reaction mass of: tetrasodium 4-amino-6-(5-(2,6-difluoropyrimidin-4-ylamino)-2-sulfonatophenylazo)-5-hydroxy-3-(4-(sulfatoethylsulfonyl)phenylazo)naphthalene-2,7-disulfonate;tetrasodium 4-amino-6-(5-(4,6-difluoropyrimidin-2-ylamino)-2-sulfonatophenylazo)-5-hydroxy-3-(4-(2-sulfatoethylsulfonyl)phenylazo)naphthalene-2,7-disulfonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reactive Navy FC 63805 is considered not to be genotoxic based on an bacteria reverse mutation and a chromosome aberration assay with the test substance and an UDS test in-vitro with a close structural analogue.

Link to relevant study records

Reference

Reference 1

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:

1998-May-12 to 1998-Jun-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix aus Rattenleber "ENGLISH" S9-mix made form rat liver

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 4, 20, 100, 500, 2500, 5000 µg/plate
Concentration range in the main test (without metabolic activation): 4, 20, 100, 500, 2500, 5000 µg/plate

Vehicle / solvent:

Solvent: Dest. Wasser

Untreated negative controls:

yes

Remarks:

untreated control

Negative solvent / vehicle controls:

yes

Remarks:

0 ug/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. 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

Key result

Species / strain:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Reactive Navy FC 63805 is not mutagenic with or without metabolic activation based on the Ames test.

Executive summary:

In a reverse gene mutation assay in bacteria, strains TA 98, 100, 1535, 1537 of S. typhimurium were exposed to Reactive Navy FC 63805 at concentrations of 4, 20, 100, 500, 2500, 5000 µg/plate in the presence and absence of mammalian metabolic activation.  There was no evidence of induced mutant colonies over background. The positive controls induced the appropriate responses in the corresponding strains.

This study is classified as acceptable and satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

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 1998-05-20

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, 500.0, 1581.0, 5000.0 ug/ml
2) Concentration range in the 20h test (without metabolic activation): 125, 250, 500, 750, 1000 µg/ml
3) Concentration range in the 20h test (without metabolic activation): 500, 750 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 and third experiment). For the third assay two concentrations 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.

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)

Remarks:

3 h

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

20 h

Metabolic activation:

with

Genotoxicity:

negative

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.

The substance was negative after 3 h incubation both without and with the addition of a metabolic activation system (S9 mix). After 20 h incubation without S9 mix the aberration rate was statistically significantly increased only in the highest concentrations of 500 and 750 µg/ml including gaps. Since gaps are not associated with a break of the DNA double helix, but represent unstained DNA pieces due to disturbed chromatid condensation, the number of aberrant cells without gaps is decisive for the evaluation of a mutagenic effect. In the present case, this number was slightly higher than that of the control, but did not reach statistical significance. It is known that positive results in the CA test in vitro are of questionable biological relevance when effects occur at low survival rates.Reactive Navy FC 63805 led to a significantly reduced mitotic index at 20 h incubation at 500 µg/ml and above, i.e. the dye was cytotoxic. The slightly higher aberration rates in Exp. 1 compared to Exp. 2 correlated with a slightly stronger cytotoxicity. This correlation is an indication that the slightly higher aberration rates compared to the control were caused by the incipient cytotoxicity of the substance. They therefore do not point to a mutagenic potential.

In vitro chromosome aberration test on V79 cells mit Reactive Navy FC 63805

	20h, Exp. 1		20 h, Exp. 2
Dose level [µg/ml]	500	750	500	750
aberrant cells with gaps	7.5%*	9.0%*	4.5%*	7.5*
aberrant cells without gaps	5.5%	3.5%	3.0%	4.0%
Mitotic index	57%	36%	77%	59%

*statistically significant increase in aberration rate compared to control (p ≤ 0.05)

Chromosome aberrations in V79 cells (First experiment) Test compound: Reactive Navy Blue FC 63805 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	1	1	1	1	8,3
500,0	1+/2	1	1	1	1	5,5
Total		2	2	2	2
1581,1	2+/1	0	0	0	0	9,1
1581,1	2+/2	2	1	2	1	8,6
Total		2	1	2	1
5000,0	3+/1	2	1	4	3	9,1
5000,0	3+/2	5	4	6	5	6,2
Total		7	5	10	8
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) P = positive control (CPA) * = p ≤ 0.05 § = only 25 metaphases were evaluated 1^1= only one chromosome of the methaphase was disintegrated

Chromosome aberrations in V79 cells (Second experiment) Test compound: Reactive Navy Blue FC 63805 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
125,0	1-/1	1	1	1	1	8,7
125,0	1-/2	2	2	2	2	5,0
Total		3	3	3	3
250,0	2-/1	4	3	6	5	8,2
250,0	2-/2	2	2	2	2	10,8
Total		6	5	8	7
500,0	3-/1	8	6	11	7	6,3
500,0	3-/2	7	5	13	11	4,8
Total		15*	11	24*	18*
750,0	4-/1	6	2	6	2	3,4
750,0	4-/2	12	5	12	5	3,7
Total		18*	7	18*	7
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) P = positive control (EMS) * = p ≤ 0.05 # = only 50 metaphases were evaluated 1^1= only one chromosome of the methaphase was disintegrated

Chromosome aberrations in V79 cells (Third experiment) Test compound: Reactive Navy Blue FC 63805 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	0	0	0	0
0,0	S-/2	2	2	2	2
Total		2	2	2	2
500,0	3-/1	5	4	8	6
500,0	3-/2	4	2	5	3
Total		9*	6	13*	9
750,0	4-/1	8	5	10	6
750,0	4-/2	6	3	6	3
Total		14*	8	16*	9
400,0	#P-/1	10	10	13	13
400,0	#P-/2	9	9	10	9
Total		19*	19*	23*	22*
S = Solvent control (Medium) P = positive control (CPA) * = p ≤ 0.05 # = only 50 metaphases were evaluated 1^1= only one chromosome of the methaphase was disintegrated

Conclusions:

According to the chromosome aberration test system in vitro, Reactive Navy FC63805 was not mutagenic in V79 Chinese hamster cell lines.

Executive summary:

In a mammalian cell cytogenetics assay [Chromosome aberration], V79 hamster cells were exposed to Reactive Navy FC63805 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 µg/ml; and concentration range in the 20h test (without metabolic activation): 500, 750 µg/ml.

Reactive Navy FC63805 was tested up to the limit concentration of 5.000 ug/ml. Positive controls induced the appropriate response. There was a slightly increased incidence for chromosome aberration including gaps only at >= 500 µg/ml (20h, without metabolic activation) at dose levels with a distincly decreased mitotic index.. Under conditions of this test, Reactive Navy FC63805 was not genotoxic in V79 Chinese hamster cell lines with or without metabolic activation.

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

Reference 3

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20-Mar-2001 to 16-May-2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Qualifier:

according to guideline

Guideline:

EU Method B.18 (DNA Damage and Repair - Unscheduled DNA Synthesis - Mammalian Cells In Vitro)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5550 - Unscheduled DNA Synthesis in Mammalian Cells in Culture

GLP compliance:

yes

Type of assay:

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Species / strain / cell type:

hepatocytes:

Metabolic activation:

not applicable

Metabolic activation system:

hepatic metabolizing systems

Test concentrations with justification for top dose:

5000.0 , 30000, 1000.0, 300.0, 100.0, 30.0, 10.0, 3.0, 1.0, 0.3 and 0.1 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:

2-acetylaminofluorene

Details on test system and experimental conditions:

Approximately 1.5 x 10^5 cells were seeded in a 35 mm culture dish. The cells were allowed to attach with approx. 5 % CO2 for approximately 2 hours at 37 °C. Two culture dishes were used for each experimental point.
The test compound was dissolved in cell culture medium. The incubation of the test and control substances at various concentrations was performed at 37 °C for 16 - 20 h. Tritiated thymidine was given to the cell culture immediately after introduction of the test compound.
In order to get a better grain quantification by enlarging the nucleus, the cells were treated with 1 % sodium citrate.
The incorporation of ['H] thymidine was determined by counting the number of grains in the nucleus and the number of grains in a nucleus sized area in the cytoplasm. The difference between these examination points is expressed as net grains per nucleus. Heavily labeled S-phase cells were not included in the count. 50 cells were evaluated per slide.

Key result

Species / strain:

hepatocytes: primary rat hepatocytes

Metabolic activation:

not applicable

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(>= 300 µg/ml)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Observation:
Up to the highest evaluated concentration of 100 µg/ml no relevant reproducible increase in the incorporation of (3h) thymidine in the cells was obtained.

Conclusions:

Interpretation of results:
negative

Up to the highest evaluated concentration of 100.0 µg/ml no relevant reproducible increase in the incorporation of [3H] thymidine in the cells was obtained in either of the experiments.
Positive controls showed a distinct increase in incorporation of [3H] thymidine in the cells, thus indicating the sensitivity of the assay

Executive summary:

In an unscheduled DNA synthesis assay (UDS), primary rat hepatocyte cultures were exposed to a structural analogue of Reactive Navy FC63805.The assay was performed in two independent experiments with freshly prepared rat hepatocytes. The test compound was dissolved in cell culture medium and tested at the following concentrations:

5000, 3000, 1000, 300, 100, 30, 10, 3, 1, 0.3 and 0.1 µg/mL

As a sign of cytotoxic effects, the hepatocyte nuclei of the four highest concentrations showed incorporation of the test substance indicating a strongly decreased vitality. These concentrations were therefore not evaluated.

Up to the highest evaluated concentration of 100 µg/mL no relevant reproducible increase in the incorporation of [³H] thymidine in the cells was obtained in either of the experiments. Appropriate reference genotoxic substances used as positive controls showed a distinct increase in incorporation of [³H] thymidine in the cells, thus indicating the sensitivity of the assay.

There was no evidence that unscheduled DNA synthesis, as determined by radioactive tracer procedures was induced. The substance is therefore considered to be not genotoxic in this UDS assay.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 482 for other genotoxic mutagenicity data. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Reactive Navy FC 63805 has been tested negative in an in vivo Erythrocyte Micronucleus Test in NMRI mice (OECD 474). In addition, negative results from an in vitro Unscheduled DNA Synthesis assay in Mammalian Cells (OECD 482) has been provided for a structural analogue.

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:

1998-02-23 to 1998-05-19

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=36.6g; F=29.3g
- Assigned to test groups randomly: yes, randomization schemes 98.0371 and 98.0372
- 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: 1 week

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 200, 600 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

Remarks:

Doses / Concentrations:
200, 600 or 2000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

Male: 200 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 600 mg/kg; No. of animals: 5; Sacrifice time: 24 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: 200 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 600 mg/kg; No. of animals: 5; Sacrifice times: 24 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):

Yes, 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 Navy Blue 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

Key result

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 observing blue-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	0,45	0,08	1,20	0,1	0,03	1,0
pooled	200	24 h	10	2000	0,51	0,10	1,30	0,1	0,08	1,1
pooled	600	24 h	10	2000	0,44	0,04	1,50	0,1	0,40	1,3
pooled	2000	24 h	10	2000	0,51	0,05	1,30	0,1	0,40	1,1
pooled	50 - Endoxan	24 h	10	2000	0,50	0,08	63.80*	3,2	1,68	53,2
pooled	0 - Control	48 h	10	2000	0,45	0,04	1,50	0,1	0,08	1,0
pooled	2000	48 h	10	2000	0,47	0,08	1,00	0,1	0,06	0,7
Mut. I. = Mutagenic Index Control = Vehicle (deionized water) * = significantly different from control (p < 0.05)

Conclusions:

Reactive Navy FC63805 did not cause an increase in 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 Navy FC63805 at doses of 0, 200, 600 or 2000 mg/kg bw.  Bone marrow cells were harvested at 24 or 40 hours post-treatment.  The vehicle was deionized water.

There were no signs of toxicity during the study. Reactive Navy FC63805 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)

Mode of Action Analysis / Human Relevance Framework

In the chromosome aberration test in vitro (CA test) on V79 cells, the substance was not mutagenic after 3 h incubation both without and with the addition of a metabolic activation system (S9 mix). After 20 h incubation without S9 mix the aberration rate was statistically significantly increased only in the highest concentrations of 500 and 750 µg/ml including gaps. Since gaps are not associated with a break of the DNA double helix, but represent unstained DNA pieces due to disturbed chromatid condensation, the number of aberrant cells without gaps is decisive for the evaluation of a mutagenic effect. In the present case, this number was slightly higher than that of the control, but did not reach statistical significance.

It is known that positive results in the CA test in vitro are of questionable biological relevance when effects occur at low survival rates [1-4]. Reactive Navy FC 63805 led to a significantly reduced mitotic index at 20 h incubation from 500 µg/ml, i.e. the dye was cytotoxic (Table 1).

Table 1. In vitro chromosome aberration test on V79 cells mit Reactive Navy FC 63805

	20h, Exp. 1		20 h, Exp. 2
Dose level [µg/ml]	500	750	500	750
aberrant cellswith gaps	7.5%*	9.0%*	4.5%*	7.5*
aberrant cellswithout gaps	5.5%	3.5%	3.0%	4.0%
Mitotic index	57%	36%	77%	59%

*statistically significant increase in aberration rate compared to control (p ≤ 0.05)

The slightly higher aberration rates in Exp. 1 compared to Exp. 2 correlated with a slightly stronger cytotoxicity. This correlation is an indication that the slightly higher aberration rates compared to the control were caused by the incipient cytotoxicity of the substance. They therefore do not point to a mutagenic potential.

Overall, the in vitro CA test is therefore to be assessed as negative, there is no mutagenic potential of Reactive Navy FC 63805.

 

The Ames test was performed on five different bacterial strains without and with the addition of a metabolic activation system (rat liver S9 mix). Reactive Navy FC 63805 was clearly not mutagenic in all cases.

The dye was further tested in vivo in the micronucleus test with the limit dose of 2000 mg/kg. In this test, Reactive Navy FC 63805 was clearly not mutagenic.

References

(1) Hilliard CA, Armstrong MJ, Bradt CI, Hill RB, Greenwood SK, Galloway SM. Chromosome aberrations in vitro related to cytotoxicity of nonmutagenic chemicals and metabolic poisons. Environ. Mol. Mutagen. 31: 316-326, 1998.

(2) Galloway SM, Miller JE, Armstrong MJ, Bean CL, Skopek TR, Nichols WW. DNA synthesis inhibition as an indirect mechanism of chromosome aberrations: comparison of DNA-reactive and non-DNA reactive clastogens. Mutat. Res. 400: 169-186, 1998.

(3) ICH Topic S2A, Genotoxicity: Guidance on specific aspects of regulatory genotoxicity tests for pharmaceuticals, international conference on harmonisation of technical requirements for registration of pharmaceuticals for human use, harmonised tripartite guideline, September, 1995.

(4) Kirkland D. Chromosome aberration testing in genetic toxicology – past, present and future. Mutat. Res. 404: 173-185, 1998.

Additional information

In a reverse gene mutation assay in bacteria, strains TA 98, 100, 1535, 1537 of S. typhimurium were exposed to Reactive Navy FC 63805 at concentrations of 4, 20, 100, 500, 2500, 5000 µg/plate in the presence and absence of mammalian metabolic activation.  There was no evidence of induced mutant colonies over background. The positive controls induced the appropriate responses in the corresponding strains.

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. The substance was not mutagenic after 3 h incubation both without and with the addition of a metabolic activation system (S9 mix). After 20 h incubation without S9 mix the aberration rate was statistically significantly increased only in the highest concentrations of 500 and 750 µg/ml including gaps.re was evidence for chromosome aberration induced over background at 500 µg/ml (20h, without metabolic activation). Reactive Navy FC 63805 led to a significantly reduced mitotic index at 20 h incubation from 500 µg/ml upwards, i.e. the dye was cytotoxic.Under conditions of this test, Reactive Navy FC63805 was not considered genotoxic in V79 Chinese hamster cell lines with or without metabolic activation.

Finally, Reactive Navy FC63805 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 40 hours post-treatment.  There were no signs of toxicity during the study. Reactive Navy FC63805 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 an unscheduled DNA synthesis assay (UDS), primary rat hepatocyte cultures were exposed to a structural analogue of Reactive Navy FC63805. Concentrations up to cytotoxicity have been tested. Up to the highest evaluated concentration of 100 µg/mL no relevant reproducible increase in the incorporation of [³H] thymidine was obtained. The substance is therefore considered to be not genotoxic in this UDS assay.

In a weight of evidence, Reactive Navy FC63805 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.