[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Deviations:

no

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

copper(II) 3,3',3'',3'''-(29H,31H-phthalocyanine-2(or 3),9(or 10),16(or 17),23(or 24)-tetrayltetrasulfonyl)tetrapropane-1-sulfonate and its amides with (2-hydroxypropyl)amine, sodium salts

Molecular formula:

Cannot be adequately assigned

IUPAC Name:

copper(II) 3,3',3'',3'''-(29H,31H-phthalocyanine-2(or 3),9(or 10),16(or 17),23(or 24)-tetrayltetrasulfonyl)tetrapropane-1-sulfonate and its amides with (2-hydroxypropyl)amine, sodium salts

Test material form:

solid: particulate/powder

Details on test material:

Molecular formula: C50H52CuN10Na2O20S8 (main component)
Molecular weight: 1479.02 (main component)
Purity of test item: 93.1%
Impurity
Water: 6.5%
EDTA-4Na: 0.4%
Supplier: FUJIFILM Corporation
Lot number: M-002P
Storage conditions: Store in the dark at room temperature.

Specific details on test material used for the study:

Not specified

Method

Target gene:

Human lymphocytes.

Species / strain

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

Controls

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 8C 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.

Results and discussion

Test results

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.

Applicant's summary and conclusion

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.