepared per culture.
3-Hour Treatment in the Absence of S9 Mix
Dye-2019 was added to each culture in 500 μL aliquots. Water was used as the vehicle
control. Mitomycin C was the positive control and was added to the appropriate cultures in
50 μL aliquots.
Following 3-hour treatment, cultures were centrifuged at 500g for 5 minutes and the
supernatant removed. Cultures were then re-suspended in saline and centrifuged at 500g for
5 minutes. The saline was then removed and the cell pellets re-suspended in fresh medium
(final volume of 5 mL). They were then incubated for 18 hours until the scheduled harvest
time.
3-Hour Treatment in the Presence of S9 Mix
Dye-2019 was added to each culture in 500 μL aliquots. Water was used as the vehicle
control. Cyclophosphamide was the positive control and was added to the appropriate
cultures in 50 μL aliquots.
Following 3-hour treatment, cultures were centrifuged at 500g for 5 minutes and the
supernatant removed. Cultures were then re-suspended in saline and centrifuged at 500g for
5 minutes. The saline was then removed and the cell pellets re-suspended in fresh medium
(final volume of 5 mL). They were then incubated for 18 hours until the scheduled harvest
time.
21-Hour Treatment in the Absence of S9 Mix
Dye-2019 was added to each culture in 500 μL aliquots. Water was used as the vehicle
control. Mitomycin C was the positive control and was added to the appropriate cultures in
50 μL aliquots.
Following the end of the treatment period the cultures were harvested.
Microscopic Examination
The prepared slides were examined by light microscopy using a low power objective. The
proportion of mitotic cells per 1000 cells in each culture was recorded (except for when clear
evidence of overt toxicity was observed, or in cultures where there were no signs of
cytotoxicity).
From these results the concentration causing a mitotic index of 45% 5% when compared
with the vehicle control value was the highest concentration selected for metaphase analysis.
Intermediate and low concentrations were also selected.
Where no significant increase in toxicity was observed (i.e. no significant reduction in mitotic
index), the maximum concentration tested (2000 μg/mL) was the highest concentration
selected for metaphase analysis. Lower concentrations were also selected.
The selected slides were then coded. Metaphase cells were identified using a low power
objective and examined at a magnification of x1000 using an oil immersion objective. From
each culture 150 metaphase figures were examined, however, this number was reduced in
cultures showing a high level of aberrant cells, where 15 cells with structural aberrations
(excluding gaps) were observed. Chromosome aberrations were scored according to the
classification of the ISCN (2009). Only cells with 44 - 48 chromosomes were analyzed. The
vernier readings of all aberrant metaphase figures were recorded. A peer review of the
metaphase analysis was performed by the analysis of 10 metaphases for the vehicle, highest
concentration selected and positive control for each exposure condition.
Traditionally gaps have been excluded from the quantitation of chromosome aberrations.
Some gaps, however, have been shown to be real discontinuities in DNA (Heddle and
Bodycote, 1970, Satya-Prakash et al., 1981). In this study the total number of cells
containing aberrations both with and without gaps has been calculated.
The incidence of polyploid and endoreduplicated cells (i.e. the ploidy status) were each
recorded as a percentage of the 150 metaphases analyzed per slide, independently from the
analysis for chromosome aberrations.
The number of aberrant metaphase cells in each test item group was compared with the
vehicle control value using the mid-p one-tailed Fisher exact test for an increase
(Richardson et al., 1989). Statistical significance was declared at 5%.
A Cochran-Armitage test for trend (Armitage, 1955) was applied to the control and all test
item groups. If this was significant at the 1% level, the test was reiterated excluding the
highest concentration group - this process continued until the trend test was no longer
significant.
The data was analyzed using the SAFEStat Chromosome Aberrations application.
Stability, Homogeneity and Formulation Analysis
The stability and homogeneity of the test item in the vehicle were not determined as part of
this study.
Analysis of achieved concentration was not performed as part of this study. - Evaluation criteria:
- Acceptance Criteria
The following criteria were applied for assessment of assay acceptability:
The concurrent vehicle control was considered acceptable for addition to the
laboratories historical vehicle control database (lie below or close to the upper control
limit). Where concurrent vehicle control data fell outside the 95% confidence limit it
may be acceptable for inclusion in the historical control distribution as long as the
data are not extreme outliers and there is evidence that the test system is ‘under
control’ and there is evidence of no technical or human failure.
Concurrent positive controls induced a response that were compatible with the
laboratories historical positive control database and produced statistically significant
increases compared with the concurrent vehicle control.
The criteria for selection of the top dose concentration were consistent with those
outlined previously.
Tests that did not fulfill the required criteria were rejected and therefore are not reported. - Statistics:
- No statistically significant increases in polyploid or endoreduplicated metaphases were
observed when compared with the vehicle control. - Key result
- Species / strain:
- lymphocytes: Human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- True negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Osmolality and pH Measurements
The osmolality and pH of Dye-2019 in medium were measured by analysing samples of
HML media, dosed at 10% (v/v), with either the vehicle (water) or a Dye-2019 formulation at
20 mg/mL (to give a final concentration of 2000 μg/mL). For medium dosed with Dye-2019
at 2000 μg/mL; no fluctuations in osmolality of the medium of more than 50 mOsmol/kg and
no fluctuations in pH of more than 1.0 unit were observed compared with the vehicle control.
The maximum final concentration tested in the preliminary toxicity test was 2000 μg/mL as
this is the standard limit concentration within this test system as recommended in the current
OECD Guideline 473 (2016).
Culture of Lymphocytes
Human blood was collected from two healthy, non-smoking, adult (between 26-35 years of
age) donors, pooled (in equal volumes from each donor) and diluted with HML media. As
lymphocytes do not normally undergo cell division, they were stimulated to do so by the
addition of phytohaemagglutinin (PHA), a naturally occurring mitogen. Cultures were
established from the pooled sample and dispensed as 5 mL aliquots (in sterile universal
containers) so that each contained blood (0.4 mL), HML media (4.5 mL) and PHA solution
(0.1 mL). All cultures were then incubated at 34 to 39°C, and the cells re-suspended (twice
daily) by gentle inversion.
Media
The following media, obtained from a suitable supplier, was used:
HML media RPMI 1640, supplemented with 10% fetal calf serum, 0.2 IU/mL sodium
heparin, 20 IU/mL penicillin / 20 μg/mL streptomycin and 2.0 mM
L-glutamine.
Preliminary Toxicity Test
In all exposure conditions the highest concentration tested was 2000 μg/mL and no
precipitate was observed by eye at the end of treatment at 2000 μg/mL as assessed in
concurrently treated HML media-only cultures.
In the absence of S9 mix following 3-hour treatment, Dye-2019 caused no biologically
significant reduction in the mitotic index at 2000 μg/mL, the highest tested concentration,
when compared with the vehicle control.
In the presence of S9 mix following 3-hour treatment, Dye-2019 caused a reduction in the
mitotic index to 64% at 2000 μg/mL, the highest tested concentration, when compared with
the vehicle control.
In the absence of S9 mix following 21-hour treatment, Dye-2019 caused no biologically
significant reduction in the mitotic index at 2000 μg/mL, the highest tested concentration,
when compared with the vehicle control.
The concentrations used in the main test were based upon these data.
Main Test
In all treatment conditions the highest concentration tested was 2000 μg/mL since
precipitation did not limit the top concentration tested.
3-Hour Treatment in the Absence of S9 Mix
Toxicity Data
Dye-2019 caused no reduction in the mitotic index at 2000 μg/mL, compared with the mean
vehicle control value. The concentrations selected for metaphase analysis were 500, 1000
and 2000 μg/mL.
Metaphase Analysis
Dye-2019 caused no statistically significant increases in the proportion of cells with
chromosomal aberrations (excluding gaps) at any analyzed concentration, when compared
with the vehicle control. There was no evidence of a linear dose-concentration relationship.
All mean values (excluding gaps) for the vehicle control (water), and all Dye-2019 treatment
concentrations were within the laboratory historical 95% confidence limits.
The positive control compound, Mitomycin C, caused statistically significant increases
(p<0.001) in the proportion of aberrant cells and was within the laboratory historical 95%
confidence limits. This demonstrated the sensitivity of the test system.
3-Hour Treatment in the Presence of S9 Mix
Toxicity Data
Dye-2019 caused no reduction in the mitotic index at 2000 μg/mL, compared with the mean
vehicle control value. The concentrations selected for metaphase analysis were 500, 1000
and 2000 μg/mL.
Metaphase Analysis
Dye-2019 caused no statistically significant increases in the proportion of cells with
chromosomal aberrations (excluding gaps) at any analyzed concentration, when compared
with the vehicle control. There was no evidence of a linear dose-concentration relationship.
All mean values (excluding gaps) for the vehicle control (water), and all Dye-2019 treatment
concentrations were within the laboratory historical 95% confidence limits.
The positive control compound, Cyclophosphamide, caused statistically significant increases
(p<0.001) in the proportion of aberrant cells and was within the laboratory historical 95%
confidence limits. This demonstrated the efficacy of the S9 mix and the sensitivity of the test
system.
21-Hour Treatment in the Absence of S9 Mix
Toxicity Data
Dye-2019 caused no biologically significant reduction in the mitotic index at 2000 μg/mL,
compared with the mean vehicle control value. The concentrations selected for metaphase
analysis were 500, 1000 and 2000 μg/mL.
Metaphase Analysis
Dye-2019 caused no statistically significant increases in the proportion of cells with
chromosomal aberrations (excluding gaps) at any analyzed concentration, when compared
with the vehicle control. There was no evidence of a linear dose-concentration relationship.
All mean values (excluding gaps) for the vehicle control (water), and all Dye-2019 treatment
concentrations were within the laboratory historical 95% confidence limits.
The positive control compound, Mitomycin C, caused statistically significant increases
(p<0.001) in the proportion of aberrant cells and was within the laboratory historical 95%
confidence limits. This demonstrated the sensitivity of the test system. - Conclusions:
- Dye-2019 has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system
- Executive summary:
A study was performed to assess the ability of Dye-2019 to cause structural chromosome aberrations in human lymphocytes cultured in vitro.
Human lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin (PHA), and exposed to the test item both in the absence and presence of exogenous metabolic activation (S9 mix). Vehicle and positive control cultures were also included where appropriate. Two hours before the end of the incubation period, cell division was arrested using Colcemid®, the cells harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage.
The study consisted of a preliminary toxicity test and a main test. In both types of tests the cells were treated for 3 and 21 hours in the absence of S9 mix and for 3 hours in the presence of S9 mix. The mitotic index was assessed for all cultures to determine cytotoxicity. Based on the data from the preliminary toxicity test, test item concentrations were selected for the main test.
In the main test, justification for the highest analyzed concentration was based on the limit final concentration (2000 μg/mL) for this test system, where relatively no cytotoxicity was observed.
The following test item concentrations were selected for metaphase analysis:
In the absence of S9 mix, 3-hour treatment: 500, 1000 and 2000 μg/mL.
In the presence of S9 mix, 3-hour treatment: 500, 1000 and 2000 μg/mL.
In the absence of S9 mix, 21-hour treatment: 500, 1000 and 2000 μg/mL.
Under the conditions described above, Dye-2019 caused no statistically significant increases
in the proportion of metaphase figures containing chromosomal aberrations (excluding gaps),
at any analyzed concentration, when compared with the vehicle control. There was no evidence of a linear dose-concentration relationship. The mean proportion of cells with chromosomal aberrations (excluding gaps) for the vehicle and test item treated cultures were within the laboratory historical 95% confidence limits.
No statistically significant increases in the proportion of polyploid or endoreduplicated metaphase cells were observed during metaphase analysis, under any treatment condition, when compared with the vehicle control.
Both positive control compounds caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.
It was concluded that Dye-2019 has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- September 9 2020 to October 16 2020
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- Not specified
- Target gene:
- Histidine and tryptophan.
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9mix was prepared immediately before use and contained 0.1 ml of the S9 fraction prepared from Sprague-Dawley rat liver (all males, 7 weeks of age, and 201-241 g of body weight) intraperitoneally administered with phenobarbital (PB) and 5,6-benzoflavone (BF)* for enzyme induction (IEDA TRADING CORPORATION, Lot No. RAA202005A, manufactured on May 29, 2020, purchased on July 28, 2020, stored at or below -80°C), 8 µmol of MgC12, 33 µmol of KCl, 5 µmol of glucose-6-phosphate, 4 µmol of NADPH, 4 µmol of NADH, and 100 µmol of Na-phosphate buffer in 1 ml of final solution.
- Test concentrations with justification for top dose:
- Prior to the main test, we conducted a dose-finding test for each strain using the test item at concentrations of 62, 185, 556, 1667, and 5000 µg/plate. The results showed that the test item did not increase the number of revertant colonies more than 2-fold compared to the negative control, in the presence or absence of metabolic activation, at all concentrations tested and for all the strains used.
It should be noted that the test item did not show any growth inhibition or precipitation at any of the doses tested, regardless of metabolic activation or strain used.
Based on these results, we selected a series of 5 concentrations comprising the highest concentration of 5000 µg/plate and lower concentrations with a common ration of 2 (313, 625, 1250, 2500 and 5000 µg/plate)as test item doses for the main study. - Vehicle / solvent:
- Because the test item is soluble in water and stable in solution concentrations below 50 mg/ml
(maximum test concentration), water was used as the solvent. - Negative solvent / vehicle controls:
- yes
- Remarks:
- Water
- Positive controls:
- yes
- Remarks:
- DMSO
- Positive control substance:
- 9-aminoacridine
- sodium azide
- benzo(a)pyrene
- other: 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide, 2-Aminoanthracene
- Details on test system and experimental conditions:
- Pre-culture of bacterial strains
The bacterial suspension (composition ratio: bacterial suspension / DMSO = 8.0/0. 7) was stored at -80°C or less. The pre-culture medium was inoculated 40 µl of bacterial suspension into an Erlenmeyer flask (capacity, 100 ml) containing 20 ml of the pre-culture medium (NUTRIENT BROTH No. 2 OXOID LTD; Lot 2300633). It was stored at 7°C for 8 h and 20 min for the dose finding test, 9 h and 10 min for the main test, and the temperature was then raised to 37°C over a period of 1 h and incubation with shaking (reciprocal shaking, 130 times/min) was perfom1ed for 8 hat 37°C.
The pre-cultured bacterial suspensions were stored in an ice bath during 18 min for the dosefinding test and 12 min for the main test, and used within 4 h.
Viable cells in the pre-cultured bacterial suspension were confirmed to be at least 1 x 109/ml by using a spectrophotometer (O.D. at 660 nm)
Test method
The test was conducted using the pre-incubation method and following the procedure described below.
A 0.1 ml solution of the test item was added to glass test tube (NIPRO CORPORATION) sterilized by a dry-heat. Subsequently, we added 0.5 ml of 0.1 M Na-phosphate buffer (for samples without metabolic activation) or S9mix (for samples with metabolic activation) and 0.1 ml of the pre-cultured bacterial suspension. Thereafter, the mixture was pre-incubated at 37°C for 20 min with reciprocal shaking at a rate of 100 times/min at a constant temperature (Thermo Minder
EX/Personal 11, TAITEC CORPORATION). After adding 2 ml of top agar and stirring well, the mixture was poured onto a minimal glucose agar plate and uniformly spread. After confirming that the medium surface had solidified, the plate containing the mixture was incubated at 37°C for 48 h (in the main test it was also subsequently stored at 4°C for 22 h). In the negative control test, solvent was used instead of the test item solution and tested identically. In the positive control test, the positive control substances were used instead of the test item solution and processed and tested identically. Three plates were used per a dose in all tests. - Evaluation criteria:
- The test item is considered positive totally when the mean number of revertant colonies counted increase in a dose-dependent manner or show reproducibility.
- Statistics:
- Not applicable.
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- True negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- The mutagenicity of the test item was tested in four strains of Salmonella typhimurium
(TAl00, TA1535, TA98, and TA1537) and one strain of Escherichia coli (WP2uvrA). The results showed that the test item did not increase the number of revertant colonies more than 2-fold compared to the negative control, in the presence or absence of metabolic activation, at all concentrations tested and for all strains used. - Conclusions:
- The test item was concluded to be non-mutagenic for the five bacterial strains used.
- Executive summary:
Bacterial reverse mutation test was carried out in accordance with OECD test guideline 471, in compliance with GLP. A dose finding test was performed at 5 doses of 5,000 µg/plate (highest dose) with a common ratio of 3. The main test was performed at 5 doses of 5,000 µg/plate (highest dose) with a common ratio of 2.
The mutagenicity of the test item was tested in four strains of Salmonella typhimurium
(TAl00, TA1535, TA98, and TA1537) and one strain of Escherichia coli (WP2uvrA), both with and without metabolic activation using the S9 mix, comprising of S9 microsomal fraction prepared from rats induced with phenobarbital (PB) injection of 5,6-benzoflavone (BF) ( concentration of S9 in S9mix: 10% v/v).
The test method used was the pre-incubation method, which was performed at 37°C for 20 min., reciprocal shaking of 100 times/min, followed by incubation at 3 7°C for 48 h.
The results showed that the test item did not increase the number of revertant colonies more than 2-fold compared to the negative control, in the presence or absence of metabolic activation, at all concentrations tested and for all strains used. The test item was concluded to be non-mutagenic for the five bacterial strains listed.
Referenceopen allclose all
Summary of mutagenicity test
(Dose-finding test)
Test Period | From: 16 Sep 2020 | To: 18 Sep 2020 | ||||
|
| Number of revertant (colonies/plate) [(mean)/SD] | ||||
Base pair substituted type | Frame shift type | |||||
TA 100 | TA1535 | WP2uvrA | TA98 | TA1537 | ||
S9 mix (-) | Negative control (water) | 142 142 (151) 169 15.6 | 10 11 (11) 12 1.0 | 44 38 (39) 35 4.6 | 33 20 (29) 33 7.5 | 11 10 (9) 7 2.1 |
62 | 150 171 (162) 166 11.0 | 10 13 (11) 11 1.5 | 32 41 (37) 37 4.5 | 23 18 (19) 16 3.6 | 11 23 (14) 8 7.9 | |
185 | 135 150 (141) 138 7.9 | 7 15 (10) 7 4.6 | 30 34 (32) 32 2.0 | 24 19 (20) 16 4.0 | 10 8 (12) 18 5.3 | |
556 | 176 142 (154) 145 18.8 | 9 7 (8) 8 1.0 | 39 33 (35) 34 3.2 | 19 12 (15) 15 3.5 | 14 7 (9) 7 4.0 | |
1667 | 130 128 (126) 120 5.3 | 14 8 (9) 6 4.2 | 49 42 (44) 41 4.4 | 25 13 (20) 23 6.4 | 6 16 (10) 9 5.1 | |
5000 | 122 127 (121) 113 7.1 | 8 14 (11) 12 3.1 | 29 30 (35) 45 9.0 | 25 17 (24) 29 6.1 | 10 15 (14) 16 3.2 | |
S9 mix (+) | Negative control (water) | 182 137 (151) 135 7.0 | 7 11 (10) 11 2.3 | 45 33 (38) 37 6.1 | 24 42 (30) 24 10.4 | 12 23 (16) 14 5.9 |
62 | 213 202 (207) 206 | 9 9 (8) 7 1.2 | 33 32 (38) 48 9.0 | 48 41 (42) 38 5.1 | 19 14 (16) 14 2.9 | |
185 | 176 182 (185) 196 10.3 | 10 10 (10) 11 0.6 | 34 39 (37) 37 2.5 | 30 22 (31) 42 10.1 | 12 14 (12) 9 2.5 | |
556 | 159 151 (152) 145 7.0 | 11 13 (10) 7 3.1 | 37 30 (39) 49 9.6 | 34 24 (29) 30 5.0 | 20 15 (15) 11 4.5 | |
1667 | 131 126 (127) 125 3.2 | 11 9 (12) 15 3.1 | 43 44 (41) 37 3.8 | 24 30 (28) 31 3.8 | 16 14 (15) 16 1.2 | |
5000 | 140 136 (140) 145 4.5 | 10 12 (10) 7 2.5 | 36 32 (36) 39 3.5 | 30 27 (27) 25 2.5 | 21 10 (16) 18 5.7 | |
Positive control S9 mix (-) | Name | AF-2 | NaN3 | AF-2 | AF-2 | 9-AA |
Concentration (µg/plate) | 0.01 | 0.5 | 0.01 | 0.1 | 80 | |
Number of Revertant (colonies/plate) | 313 327 (330) 351 19.2 | 298 275 (301) 331 28.1 | 105 115 (114) 123 9.0 | 475 519 (493) 485 23.1 | 819 740 (784) 794 40.4 | |
Positive control S9 mix (+) | Name | B[a]P | 2-AA | 2-AA | B[a]P | 2-AA |
Concentration (µg/plate) | 5 | 2 | 10 | 5 | 2 | |
Number of Revertant (colonies/plate) | 1462 1517 (1432) 1317 103.3 | 129 156 (140) 135 14.2 | 848 898 (891) 927 40.0 | 594 547 (591) 632 42.6 | 182 169 (179) 187 9.3 |
Abbreviations •: Inhibition of growth of the bacteria
P: Precipitation
( ): Mean values
AF-2:2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide
NaN3:Sodium azide
9-AA:9-Aminoacridine hydrochloride hydrate
2-AA:2-Aminoanthracene
B[a]P: Benzo[a]pyrene
Summary of mutagenicity test
Main test
Test Period | From: 25 Sep 2020 | To: 28 Sep 2020 | ||||
|
| Number of revertant (colonies/plate) [(mean)/SD] | ||||
Base pair substituted type | Frame shift type | |||||
TA 100 | TA1535 | WP2uvrA | TA98 | TA1537 | ||
S9 mix (-) | Negative control (water) | 150 188 (166) 159 19.9 | 10 8 (11) 13 2.5 | 32 22 (31) 38 8.1 | 13 11 (15) 20 4.7 | 20 11 (15) 7 4.5 |
313 | 161 167 (153) 131 19.3 | 13 10 (12) 12 1.5 | 37 38 (32) 22 9.0 | 19 12 (17) 19 4.0 | 18 18 (19) 20 1.2 | |
625 | 165 140 (153) 154 12.5 | 7 8 (7) 7 0.6 | 16 30 (23) 22 7.0 | 8 12 (14) 22 7.2 | 12 10 (13) 18 4.2 | |
1250 | 144 148 (145) 144 2.3 | 7 8 (9) 7 2.1 | 33 35 (29) 20 3.2 | 11 9 (11) 14 2.5 | 13 11 (12) 11 1.2 | |
2500 | 169 148 (158) 156 10.6 | 9 13 (12) 14 2.6 | 39 36 (33) 23 8.5 | 25 17 (22) 24 4.4 | 17 9 (16) 22 6.6 | |
5000 | 144 138 (140) 137 3.8 | 11 11 (10) 8 1.7 | 28 30 (32) 38 5.3 | 16 20 (16) 12 4.0 | 14 15 (16) 19 2.6 | |
S9 mix (+) | Negative control (water) | 192 184 (185) 179 6.6 | 8 10 (9) 8 1.7 | 24 35 (36) 49 12.5 | 20 35 (32) 42 11.2 | 30 16 (23) 24 7.0 |
313 | 167 171 (168) 167 | 9 11 (9) 7 2.0 | 46 23 (34) 34 11.5 | 24 37 (27) 19 9.3 | 35 23 (27) 22 7.0 | |
625 | 187 171 (167) 142 22.8 | 8 9 (8) 8 0.6 | 37 19 (29) 32 9.3 | 24 24 (24) 23 0.6 | 23 19 (20) 18 2.6 | |
1250 | 158 187 (156) 124 31.5 | 10 8 (10) 11 1.5 | 34 34 (35) 38 2.3 | 30 32 (27) 19 7.0 | 20 18 (17) 13 3.6 | |
2500 | 142 153 (154) 167 12.5 | 10 12 (11) 10 1.2 | 39 26 (30) 25 7.8 | 26 29 (23) 19 7.0 | 20 26 (23) 22 3.1 | |
5000 | 146 158 (157) 167 10.5 | 16 10 (12) 11 3.2 | 37 48 (44) 47 6.1 | 24 12 (19) 20 6.1 | 26 23 (24) 22 2.1 | |
Positive control S9 mix (-) | Name | AF-2 | NaN3 | AF-2 | AF-2 | 9-AA |
Concentration (µg/plate) | 0.01 | 0.5 | 0.01 | 0.1 | 80 | |
Number of Revertant (colonies/plate) | 427 417 (431) 449 16.4 | 453 441 (448) 450 6.2 | 102 115 (115) 129 13.5 | 447 491 (488) 525 39.1 | 949 983 (909) 794 100.7 | |
Positive control S9 mix (+) | Name | B[a]P | 2-AA | 2-AA | B[a]P | 2-AA |
Concentration (µg/plate) | 5 | 2 | 10 | 5 | 2 | |
Number of Revertant (colonies/plate) | 1726 1508 (1607) 1587 110.4 | 302 281 (278) 252 25.1 | 743 934 (818) 776 102.1 | 652 728 (716) 769 59.4 | 162 223 (179) 151 38.8 |
Abbreviations •: Inhibition of growth of the bacteria
P: Precipitation
( ): Mean values
AF-2:2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide
NaN3:Sodium azide
9-AA:9-Aminoacridine hydrochloride hydrate
2-AA:2-Aminoanthracene
B[a]P: Benzo[a]pyrene
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
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