reaction mass of: trisodium 3-(5-(2,6-difluoropyrimidin-4-ylamino)-2-sulfonatophenylazo)-5-(4-fluoro-6-morpholin-4-yl-1,3,5-triazin-2-ylamino)-4-hydroxy-2,7-naphthalenedisulfonate; trisodium 3-(5-(4,6-difluoropyrimidin-2-ylamino)-2-sulfonatophenylazo)-5-(4-fluoro-6-morpholin-4-yl-1,3,5-triazin-2-ylamino)-4-hydroxy-2,7-naphthalenedisulfonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance has been tested negative in an Ames test (OECD 471) and in an in vitro Chromosome aberration test (OECD 473) with metabolic activation.

Without metabolic activation, the test substance was positive in the in vitro Chromosome aberration test (OECD 473). As seen in the in vivo test, this was a false positive effect most likely due to the depletion of phase II enzymes.

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:

1998-Jun-10 to 1998-Jun-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

none

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (rat liver)

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 50, 160, 500, 1600, 5000 µg/plate
Concentration range in the main test (without metabolic activation): 50, 160, 500, 1600, 5000 µg/plate

Vehicle / solvent:

Vehicle(s)/solvent(s) used: double-distilled water, stability confirmed over 15 days

Untreated negative controls:

yes

Remarks:

untreated control

Negative solvent / vehicle controls:

yes

Remarks:

0 µg/plate

Positive controls:

yes

Remarks:

Without metabolic activation: sodium-azide for strain TA 100 and TA 1535, 9-aminoacridine for strain TA 1537, 2-nitrofluorene for strain TA 98, MNNG for strain WP2uvrA. With metabolic activation: 2-aminoanthracene for all strains

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: incubaton for 48 h

SELECTION AGENT (mutation assay): revert mutation

NUMBER OF REPLICATIONS:
-3 plates per strain per concentration

Evaluation criteria:

The test compound is classified as mutagenic if:
- The test compound 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.
OR
- The test compound induces a dose-related 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 in at least two to three concentrations of the test compound at complete bacterial background lawn.

Statistics:

means and standard deviations reported

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

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:

not determined

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:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Based on the results in an Ames test, Reactive Red FC73270 is not mutagenic with or without metabolic activation (5 strains tested up to 5000 µg/plate).

Executive summary:

In a reverse gene mutation assay in bacteria, strains TA 98, 100, 1535, 1537 of S. typhimurium and WP2 uvrA E. coli were exposed to Reactive Red FC 73270 at concentrations of 50, 160, 500, 1600, 5000 µg/plate in the presence and absence of mammalian metabolic activation (S9 mix).  There was no evidence of induced mutant colonies over background. The positive controls induced the appropriate responses in the corresponding strains. Based on the results, Reactive Red FC73270 is not classified as mutagenic accroding to the CLP Regulation.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998-04-21 to 1998-05-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5375 (In Vitro Mammalian Chromosome Aberration)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

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 from rat liver

Test concentrations with justification for top dose:

1) Concentration range in the 3h test (with and without metabolic activation): 158.1, 500, 1581, 5000 µg/ml
2) Concentration range in the 20h test (without metabolic activation): 125, 250, 500, 750, 1000, 1750, 2500 µg/ml

Vehicle / solvent:

culture medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

DURATION
- Preincubation period:
- Exposure duration: The first experiment with 3 hours treatment time of the test substance was performed in the presence and in the absence of S9-mix. Cultured cells were seeded onto slides (2 per dose level) then treated for either 3 hours (with and without S9-mix in the first experiment) or for 20 hours (without 89-mix in the second experiment).

STAIN (for cytogenetic assays):
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®

NUMBER OF CELLS EVALUATED: The slides were coded and 25 ~ 100 metaphases per experimental group and cell culture were examined.

DETERMINATION OF CYTOTOXICITY
- Method: The meta phases 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 intrachanges, dicentrics, pulverization and ring formation. Furthermore the rate 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.

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 rate (without gaps).
- The test compound is classified as non-mutagenic if the tests are negative bath with and without metabolic activation.

Statistics:

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

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

500 µg/ml (20h); 5.000 µg/ml (3h)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

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.

Evaluation of the solubility of the test substance in cell culture medium showed that 5000 µg/ml was the highest concentration tolerated by the test system. Microscopically visible precipitation of the test compound was observed 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 10 µg/ml.
These results show that the test substance was only slight toxic to the,V79 cells in the absence and in the presence of metabolic activation after 3 hours treatment time. Survival declined in a dose-related manner reaching 63.7 %, respectively 66.1 % of the solvent control value.
Following treatment for 20 hours in the absence of S9 metabolic activation, toxicity was observed at 500 µg/ml and above reaching 8.8 % of the solvent control value at the highest dose level, 5000 µg/ml.

Chromosome aberrations in V79 cells (First experiment) Test compound: Reactive Red FC 73270 Preparation: 3 h treatment time (100 metaphases were analysed)
Dose [µg/ml]	Culture without S9-mix	No. of phases with aberrations		No. of aberrations		MI %
		incl. gaps	excl. gaps	incl. gaps	excl. gaps
0	S+/1	3	3	3	3	6.7
0	S+/2	1	1	1	1	8.9
Total		4	4	4	4
500	1+/1	0	0	0	0	7.6
500	1+/2	1	1	1	1	7.6
Total		1	1	1	1
1581.1	2+/1	2	1	2	1	6.8
1581.1	2+/2	2	1	2	1	5.3
Total		4	2	4	2
5000	3+/1	6	2	6	2	6.2
5000	3+/2	4	3	4	3	6.0
Total		10	5	10	5
1750	#P+/1	11	11	15	15	6.6
1750	#P+/2	11	11	12	12	6.7
Total		22*	22*	27*	27*
S = Solvent control (Medium MEM) P = positive control (EMS) * = p ≤ 0.05 # = only 50 metaphases were evaluated

Chromosome aberrations in V79 cells (First experiment) Test compound: Reactive Red FC 73270 Preparation: 3 h treatment time (100 metaphases were analysed)
Dose [µg/ml]	Culture without S9-mix	No. of phases with aberrations		No. of aberrations		MI %
		incl. gaps	excl. gaps	incl. gaps	excl. gaps
0,0	S+/1	4	2	4	2	8,8
0,0	S+/2	1	0	1	0	6,6
Total		5	2	5	2
500,0	1+/1	0	0	0	0	7,2
500,0	1+/2	3	2	3	2	3,6
Total		3	2	3	2
1581,1	2+/1	2	2	2	2	7,1
1581,1	2+/2	4	2	4	2	4,8
Total		6	4	6	4
5000,0	3+/1	7	6	8	7	6,2
5000,0	3+/2	2	2	2	2	9,0
Total		9	8	10	9
3,0	§P+/1	11	10	14	12	2,9
3,0	§P+/2	10	10	21	17	4,4
Total		21*	20*	35*	29*
S = Solvent control (Medium MEM) P = positive control (CPA) * = p ≤ 0.05 # = only 25 metaphases were evaluated

Chromosome aberrations in V79 cells (Second experiment) Test compound: Reactive Red FC 73270 Preparation: 20 h treatment time (100 metaphases were analysed)
Dose [µg/ml]	Culture without S9-mix	No. of phases with aberrations		No. of aberrations		MI %
		incl. gaps	excl. gaps	incl. gaps	excl. gaps
0,0	S-/1	1	1	1	1	10,9
0,0	S-/2	2	2	2	2	8,9
Total		3	3	3	3
250,0	1-/1	0	0	0	0	8,8
250,0	1-/2	4	3	4	3	8,3
Total		4	3	4	3
500,0	2-/1	6	3	6	3	5,0
500,0	2-/2	6	2	6	2	6,1
Total		12*	5	12*	5
1000,0	3-/1	25	17	33	21	2,8
1000,0	3-/2	17	15	21	18	2,4
Total		42*	32*	54*	39*
500,0	#P-/1	8	8	12	12	4,9
500,0	#P-/2	11	8	13	10	5,4
Total		19*	16*	25*	22
S = Solvent control (Medium MEM) P = positive control (EMS) * = p ≤ 0.05 # = only 50 metaphases were evaluated 1^1= only one chromosome of the methaphase was disintegrated

Conclusions:

In conclusion Reactive Red FC 73270 does induce chromosome mutations (=aberrations) in V79 Chinese hamster cells in absence of metabolic activation system, under the experimental conditions described.
According to the chromosome aberration test system in vitro, Reactive Red FC73270 was mutagenic.

Executive summary:

In a mammalian cell cytogenetics assay [Chromosome aberration], V79 hamster cells were exposed to Reactive Red FC73270 at a concentration range of 158.1, 500.0, 1581.0, 5000.0 ug/ml (in the 3h test (with and without metabolic activation): ; concentration range in the 20h test (without metabolic activation): 125, 250, 500, 750, 1000, 1750, 2500 µg/ml.

Reactive Red FC73230 was tested up to the limit concentration of 5.000 µg/ml. Positive controls induced the appropriate response. There was evidence for chromosome aberration induced over background at >= 500 µg/ml (20h, with and without metabolic activation). Cytotoxic effects have been reported at 1000 µg/ml (20h, without metabolic activation). Under conditions of this test, Reactive Red FC73230 was genotoxic in V79 Chinese hamster cell lines without metabolic activation.

According to the chromosome aberration test system in vitro, Reactive Red FC73270 was mutagenic in V79 Chinese hamster cell lines.

This study is classified as acceptable and satisfies the requirement for Test Guideline OECD 473 for in vitro cytogenetic mutagenicity data. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test substance has been tested negative in an in vivo Erythrocyte Micronucleus Test in NMRI mice (OECD 474).

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998-08-17 to 1998-09-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)

Qualifier:

according to guideline

Guideline:

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

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:

TEST ANIMALS
- Source: Harlan Winkelmann Gartenstr. 27; D-33178 Borchen
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: M=34.6g; F=27.g
- Assigned to test groups randomly: yes, randomization schemes 98.0665 and 98.0666
- Fasting period before study:- Housing: in fully air-conditioned rooms in makrolon cages type 3 (five animals per cage) on soft wood granulate
- Diet (e.g. ad libitum): rat/mice diet ssniff RIM-H (V 1534), ad libitum ssniff GmbH, Postbox 2039, 59480 Soest
- Water (e.g. ad libitum): tap water in plastic bottles, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22± 3 degrees C
- Humidity (%): 50± 20%
- Light/dark cycles: 12 hours daily

Route of administration:

oral: gavage

Vehicle:

deionized water

Details on exposure:

The test substance was administered once orally by gavage to the test animals at doses of 0 or 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:

According to the test procedure the animals were killed 24 or 48 hours after administration

Frequency of treatment:

once

Post exposure period:

24 or 48h

Dose / conc.:

2 000 mg/kg bw/day

No. of animals per sex per dose:

Male: 0 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 0 mg/kg; No. of animals: 5; Sacrifice time: 48 hours
Male: 2000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 2000 mg/kg; No. of animals: 5; Sacrifice time: 48 hours
Female: 0 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 0 mg/kg; No. of animals: 5; Sacrifice times: 48 hours
Female: 2000 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 2000 mg/kg; No. of animals: 5; Sacrifice times: 48 hours

Control animals:

yes, concurrent vehicle

Positive control(s):

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.

Tissues and cell types examined:

bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In a preliminary dose range finding study, oral administration of 2000 mg Reactive Red FC73270 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.

DETAILS OF SLIDE PREPARATION: Animals were killed by carbon dioxide asphyxiation 24 or 48 hours after dosing. For each animal, about 3 ml fetal bovine serum was poured into a centrifuge tube. Both femora were removed and the banes freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. 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-Grünwald' 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

METHOD OF ANALYSIS:
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.

Evaluation criteria:

A substance is considered as positive if there is a significant dose-related increase in the number of micronucleated polychromatic erythrocytes for at least one of the time points compared with the concurrent negative control group. A test substance producing no significant dose-related increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Statistics:

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

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

All animals survived after treatment, no signs of toxicity were observed besides observingred-colored feces. Upon dissection there were no macroscopic findings.

Sex	Dose [mg/kg b.w.]	killing time	Number of animals	Poly counted	Poly/Ery Mean	Poly/Ery SD Mean	Poly with MN Mean	Poly with MN [%] Mean	Poly with MN SD Mean	Mut. I.
pooled	0 - Control	24 h	10	2000	0,50	0,07	2,00	0,1	0,04	1,0
pooled	2000	24 h	10	2000	0,49	0,05	1,70	0,1	0,05	0,9
pooled	50 - Endoxan	24 h	10	2000	0,49	0,10	59.70*	3,0	0,64	29,9
Mut. I. = Mutagenic Index Control = Vehicle (deionized water) * = significantly different from control (p < 0.05)

Conclusions:

Interpretation of results (migrated information): negative
Reactive Red FC73270 did not lead to a substantial increase of micronucleated polychromatic erythrocytes and is not mutagenic in the micronucleus test under the conditions.

Executive summary:

In a NMRI mouse bone marrow micronucleus assay, 5 animals/sex/dose were treated via oral gavage with Reactive Red FC73270 at doses of 0 or 2000 mg/kg bw.  Bone marrow cells were harvested at 24 or 48 hours post-treatment.  The vehicle was deionized water.

There were no signs of toxicity during the study. Reactive Red FC73270 was tested at an adequate dose based on previous studies. The positive control induced the appropriate response. There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow.

This study is classified as acceptable and satisfies the requirement for Test Guideline OECD 474 for in vivo cytogenetic mutagenicity data.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In a reverse gene mutation assay in bacteria, strains TA 98, 100, 1535, 1537 of S. typhimurium and WP2 uvrA E. coli were exposed to Reactive Red FC 73270 at concentrations of 50, 160, 500, 1600, 5000 µg/plate in the presence and absence of mammalian metabolic activation (S9 mix).  There was no evidence of induced mutant colonies over background. The positive controls induced the appropriate responses in the corresponding strains. Based on the results, Reactive Red FC73270 is not classified as mutagenic accroding to the CLP Regulation.

In an in vitro Chromosome aberration assay (OECD 473), V79 hamster cells were exposed to the test item for 3h & 20h up to the limit concentration of 5000 µg/ml. There was evidence for chromosome aberration induced over background at 500 µg/ml (20h, without metabolic activation). Cytotoxic effects have been reported after exposure to 1000 µg/ml (20h, with and without metabolic activation). Under conditions of this test, Reactive Red FC73270 was genotoxic in V79 Chinese hamster cell lines without metabolic activation.

Finally, Reactive Red FC73270 was tested in a NMRI mouse bone marrow micronucleus assay at concentrations of up to 2000 mg/kg bw. Bone marrow cells were harvested at 24 or 48 hours post-treatment.  There were no signs of toxicity during the study. Reactive Red FC73270 was tested at an adequate dose based on previous studies. The positive control induced the appropriate response. There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow.

In a weight of evidence, Reactive Red FC73270 is not considered to be genotoxic.

Justification for classification or non-classification

The test item is not considered genotoxic based on a weight of evidence assessment of the results of an appropriate testing battery.