3-(2,4-bis(4-((5-(4,6-bis(2-aminopropylamino)-1,3,5-triazin-2-ylamino)-4-hydroxy-2,7-disulfonaphthalen-3-yl)azo)phenylamino)-1,3,5-triazin-6-ylamino)propyldiethylammonium lactate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

AMES test

The test was performed according to OECD Guideline 471 (Bacterial Reverse Mutation Assay). Under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Chromosomal aberration

The test was carried out according to OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test). During the in vitro chromosome aberration study in mammalian cells the substance did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line). Therefore, the test substance is classified to be non-mutagenic in this chromosome aberration study.

Mammalian cell gene mutation

The test item was tested in a Mammalian Cell Gene Mutation Test (HPRT test) in CHO-K1 cells, according to OECD Guideline 476 (updated 2016).
The test item tested up to the maximum recommended concentration (2000 µg/mL) without and with metabolic activation system over a 5-hour treatment period did not induce statistically and biologically significant increases in mutant frequency compared to the solvent control.

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 20th to July 5th, 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

May 26th, 1983

Deviations:

yes

Remarks:

Salmonella Typhimurium Reverse Mutation Assay Modified Version for Azo-Dyes

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

Test I (plate incorporation test): rat liver S9 mix
Test II (pre-incubation test): hamster liver S9 mix

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 33.3, 100.0, 333.3, 1000.0, 2500.0, 5000.0 μg/plate
Concentration range in the main test (without metabolic activation): 33.3, 100.0, 333.3, 1000.0, 2500.0, 5000.0 μg/plate

Vehicle / solvent:

Bi-distilled water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-o-phenylene-diamine

Remarks:

without 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

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:

congo red

other: 2-aminoanthracene

Remarks:

with metabolic activation - hamster liver S9 mix

Details on test system and experimental conditions:

According to the results of the pre-experiment, the concentrations applied in the main experiment were chosen. The maximum concentration was 5000.0 μg/plate. According to the dose selection criteria the test article was tested at the following concentrations: 33.3, 100.0, 333.3, 1000.0, 2500.0, 5000.0 μg/plate. For each strain and dose level, including the controls, three plates were used. The following materials were mixed in a test tube and poured into the selective agar plates:
- 100 μl test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control).
- 500 μl S9 mix (for the test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation).
- 100 μl bacteria suspension.

Rationale for test conditions:

A pre-experiment on toxicity was run on TA 98 and TA 100. The plates incubated with the test article showed normal background growth up to 5000.0 μg/plate with and without S9 mix in all strains used.

Evaluation criteria:

A test article is considered mutagenic if in strains TA 98 and TA 100 the number of reversions is at least twice higher and in strains TA 1535 and TA 1537 at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.

Species / strain:

S. typhimurium TA 1535

Remarks:

exp. I and II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Remarks:

exp. I and II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Remarks:

exp. I and II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Remarks:

exp. I and II

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No toxic effects, evident as a reduction in the number of revertants, occured in the test groups with and without metabolic activation up to the highest investigated dose. An isolated and moderate reduction of revertant colonies occured in the strain TA 98 in the absence of metabolic activation at 2500 μg/plate in the second experiment. This effect was considered as a fluctuation of the frequency of spontaneous reversions rather than a true toxic effect. The reduction was not observed at any other concentration of the test article and did not occur in the first experiment.
The plates incubated with the test article showed normal background growths up to 5000.0 μg/plate with and without S9 mix in all strains used.
No substantial increase in revertant colony numbers of any of the four tester strains were observed following treatment with the test substance at any concentration level, neither in the presence nor absence of metabolic activation mix. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagen were used as positive controls. They showed a distinct increase in induced revertant colonies.

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 01st to September 30th, 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

Hprt and xprt

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Sub-line (K1) of Chinese hamster ovary cell line CHO, provided by ECACC

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Components of Media
Name: Ham's F12 medium
Supplier: BIOWEST SAS France
Lot Number: MS00O8, MS00VK
Expiry date: September 29, 2021, March 18, 2022
Storage condition: At 2-8 °C
Name: Fetal Bovine Serum
Batch Number: BCCC8749
Supplier: Sigma-Aldrich Chemie GmbH Germany
Expiry date: December 2024
Appearance: liquid
Storage condition: In freezer at (-20 °C)
Name: Antibiotic-Antimycotic
Batch Number: 0000080388
Appearance: suspension
Supplier: Sigma-Aldrich Chemie GmbH Germany
Expiry dates: December 2021
Storage condition: In freezer at (-20 °C)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Trinova Biochem GmbH (Rathenau Strasse 2; D-35394 Giessen, Germany;
Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA)
- method of preparation of S9 mix: The complete S9 Mix was freshly prepared containing components with the following ratios: HEPES 20 mM, KCl 330 mM, MgCl2 50 mM, NADP 40 mM, D-Glucose-6-phosphate (Monosodium salt) 50 mM
- concentration or volume of S9 mix and S9 in the final culture medium: 0.3 mL/mL

Test concentrations with justification for top dose:

Based on the result of the preliminary cytotoxicity assay, the concentration levels for the performed Mutation Assay were chosen according to the maximum recommended for lower- cytotoxic substances in OECD Guideline 476 (updated 2016): 125, 250, 500, 1000 and 2000 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Aqua ad injectabilia

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 2 replicates with s9-mix and 2 replicates without s9-mix

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 5 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: relative increase in cell count (RICC)
- Any supplementary information relevant to cytotoxicity: A non GLP Pre-test on Toxicity was performed between August 10-16, 2021. Treatment concentrations for the mutation assay were selected on the basis of the result of Pre-test on Toxicity. During the Pre-test on Toxicity (cytotoxicity assay), the cultures (more than 50 % confluent) were trypsinised and cell suspensions were prepared in Ham's F12-10 medium. Cells were seeded into dishes (diameter is approx. 150 mm) at 10 x106 cells each and incubated with culture medium. After 24 hours the cells were treated with the suitable concentrations of the test item in absence or in presence of S9 mix (40 µL/mL) and incubated at 37 °C for 5 hours. At the treatment the cultures were washed three times with Ham's F12-5 medium and the cells were covered with trypsin-EDTA solution. In the control and test item-treated cultures the cells were counted using a Bürker chamber. Measurement of CE after treatment, the cell concentration was adjusted to 40 cells/mL with Ham's F12-10 medium. For each concentration of test solution or control solution, 5 mL was plated in parallel into 3 sterile dishes (diameter is approx. 60 mm). The dishes were incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air for 6 days for colony growing. Colonies were then fixed with methanol and stained with Giemsa and counted. Additionally, 4 cultures (10 x106 cells/dish) were set up for determining the initial cell count. Cytotoxicity was determined by RS (relative survival), where the cloning efficiency (CE) of cells plated immediately after treatment adjusted by any loss of cells during treatment as compared with adjusted cloning efficiency in negative controls (assigned a survival of 100%). Precipitation of the test item in the final culture medium was visually examined at beginning and end of the treatments. In addition, pH and osmolality was considered for dose level selection. The results of Pre-test on cell toxicity were used for dose selection of the test item concentrations used in the Main Mutation Assay. Five concentrations were selected for the treatment without and with metabolic activation system, respectively.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The Main Mutation Assay was performed in the presence and in the absence of S9 mix. For the 5-hour treatment, approximately 10 x106 cells/dish was used and incubated for 24 hours before treatment at 37 °C in a humidified atmosphere of 5 % CO2. Duplicate cultures were used at each concentration and for the solvent control cultures as well as the positive controls for treatment without and with S9 mix. On the day of treatment, the culture medium of exponentially growing cell cultures was replaced with medium (Ham's F12-5) containing the test item. The exposure period was 5 hours. Following the exposure period, the cells were washed three times with Ham's F12-5 medium and detached with trypsin-EDTA solution and counted using a Bürker chamber. Solubility of the test item in the cultures was assessed by the naked eye, at the beginning and end of treatment. In samples where sufficient cells survive, cell number was adjusted to 105 cells/mL. Cells were transferred to dishes for growth through the
expression period or diluted to be plated for survival.

Evaluation criteria:

Assay Acceptance Criteria
The assay was considered valid as all the following criteria were met:
• The mutant frequency of concurrent negative controls is within the 95% control limits of
the distribution of the laboratory’s historical negative control database.
• The positive control chemicals induced a statistically significant and biologically
relevant increase in mutant frequency compared to the concurrent negative control. The
increases are compatible with the laboratory historical positive control data base.
• Adequate number of cells and concentrations were analysable.
• Two experimental conditions with and without metabolic activation were tested.
• The highest concentration was adequate (10 mM).
• The cloning efficiency of the negative controls is between the range of 60 % to 140 %
on Day 1 and 70 % to 130 % on Day 8.

Evaluation of Results
Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly
positive if, in any of the experimental conditions examined:
• at least one of the test concentrations exhibits a statistically significant increase
compared with the concurrent negative control,
• any of the results are outside the distribution of the laboratory historical negative control
data (based 95% control limit),
• the increase of mutant frequency is concentration-related when evaluated with an
appropriate trend test.
Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative
if, in all experimental conditions examined:
• none of the test concentrations exhibits a statistically significant increase compared with
the concurrent negative control,
• there is no concentration-related increase when evaluated with an appropriate trend test,
all results are inside the distribution of the historical negative control data (based 95%
control limit.

Statistics:

Statistical Analysis was performed with SPSS PC+ software for the following data:
• mutant frequency between the negative (solvent) control group and the test item or
positive control item treated groups.
• mutant frequency between the laboratory historical negative (solvent) control group and
concurrent negative (solvent) control, the test item or positive control item treated
groups.
• The data were checked for a linear trend in mutant frequency with treatment dose using
the adequate regression analysis by Microsoft Excel software.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At concentration of 2000 µg/mL a very mild cytotoxic effect (16-17%) were observed, confirming the response seen in the preliminary cytotoxicity assays.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

It is concluded that the test item is not mutagenic in this In Vitro Mammalian Cell Gene Mutation Test performed with Chinese hamster ovary cells.

Executive summary:

The test item, Blue PE 3811/Cartasol Blue K5R was tested in a Mammalian Cell Gene Mutation Test (HPRT test) in CHO-K1 cells. The purpose of this study was to determine whether the test item or its metabolites can induce forward mutation at the hypoxanthine-guanine phosphoribosyl transferase enzyme locus (hprt) in cultured Chinese hamster cells.
Based on the result of the preliminary cytotoxicity assay, the concentration levels for the performed Mutation Assay were chosen according to the maximum recommended for lower- cytotoxic substances in OECD Guideline 476 (updated 2016).
The Mutation Assay was performed at the concentrations and treatment intervals given below:
Mutation Assay 5-hour treatment period without S9-mix: 125, 250, 500, 1000 and 2000 µg/mL
Mutation Assay 5-hour treatment period with S9-mix:125, 250, 500, 1000 and 2000 µg/mL
Following treatment, phenotypic expression was allowed for 8 days followed by a mutant selection period for another 8 days.
In the absence and in the presence of metabolic activation, no cytotoxicity was observed at concentrations of 125, 250, 500 and, 1000 µg/mL. At concentration of 2000 µg/mL a very mild cytotoxic effect (16 and 17%) were observed.
In conclusion, Blue PE 3811/Cartasol Blue K5R tested up to the maximum recommended concentration (2000 µg/mL) without and with metabolic activation system over a 5-hour treatment period did not induce statistically and biologically significant increases in mutant frequency compared to the solvent control.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 30th to August 22nd, 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

December 29th, 1992

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

May 26th, 1983

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix obtained from livers of 8 - 12 weeks old male rats

Test concentrations with justification for top dose:

Concentration range in exp. I (with metabolic activation): 3 - 5000 µg/ml
Concentration range in exp. II (with metabolic activation): 3 - 100 µg/ml
Concentration range in exp. I (without metabolic activation): 3 - 5000 µg/ml
Concentration range in exp. II (without metabolic activation): 0.5 - 20 µg/ml

Vehicle / solvent:

Deionized water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

The chromosomes were prepared 18h and 28h after the start of the treatment with the test article. The treatment interval was 4h with metabolic activation, 18h and 28h without metabolic activation. In each experimental group two parallel cultures were set up.
Per culture 100 metaphases were scored for structural chromosome aberrations. The concentration range of the test article had been determined in a pre-test using the reduction of cell numbers as indicator for toxicity response. Test article concentrations from 25 – 5000 μg/ml were applied for the treatment of the cultures.
In the absence and the presence of S9 mix precipitation of the test article was observed 4h after start of treatment at concentrations of 25 μg/ml and above. In the absence of S9 mix toxic effects could be observed after treatment with 100 μg/ml and above. In the presence of S9 mix test article induced cytotoxicity was observed after treatment with 1000 μg/ml and above.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

exp. II at the 18h interval with 20 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

exp. I at 28h with 10 µg/ml; exp. II at 18h with 100 µg/ml.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In the absence of S9 mix, toxic effects evidenced by a reduction of the mitotic indices were observed in experiment II at the 18h interval after treatment with 20.0 μg/ml (40.5 % of the corresponding solvent control). In the presence of S9 mix toxic effects were observed in experiment I at the 28h interval after treatment 10 μg/ml (57.1 %) and in experiment II at the 18h interval after treatment with 100 μg/ml (69.2 %).
However, evaluation of cultures after treatment with higher concentrations was not feasible due to heavy precipitation of the test article. Precipitation of the test article in culture medium occurred from 5 – 5000 μg/ml in the absence of S9 mix and from 30 – 5000 μg/ml in the presence of S9 mix.
In both independent experiments, neither in the absence nor in the presence of S9 mix biologically relevant test article induced increases in cells carrying structural chromosome aberrations were observed. However, in experiment I in the absence of S9 mix, a single statistically significant increase (2.0 % aberrant cells) due to a low solvent control value (0.0 %) must be regarded as statistically anomaly without biological relevance since the value is within the historical control data range (0.0% - 4.0 %).
In both experiments, no reproducible biologically relevant increase in the frequencies of polyploidy metaphases was found after treatment with the test article as compared to the frequencies of the controls. EMS (600 μg/ml) and CPA (0.71 μg/ml) were used as positive controls and induced significant increases in the number of cells carrying structural chromosome aberrations.

Conclusions:

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the substance did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line). Therefore, the test substance is classified to be non-mutagenic in this chromosome aberration study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

According to the CLP Regulation (EC) No. 1272/2008, Annex I: 3.5.2.2., for the purpose of the classification for germ cell mutagenicity, substances are allocated in one of two following categories:

- substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans or

- substances, which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

The test substance is negative in the three in vitro tests. Therefore, it is not classified as genotoxic according to the CLP Regulation (EC) No. 1272/2008.