REAKTIV-ORANGE DYPR 1410

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reactive Orange DYPR 1410 is considered not to be mutagenic in the bacterial reverse mutation assay including the modification for azo-dyes or in mammalian cells. Reactive Orange DYPR 1410 produced a false positive result in the in-vitro chromosome assay in V79 cells.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 December 1998 to 17 March 1999

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Solubility: dissolved in cell culture medium
Stability in the solvent: not necessary because the test substance was dissolved directly into the test system (MEM)
Concentration of stock solution: 5 mg/ml

Target gene:

Chromosome aberration.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Large stocks of the mycoplasma-free V79 cell line are stored in liquid nitrogen in the cell bank of "Genetic Toxicology", thus permitting repeated use of the same cell culture batch for numerous experiments. The identical characteristics of the cells ensure comparability of the experimental parameters.
Thawed stock cultures were kept at approx. 37 °C and approx. 4 % CCX, in 175 cm2 plastic flasks. About 5 x 10s to 1 x 10s cells were seeded into each flask in 30 ml of MEM-medium supplement with approx. 10 % (v/v) FCS (fetal calf serum) containing approx. 2 mM L-glutamine and approx. 0.1 % (w/v) neornycinsulfate. The cells were subcultured twice a week.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Colcemid

Metabolic activation:

with and without

Metabolic activation system:

liver homogenate fraction (S9-mix)

Test concentrations with justification for top dose:

First experiment with 3 h treatment time:
without S9-mix: 250#, 500, 1000, 2500 and 5000 μg/ml
with S9-mix: 250#, 500, 1000, 2500 and 5000 μg/ml

Second experiment with 20 h treatment time:
without S9-mix: 25#, 50, 100, 250 and 375* μg/ml

* not evaluated because of high toxicity
# not used because higher concentrations were evaluated

Vehicle / solvent:

Dissolved in cell culture medium at appropriate concentrations immediately before use.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Solvent controls: cultures treated with the solvent
Positive controls: a: without metabolic activation: EMS (ethyl methane sulfonate)
b: with metabolic activation: CPA (cyclophosphamide) = Endoxan®
Formulation of test compound: dissolved in cell culture medium at appropriate concentrations immediately before use
Formulation of reference compounds: EMS dissolved in cell culture medium on the day of treatment
final concentration; 1.5 mg/ml (3 h treatment) final concentration: 0.4 mg/ml (20 h treatment)
CPA dissolved in cell culture medium on the day of treatment, final concentration in cell culture medium: 3.0 μg/ml
Source of cells: cell bank of "Genetic Toxicology", HMR Germany, ProTox
Test organism: cell line V79 of Chinese hamster lung fibroblasts
Cell culture medium: MEM (minimal essential medium) with Hanks-salts and 25 mM Hepes-buffer
Experimental conditions In vitro: approx. 37 °C and approx. 4 % CO2 in plastic flasks

Preparation and storage of a liver homogenate fraction (S9)
The S9 fraction was prepared by the testing facility according to Ames et. al (1975). Male Sprague Dawley rats (200-300 g), supplied by Harlan Winkelmann, Gartenstrasse 27, 33178 Borchen, Germany, received a single intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight) 5 days before killing. The livers were removed from at least 5-6 animals using cold sterile solutions at approx. 0 to 4 °C and glassware, and were then pooled and washed in approx. 150 mM KCI (approximately 1 ml/g wet liver). The washed livers were cut into small pieces and homogenized in three volumes of KCI. The homogenate was centrifuged at approx. 9000 g for 10 minutes. The supernatant, the S9 fraction, was divided into small portions, rapidly frozen and stored at approx. - 80 °C for not longer than six months. The protein content was determined for every batch. Also for every batch of S9 an independent validation was performed with a minimum of two different mutagens, e.g., 2-aminoanthracene and dimethylbenzanthracene to confirm metabolic activation by microsomal enzymes.

Preparation of S9-mix
Sufficient S9 fraction was thawed to room temperature immediately before each test. An appropriate quantity of S9 fraction (batch no. 98/1, protein concentration 51.8 g/l) was mixed with S9 cofactor solution to yield a final protein concentration of 0.3 mg/ml in the cultures which was kept on ice until used. This preparation is termed S9-mix The concentrations of the different cofactors of the S9-mix were:

8 mM MgCI2
33 mM KCI
5 mM glucose-6-phosphate
5 mM NADP
100mM phosphate buffer pH 7.4

Cell culture
Large stocks of the mycoplasma-free V79 cell line are stored in liquid nitrogen in the cell bank of "Genetic Toxicology", thus permitting repeated use of the same cell culture batch for numerous experiments. The identical characteristics of the cells ensure comparability of the experimental parameters.
Thawed stock cultures were kept at approx. 37 °C and approx. 4 % CCX, in 175 cm2 plastic flasks. About 5 x 10s to 1 x 10s cells were seeded into each flask in 30 ml of MEM-medium supplement with approx. 10 % (v/v) FCS (fetal calf serum) containing approx. 2 mM L-glutamine and approx. 0.1 % (w/v) neomycinsulfate. The cells were subcultured twice a week.

Toxicity experiments and dose range finding
A preliminary toxicity test was undertaken in order to select appropriate dose levels for the
cytogenetic assay. Cell cultures were subjected to the same treatment conditions as in the main experiment. Cytotoxic effects were determined by photometric measurement of V79 cell cultures grown in microwell plates and stained with crystal violet. The relative cell density in the microwell plates was nearly the same as in the Quadriperm® dishes.

The test included the following treatments:

Solvent control : the maximum final concentration of organic solvents was approx. 1 % (v/v).
Test compound : the highest dose level for the preliminary toxicity test was determined by the solubility of the test compound up to the maximum of 10 mM or 5000 ug/ml.
Treatments were performed both in the presence and absence of S9 metabolic activation system using a duplicate cell culture at each test point.

Rationale for dose selection

The concentrations for the mutagenicity assay were based on the results of the toxicity experiment.
For non-toxic, freely soluble test compounds, the top dose is 10 mM or 5000 ug/ml according to International testing guidelines,
For relatively insoluble test compounds, that are not toxic at concentrations lower than the insoluble concentration, the highest dose used should be a concentration above the limit of solubility in the final culture medium after the end of the treatment period. In the case of toxic effects, the highest dose level should reduce the survival rate to approximately 20 - 50 % and/or the mitotic index to approximately 50 % compared with the corresponding solvent control.
For toxic compounds additional concentrations may be included in the treatment series. According to the criteria described above, three adequately spaced dose levels extending over at least one decadic logarithm were evaluated. For each experimental point two cultures were used for each concentration.

Mutagenicity test

Two independent experiments were conducted. The first experiment with 3 hours treatment time of the test substance was performed in the presence and the absence of S9-mix. Cultured celis were seeded onto slides (duplicate culture) then treated for either 3 hours (with and without S9-mix in the first experiment) or for 20 hours (without S9-mix in the second experiment). Celcemide was then added to arrest cell division and the chromosomes were stained and examined. In both assays, cells were sampled 20 hours after the start of treatment. For both assays, at least three dose levels were evaluated for chromosome aberrations. Where negative or equivocal results were obtained, cells were treated and also examined 20 hours after the start of treatment.

Before treatment, the pH values and osmolality of the treatment medium were determined. If necessary the pH was adjusted to pH 7.3 with NaOH or HCI. Any effects on the osmolality during the study were described in the study report.

Two-day old, exponentially growing stock cultures which were over 50 % confluent were trypsinised and a single cell suspension (culture) was prepared. The trypsin concentration was approx. 0.25 % (v/v) in Ca-Mg-free salt solution. Two slides were placed in Quadriperm® dishes which were then seeded with cells to yield 2-3 x 10-3 cells/slide. Thus for each dose level and treatment time, duplicate cultures were used. The Quadriperm® dishes contained 5 ml MEM with approx. 10 % (v/v) FCS.

After 48 h, the medium was replaced with one containing approx. 10 % (v/v) FCS and the test compound, or positive control, or solvent and in the presence of metabolic activation additional 2 % ( v/v) S9-mix.
For the 3 hour treatment time, the medium was replaced by normal medium following two rinses. In the repeat experiment the cells were exposed to the treatment medium without S9-mix for 20h.

18 h after the start of the treatment, colcemide was added (approx. 0.05 ug/ml/culture medium) to the cultures to arrest mitosis and 2 h later (20 h after the start of treatment) metaphase spreads were prepared as follows:
The cultures were made hypotonic by adding about 4 ml of approx. 0.075 M potassium chloride solution at around 37 °C, The cells were then incubated for 20 minutes at approx. 37 °C. The next step was the addition of 1.5 ml fixative.

Then the liquid was replaced by 5 ml fixative (methanol: glacial acetic acid, 3 :1). After 10 minutes the procedure was repeated. After at least another 10 minutes, the slides were taken out and airdried for 24 hours. The chromosomes were stained as follows:
- staining for 10 minutes in approx. 2 % (w/v) orcein solution
- rinsing 3 times in distilled water
- rinsing twice in acetone
- brief rinsing in acetone/xylene
- 2 minutes in acetone/xylene
- 5 minutes in xylene
- 10 minutes in xylene
- embedding in Entellan® or Corbit®

Duplicate cultures were prepared from each experimental group,

For both treatment times the solvent and the positive controls were prepared 20 h after treatment in the same way.

Evaluation of data - Analysis of metaphases

The slides were coded and 25 - 100 metaphases per experimental group and cell culture were examined. The set of chromosomes was examined for completeness and the various chromosomal aberrations were assessed. The chromosomal aberrations were classified as shown in chapter 9.1. Only metaphases with 22 +/- 2 chromosomes are included in the analysis. The metaphases were examined for the following aberrations: chromatid gap, chromosome gap. chromatid break, chromosome break, minute, double minute, chromatid deletion, chromosome deletion, chromatid exchanges Including intrachanges, chromosome exchanges including inirachangesj dicentrics, pulverization and ring formation. Furthermore the incidence of polyploid metaphases was determined in 1000 cells of each cell culture.
Additionally a mitotic index was determined by counting the number of cells undergoing mitosis in a total of 1000 cells. The mitotic index is given in per cent.

After the metaphases had been evaluated, the code was broken. The values for the control group were compared with the results from the dose groups and the positive control at each sampling time.

Evaluation criteria:

Criteria for a valid assay
The assay was considered valid if the following criteria are met:
The solvent control data were within the laboratory's normal control range for the spontaneous mutant frequency
The positive controls induced increases in the mutation frequency which were both statistically significant and within the laboratory's normal range

Criteria for a positive response
The evaluation of the results was performed as follows:
The test compound is classified as mutagenic if it induces a statistically significant increase in the aberration rate (without gaps) with one or more of the concentrations tested as compared with the solvent controls.
The test compound is classified as mutagenic if there Is a concentration-related increase in the aberration rale (without gaps).
The test compound is classified as non-mutagenic if the tests are negative both with and without metabolic activation

Statistics:

The Biometry of the results was performed with a one-sided Fisher - Exact test.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Solubility and preliminary toxicity testing
Reaktiv-Orange DYPR 1410 was dissolved in cell culture medium. Evaluation of the solubility of that solution in cell culture medium showed that 5000 μg/ml was the highest practicable concentration and produced no macroscopic precipitate. Microscopic visible precipitation was observed at the 3h treatment time from a dose level of 2000 μg/ml up to 5000 μg/ml and at the fixation interval of 20h at a concentration of 1000 μg/ml and above.
Accordingly, the preliminary toxicity study was carried out using a maximum concentration of 5000 μg/ml and a range of lower dose levels down to 100 μg/ml.
Following treatment for 3 hours in the absence of S9 metabolic activation, several toxicity was observed at 2000 μg/ml and above. Survival declined in a dose-related manner reaching 51.9 % of the solvent control value at the highest dose level, 5000 μg/ml. After 20 hours treatment survival was reduced to 6.8 % of the solvent control value at a concentration of 500 μp/ml and 12.9 % of the solvent control value at a concentration of 5000 μg/ml.
In the presence of S9 metabolic activation mild toxicity was observed at 2000 μg/ml and above. At a concentration of 5000 μg/ml survival was reduced to 69.4 % of the solvent control value.
Before treatment, the pH values and osmolality of the treatment media were determined. The addition of test compound solutions did not have any effect on these parameters.

Mutagenicity test
In the main experiments cytotoxicity was also evaluated by treatment of cells seeded in microwell plates. Survival was reduced in a dose-related manner reaching 40.1 % of the solvent control value without S9-mix at the 3 hours treatment time and 53.9 % in the presence of S9-mix at the highest Concentration tested. 5000 μg/ml. At the 20 hours treatment time survival was reduced in a dose-related manner reaching 17.9 % of the solvent control value without metabolic activation at the concentration of 375 μg/ml.
In the main and in the repeat experiments the mitotic index was reduced (indication of toxicity) after treatment with the highest dose levels.
After treatment with the test compound there was no relevant increase in the number of polyploid cells as compared with the solvent controls.
The test compound Reaktiv-Orange DYPR 1410 was assessed for its mutagenic potential in vitro in the chromosome aberration test in two independent experiments.
There was an enhancement of the aberration rates at a non-toxic concentration of 2500 μg/ml at the 3 h treatment time with and without S9-mix. Also at the sampling time of 3 h with and without S9-mix at the toxic concentration of 5000 μg/ml the aberration rates were enhanced. These data were found significantly enhanced in the Fisher's exact-test. Because of the lower dose range at the 20 h treatment time no enhancement of the aberration rates was found. The sensitivity of the test system was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control compounds.

Remarks on result:

not determinable because of methodological limitations

In the chromosomal aberration test strong positive effects were obtained with and without S-9 mix at doses which induced precipitations. However, it is a well documented effect for vinyl sulphone dyes to result in a false positive in vitro assay for chromosome aberration. This is due to GSH depletion within the in-vitro test system -with a very limited amount of phase 2 enzymes- caused by the Michael Addition at the vinyl sulphone structures. However, it was shown in many (>100) studies that this effect is not noted in in-vivo studies (as is the case with the substance here). One explanation is that the cause for this is that in vivo, the relation of the Michael Addition and the GSH is much in favour for the repair system. As such, these effects are not noted. In addition, the chromosome aberration test was only positive at the 3 h at doses and precipitates were seen. It is well-known that these precipitates can cause a false positive reaction. Therefore, the chromosome aberration text in vitro cannot be assessed as unequivocally positive.

Conclusions:

Reaktiv-Orange DYPR 1410 was apparently cytogenic in this chromosome aberration test system in vitro with cells of the V79 Chinese hamster cell line under the conditions described in this report.
It is a well documented effect for vinyl sulphone dyes to result in a false positive in vitro assay for chromosome aberration. This is due to GSH depletion within the in-vitro test system, with a very limited amount of phase 2 enzymes, caused by the Michael Addition at the vinyl sulphone structures. However, it was shown in many (>100) studies that this effect is not noted in in-vivo studies (as is the case with the substance here). One explanation is that the cause for this is that in vivo, the relation of the Michael Addition and the GSH is much in favour for the repair system. As such, these effects are not noted.

Executive summary:

The study was conducted in compliance with OECD Guideline For Testing Of Chemicals, 473 "Genetic Toxicology: In VitroMammalian Chromosome Aberration Test". U.S. EPA: OPPTS 870.5375 Health Effects Test Guidelines In Vitro Mammalian Chromosome Aberration Test and EEC Directive 92f69, L3S3 A, Annex B. 10., p. 148 -150. This study was conducted in compliance with the Principles of Good Laboratory Practice (GLP).

 

In this study the potential of Reaktiv-Orange DYPR 1410 to induce chromosome aberrations was investigated in V 79 cells of the Chinese hamster lungin vitro.For each experiment duplicate cultures were used for each concentration,

 

The test compound was dissolved in cell culture medium and tested at the following concentrations:

 

First experiment with 3 h treatment time:

without S9-mix: 250#, 500, 1000, 2500 and 5000 μg/ml

with S9-mix: 250#, 500, 1000, 2500 and 5000 μg/ml

 

Second experiment with 20 h treatment time:

 

without S9-mix: 25#, 50, 100, 250 and 375* μg/ml

 

*          not evaluated because of high toxicity

#         not used because higher concentrations were evaluated

 

The concentration ranges were based on the results of preliminary testing for solubility and toxicity. The highest concentration produced a distinct lowering of the mitotic index. Higher concentrations were not applied because of the 5000 μg/ml limitation (OECD guideline).

At the 3h sampling time the concentrations of 2500 and 5000 µg/ml with and without metabolic activation the test compound induced a significant increase in the number of chromosome aberrations.

 

Appropriate reference mutagens used as positive controls showed a significant increase in chromosome aberrations, thus indicating the sensitivity of the assay, and the efficacy of the S9-mix.

 

In conclusion, Reaktiv-Orange DYPR 1410 appears to induce chromosome mutations (=aberrations) in V79 Chinese hamster cells, both in the presence as well as in the absence of a metabolic activation system, under the experimental conditions described.

In the chromosomal aberration test strong positive effects were obtained with and without S-9 mix at doses which induced precipitations. However, it is a well documented effect for vinyl sulphone dyes to result in a false positive in vitro assay for chromosome aberration. This is due to GSH depletion within the in-vitro test system -with a very limited amount of phase 2 enzymes- caused by the Michael Addition at the vinyl sulphone structures. However, it was shown in many (>100) studies that this effect is not noted in in-vivo studies (as is the case with the substance here). One explanation is that the cause for this is that in vivo, the relation of the Michael Addition and the GSH is much in favour for the repair system. As such, these effects are not noted. In addition, the chromosome aberration test was only positive at the 3 h doses and precipitates were seen. It is well-known that these precipitates can cause a false positive reaction. Therefore, the chromosome aberration text in vitro cannot be assessed as unequivocally positive.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 07 October 2021 to 11 February 2022

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING

On the day of the experiment (immediately before treatment), the test item was dissolved in water. The final concentration of water in the culture medium was 10% (v/v). The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.
All formulations were prepared freshly before treatment and used within two hours of preparation.

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-Naphtoflavone induced Rat liver S9

Test concentrations with justification for top dose:

exposure
period S9
mix concentrations
in µg/mL
Main experiment
4 hours - 98.8 197.5 395 790 1580 3160
4 hours + 98.8 197.5 395 790 1580 3160
The cultures at the lowest concentration with and without metabolic activation were not continued as a minimum of only four analysable concentrations is required by the guidelines.

Vehicle / solvent:

Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: solubility properties

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation, Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 6-Thioguanine

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: >1,5x10exp. 6

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

The gene mutation assay is considered acceptable if it meets the following criteria:
a) The mean values of the numbers of mutant colonies per 106 cells found in the solvent controls of both parallel cultures remain within the 95% confidence interval of the laboratory historical control data range.
b) Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistical significant increase compared with the concurrent solvent control.
c) Two experimental conditions (i.e. with and without metabolic activation) were tested unless one resulted in positive results.
d) An adequate number of cells and concentrations (at least four test item concentrations) are analysable even for the cultures treated at concentrations that cause 90% cytotoxicity during treatment.
e) The criteria for the selection of the top concentration are fulfilled (see 3.5.2 Dose selection).

Statistics:

The statistical analysis was performed on the mean values of culture I and II for the main experiment.
A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
A t-Test was performed only for the positive controls since all mean mutant frequencies of the groups treated with the test item were well within the 95% confidence interval of our laboratory’s historical negative control data.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the main experiment in the absence of S9 mix, cytotoxicity was observed at the highest applied concentration (3160 µg/mL).

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected (pH 7.35 in the solvent control versus pH 7.4 at 3160 µg/mL)
- Effects of osmolality: No relevant increase (293 mOsm in the solvent control versus 308.mOsm at 3160 µg/mL)
- Evaporation from medium: Not examined
- Precipitation: No precipitation in the main experiment
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES:
Test item concentrations between 24.7 µg/mL and 3160 µg/mL were used in the pre-experiment with and without metabolic activation following 4 hours treatment. The highest concentration was chosen with respect to the OECD guideline 476 (2000 µg/mL) adjusted with the given correction factor of 1.58 (correction for purity of the test item).
The test medium was checked for phase separation and precipitation at the end of the treatment period (4 hours) before the test item was removed. Precipitation occurred at 1580 µg/mL and above without metabolic activation and at 3160 µg/mL with metabolic activation.
No relevant cytotoxic effect, indicated by a relative cloning efficiency of 50% or below was observed at any concentration with and without metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA: Complies

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the main experiment in the absence of S9 mix, cytotoxicity was observed at the highest applied concentration (3160 µg/mL). Relevant cytotoxic effects indicated by a relative adjusted cloning efficiency I (survival rate) below 50% (mean value of both parallel cultures) were noted at 3160 µg/mL, achieving a relative adjusted cloning efficiency I between 10 and 20% (mean value 15.3%).

Summary Table

				relative	relative	rel. adjusted	(MF)	95%	statistical
	conc.	P/	S9	cloning	cell	cloning	mutant	control	analysis*
	µg/mL	PS	mix	efficiency I	density	efficiency I	colonies/	limit	t-test	linear
				%	%	%	10^6 cells			regression
Column	1	2	3	4	5	6	7	8	9	10
Main Experiment / 4 h treatment				mean values of culture I and II
Solvent control with water			-	100.0	100.0	100.0	8.5	2.9 – 22.4
Positive control (EMS)	300.0		-	95.4	88.1	82.9	198.2	−	0.000^S
Test item	98.8	-	-	98.3	89.2	87.9	#	2.9 – 22.4
Test item	197.5	-	-	96.4	80.0	77.1	8.4	2.9 – 22.4	n.c.	0.433
Test item	395.0	-	-	99.5	90.4	90.1	8.1	2.9 – 22.4	n.c.
Test item	790.0	-	-	91.5	76.9	71.4	10.1	2.9 – 22.4	n.c.
Test item	1580.0	-	-	75.9	80.1	61.3	10.0	2.9 – 22.4	n.c.
Test item	3160.0	-	-	19.8	77.8	15.3	6.7	2.9 – 22.4	n.c.
Solvent control with water			+	100.0	100.0	100.0	10.0	2.9 – 23.7
Positive control (DMBA)	2.3		+	78.1	93.9	72.5	157.9	−	0.000^S
Test item	98.8	-	+	94.5	100.3	94.9	#	2.9 – 23.7
Test item	197.5	-	+	89.8	84.9	76.5	8.7	2.9 – 23.7	n.c.	0.776
Test item	395.0	-	+	85.4	88.2	74.6	8.1	2.9 – 23.7	n.c.
Test item	790.0	-	+	95.5	92.3	88.2	7.8	2.9 – 23.7	n.c.
Test item	1580.0	-	+	83.8	86.7	72.4	16.6	2.9 – 23.7	n.c.
Test item	3160.0	-	+	79.5	89.4	70.8	8.4	2.9 – 23.7	n.c.

*                 statistical analysis based on the mean values of culture I and II

n.c.             not calculated (mean MF below the upper limit of the 95% control limit)

P/PS           Precipitation/Phase separation at the end of treatment

S                 Significant trend (p < 0.05)

MF             Mutant frequency

#                 cultures not continued as a minimum of only four analyzable concentrations are required

Conclusions:

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, Reactive Orange DYPR 1410 is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The study was performed to investigate the potential of Reactive Orange DYPR 1410, dissolved in water, to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

The treatment period was 4 hours with and without metabolic activation.

The maximum test item concentration of the pre-experiment (3160 µg/mL) was chosen with respect to the OECD guideline 476 (2000 µg/mL) adjusted with the given correction factor of 1.58 (correction for purity of the test item).

In the main experiment in the absence of S9 mix, cytotoxicity was observed at the highest applied concentration (3160 µg/mL). Relevant cytotoxic effects indicated by a relative adjusted cloning efficiency I (survival rate) below 50% (mean value of both parallel cultures) were noted at 3160 µg/mL, achieving a relative adjusted cloning efficiency I between 10 and 20% (mean value 15.3%).

In the main experiment in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration (3160 µg/mL).

No precipitation occurred up to the highest applied concentration after four hours treatment with and without S9 mix.

No substantial and dose dependent increase of the mutation frequency was observed in the main experiment.

The tested concentrations are described in chapter 3.5.2 (Table 1). The evaluated experimental points and the results are summarised in Table 3.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 November 1998 to 08 January 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine or tryptophan mutation

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

liver extract (S9-mix)

Test concentrations with justification for top dose:

Concentrations for all studies were 50, 150, 500, 1600 and 5000 µg/plate.

Vehicle / solvent:

deionized water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: MNNG, 2-aminoanthracene

Details on test system and experimental conditions:

The first mutation test was performed as the plate incorporation method in both the presence and absence of S9-mix using all bacterial tester strains and a range of concentrations of the test substance. Positive and negative controls as well as solvent controls were included in each test. Triplicate plates were used. This mutation experiment also assessed the toxicity of the test substance by evaluation of the bacterial lawn in order to select a suitable range of dose levels for a second mutation test which was performed as the preincubation method. The highest concentration was usually 50 mg/ml of the test substance in the chosen solvent, which provided a final concentration of 5000 pg/plate. Further dilutions of 1600, 500, 160 and 50 pg/plate were used, A reduced rate of spontaneously occurring colonies and visible thinning of the bacterial lawn were used as toxicity indicators. Thinning of the bacterial lawn was evaluated microscopically.

If the total number of concentrations selected for evaluation in the plate incorporation test does not allow for a statement of genotoxicity of the test substance to be made, an additional plate incorporation test, based on the toxicity results of the first test, has to be performed. As negative or equivocal results were obtained, a second mutation experiment must be performed on the basis of toxicity results in the plate incorporation test as a preincubation test.

For the plate incorporation test top agar was prepared for the Salmonella strains by mixing 100 ml molten agar (0.6 % (w/v) agar, 0.5 % (w/v) NaCI) with 10 ml of a 0.5 mM histidine-biotin solution. With E. coir histidine was replaced by tryptophan (2.5 ml, 0.5 mM). The following ingredients were added (in the following order) to 2 ml of top agar at approx. 48 °C:
0.5 ml S9-mix (if required) or buffer
0.1 ml of an overnight nutrient broth culture of the bacterial tester strain
0,1 ml test compound solution (dissolved in deionized water)

For the preincubation method this top-agar ingredients were preincubated for approximately 20 minutes at approx. 30 °C and the top agar composition as described was added accordingly.
After mixing, the liquid was poured into a petri dish containing a 25 ml layer of minimal agar (1.5% (w/v) agar, Vogel-Bonner E medium with 2 % (w/v) glucose). After incubation for approximately 48 hours at approx. 37 °C in the dark, colonies (his* and trp* revertants) were counted with an automatic colony counter (Artec counter Model 880). The counter was calibrated for each test by comparison of manual count data of three control plates with automatic data of the colony counter. A correction factor was determined to compensate for differences between manual and automatic count. This correction factor was used to automatically adjust the observed number of colonies on each plate to more accurately reflect the actual number of colonies present

Evaluation criteria:

The assay is considered valid if the following criteria are met:
- the solvent control data are within the laboratory's normal control range for the spontaneous mutant frequency
- the positive controls induced increases in the mutation frequency which were both statistically significant and within the laboratory's normal range


Criteria for a positive response

A test compound is classified as mutagenic if it has either of the following effects:
- it produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
- it induces a dose-related increase in the mean number of revertanls per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn.

If the test substance does not achieve either of the above criteria, it is considered to show no evidence of mutagenic activity in this system.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Sterility checks and control plates

Sterility of S9-mix and the test compound were indicated by the absence of contamination on the test material and S9-mix sterility check plates. Control plates (background control and positive controls) gave the expected number of colonies, i.e. values were within the laboratory's historical control range.

Solubility and toxicity

The test compound was dissolved in deionized water and a stock solution of 50 mg/ml was prepared for the highest concentration, which provided a final concentration of 5000 µg/plate. Further dilutions of 1600, 500, 160 and 50 µg/plate were used in all experiments.

Additionally a repeat of the preincubation test was performed with the strain TA 100 with metabolic activation. The concentrations were 50, 160, 500,1600, 3000 and 5000 µg/plate.

The test compound did not precipitate on the plates up to the highest investigated dose of 5000 ug/plate.

The test compound proved to be not toxic to the bacterial strains.

Mutagenicity

In all independent mutation tests Reaktiv-Orange DYPR 1410 was tested for mutagenicity with the same concentrations. The number of colonies per plate with each strain as well as mean values of 3 plates are given.

Because of the non-mutagenic effects of the positive control with the strain TA 100 in the presence of S9-mix in the first preincubation test, a second preincubation test was performed. Because of slight increases only at the highest concentration in this test, a third preincubation test was performed with this strain and a concentration of 3000 µg/plate was included in the treatment series.

The slight mutagenic effects were not confirmed.

The test compound did not cause a significant increase in the number of revertant colonies at any dose level with any of the tester strains either in the absence or in the presence of S9-mix in either mutation test. No dose-dependent effect was obtained.

All positive controls produced significant increases in the number of revertant colonies. Thus, the sensitivity of the assay and the efficacy of the exogenous metabolic activation system were demonstrated.

Conclusions:

The results lead to the conclusion that Reaktiv-Orange DYPR 1410 is not mutagenic in these bacterial test systems either in the absence or in the presence of an exogenous metabolizing system.

Executive summary:

The present study was conducted in compliance with EEC Directive 92/69, L 383 A, Annex B 14 and EEC Directive 92/69, L 333 Annex B 13, OECD Guideline For Testing Of Chemicals, 471 Bacterial Reverse Mutation Test Adopted and U.S. EPA: OPPTS 870.5100 Health Effects Test Guidelines Bacterial Reverse Mutation Test. This study was conducted in compliance with GLP.

Reaktiv-Orange DYPR 1410 was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium and Escherichia coli WP2uvrA.

Four independent experiments were conducted (one plate incorporation test and three preincubation tests), each in the absence and in the presence of a metabolizing system derived from a rat liver homogenate.

For all studies the compound was dissolved in deionized water, and each bacterial strain was exposed to 5 dose levels, in a third preincubation test to 6 dose levels.

Concentrations for all studies were 50, 150, 500, 1600 and 5000 µg/plate.

Because of the non-mutagenic effects of the positive, control with the strain TA 100 in the presence of S9-mix in the first preincubation test, a second preincubation test was performed. Because of slight increases only at the highest concentration in this test, a third preincubation test was performed with this strain and a concentration of 3000 µg/plate was included in the treatment series.

Control plates without mutagen showed that the number of spontaneous revertant colonies was within the laboratory's historical control range. All the positive control compounds showed the expected increase in the number of revertant colonies.

The test compound proved to be not toxic to the bacterial strains,

Mutagenicity: In the absence and in the presence of the metabolic activation system Reaktiv-Orange DYPR 1410 did not result in relevant increases in the number of revertants in any of the bacterial strains.

Summarizing, it can be stated that Reaktiv-Orange DYPR 1410 was not mutagenic in this bacterial mutation test at any dose level in either the absence or in the presence of an exogenous metabolic activation.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-10-06 till 2021-11-24

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 (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Experiment I: Phenobarbital/Beta-naphthoflavone induced rat liver S9
Experiment II, IIa, IIb: non-induced hamster liver s9 mix

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment IIa and IIb: 333; 1000; 2000; 3000; 4000; and 5000 µg/plate

Vehicle / solvent:

Solvent used: deionised water
Justification for choice of solvent: best suitable solvent, because of its solubility properties and its relative nontoxicity to the bacteria

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

congo red

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar plate incorporation; pre-incubation

DURATION:
Preincubation period: 30 Minutes
exposure duration: 72 hours

NUMBER OF REPLICATIONS: 3 plates for each concentration including the controls

DETERMINATION OF CYTOTOXICITY: Evident as a reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:

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

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

- An increase exceeding the threshold at only one concentration is judged as biologically
relevant if reproduced in an independent second experiment.

- A dose dependent increase in the number of revertant colonies below the threshold is
regarded as an indication of a mutagenic potential if reproduced in an independent second
experiment. However, whenever the colony counts remain within the historical range of
negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

intense colouring of the agar plates was observed in the preincubation assay from 2000 µg/plate onwards

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

intense colouring of the agar plates was observed in the preincubation assay from 2000 µg/plate onwards

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

intense colouring of the agar plates was observed in the preincubation assay from 2000 µg/plate onwards

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

intense colouring of the agar plates was observed in the preincubation assay from 2000 µg/plate onwards

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Remarks:

A slight increase in mutation frequency (2.06-fold) was observed during manual count of the extremely coloured plate at 5000 µg/plate with hamster S9 with a high variability in the replicates (SD +/- 13). This was not confirmed in the 2nd experiment.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

intense colouring of the agar plates was observed in the preincubation assay from 2000 µg/plate onwards

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST SPECIFIC CONFOUNDING FACTORS
Effects of pH: None
Water solubility: soluble
Precipitation: No precipitation of the test item occurred in the overlay agar in the test tubes and in the overlay agar on the incubated agar plates, but an intense coloring was observed in the overlay agar on the incubated agar plates in experiment II at 5000 µg/plate, in experiment IIa from 3000 to 5000 µg/plate and in experiment IIb from 2000 to 5000 µg/plate. This extreme coloring prevented automatic counting; all densely colored plates had to be evaluated manually.

COMPARISON WIT HISTORICAL CONTROL DATA: performed, no deviations
ADDITIONAL INFORMATION ON CYTOTOXICITY: none

Summary of Experiment I

Study Name: 2190001	Study Code: ICCR 2190001
Experiment: 2190001 VV Plate	Date Plated: 15.10.2021
Assay Conditions:	Date Counted: 18.10.2021

 

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ±SD)TA 1535	Revertant Colony Counts (Mean ±SD)TA 1537	Revertant Colony Counts (Mean ±SD)TA 98	Revertant Colony Counts (Mean ±SD)TA 100	Revertant Colony Counts (Mean ±SD)WP2 uvrA
Without Activation	Deionised water		12 ± 3	8 ± 2	29 ± 9	106 ± 15	41 ± 9
	Untreated		14 ± 3	12 ± 4	20 ± 5	116 ± 9	42 ± 2
	Reactive	3 µg	10 ± 1	10 ± 3	22 ± 3	118 ± 27	40 ± 9
	Orange DYPR	10 µg	9 ± 3	11 ± 4	33 ± 8	123 ± 6	41 ± 5
	1410	33 µg	15 ± 3	11 ± 1	28 ± 5	101 ± 6	48 ± 9
		100 µg	14 ± 3	13 ± 4	23 ± 2	126 ± 16	46 ± 11
		333 µg	11 ± 5	14 ± 4	35 ± 6	110 ± 8	33 ± 2
		1000 µg	8 ± 3	9 ± 3	30 ± 8	103 ± 6	43 ± 10
		2500 µg	7 ± 3	8 ± 3	25 ± 9	117 ± 32	49 ± 4
		5000 µg	10 ± 4	9 ± 4	21 ± 7	103 ± 28	42 ± 1
	NaN3	10 µg	1082 ± 44			1804 ± 47
	4-NOPD	10 µg			842 ± 43
	4-NOPD	50 µg		99 ± 6
	MMS	2.0 µL					913 ± 11
With Activation	Deionised water		17 ± 6	13 ± 2	47 ± 3	111 ± 6	58 ± 6
	Untreated		11 ± 3	17 ± 4	48 ± 12	124 ± 8	62 ± 5
	Reactive	3 µg	12 ± 4	16 ± 6	44 ± 7	119 ± 7	57 ± 6
	Orange DYPR	10 µg	19 ± 2	13 ± 5	41 ± 7	122 ± 3	56 ± 14
	1410	33 µg	17 ± 5	12 ± 4	46 ± 3	136 ± 27	55 ± 3
		100 µg	15 ± 5	16 ± 5	48 ± 8	130 ± 6	64 ± 5
		333 µg	18 ± 7	12 ± 7	50 ± 3	139 ± 13	74 ± 4
		1000 µg	15 ± 4	11 ± 3	49 ± 7	148 ± 15	67 ± 11
		2500 µg	16 ± 2	10 ± 3	32 ± 5	114 ± 5	65 ± 4
		5000 µg	12 ± 2	9 ± 2	26 ± 1	112 ± 3	42 ± 6
	2-AA	2.5 µg	329 ± 17	409 ± 43	2880 ± 340	3971 ± 115
	2-AA	10.0 µg					326 ± 46

 

Summary of Experiment II

Study Name: 2190001	Study Code: ICCR 2190001
Experiment: 2190001 HV2 Pre	Date Plated: 29.10.2021
Assay Conditions:	Date Counted: 04.11.2021

 

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ±SD) TA 1535	Revertant Colony Counts (Mean ±SD) TA 1537	Revertant Colony Counts (Mean ±SD) TA 98	Revertant Colony Counts (Mean ±SD) TA 100	Revertant Colony Counts (Mean ±SD) WP2 uvrA
Without Activation	Deionised water		12 ± 1	14 ± 2	28 ± 3	139 ± 8	57 ± 2
	Untreated		15 ± 5	15 ± 3	27 ± 8	158 ± 8	59 ± 9
	Reactive	33 µg	14 ± 2	11 ± 1	29 ± 2	153 ± 9	54 ± 4
	Orange	100 µg	14 ± 5	12 ± 2	35 ± 7	128 ± 6	64 ± 6
	DYPR 1410	333 µg	16 ± 2	13 ± 3	28 ± 3	153 ± 7	53 ± 5
		1000 µg	13 ± 4	10 ± 3	22 ± 8	163 ± 14	61 ± 2
		2500 µg	14 ± 5	11 ± 4	23 ± 4	149 ± 8	67 ± 17
		5000 µg	14 ± 6^D M	9 ± 3^D M	19 ± 6^D M	120 ± 4^D M	65 ± 10^D M
	NaN3	10 µg	1280 ± 127			2141 ± 33
	4-NOPD	10 µg			549 ± 390
	4-NOPD	50 µg		131 ± 8
	MMS	2 µL					1143 ± 36
With Activation	Deionised water		15 ± 5	21 ± 5	52 ± 11	117 ± 9	50 ± 8
	Untreated		14 ± 6	24 ± 8	52 ± 11	117 ± 4	47 ± 1
	Reactive	33 µg	16 ± 3	25 ± 6	50 ± 6	115 ± 5	41 ± 6
	Orange	100 µg	15 ± 1	19 ± 2	53 ± 12	138 ± 8	47 ± 8
	DYPR 1410	333 µg	17 ± 2	22 ± 1	52 ± 5	151 ± 2	54 ± 7
		1000 µg	16 ± 4	13 ± 2	46 ± 6	186 ± 5	85 ± 5
		2500 µg	14 ± 1	14 ± 2	56 ± 8	150 ± 14	84 ± 17
		5000 µg	12 ± 3^D M	15 ± 3^D M	34 ± 4^D M	205 ± 6^D M	103 ± 13^D M
	2-AA	2.5  µg				2842 ± 334
	2-AA	2.5 µg	288 ± 37	320 ± 49
	2-AA	10 µg					770 ± 49
	Congo red	500 µg			437 ± 18

 

 

Summary of Experiment II a/b

 

Study Name: 2190001	Study Code: ICCR 2190001
Experiment: 2190001 HV2a/b Pre	Date Plated:       09.11.2021                              18.11.2021*
Assay Conditions: Pre-Incubation	Date Counted:                     12.11.2021                               24.11.2021*

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD) TA 100	Revertant Colony Counts (Mean ±SD) WP2 uvrA*
With Activation	Deionised water			130 ± 15	43 ± 8
	Untreated			127 ± 10	51 ± 9
	Reactive Orange	333 µg		154 ± 11	42 ± 6
	DYPR 1410	1000 µg		199 ± 13	53 ± 6
		2000 µg		200 ± 18	59 ± 5^D M
		3000 µg		191 ± 14^D M	74 ± 9^D M
		4000 µg		187 ± 8^D M	77 ± 1^D M
		5000 µg		180 ± 16^D M	86 ± 5^D M
	2-AA	2.5  µg		1624 ± 182
	2-AA	10 µg			820 ± 47

* Data generated in experiment IIb - the data in the solvent control of strain WP2 uvrA in experiment IIa did not reach the lower limit of the historical control data range this part of  was invalid and therefore repeated in experiment IIb. Also in this experiment the data of the solvent control is in the lower range of the historicol controls (mean +/- SD: 52 +/- 6.7) but above the minimum value of the range.

 

Key to Plate Postfix Codes:              

M: Manuel Count

D:  Densely Colored Plate

 

Key to positive controls:

NaN3: sodium azide

4 -NOPD: 4 -nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

2-AA: 2 -aminoanthracene

Congo red

Conclusions:

Reactive Orange DYPR 1410 did not induce a reproducible biologically relevant increase in the number of revertants exceeding the threshold of at least twice (strains Salmonella typhimurium TA 98, TA 100, and Escherichia coli WP2 uvrA) or thrice (strains Salmonella typhimurium TA 1535 and TA 1537) the colony count of the corresponding solvent control either in the presence or absence of metabolic activation systems (rat or hamster liver S9 mix).
Therefore, Reactive Orange DYPR 1410 is considered not to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of Reactive Orange DYPR 1410 to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II, IIa and IIb, which is reported as part of experiment IIa) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in four independent experiments. Experiments I and II were performed with and without liver microsomal activation, Experiment IIa and IIb with liver microsomal activation only. Experiment I was performed with induced rat liver S9 mix as an exogenous metabolic activation system, experiments II, IIa and IIb were performed with non-induced hamster liver S9 mix. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:        3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:                                 33; 100; 333; 1000; 2500; and 5000 µg/plate

To validate the minimal increases in strains TA 100 (1.75-fold) and WP2 uvrA (2-06-fold) in the presence of non-induced hamster liver S9 mix, a confirmatory experiment IIa was performed as a pre-incubation assay with both strains in the presence of non-induced hamster liver S9 mix. Since the data in the solvent control of strain WP2 uvrA did not reach the lower limit of the historical control data range this part of experiment IIa was invalid and therefore repeated as experiment IIb. The following concentrations were tested in experiment IIa and IIb:

333; 1000; 2000; 3000; 4000; and 5000 µg/plate

No precipitation of the test item occurred in the overlay agar in the test tubes and in the overlay agar on the incubated agar plates, but an intense coloring was observed in the overlay agar on the incubated agar plates in experiment II at 5000 µg/plate, in experiment IIa from 3000 to 5000 µg/plate and in experiment IIb from 2000 to 5000 µg/plate which prevented automatic counting. These plates had to be evaluated manually.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabolic activation.

In experiment I no substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Reactive Orange DYPR 1410 at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
In experiment II a single minial increase by 2.06-fold in revertant colony numbers was observed following treatment with Reactive Orange DYPR 1410 in experiment II in strain WP2 uvrA in the presence of non-induced hamster S9 mix with a high variability within the replicated (+/- 13) in the densely coloure plates. The threshold of twofold the number in revertant colony count of the corresponding solvent control was exceeded at 5000 µg/plate. Furthermore, a minor increase in revertant colony count, not reaching the threshold of two-fold the number in revertant colony count of the corresponding solvent control, occurred in strain TA 100 in the presence of non-induced hamster S9 mix.
To validate both increases observed in experiment II, a confirmatory experiment IIa/b was performed as a pre-incubation assay with six adequately spaced concentrations using strains TA 100 and WP2 uvrA with non-induced hamster liver S9 mix. The increase in strain TA 100 was not reproduced in experiment IIa and can therefore be stated as biologically irrelevant. Also in  strain WP2 uvrA the mutation frequency did not exceed the 2-fold threshold at 5000 µg/plate, although it reached exactly 2-fold. This is attributed to the fact that the solvent control (42.7 +/- 8.0) is also in this repeat experiment below the mean +/- SD value (52 +/- 6.7) of the historical control data, but above the minimum value of the control solvent data for this strain.

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease in induced revertant colonies.

Hence, Reactive Orange DYPR 1410 did not induce a reproducible biologically relevant increase in the number of revertants exceeding the threshold of twice (strains Salmonella typhimurium TA 98, TA 100, and Escherichia coli WP2 uvrA) or thrice (strains Salmonella typhimurium TA 1535 and TA 1537) the colony count of the corresponding solvent control either in the presence or absence of metabolic activation systems (rat or hamster liver S9 mix).
Therefore, Reactive Orange DYPR 1410 is considered not to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Reactive Orange DYPR 1410 is not clastogenic or aneugenic in the mouse micronucleus test.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 April 1999 - 29 April 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species of animals: mouse
Strain of animals: HsdWin:NMRI
Origin (supplier) of animals: Harlan Winkelmann GmbH Gartenstrasse 27, 33178 Borchen
Animal identification: fur marking with KMnO4 and cage numbering

Body weight at start of study

Male animals
mean= 37,0 g
min= 33 g
max= 44 g
n= 15

Female animals:
mean = 25,7 g
min= 23 g
max= 28 g
n= 15

Age at the start of study; male/female animals approximately 8 weeks
Animal maintenance: in fully air-conditioned rooms in makrolon cages type 3 (five animals per cage) on soft wood granulate
Room temperature: 22 ± 3°C
Relative humidity: 50 ± 20 %
Lighting times: 12 hours daily
Acclimatization: 5 days under study conditions
Food: rat/mice diet ssniff® R/M-H (V 1534), ad libitum ssniff GmbH, Postbox 2039, 59480 Soest
Water: tap water in plastic bottles, ad libitum

Route of administration:

oral: gavage

Vehicle:

Deionized water

Details on exposure:

Test groups
In a preliminary dose range finding study, oral administration of 2000 mg Reaktiv Orange DYPR 1410 per kg body weight did not cause any toxic effects in male and female mice over an observation period of 7 days . This dose level was therefore the regularly limit dose, selected as the highest dose for the main study.

The test substance was administered twice at an interval of 24 hours orally by gavage to the test animals at a dose of 2000 mg per kg body weight. The vehicle, deionized water, was administered in the same way to the negative control groups. The study included a concurrent positive control using Endoxan which was administered once orally by gavage at a dose of 50 mg per kg body weight.

Following dosing, the animals were examined regularly for mortality and clinical signs of toxicity.

Duration of treatment / exposure:

48 hours

Frequency of treatment:

Twice

Post exposure period:

24 hours

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

administered twice with an interval of 24 h at a volume of 10 ml/kg bw

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (Endoxan®) was used as positive control substance and was administered once orally at a dose of 50 mg per kg body weight.

Tissues and cell types examined:

polychromatic erythrocytes

Details of tissue and slide preparation:

Animals were killed by carbon dioxide asphyxiation 24 hours after dosing. For each animal, about 3 ml fetal bovine serum was poured into a centrifuge tube. Both femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was farmed. The mixture was then centrifuged for 5 minutes at approx. 1200 rpm, after which almost all the supernatant was discarded. One drop of the thoroughly mixed sediment was smeared onto a cleaned slide, identified by project code and animal number and air-dried for about 12 hours.
Staining was performed as follows:
- 5 minutes in methanol
- 5 minutes in May-Grunwald's solution
- brief rinsing twice in distilled water
- 10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2 (Weise)
- rinsing in distilled water
- drying
- coating with Entellan®

Evaluation criteria:

Both biological and statistical significances were considered together for evaluation purposes.
A substance is considered positive if there is a significant increase in the number of micronucleafed polychromatic erythrocytes compared with the concurrent negative control group. A test substance producing no significant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

2000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. In addition, the ratio of polychromatic erythrocytes to 200 total erythrocytes was determined. Main parameter for the statistical analysis, i.e. validity assessment of the study and mutagenicity of the test substance, was the proportion of polychromatic erythrocytes with micronuclei out of the 2000 counted erythrocytes. All bone marrow smears for evalua¬tion were coded to ensure that the group from which they were taken remained unknown to the investigator.
A one-sided Wilcoxon-Test was evaluated to check the validity of the study. The study was considered as valid in case the proportion of polychromatic erythrocytes with micronuclei in the positive control was significantly higher than in the negative control (p=0.05).
If the validity of the study had been shown the following sequential test procedure for the examination of the mutagenicity was applied: Based on a monotone dose-relationship one-sided Wilcoxon tests were performed starting with the highest dose group. These test were performed with a multiple level of significance of 5%

Statistics:

A one-sided Wilcoxon-Test was evaluated to check the validity of the study

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

No signs of toxicity were observed, but faeces and urine were red discolored up to 24 hours after second application

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Mice were treated twice with 2000 mg Reaktiv Orange DYPR 1410 per kg body weight to study the induction of micronuclei in bone marrow cells.
All animals survived after treatment. No signs of toxicity were observed, but feces and urine were red discolored up to 24 hours after second application.
The dissection of the animals revealed no test substance related macroscopic findings.

The bone marrow smears were examined for the occurrence of micronuclei in red blood cells.
The incidence of micronucleated polychromatic erythrocytes in the dose group of Reaktiv Orange DYPR 1410 was within the normal range of the negative control groups. No statistically significant increase of micronucleated polychromatic erythrocytes was observed. The ratio of polychromatic erythrocytes to total erythrocytes remained essentially unaffected by the test compound and was not less than 20% of the control values.

Cyclophosphamide (Endoxan) induced a marked and statistically significant increase in the number of polychromatic erythrocytes with micronuclei, thus indicating the sensitivity of the test system.

The present study was conducted in compliance with OECD Guideline For Testing Of Chemicals, 474 Genetic Toxicology, Mammalian Erythrocyte Micronucleus Test, Adopted 21^st, July 1997 and U.S. EPA: OPPTS 870.5395 Health Effects Test Guidelines; Mammalian Erythrocyte Micronucleus Test, August 1998 and EEC Directive 92/69, L 383 A, Annex B. 12., p. 154 -156

 

The micronucleus test was carried out with Reaktiv Orange DYPR 1410. The test compound was suspended in deionized water and was given twice at an interval of 24 hours as an orally dose of 2000 mg per kg body weight to male and female mice, based on the results of a previous dose range finding assay (see preliminary study). According to the test procedure the animals were killed 24 hours after administration.

 

Cyclophosphamide was used as positive control substance and was administered once orally at a dose of 50 mg per kg body weight.

The number of polychromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment withReaktiv Orange DYPR 1410and was not less than 20% of the control value.

 

Cyclophosphamide induced a marked statistically significant increase in the number of poly-chromatic cells with micronuclei, indicating the sensitivity of the test system. The ratioof polychromatic erythrocytes to total erythrocytes was not changed to a significant extent.    

 

Under the conditions of the present study the results indicate that Reaktiv Orange DYPR 1410 is not cytogenic in the micronucleus test.

Conclusions:

The results lead to the conclusion that Reaktiv Orange DYPR 1410 did not lead to a substantial increase of micronucleated polychromatic erythrocytes and is not cytogenic in the micronucleus test.

Executive summary:

A micronucleus test was conducted according to OECD Guideline 474 with Reaktiv Orange DYPR 1410. The test compound was suspended in deionized water and was given twice at an interval of 24 hours as an dose of 2000 mg per kg body weight to male and female mice, based an the results of a previous dose range finding assay. According to the test procedure the animals were killed 24 hours alter administration.

Endoxan® was used as positive control substance and was administered once orally at a dose of 50 mg per kg body weight.

The number of polychromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment with Reaktiv Orange DYPR 1410 and was not less than 20% of the control value.

Endoxan® induced a marked statistically significant increase in the number of poly-chromatic cells with micronuclei, indicating the sensitivity of the test system. The ratio of polychromatic erythrocytes to total erythrocytes was not changed to a significant extent.

Under the conditions of the present study the results indicate that Reaktiv Orange DYPR 1410 is not clastogenic or aneugenic in the micronucleus test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Kirkland et al (2005) demonstrated an extremely high false-positive rate for in-vitro clastogenicity tests, particularly in mammalian cell tests, when compared to rodent carcinogenicity study results (Poth, A (2008)). “Certain characteristics of the commonly used rodent cell lines (CHO, CHL, V79, L5178Y, etc.), such as their p53 status, their karyotypic instability and their DNA repair deficiencies, are recognized as possibly contributing to the high rate of irrelevant positives. Also the need for exogenous metabolism with the cell systems is expected to contribute to these irrelevant positive findings, as metabolites produced by S9 used as a metabolic source in cell culture may be quite different from those produced by normal human liver metabolism.” Accordingly, “the high false-positive rate of the established in-vitro mammalian cell tests means that an increased number of compounds are subjected to earlier and additional in-vivo genotoxicity testing.”

Furthermore, it is well known that vinyl-sulphone compounds often result in false positive test results in in-vitro tests for clastogenicity (Dearfield KL et al. (1991); Warra TJ et al. (1990)). This is due to the fact that these chemical agents react via the Michael addition reaction. Chemical reactivity via Michael addition is essential for many of the uses for which these compounds are important. As in the currently assessed dye, Reactive Orange DYPR 1410, vinyl sulphone moieties are used in fiber-reactive dyes (MacGregor et at. (1980)). These compounds are known to deplete glutathione in in‑vitro test systems, in which the concentration of phase II enzymes is very low. Glutathione plays a role in the detoxification of many compounds. Conjugation with glutathione via Michael addition and subsequent excretion is the most common bio-elimination route for these compounds. Since in-vitro systems have low levels of glutathione, the glutathione depletion leads to a false positive result in the in-vitro test system, which is not the case in the in-vivo test system, where glutathione is present in adequate amounts.

Hence, the in-vivo test produces more reliable data for this kind of substance.

 

References

Dearfield KL et al. (1991). Genotoxicity in mouse lymphoma cells .of chemicals capable of Michael addition. Mutagenesis 1991;6(6):519-525

Kirkland et al (2005). Evaluation of the ability of a battery of three in-vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. I. Sensitivity, specificity and relative predictivity. Mutat Res. 2005 July 4;584(1–2):1–256

MacGregor et at. (1980). Mutagenicity tests of fabric-finishing agents in Salmonella typhimurium: fiber-reactive wool dyes and cotton flame retardants. Environ. Mutagenesis 1980;2:405-418

Poth, A (2008). Challenges in Testing for Genotoxycity. Genetic Toxicology and Cell Biology, RCC Cytotest Cell Research GmbH

Additional information

The bacterial reverse mutation tests showed that the substance does not have any mutagenic properties in different Salmonella and E. coli strains or in mammalian cells.

In the in-vitro chromosome aberration assay in V79 cells, apparent positive effects were obtained with and without S-9 mix at doses which induced precipitations. This false positive effect could either be due to the precipitations observed, due to the cell system used or due to a GSH depletion in the in-vitro test system.

It is a well documented effect for vinyl sulphone dyes to result in a false positive in vitro assay for chromosome aberration. This is due to GSH depletion within the in-vitro test system -with a very limited amount of phase 2 enzymes- caused by the Michael Addition at the vinyl sulphone structures. 

As the in vivo micronucleus test in mice was clearly negative at the limit dose level Reactive Orange DYPR 1410 is not considered to be clastogenic or aneugenic.

Justification for classification or non-classification

The above studies have all been ranked reliability 1 according to the Klimisch et al system. This ranking was deemed appropriate because the studies were conducted to GLP an in compliance with agreed protocols. Sufficient dose ranges and numbers are detailed; hence it is appropriate for use based on reliability and animal welfare grounds. As the effects are considered adaptive rather than toxicological, no classification is proposed.

The above results triggered no classification under the Dangerous Substance Directive (67/548/EEC) and the CLP Regulation (EC No 1272/2008). No classification for prolonged effects is therefore required.