Tetrasodium 4-amino-5-hydroxy-6-[[2-methoxy-5-[[2-(sulphonatooxy)ethyl]sulphonyl]phenyl]azo]-3-[[4-[[2-(sulphonatooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2,7-disulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the data from Reactive Blue 250 and its structural analogues, it is concluded that Reactive Blue 250 is not genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-04-23 until 2014-07-08

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

Principles of method if other than guideline:

first experiment 4 hours treatment with and without metabolic activation
second experiment 24 hours treatment without metabolic activation, 4 hours treatment with metabolic activation

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

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:

Experiment I:
without metabolic activation: 525.0; 1050; 2100; 3150; 4200 µg/mL
with metabolic activation: 1050; 2100; 4200; 6300; 8400 µg/mL
Experiment II:
without metabolic activation: 134.2; 268.4; 536.9; 805.3; 1073.8 µg/mL
with metabolic activation: 268.4; 536.9; 1073.6; 2147.5; 4295.0 µg/mL

Vehicle / solvent:

deionised water

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:

A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces reproducibly with one of the concen¬trations a mutation frequency that is three times higher than the spontaneous mutation fre¬quency in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
In a case by case evaluation this decision depends on the level of the correspon¬ding solvent control data.

Statistics:

A linear regression (least squares) 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 was compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected (pH 7.32 measured in the solvent control versus pH 7.36 measured at 8590 µg/mL)
- Effects of osmolality: no relevant increase (287 measured in the solvent control versus 329 measured at 8590 µg/mL)
- Evaporation from medium: Not examined
- Water solubility:123g/L (without correction for purity)
- Precipitation: No precipitation of the test item was observed up to the maximum concentration in all experiments.
- Other confounding effects: None


RANGE-FINDING/SCREENING STUDIES:
According to the current OECD Guideline for Cell Gene Mutation Tests at least four analysable concentrations should be used in two parallel cultures. For freely-soluble and non-cytotoxic test items the maximum concentration should be 5 mg/mL, 5 µL/mL or 10 mM, whichever is the lowest. For cytotoxic test items the maximum concentration should result in approximately 10 to 20% relative survival or cell density at subcultivation and the analysed concentrations should cover a range from the maximum to little or no cytotoxicity. Relatively insoluble test items should be tested up to the highest concentration that can be formulated in an appropriate solvent as solution or homogenous suspension. These test items should be tested up or beyond their limit of solubility. Precipitation should be evaluated at the beginning and at the end of treatment by the unaided eye.

The range finding pre-experiment was performed using a concentration range of 67.1 to 8590 µg/mL to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24-hours treatment) of metabolic activation. The highest applied concentration in the pre-test on toxicity (8590 µg/ml) was equal to 5 mg/mL of the pure substance.

Relevant toxic effects occurred after 4 hours treatment at 4295 µg/mL and above with metabolic activation. Following 4 hours treatment without metabolic activation toxic effects were noted at 2147.5 µg/mL and above. Another low value of the cloning efficiency was noted at 536.9 µg/mL following 4-hour treatment without metabolic activation. This effect was judged as irrelevant fluctuation rather than a true cytotoxic effect however, as the relative cloning efficiency remained above 50% at the next higher concentration. Following 24 hours treatment without metabolic activation a strong toxic effect occurred at 536.9 µg/mL. At all higher concentrations the cell growth was completely inhibited.

The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. No precipitation or phase separation was observed up to the maximum concentration with and without metabolic activation following 4 and 24 hours treamtment.

There was no relevant shift of the osmolarity and pH value of the medium even at the maximum concentration of the test item.
The dose range of the first experiment was set according to the data generated in the pre-experiment. The dose range of the second experiment was adjusted to data produced in the pre-experiment (without metabolic activation) and in the first experiment (with metabolic activation). The individual concentrations were generally spaced by a factor of 2.0. A narrower spacing was used at higher concentrations to cover the cytotoxic range more closely.

To overcome problems with possible deviations in toxicity the main experiments were started with more than four concentrations.


COMPARISON WITH HISTORICAL CONTROL DATA: Complies


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects, indicated by a relative cloning efficiency I or a relative cell density at first subcultivation of less than 50% in both parallel cultures, occurred in the first experiment at 1050 µg/mL and above without metabolic activation. In the second experiment relevant cytotoxic effects as described above were noted at 805.3 µg/mL and above without metabolic activation and at 4295 µg/mL with metabolic activation. The recommended cytotoxic range of approximately 10%-20% relative cloning efficiency or relative cell density was covered with and without metabolic activation. The difference in cytotoxicity noted in the first and the second experiment with metabolic activation is based on the variability of the cell density during treatment. According to the OECD 476 guideline proliferating cells should be treated so, the actual cell density varies from experiment to experiment.

Summary Table

			relative	relative	relative	mutant		relative	relative	relative	mutant
	conc.	S9	cloning	cell	cloning	colonies/	induction	cloning	cell	cloning	colonies/	induction
	µg/mL	mix	efficiency I	density	efficiency II	10^6cells	factor	efficiency I	density	efficiency II	10^6cells	factor
			%	%	%			%	%	%
Column	1	2	3	4	5	6	7	8	9	10	11	12
Experiment I / 4 h treatment			culture I					culture II
Solvent control with water		-	100.0	100.0	100.0	10.1	1.0	100.0	100.0	100.0	12.4	1.0
Positive control (EMS)	150.0	-	90.6	77.6	99.9	188.3	18.7	97.8	55.3	87.1	221.4	17.8
Test item	131.3	-	55.9	culture was not continued^#				79.5	culture was not continued^#
Test item	262.5	-	63.4	culture was not continued^#				68.7	culture was not continued^#
Test item	525.0	-	49.9	93.0	111.9	8.3	0.8	67.6	69.6	105.6	4.9	0.4
Test item	1050.0	-	29.6	93.0	104.6	5.9	0.6	38.2	81.5	93.4	12.2	1.0
Test item	2100.0	-	32.2	95.4	90.8	21.6	2.1	31.2	91.8	100.1	19.9	1.6
Test item	3150.0	-	9.1	83.8	96.4	4.6	0.5	16.8	76.9	96.5	5.6	0.4
Test item	4200.0	-	8.1	76.5	94.8	17.6	1.7	10.0	66.3	85.7	21.6	1.7
Solvent control with water		+	100.0	100.0	100.0	16.3	1.0	100.0	100.0	100.0	7.3	1.0
Positive control (DMBA)	1.1	+	99.7	99.9	93.8	131.5	8.1	89.2	100.6	95.2	144.6	19.7
Test item	262.5	+	82.5	culture was not continued^#				100.4	culture was not continued^#
Test item	525.0	+	92.9	culture was not continued^#				69.5	culture was not continued^#
Test item	1050.0	+	81.4	120.2	76.2	4.5	0.3	63.6	130.1	50.6	6.9	0.9
Test item	2100.0	+	89.2	109.1	76.6	18.1	1.1	78.3	107.1	93.6	19.2	2.6
Test item	4200.0	+	89.0	121.4	107.7	9.9	0.6	59.1	93.5	125.9	16.6	2.3
Test item	6300.0	+	69.2	107.4	120.2	16.1	1.0	60.1	97.4	129.3	13.3	1.8
Test item	8400.0	+	62.0	94.6	100.0	9.4	0.6	47.5	88.4	99.1	11.2	1.5
Experiment II / 24 h treatment			culture I					culture II
Solvent control with water		-	100.0	100.0	100.0	5.5	1.0	100.0	100.0	100.0	23.4	1.0
Positive control (EMS)	150.0	-	95.1	84.4	98.3	450.7	82.4	96.1	83.9	78.3	639.7	27.3
Test item	33.5	-	90.6	culture was not continued^#				95.7	culture was not continued^#
Test item	67.1	-	93.1	culture was not continued^#				94.9	culture was not continued^#
Test item	134.2	-	93.1	63.8	100.8	13.6	2.5	92.5	69.5	91.5	21.9	0.9
Test item	268.4	-	95.8	67.7	108.6	8.4	1.5	84.2	50.9	89.5	9.9	0.4
Test item	536.9	-	88.4	56.9	96.8	7.9	1.5	83.2	59.8	87.3	35.4	1.5
Test item	805.3	-	10.3	51.9	101.3	10.0	1.8	8.8	40.7	88.4	31.4	1.3
Test item	1073.8	-	0.0	42.0	101.5	10.8	2.0	0.0	48.8	93.2	16.5	0.7
Experiment II / 4 h treatment
Solvent control with water		+	100.0	100.0	100.0	17.8	1.0	100.0	100.0	100.0	20.1	1.0
Positive control (DMBA)	2.2	+	98.9	99.6	105.1	184.1	10.3	100.3	58.2	93.6	264.7	13.2
Test item	268.4	+	87.9	86.9	96.3	19.4	1.1	105.6	104.8	99.0	20.5	1.0
Test item	536.9	+	91.2	124.3	93.6	15.4	0.9	99.9	107.7	100.5	15.5	0.8
Test item	1073.8	+	99.7	116.2	93.6	14.9	0.8	95.2	100.3	100.8	8.9	0.4
Test item	2147.5	+	96.6	93.0	95.9	11.6	0.7	84.2	97.1	97.4	23.1	1.1
Test item	4295.0	+	16.5	36.5	95.6	35.0	2.0	15.4	42.4	96.0	8.0	0.4
Test item	6442.5	+	0.0	3.8	culture was not continued^##			0.0	4.5	culture was not continued^##
Test item	8590.0	+	0.0	culture was not continued^##				0.0	culture was not continued^##

#       culture was not continued since a minimum of only four analysable concentrations is required

##     culture was not continued due to exceedingly severe cytotoxic effects

Conclusions:

The test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, it is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

 The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

 The main experiments were evaluated at the following concentrations:

 

exposure period	S9 mix	concentrations in µg/mL
		Experiment I
4 hours	-	525.0	1050	2100	3150	4200
4 hours	+	1050	2100	4200	6300	8400
		Experiment II
24 hours	-	134.2	268.4	536.9	805.3	1073.8
4 hours	+	268.4	536.9	1073.6	2147.5	4295.0

 

No precipitation of the test item was observed up to the maximum concentration in any of the experiments.

 

Relevant cytotoxic effects, indicated by a relative cloning efficiency I or a relative cell density at first subcultivation of less than 50% in both parallel cultures, occurred in the first experiment at 1050 µg/mL and above without metabolic activation. In the second experiment relevant cytotoxic effects as described above were noted at 805.3 µg/mL and above without metabolic activation and at 4295 µg/mL with metabolic activation. The recommended cytotoxic range of approximately 10%-20% relative cloning efficiency or relative cell density was covered with and without metabolic activation. The difference in cytotoxicity noted in the first and the second experiment with metabolic activation is based on the variability of the cell density during treatment. According to the OECD 476 guideline proliferating cells should be treated so, the actual cell density varies from experiment to experiment.

 

No relevant and reproducible increase in mutant colony numbers/10⁶ cells was observed in the main experiments up to the maximum concentration. The mutation frequency did not exceed the historical range of solvent controls, the induction factor did not reach or exceed the threshold of 3.0.

 

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

 

In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 5.5 up to 23.4 mutants per 10⁶cells; the range of the groups treated with the test item was from 4.5 up to 35.4 mutants per 10⁶ cells.

 

EMS (150 µg/mL) and DMBA (1.1 µg/mL in experiment I and 2.2 µg/mL in experiment II) were used as positive controls and showed a distinct increase in induced mutant colonies.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28. Aug. to 25. Oct. 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar 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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix from rat (Ames) and hamster (Prival)

Test concentrations with justification for top dose:

Preliminary Test: 4, 20, 100, 500, 2500, 10000 µg/plate
Main Test: 4, 20, 100, 500, 2500, 5000 µg/plate

Vehicle / solvent:

aqua bidest

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

all strains with rat S9; TA 1535, TA 1537, and TA 100 with hamster S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

all strains with rat S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: benzidine

Remarks:

TA98 with hamster S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100, TA1535 w/o S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537 w/o S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA98 w/o S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 30 min (Prival)
- Expression time (cells in growth medium): 48 to 72 hours

DETERMINATION OF CYTOTOXICITY
- Method: thinning of bacterial lawn; reduced rate of spontaneously occurring colonies (surviving fraction)

Evaluation criteria:

Cytotoxicity: reduced growth rate or thinning of bacterial lawn
Positive: - dose-related and reproducible increase in number of revertant colonies
- doubling of spontaneous mutation rate in at least one tester strains either with or without S9

Statistics:

-

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

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

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

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Reactive Black 5 is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Prival

Executive summary:

Reactive Black 5 was tested for mutagenicity with the strains TA100, TA1535, TA1537, TA98 of Salmonella typhimurium.

The mutagenicity studies were conducted in the standard plate test (Ames Test) and in a modified preincubation test (Prival Test). The studies were performed in the absence and in the presence of a metabolizing system derived from rat or hamster liver homogenate. A dose range of 6 different doses from 4 µg/plate to 5000 µg/plate was used.

Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies.

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

5000 µg/plate was chosen as top dose level for the mutagenicity study.

a) Ames test:

Mutagenicity: In the absence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of a metabolic activation system, treatment of the cells with Reactive Black 5 did not result in relevant increases in the number of revertant colonies.

b) Prival Test:

In the presence of hamster liver S9 using the preincubation method according to Prival Reactive Black 5 did not induce a significant increase in the number of revertant colonies, with any of the tester strains.

 

Summarizing, it can be stated that Reactive Black 5 is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Prival.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Test Guideline 111.1

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- pH value in water: approx. 5.5
- Solubility in water: > 100 g/L
- Stability and homogeneity in the solvent: guaranteed for 4 h in deionized water by HPLC analysis
- Concentration of stock solution: 50 mg/mL

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from rat liver (plate incorporation test) and S9-mix from hamster liver (preincubation test)

Test concentrations with justification for top dose:

Plate incorporation test:
with metabolic activation (10% rat liver):
50, 160, 500, 1600 and 5,000 µg/plate
without metabolic activation:
50, 160, 500, 1600 and 5,000 µg/plate

Preincubation test:
with metabolic activation (30% hamster liver):
16, 50, 160, 500, 1600 and 5,000 µg/plate
without metabolic activation:
16, 50, 160, 500, 1600 and 5,000 µg/plate

Vehicle / solvent:

- Vehicle used: deionized water
- solvent used for positive controls: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation for strain TA 100 and TA 1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation for strain TA 1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without metabolic activation for strain TA 98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without metabolic activation for strain WP2uvrA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation (10% rat liver) for all strains

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation (30% syrian golden hamster liver) for strain TA 100, TA 1535, TA 1537 and WP2uvrA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

congo red

Remarks:

with metabolic activation (30% syrian golden hamster liver) for strain TA 98

Details on test system and experimental conditions:

ASSAY PROCEDURE:
Each test was performed 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. The highest concentration in the first mutation experiment was 50 mg/mL of the test substance in the chosen solvent, which provided a final concentration of 5,000 µg/plate. Further dilutions of 1,600, 500, 160 and 50 µg/plate were also used. Dose levels used in the second experiment were based on findings, including toxicity, in the first experiment. Toxicity was assessed after microscopic thinning of the bacterial lawn and/or reduction of the number of spontaneously occurring mutants compared to the corresponding solvent control value.

In both tests top agar was prepared which, for the Salmonella strains, contained 100 mL agar (0.6% (w/v) agar, 0.5% (w/v) NaCI) with 10 mL of a 0.5 mM histidine-biotin solution. For E. coli histidine was replaced by tryptophan (2.5 mL, 2 mM).
The following ingredients were added (in the following order) to 2 mL of molten top agar at approximately 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 substance solution (dissolved in deionized water)

In the second mutagenicity test if appropriate these top-agar ingredients were preincubated by shaking for approximately 20 to 30 minutes at approximately 30°C. After mixing, and preincubation if appropriate, the liquid was poured into a petri dish containing a 25 mL layer of minimal agar (1.5% (w/v) agar, Vogel-BonnerE medium with 2% (w/v) glucose). After incubation for approximately 48 h at approximately 37°C in the dark, colonies (his+ or trp+ revertants) were counted by hand or by a suitable automatic colony counter. The counter was calibrated for each test by reading a test pattern plate to verify the manufacturer'srequirements for sensitivity.

Evaluation criteria:

Criteria for a valid assay
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 induce increases in the mutation frequency which are significant and within the laboratory's normal range

Criteria for a positive response
A test substance is classified as mutagenic if it has either of the following effects:
a) 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
b) it 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 substance 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 nor precipitates, but tested up to recommended limit concentrations

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:

cytotoxicity

Remarks:

In the plate incorporation test toxicity was observed with metabolic activation at concentrations of 500 to 5000 µg/plate and without S9-mix at a concentration of 5000 µg/plate. In the preincubation test no toxicity was seen.

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

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

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

SOLUBILITY AND TOXICITY: Test substance 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 5,000 µg/plate. Further dilutions of 1,600, 500, 160 and 50 µg/plate were used in all experiments. For the preincubation test the concentration of 16 µg/plate was additionally included in the treatment series. Test substance did not precipitate on the plates up to the highest investigated dose of 5,000 µg/plate.
In the plate incorporation test toxicity was observed only with the strain TA 1537 with metabolic activation at concentrations of 500 to 5,000 µg/plate and without S9-mix at a concentration of 5,000 µg/plate. In the preincubation test no toxicity was observed in the absence and in the presence of hamster liver metabolic activation.

MUTAGENICITY:
Plate incorporation test: The test substance 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 presence of rat liver S9-mix. No dose-dependent effect was obtained.
Preincubation test: In the presence of hamster liver S9-mix (30% (v/v)) using the preincubation method according to Prival the test substance did not cause a significant or dose dependent increase in the number of revertant colonies under the experimental conditions described.

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. The number of revertant colonies of the positive control in the preincubation test with the strain TA 1537 in the presence ofS9-mix was even slightly above ofthe historical control data range, but the criteria for the positive response were clearly fulfilled and the validity not influenced.

STERILITY CHECKS AND CONTROL PLATES:

Sterility of S9-mix and the test substance were indicated by the absence of contamination on the test substance 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. The number of revertant colonies of the solvent and negative controls with the strain WP2uvrA in the presence of S9-mix in both experiments was marginally below the historical control data range, which had no influence on the validity of the assay.

Conclusions:

The test substance was found to be non-mutagenic in the bacterial reverse mutation assay including the Prival modification.

Executive summary:

The test substance was investigated for its potential to induce gene mutations according to OECD Guideline 471, EPA OPPTS 870.5100, EU Method B.14 and Japan: Guidelines for Screening Mutagenicity Testing of Chemicals, Test Guidelines 111.1, in compliance with GLP.

Two independent mutagenicity studies were conducted, one with the plate incorporation method and the other as a modified preincubation test (Prival test). The studies were performed in the absence and in the presence of a metabolizing system derived from rat or hamster liver homogenate. The substance was assessed at 50 - 5,000 µg/plate in the standard plate test and 16 - 5,000 µg/plate in the modified preincubation test.

The substance did not cause a significant increase in the number of revertant colonies at any dose level in the absence or presence of metabolic activation system in either of the tests.

Under the study conditions, the substance was found to be non-mutagenic in the bacterial reverse mutation assay.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 03, 2002 to August 09, 2002

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 OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: Japanese Substance Control Law (JSCL) Test Guideline 111.1 chromosome aberration test with bacteria

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

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from rat liver (plate incorporation test) and S9-mix from hamster liver (preincubation test)

Test concentrations with justification for top dose:

Plate incorporation test:
with metabolic activation (10% rat liver):
50, 160, 500, 1,600 and 5,000 µg/plate
without metabolic activation:
50, 160, 500, 1,600 and 5,000 µg/plate

Preincubation test:
with metabolic activation (30% hamster liver):
50, 160, 500, 1,600 and 5,000 µg/plate
without metabolic activation:
50, 160, 500, 1,600 and 5,000 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without metabolic activation for strain TA 100 and TA 1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without metabolic activation for strain TA 1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without metabolic activation for strain TA 98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without metabolic activation for strain WP2uvrA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation (10% rat liver) for all strains

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation (30% syrian golden hamster liver) for strain TA 100, TA 1535, TA 1537 and WP2uvrA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

congo red

Remarks:

with metabolic activation (30% syrian golden hamster liver) for strain TA 98

Details on test system and experimental conditions:

ASSAY PROCEDURE:

Each test was performed 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. The highest concentration in the first mutation experiment was 50 mg/mL of the test substance in the chosen solvent, which provided a final concentration of 5,000 µg/plate. Further dilutions of 1,600, 500, 160 and 50 µg/plate were also used. Dose levels used in the second experiment were based on findings, including toxicity, in the first experiment. Toxicity was assessed after microscopic thinning of the bacterial lawn and/or reduction of the number of spontaneously occurring mutants compared to the corresponding solvent control value.

In both tests top agar was prepared which, for the Salmonella strains, contained 100 mL agar (0.6% (w/v) agar, 0.5% (w/v) NaCI) with 10 mL of a 0.5 mM histidine-biotin solution. For E. coli histidine was replaced by tryptophan (2.5 mL, 2 mM).
The following ingredients were added (in the following order) to 2 mL of molten top agar at approximately 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 substance solution (dissolved in deionized water)

In the second mutagenicity test if appropriate these top-agar ingredients were preincubated by shaking for approximately 20 to 30 minutes at approximately 30°C. After mixing, and preincubation if appropriate, the liquid was poured into a petri dish containing a 25 mL layer of minimal agar (1.5% (w/v) agar, Vogel-BonnerE medium with 2% (w/v) glucose). After incubation for approximately 48 h at approximately 37°C in the dark, colonies (his+ or trp+ revertants) were counted by hand or by a suitable automatic colony counter. The counter was calibrated for each test by reading a test pattern plate to verify the manufacturer'srequirements for sensitivity.

Evaluation criteria:

Criteria for a valid assay
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 induce increases in the mutation frequency which are significant and within the laboratory's normal range

Criteria for a positive response
A test substance is classified as mutagenic if it has either of the following effects:
a) 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
b) it 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 substance 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

Remarks:

(in plate incorporation test and preincubation test)

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

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(in plate incorporation test and preincubation test)

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

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(in plate incorporation test and preincubation test)

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

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(in plate incorporation test and preincubation test)

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

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(in plate incorporation test and preincubation test)

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

Additional information on results:

SOLUBILITY AND TOXICITY: Test substance was dissolved in DMSO and a stock solution of 50 mg/mL was prepared for the highest concentration, which provided a final concentration of 5,000 µg/plate. Further dilutions of 1,600, 500, 160 and 50 µg/plate were used in all experiments. Test substance did not precipitate on the plates up to the highest investigated dose of 5,000 µg/plate. Test substance proved to be not toxic to the bacterial strains.

MUTAGENICITY: In both independent mutation tests test substance was tested for mutagenicity with the same concentrations as described above. The number of colonies per plate with each strain as well as mean values of 3 plates were given.
Plate incorporation test: The test substance 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 presence of rat liver S9-mix. No dose-dependent effect was obtained.
Preincubation test: In the presence of hamster liver S9-mix (30% (v/v)) using the preincubation method according to Prival the test substance did not cause a significant increase in the number of revertant colonies under the experimental conditions described.

STERILITY CHECKS AND CONTROL PLATES:

Sterility of S9-mix and the test substance were indicated by the absence of contamination on the test substance 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. In the preincubation test the number of revertant colonies of the solvent controls with the strain TA 98 in the absence of S9-mix and the number of revertant colonies of the negative controls with the strain TA 1537 in the presence of S9-mix were out of the historical control data range, but the criteria for the negative response were fulfilled.

Conclusions:

Under the study conditions, the test substance was found to be non-mutagenic in the bacterial reverse mutation assay.

Executive summary:

An in vitro study was performed to investigate the potential of the test substance to induce gene mutations according to OECD Guideline 471, EPA OPPTS 870.5100, EU Method B.14 and Japan: Guidelines for Screening Mutagenicity Testing of Chemicals, in compliance with GLP.

Two independent mutagenicity studies were conducted, one as the standard plate test with the plate incorporation method and the other as a modified preincubation test (Prival test). The studies were performed in the absence and in the presence of a metabolizing system derived from a rat liver homogenate or a hamster liver homogenate. The test substance was tested for mutagenic effects without and with metabolic activation at five concentrations in the range of 50 - 5,000 µg/plate in both assays.

In the plate incorporation test, the test substance did not result in relevant increases in the number of revertants in any of the bacterial strains in the absence and presence of the metabolic activation (rat liver S9-mix (10% (v/v)). Also, in the preincubation test no relevant increase in the number of revertants was observed in any of the bacterial strains in the absence and presence of the metabolic activation (hamster liver S9 -mix (30% (v/v)).

 

Under the study conditions, the test substance was found to be non-mutagenic in the bacterial reverse mutation assay.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 July 2003 to 28 August 2003

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

incorporating Privall Mitchell preincubation test

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

JSCL TG III.1 Gene mutation test with bacteria

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella typhimurium:
TA98 hisD3052 Frameshift
TA100 hisG46 Base pair substitution
TA1535 hisG46 Base pair substitution
TA1537 hisC3076 Frameshift

Escherichia coli:
WP2uvrA trpE Base pair substitution

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: histidine dependent

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Additional strain / cell type characteristics:

other: tryptophan dependent

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix from rat and hamster liver

Test concentrations with justification for top dose:

50, 160, 500, 1600, 5000 µg/plate

Vehicle / solvent:

deionised water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

w/o S9 TA100, TA1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

w/o S9 TA1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

w/o S9 TA98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

w/o S9 WP2uvrA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with rat S9 10%: TA98, TA100, TA1535, TA1537, WP2uvrA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

congo red

Remarks:

with hamster S9 30% TA98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with hamster S9 30%: TA100, TA1535, TA1537, WP2uvrA

Details on test system and experimental conditions:

Test groups
plate incorporation test:
with metabolic activation(10 % rat liver): 50,160, 500, 1600 and 5000 μg/plate b:
without metabolic activation: 50, 160, 500, 1600 and 5000 μg/plate

preincubation test:
with metabolic activation (30 % hamster liver): 50, 160, 500, 1600 and 5000 μg/plate
without metabolic activation: 50, 160, 500, 1600 and 5000 μg/plate

Control groups
negative controls:
a: untreated controls
b: solvent controls (0 μg/plate)

positive controls:
without metabolic activation: sodium-azide for strain TA 100 and TA 1535, 9-aminoacridine for strain TA 1537, 2-nitrofluorene for strain TA 98, 4-nitroquinoline-N-oxide for strain WP2uvrA
with metabolic activation (10 % rat liver): 2-aminoanthracene for all strains
with metabolic activation (30 % syrian golden hamster liver): 2-aminoanthracene for strain TA 100, TA 1535, TA 1537 and WP2uvrA, congo red for strain TA 98

Formulation of test compound: dissolved in deionised water at appropriate concentrations immediately before use.

Formulation of reference compounds
Sodium-azide dissolved in deionised water final concentration: 1.0μg/plate for strain TA 1535, 2.0μg/plate for strain TA 100
9-aminoacridine dissolved in DMSO final concentration: 50.0μg/plate for strain TA 1537
2-nitrofluorene dissolved in DMSO final concentration: 2.5μg/plate for strain AT 98
4-nitroquinoline-N-oxide dissolved in DMSO final concentrations: 2.0μg/plate (plate inc.), 0.5μg/plate (preinc.) for strain WP2uvrA
2-aminoanthracene dissolved in DMSO final concentrations: (10% (v/v) rat liver S9-mix): 1.5μg/plate for strains TA 98, TA 100, TA 1535 and TA 1537, 20.0μg/plate for strain WP2uvrA
2-aminoanthracene dissolved in DMSO final concentrations (30% (v/v) hamster liver S9-mix): 1.0μg/plate for strain TA 100, TA 1535 and TA 1537, 30.0μg/plate for strain WP2uvrA
Congo red dissolved in deionised water final concentration: 250μg/plate for strain TA98
The frozen stock solutions of each compounds were diluted progressively up to the final concentration on the day of treatment.

Source of bacteria: stock cultures in the bank of “Genetic Toxicology”, Aventis Pharma Germany, ProTox prepared from the original bacteria strains.

Test organism: Salmonella typhimurium strains – TA 98 hisD3052 rfa uvrB +R, TA 100 hisG46 rfa uvrB +R, TA 1535 hisG46 rfa uvrB, TA 1537 hisC3076 rfa uvrB and Escherichia coli WP2uvrA pKM101

Experimental conditions in vitro: approx. 37°C in an incubator.

Preparation and storage of a liver homogenate fraction (S9)
The S9 fraction of Spraque Dawley rat liver induced with Aroclor 1254 was obtained by Molecular Toxicology, Inc., 157 Industrial Park Dr. Boone, NC 28607, [phone]. The protein content for every batch was guaranteed by a Quality Control & Production Certificate by the supplier. Also for every batch of S9 an independent validation was performed in the laboratory with a minimum of two different mutagens, e.g. 2-aminoanthracene and benzo(a)pyrene, to confirm metabolic activation by microsomal enzymes.
The S9 fraction of Syrian golden hamster liver was prepared by the department conducting the study according to Prival et. al (1982). Male Syrian golden hamsters (7-8 weeks old), were supplied by Harlan Winkelmann, Gartenstrasse 27, 33178 Borchen, Germany. Liver preparations were performed from the liver of non pretreated Syrian hamsters. The livers were removed from 10 male Syrian hamsters (7-8 weeks old) using cold sterile solutions at approx. 0 to 4 °C and glassware, and were then pooled and washed in approx. 150 mM KC1 (approximately 1 ml/g wet liver). The washed livers were cut into small pieces and homogenized in three volumes of KC1. The homogenate was centrifuged at approx. 9000g for 10 minutes. The supernatant was the S9 fraction. This was divided into small portions, rapidly frozen and stored at approx. - 80 °C. 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 congo red, to confirm metabolic activation by microsomal enzymes.

Preparation of S9-mix
Sufficient S9 fraction was thawed at room temperature immediately before each test. One volume of Moltox. S9 fraction (batch no. 1530 for the plate incorporation test, protein concentration 36.1 g/l) was mixed with 9 volumes of the S9 cofactor solution, which was kept on ice until used. This preparation is termed S9-mix. The concentrations of the different compounds in the rat liver S9-mix were:

8 mM MgCl2
33 mM KC1
5 mM glucose-6-phosphate
4mM NADP
100 mM phosphate buffer pH 7.4

According to the modification proposed by Prival (8) the test substance and the tester strains were
preincubated for 20 to 30 minutes with 30 % (v/v) Syrian golden hamster S9-mix.
Three volumes of S9 fraction (batch no. 2002/1 for the preincubation, protein concentration
45 g/l) were mixed with 7 volumes of the S9 cofactor solution.

This preparation is termed S9-mix. The hamster liver S9-mix consists of:

8 mM MgCl2
33 mM KC1
20 mM glucose-6-phosphate
2.8 units/ml glucose-6-phosphate dehydrogenase
4mM NADP+
2 mM NADH
2 mM FMN (Riboflavine-5’-phosphate-sodium-salt)
100 mM phosphate buffer pH 7.4

Bacteria
The strains of Salmonella typhimurium were obtained from Professor B.N. Ames, University of California, U.S.A. The strain E. coli was obtained from E.coli Genetic Stock Center, Yale University, New Haven, U.S.A.

Bacteria were grown overnight in nutrient broth (25 g Oxoid Nutrient Broth No. 2 /liter) at approx. 37 °C. The amount of bacteria in the cell suspension was checked by nephelometry. Inoculation was performed with stock cultures which had been stored in liquid nitrogen. Each new stock of the different bacterial strains was checked with regard to the respective biotin and histidine requirements, membrane permeability, ampicillin resistance, tetracyclin resistance, crystal violet sensitivity, UV resistance and response to diagnostic mutagens.

ASSAY PROCEDURE
An independent mutation test was performed using the plate incorporation method. When results were negative or equivocal, a second test was conducted. This included a preincubation step if the first test was clearly negative. Preincubation involved incubating the test substance, S9-mix and bacteria for a short period before pouring this mixture onto plates of minimal agar.
Each test was performed 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. The highest concentration in the first mutation experiment was 50 mg/ml of the test substance in the chosen solvent, which provided a final concentration of 5000μg/plate. Further dilutions of 1600, 500, 160 and 50μg/plate were also used. Dose levels used in the second experiment were based on findings, including toxicity, in the first experiment. Toxicity was assessed after microscopic thinning of the bacterial lawn and/or reduction of the number of spontaneously occurring mutants compared to the corresponding solvent control value.

In both tests top agar was prepared which, for the Salmonella strains, contained 100 ml agar (0.6 % (w/v) agar, 0.5 % (w/v) NaCl) with 10 ml of a 0.5 mM histidine-biotin solution. For E. coli histidine was replaced by tryptophan (2.5 ml, 2.0 mM). The following ingredients were added (in the following order) to 2 ml of molten 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 deionised water)

In the second mutagenicity test if appropriate these top-agar ingredients were preincubated by shaking for approximately 20 to 30 minutes at approx. 30 °C.

After mixing, and preincubation if appropriate, 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+ or trp+ revertants) were counted by hand or by a suitable automatic colony counter. The counter was calibrated for each test by reading a test pattern plate to verify the manufacturer's requirements for sensitivity.

Evaluation criteria:

Criteria for a valid assay
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 induce increases in the mutation frequency which are 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 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
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.

Statistics:

No data

Species / strain:

E. coli WP2 uvr A pKM 101

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

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

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

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

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

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.
The number of revertant colonies of the solvent controls with the strain TA 100 in the absence of S9-mix in the plate incorporation test was below the historical control data range, but the criteria for the negative response were fulfilled.
The number of revertant colonies of the positive controls with the strains TA 1535, TA 98 and WP2 uvrA in the presence of S9-mix in the preincubation test was above the historical control data range, but the criteria for the positive response were fulfilled. Also in the preincubation test the number of revertant colonies with the strain TA 1537 was above the historical control data range, but the criteria for the negative/positive response were fulfilled.

SOLUBILITY AND TOXICITY
The test substance 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 5000μg/plate. Further dilutions of 1600, 500, 160 and 50μg/plate were used in all experiments.
The test substance did not precipitate on the plates up to the highest investigated dose of 5000μg/plate.

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

MUTAGENICITY
The number of colonies per plate with each strain as well as mean values of 3 plates were given.

Plate incorporation test:
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 presence of rat liver S9-mix in either mutation test. No dose-dependent effect was obtained.
Preincubation test:
In the presence of hamster liver S9-mix (30 % (v/v)) using the preincubation method according to Prival the test compound did not cause a significant increase in the number of revertant colonies under the experimental conditions described.

Conclusions:

The results lead to the conclusion that the substance is not mutagenic in the absence and presence of metabolic activation using the standard Ames Test procedure (plate incorporation test) and the preincubation test as described.

Executive summary:

The present study was conducted in compliance with OECD Guideline For Testing Of Chemicals, 471 Bacterial Reverse Mutation Test Adopted: 21-July-1997andU.S. EPA: OPPTS 870.5100 Health Effects Test Guidelines Bacterial Reverse Mutation Test, Aug-1998andEC Directive 2000/32/EC, L 136, Annex 4D, B.13/B.14andJapanese Substance Control Law (JSCL) Test Guideline III.l Gene Mutation Test with bacteria.The study is based on the Principles ofGood Laboratory Practice (GLP).

 

The test substance was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537, TA 98 of Salmonella typhimurium and with Escherichia coli WP2uvrA. Two independent mutagenicity studies were conducted, one as the standard plate test with the plate incorporation method and the other as a modified preincubation test (Prival test). The studies were performed in the absence and in the presence of a metabolizing system derived from a rat liver homogenate or a hamster liver homogenate.

For all studies, the compound was dissolved in deionised water, and each bacterial strain was exposed to 5 dose levels. Doses for both studies ranged from 50 to 5000μg/plate.

The substance did not precipitate on the plates up to the highest investigated dose of 5000μg/plate.

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

The number of revertant colonies of the solvent controls with the strain TA 100 in the absence of S9-mix in the plate incorporation test was below the historical control data range, but the criteria for the negative response were fulfilled.

The number of revertant colonies of the positive controls with the strains TA 1535, TA 98 and WP2 uvrA in the presence of S9-mix in the preincubation test was above the historical control data range, but the criteria for the positive response were fulfilled. Also in the preincubation test the number of revertant colonies with the strain TA 1537 was above the historical control data range, but the criteria for the negative/positive response were fulfilled.

Toxicity: In both studies toxicity was not observed either with or without metabolic activation.

 

Plate incorporation test:

Mutagenicity: In the absence of the metabolic activation system the test compound did not result in relevant increases in the number of revertants in any of the bacterial strains. Also in the presence of rat liver activation system (10 % (v/v)), treatment of the cells did not result in relevant increases in the number of revertant colonies.

 

Preincubation test:

In the absence and in the presence of hamster liver S9-mix (30 % (v/v)) using the preincubation method according to Prival the test substance did not result in relevant increases in the number of revertant colonies with any of the tester strains.

 

Summarizing, it can be stated that the substance is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Prival at the dose levels investigated.

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Principles of method if other than guideline:

B.N. Ames, W.W. Durston, E. Yamasaki and F.D. Lee, Carcinogens are mutagens. A simple test system combining liver homogenate for activation and bacteria for detection, Proc. Nat. Acad. Sci. USA 70 (1973) 2281 - 2285.
B.N. Ames, J. McCann and E. Yamasaki: Methods for detecting carcinogens and mutagens with the Salmonella / mammalian-microsome mutagenicity test, Mutation Res. 31 (1975) 347 - 364.
M.H.L. Green and W.J. Murfel: Mutagen testing using trp+ reversion in Escherichia coli, Mutation Res. 38 (1976) 3 - 32.
A.P. Alvares, D.R. Bickers and A. Kappas: Polychlorinated biphenyls: a new type of inducer of cytochrome P 448 in the liver. Proc. Nat. Acad. Sci. USA 70 (1973) 1321 - 1325.
J. McCann, N.E. Springarn, J. Kobory and B.N. Ames: Detection of carcinogens as mutagens: bacterial tester strains with R factor plasmids, Proc. Nat. Acad. Sci. USA 72 (1975) 979 - 983.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1538

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix from rat liver

Test concentrations with justification for top dose:

First experiment: 4, 20, 100, 500, 2500, 10000 µg/plate
Second experiment: 20, 100, 500, 2500, 5000, 10000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: none

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: N-Methyl-N'-nitro-N-nitrosoguanidine, 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

Top agar is prepared for the Salmonella strains by mixing 100 ml agar (0.6 % % agar, 0.5 % NaCl) with 10 ml of a 0.5 mM histidine-biotin solution . With E. coli histidine is replaced by tryptophan' (2.5 ml , 0.5 mM) . The following ingredients are added (in order) to 2 ml of molten top agar at 45° C:
0.1 ml of an overnight nutrient broth culture of the bacterial tester strain
0.1 ml test compound solution
0.5 ml S-9 Mix (if required) or buffer
After mixinq, the liquid is poured into a petridish with minimal agar (1.5 % agar, Vogel-Bonner E medium with 2 % glucose). After incubation for 48 to 72 hours at 37° C in the dark, colonies (his+ revertants) are counted.

Evaluation criteria:

Cytotoxicity: reduced growth rate or thinning of bacterial lawn
Positive: - dose-related and reproducible increase in number of revertant colonies
- doubling of spontaneous mutation rate in at least one tester strains either with or without S9

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:

up to 10000 ug/plate

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:

up to 10000 ug/plate

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:

up to 10000 ug/plate

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:

up to 10000 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

up to 10000 µg/plate

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:

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

Remarks:

up to 10000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Reactive Blue 250 is not mutagenic in these bacterial test systems neither with or without exogenous metabolic activation at the dose levels investigated.

Executive summary:

Reactive Blue 250 was tested for mutagenicity with the strains Salmonella typhimurium TA100, TA1535, TA1537, TA1538, TA98 and Escherichia coli WP2uvrA.

The mutagenicity studies were conducted in the absence and in the presence of a metabolizing system derived from rat liver homogenate. A dose range of 6 different doses from 20 µg/plate to 10000 µg/plate was used.

Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies.

Toxicity: the test compound proved to be not toxic o f the bacterial strains. On the basis of the preliminary test results, 10000 µg/plate was chosen as the top dose level.

Mutagenicity: In the absence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of a metabolic activation system, treatment of the cells with the test item did not result in relevant increases in the number of revertant colonies.

Sumarizing, it can be stated that Reactive Blue 250 is not mutagenic in these bacterial test systems either with or without exogenous metabolic activation at the dose levels investigated.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Based on the data from Reactive Blue 250 and its structural analogues, it is concluded that Reactive Blue 250 is not genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 24, 2002 to August 21, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Gartenstrasse 27, 33178 Borchcn
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: Male animals-mean = 179.7 g (= 100 %) (min = 175 g (-2.6 %), max = 193 g (+7.4 %)); female animals- mean = 140.2 g (=100 %) (min = 135 g (-3.7%), max = 150 g (+7.0%))
- Assigned to test groups randomly: Yes, under following basis: randomization schemes 2002.0403 and 2002.0404
- Housing: Five animals per cage in transparent macrolon cages (type IV) on soft wood granulate in an air conditioned room.
- Diet (e.g. ad libitum): Rat/mice diet ssniff R/M-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 d under study conditions
- Animal identification: Fur marking with KMnO4 and cage numbering

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C (except short lasting deviations due to disturbances of air condition)
- Humidity (%): 50% (except short lasting deviations due to disturbances of air condition)
- Photoperiod (hrs dark / hrs light): 12 h light/dark cycle

IN-LIFE DATES: From July 30, 2002 to August 01, 2002

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: sesame oil
- Concentration of test material in vehicle: 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SUSPENSION: The test substance was suspended in sesame oil at a appropriate concentration. A magnetic stirrer was used to keep the preparation homogeneous until dosing had been completed.

Duration of treatment / exposure:

2 d

Frequency of treatment:

twice at an interval of 24 h

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5/sex/group.

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive control: Cyclophosphamide
Dissolved in: distilled water
Dose: 40 mg/kg bw
Route and frequency of administration: Oral (gavage), once
Volume Administered: 10 mL/kg bw

Tissues and cell types examined:

- 2,000 polychromatic erythrocytes were counted for each animal.
- The number of cells with micronuclei was recorded, not the number of individual micronuclei.
- The ratio of polychromatic erythrocytes to 200 normochromatic 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.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on the results of the acute oral toxicity study the dose of 2,000 mg/kg bw was selected as the limit dose, for the main study.

-Extraction of the bone marrow: Animals were killed by carbon dioxide asphyxiation 24 h after dosing. One femora was removed and the bone freed of muscle tissue. The proximal end of the femora was opened, the bone marrow flushed into a centrifuge tube containing about 3 mL of fetal bovine serum and a suspension was prepared. The mixture was then centrifuged for 5 minutes at approximately 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 h.

-Staining procedure: The slides were stained 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 test substance is considered as positive if there is a significant dose- related increase in the number of micronucleated polychromatic erythrocytes 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-clastogenic in this system.

Statistics:

Assuming the study is valid based on a monotone-dose-relationship, one-sided Wilcoxon tests were performed initially comparing control values with those of the highest dose group. A significance level of 5% is adopted for all tests.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

-All animals survived after treatment. No signs of toxicity were observed.
- All treated animals showed blue-black discolored feces 24 h after the first administration.
-The dissection of the animals revealed an blue-black colored content of the gastro-intestinal tract.

Conclusions:

Under the study conditions, the test substance did not induce micronuclei in bone marrow cells of the rat. Therefore, the test substance was not considered to be clastogenic in this micronucleus assay.

Executive summary:

A study was conducted to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the rat according to OECD Guideline 474, EPA OPPTS 870.5395 and EU Method B.12, in compliance with GLP.

The test substance was suspended in sesame oil and given twice at an interval of 24 h as a oral dose of 2,000 mg/kg bw/d to male and female rats. The dose was selected based on the results of a previous rat acute oral toxicity study. The animals were sacrificed 24 h after the last administration and bone marrow cells were collected for micronuclei analysis. After treatment with the test substance, 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 and differed less than 20% from the control value.

Under the study conditions, the test substance did not induce micronuclei in bone marrow cells of the rat. Therefore, the test substance was not considered to be clastogenic in this micronucleus assay.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 to 19. July 1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (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: Hoechst AG
- Age at study initiation: 7 to 9 weeks
- Weight at study initiation: males: 25 to 33 g; females: 22 to 29 g
- Assigned to test groups randomly: yes, computer based randomization scheme
- Fasting period before study: -
- Housing: 5 animals/cage
- Diet: Altromin 1324 rat and mouse diet ad libitum
- Water: tap ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24
- Humidity (%): 45 to 65
- Air changes (per hr): -
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 09 to 19. July 1984

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: freshly 25% suspension in water: 12500 mg TS mixed with water by means of a magnetic stirrer and filled up to 50 mL in a volumetric flask.

Dose level: 2000 and 10000 mg/kg bw
Dose concentration: 250 mg/mL
Dose volume: 8 and 40 mL/kg bw

Duration of treatment / exposure:

single dose

Frequency of treatment:

once

Post exposure period:

Vehicle control and high dose: 24, 48, and 72 hours
Positive control and low dose: 24 hours

Dose / conc.:

2 000 mg/kg bw (total dose)

Remarks:

Concentration: 250 mg/mL

Dose / conc.:

10 000 mg/kg bw (total dose)

Remarks:

Concentration: 250 mg/mL

No. of animals per sex per dose:

5 per killing time point

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (Endoxan - batch 0378)

Tissues and cell types examined:

bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
maximum feasible dose

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
single administration
sampling 24, 48, and 72 hours after administration (vehicle control and high dose group)

DETAILS OF SLIDE PREPARATION:
Bone marrow was flushed into a tube containing fetal bovine serum and centrifuged (5 min, 1000 rpm)
Air dried smears were stained with May-Grünwald-Giemsa:
- 5 minutes Methanol
- 3 minutes May-Grünwald-solution
- 2 minutes May-Grünwald-solution, diluted with aqua dest. 1 + 1
- 2x rinsed in aqua dest.
- 10 minutes stained with 1 part Giemsa-solution + 6 parts buffer solution pH 7.2 according to Weise
- rinsed in aqua dest.
- air dried
- coating with Entellan


METHOD OF ANALYSIS: (blinded scoring)
1000 polychromatic erythrocytes per animal were scored for incidence of micronuclei. The number of cells with micronuclei was recorded, not the number of individual micronuclei.
1000 mature erythrocytes were scored for incidence of micronuclei as inspection measure
the ratio of polychromatic to normochromatic erythrocytes was determined

Evaluation criteria:

statistically significant difference in number of cells with micronuclei in dose groups compared to respective vehicle control groups

Statistics:

incidence of cells with micronuclei (polychromatic or normochromatic) compared to the vehicle control groups by means of binominal distribution (computer program Diamant) with a level of significance of 95%

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

diarrhea, narrowed lids, squatted posture, discolorations (eyes, extremities, muzzle, tail, ears, skin, urine, feces)

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Necropsy findings: dark to light bluish staining of: skin, tongue, peritoneum, fatty tissue, muscles, stomach and intestines, urinary bladder, testes, ovaries, adrenals

Conclusions:

No significant increase in number of cells with micronuclei at 2000 and 10000 mg/kg body weight Reactive Black 5. Administration of the substance up to the maximum feasible dose did not result in chromosomal alterations.

Executive summary:

Reactive Black 5 was administered once orally by gavage as single doses of 0 (vehicle control), 2000 and 10000 mg/kg bodyweight to male and female NMRI mice (5 mice per sex and killing time point). 10000 mg/kg bw had been shown in a preliminary study to be the maximum feasible dose. A positive control group received Endoxan at an oral dose of 50 mg/kg body weight.

Animals were killed 12, 24, or 48 hours after treatment by carbon dioxide asphyxiation. The bone marrow obtained from femora of the animals was prepared, placed on microscopic slides and stained. 1000 poly- and 1000 normochromatic erythrocytes were screened for micronuclei. In addition, the ratio of polychromatic to normochromatic erythrocytes was determined.

Under the conditions of the present study, Reactive Black 5 caused no significant increase in the number of cells with micronuclei in the bone marrow cells of treated animals as compared with the control group. Endoxan however produced a marked increase in number of cells with micronuclei..

The results indicate that, under the conditions of the present study, Reactive Black 5 is not mutagenic in the in vivo micronucleus test.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 07, 2005 to June 09, 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH Guidelines S2A and S2B

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Remarks:

CRL: NMRI BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Europe) Laboratories, Budapest, HUNGARY
- Age at study initiation: young adult mice
- Weight at study initiation: male: 26.8-32.1 g and female: 22.5-25.8 g
- Assigned to test groups randomly: yes
- Housing: group caging (5 animals/cage), type II polypropylene/polycarbonate cages with laboratory bedding
- Diet: SSNIFF RIM-Z+H diet for rats and mice (ad libitum)
- Water: ad libitum
- Acclimation period: 6 d

ENVIRONMENTAL CONDITIONS
- Temperature: 22±3°C
- Humidity: 30-70 %
- Photoperiod: 12 h dark/light

IN-LIFE DATES: From: June 07, 2005 To: June 09, 2005

Route of administration:

oral: gavage

Vehicle:

Vehicle used for test substance: distilled water
- Concentration of test substance in vehicle: 20% (w/v)
- Batch no.: 9240604
- Date of expiry: June, 2007
- Storage condition: at room temperature

Vehicle of positive control: 0.9% NaCl infusion
- Batch no.: 5671002
- Expiry: October, 2005
- Storage condition: at room temperature

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was dissolved in distilled water for the treatment. The necessary amount of the test substance was weighed into a calibrated volumetric flask and distilled water was added and stirred to obtain a homogenous formulation. It was diluted to the final volume with the distilled water. The solution was prepared fresh each day of dosing and used within 2 h. The test substance was used for treatment at concentration of 20% (w/v). Cyclophosphamide (positive control) was dissolved in 0.9% NaCI infusion for treatment.

Duration of treatment / exposure:

48 h

Frequency of treatment:

twice

Post exposure period:

24 h after the last dosing

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5/sex

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: intraperitoneally
- Doses: 60 mg/kg
- Animals used: 5/sex

Tissues and cell types examined:

The slides were examined in a blind manner. Two thousand polychromatic erythrocytes were scored per animal to asses the micronucleatcd cells. The frequency of micronucleated cells were expressed as percent of micronucleated cells based on the first 2000 polychromatic erythrocytes counted in
the optic field. Multiple micronuclei cells were not registered. The proportion of immature among total (irnmature+mature) erythrocytes were
determined for each animal by counting a total of at least 200 immature erythrocytes.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on the results of the acute oral toxicity study the dose of 2,000 mg/kg bw was selected as the limit dose, for the main study.

TREATMENT AND SAMPLING TIMES: The test substance was administered twice at an interval of 24 h orally by gavage to the test animals at a dose 2,000 mg/kg bw. The treatment volume was 0.1 mL/kg bw. The vehicle, distilled water was administered in the same way to the negative control groups. The mice were examined for visible signs of reactions to treatment, immediately after dosing, and at intervals until sacrifice. Sampling was made once at 24 h after the last dosing. Five male and female animals per group were used for sampling.
Cyclophosphamide (positive control) was administered intraperitoneally with a treatment volume: 0.1 mL/kg bw. Sampling was performed 24 h after the beginning of the treatment and five male and female animals were used for sampling.

DETAILS OF SLIDE PREPARATION: Bone marrow was obtained from two exposed femurs of the mice from animals immediately after sacrificing. The bone marrow was flushed with foetal calf serum. The cells were concentrated by a gentle centrifugation, then spread on a standard microscopic slide. Slides were fixed and after that stained with Giemsa. Two slides were prepared from each animal.

EVALUATION OF THE RESULTS: The micronucleated polychromatic erythrocytes referring to 2,000 polychromatic erythrocytes and the ratio of polychromatic erythrocytes to normochromatic erythrocytes (PCE/NCE) were listed for each animal. The frequency of micronucleated cells of treated male and female groups were compared with those of the vehicle control groups.

Evaluation criteria:

A positive result was one that gave a significant increase (at p<0.05, p<0.01) of the frequency of micronucleated polychromatic erythrocytes at least in two treated groups in one sex.
This increase would be classified as significant if observed:
- at adjacent dose levels: in the same experiment, dose- dependency,
- at adjacent time points: in the same experiment, same dose level, that is time dependency,
- in two experiments, in the same dose level and time point, that is reproducibility.
Both biological and statistical significance were considered together for evaluation purposes.
The historical range for this laboratory was also considered when evaluating the biological significance of small increases.

Statistics:

Dose dependent increase in the number of micronucleated polychromatic erythrocytes was evaluated by Kruskal-Wallis Non Parametric ANOVA test at 1 and 5% probability levels.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Clinical signs and mortality:

No animals died during the study. Animals did not show any toxicity symptom in the test substance treated group or in the vehicle or positive control groups. Due to the effect of test substance the faeces and tissue of abdominal organs of mice were found to be discoloured with a reddish appearance. The presence of red colour in the tissue after an oral exposure demonstrates systematic exposure to the test substance.

 

Frequency of micronucleated polychromatic erythrocytes:

The frequency of micronucleated polychromatic erythrocytes for the vehicle control mice was within an acceptable range and compatible with the historical control data. Cyclophosphamide treated mice (60 mg/kg bw) showed large, statistically significant increase in the micronucleated polychromatic erythrocytes number compared to the vehicle control, demonstrating an acceptable sensitivity of the test. The repeated administration of the test substance (twice at an interval of 24 h) at 2,000 mg/kg bw did not induce biologically relevant increase in the frequency of micronucleated polychromatic erythrocytes (MPCEs) in male or female mice at 24 h after the treatment compared to the vehicle control. In the female animals, at 24 h after treatment with 2,000 mg/kg test substance there was small, statistically significant, but biologically not important increase in the number of micronucleated polychromatic erythrocytes (MPCEs). These minor increases were in the range of historical negative control value. No significant difference was found in the ratio of polychromatic and normochromatic erythrocytes after treatment.

 

TABLE 1 – Results in male mice

Groups	Sampling time (hour)	Total number of PCEs analyzed	PCE/NCE		MPCE
			mean	± SD	mean	± SD
Vehicle Control	24	10000	1.12	0.07	3.80	0.45
2000 mg/kg	24	10000	0.95	0.10	4.40	0.89
Positive Control (60 mg/kg)	24	10000	0.38	0.09	38.60**	4.16

TABLE 2 – Results in female mice

Groups	Sampling time (hour)	Total number of PCEs analyzed	PCE/NCE		MPCE
			mean	± SD	mean	± SD
Vehicle Control	24	10000	1.32	0.15	3.80	0.45
2000 mg/kg	24	10000	1.08	0.29	4.80*	0.84
Positive Control (60 mg/kg)	24	10000	0.38	0.08	37.40**	2.07

PCP = Polychromatic Erythrocyte

NCE = Normochromatic Erythrocyte

MPCE = Number of Micronucleated Polychromatic Erythrocyte referring to 2000PCE

Vehicle Control -Distilled water

Positive Control = 60 mg/kg Cyclophosphamide

*            = p < 0.05

**          = p < 0.01

Kruskal-Wallis Non Parametric ANOVA

Conclusions:

Under the test conditions, the test substance did not induce micronuclei in bone marrow cells of the treated mice. Therefore, the test substance was not considered to be clastogenic in this micronucleus assay.

Executive summary:

A study was conducted to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of mice according to OECD Guideline 474 and ICH Guidelines S2A and S2B, in compliance with GLP.

The substance was dissolved in distilled water and given twice at an interval of 24 h as an oral dose of 2,000 mg/kg bw to male and female rats. The dose was selected based on the results of a preliminary acute oral toxicity study. A vehicle control group treated with distilled water and a positive control group treated with cyclophosphamide (60 mg/kg bw intraperitoneally) were also included. The animals were sacrificed 24 h after the last administration and bone marrow cells were collected for micronuclei analysis. Two thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the micronucleated cells. The ratio of polychromatic to normochromatic erythrocytes (PCE/NCE) was used to assess the toxicity of the test substance.

The oral administration did not induce any biologically relevant increase in the frequency of micronucleated polychromatic erythrocytes (MPCEs) in male or female mice compared to the vehicle control. No biologically important alteration in the ratio of polychromatic to normochromatic erythrocytes occurred in the treated groups when compared to the vehicle control. Treatment with the positive control, cyclophosphamide, caused significant increase in the number of micronucleated polychromatic erythrocytes (MPCEs), thus validating the test.

Under the study conditions, the test substance did not induce micronuclei in bone marrow cells of the treated mice. Therefore, the test substance was not considered to be clastogenic in this micronucleus assay (Beres, 2005).

Reference 4

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21. to 24. March 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

chromosome aberration assay

Species:

hamster, Chinese

Strain:

other: Han: Chin

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Zentralinstitut fur Versuchstiere, Hannover
- Age at study initiation: 10 - 14 weeks
- Weight at study initiation: males: mean = 29.2 g (25 - 33 g)
females: mean = 27.29 (24 - 31 g)
- Assigned to test groups randomly: yes, computer based randomization scheme
- Fasting period before study: no data
- Housing: one animal per cage
- Diet: Altromin 7010 hamster diet (Altromin GmbH, Lage/lippe), ad libitum
- Water: tap water in plastic bottles ad libitum
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24
- Humidity (%): 45 to 65
- Air changes (per hr): -
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 21. to 24. March 1988

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: sesame oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: 50% suspension in sesame oil

Duration of treatment / exposure:

single dose

Frequency of treatment:

once

Post exposure period:

12, 24 and 48 hours

Dose / conc.:

5 000 mg/kg bw (total dose)

Remarks:

Concentration: 500 mg/mL

No. of animals per sex per dose:

Control and Reactive Black 5: 5 males and 5 females per killing time point
Positive control: 5 males and 5 females

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (Endoxan: batch 05459)
- Justification for choice of positive control(s): availability of historical data
- Route of administration: oral
- Doses / concentrations: 50 mg/kg body weight (5 mg/mL)

Tissues and cell types examined:

bone marrow cells from femur: 50 metaphases/animal

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: MTD based on preliminary study


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The test substance was administered orally by gavage to the test animals at a single dose of 5000 mg/kg bodyweight. Sesame oil was administered in the same way to the negative control group. A positive control group, introduced after exactly 24 hours to run parallel with the negative control and the dose groups, received Endoxan at an oral dose of 50 mg/kg bodyweight. Two hours before killing by carbon dioxide asphyxation (12, 24, or 48 hours after treatment ), the hamsters each received an intraperitoneal injection of 3.3 mg demecolcin (Colcemid) per kg bodyweight.

DETAILS OF SLIDE PREPARATION:
After killing, both femora were removed and the bones completely stripped of muscle tissue. After removal of the epiphyses, the bone marrow was flushed in alternate directions out of the diaphysis into a centrifuge tube by means of a syringe containing Hanks solution (2 ml/femur) at the temperature of 37°C, mixed and centrifuged for 5 minutes at 1000 rpm after which all but a few drops of the supernatant was drawn off by pipette and sediment resuspended by tapping.
For hypotonic treatment, approximately 5 ml of 0.075 M potassium chloride solution at 37°C was quickly added and suspended. This suspension was then allowed to incubate for 10 minutes in a water bath at 37°C. Addition of 1.5 ml fixative (methanol : glacial acetic acid 3 + 1) and flow through with air. After re-centrifugation for five minutes at 1000 rpm, all but one drop of the supernatant was drawn off by pipette. The sediment was carefully covered with a layer composed of 2.5 ml fixative. After 20 minutes the fixation was removed carefully with a pipette and suspended in 2.5 ml fixative. After another 30 minutes, the mixture was centrifuged, after which the liquid was removed by pipette and fresh fixative added. The tubes were covered and kept for at least 12 hours (overnight) in a refrigerator at 4°C.
After re-centrifuging for 5 minutes at 1000 rpm, all but one drop of the liquid was removed by pipette and a new suspension formed with a small quantity of freshly prepared fixative. A few drops of this suspension were placed with a Pasteur pipette onto clean microscopic slides which had been stored in distilled water at 4°C, the drops were then briefly passed through a Bunsen flame and air-dried for 24 hours. Staining was performed as follows:
- staining for 10 minutes in 2% 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 Eukitt


METHOD OF ANALYSIS:

After the slides had been coded, 50 metaphases per animal were examined. The set of chromosomes was examined for completeness and the various chromosomal aberrations were assessed. The chromosomal aberrations were classified. The metaphases were examined for the following aberrations: gap (g), break (b), fragment (f), minute (m), deletion (d), exchanges including intrachanges (ex), dicentricity (di), chromosome disintegration (cd) ring (ri) and polyploidy (pp). In addition, metaphases with 5 and more aberrations were classified separatly as multiple aberrations (ma).
After the metaphases had been evaluated, the code was lifted. The values for the control group were compared at each killing time with the results from the dose groups and the positive control.

Evaluation criteria:

1. Structural aberrations
Gap: Non stained segment (achromatic gap) of chromatide without dislocation of the apparently separate part, irrespective of size of the non-stained area.
Break: A visible fracture of the chromatide structure where the broken piece is laterally dislocated or shifted in the longitudinal axis but can still be assigned to the corresponding centric part.
Fragment: Acentric part of a chromosome which may appear individually, regardless of their size.
Minute: Small chromatide body with a diameter smaller than the width of the chromatide.
Deletion: Terminal or interstitial losses of part of the chromatide.
Exchange: These are exchange aberrations, subdivided into intrachanges (the union of parts that can combine, within a chromosome) and interchanges (the union of parts that can combine from two or more chromosomes). Dicentric chromosomes and ring chromosomes are included in this group.

The chromatide aberrations specified above can also occur as iso-chromatide aberrations (e.g. isochromatid break)

2. Numerical aberrations
Aneuploidy: A deviation from the typical number of individual chromosomes in a set of chromosomes; a decrease in the number is known as hypoploidy an a increase as hyperploidy.
Polyploidy: More than two sets of chromosomes.

3. Additional criterion:
Chromosomal disintegration: where all or most of the chromosomes are irregular particles. If exchange figures occur in the metaphases, they are only included in this aberration group.

Statistics:

Comparison of no of aberrations of treated and control groups

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

feces black colored; urine greyblue colored

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 5000 mg/kg bw (highest applicable dose)
- Clinical signs of toxicity in test animals: no clinical signs, no deaths
- Evidence of cytotoxicity in tissue analyzed: -


RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels: not different from vehicle control

Conclusions:

Reactive Black 5 is not mutagenic in the in vivo chromosome aberration test in bone marrow cells of the Chinese hamster.

Executive summary:

Reactive Black 5 was administered once orally by gavage in a single dose of 5000 mg/kg bodyweight to male and female Chinese hamsters. This dose had been shown in a preliminary study to be the maximum tolerated dose.

A positive control group, induced exactly 24 hours later to run parallel with the negative control and the dose group, received Endoxan in an oral dose of 50 mg/kg bodyweight.

Animals from each group were killed 12, 24 and 48 hours after treatment by carbon dioxide asphyxiation. 5 males and 5 females from each group were killed at each of these times.

The bone marrow obtained from femora of the animals was prepared, placed on microscopic slides and stained, after which 50 metaphases per animal were evaluated. The completeness in the number of chromosomes and the various chromatic and chromosomal aberrations were assessed.

Under the conditions of the present study, Reactive Black 5 caused no significant increase in the aberration rate in the bone marrow cells of the treated animals as compared with the control group.

Endoxan however produced a marked increase in the aberration rate in the test animals.

The results indicate that, under the conditions of the present study, Reactive Black 5 is not mutagenic in the in vivo chromosome aberration test in bone marrow cells of the Chinese hamster.

Reference 5

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 November to 30 November 1989

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

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: NMRI mouse
Strain: Hoe: NMRKf (SPF71)
Origin: HOECHST AG, Kastengrund, SPF breeding colony
Initial age at test: 7 weeks
Number of animals: 70 (35 males / 35 females)
Bodyweight at start of study: males : x= 30.4 g (27 - 37 g) females: x= 23.7 g (21 - 28 g)
Acclimatization: at least 5 days
Food / water: rat/mice diet Altromin 1324 (Altromin-GmbH, Lage/Lippe), ad libitum, tap water in plastic bottles, ad libitum
Housing: in fully air-conditioned rooms in Macrolon cages (Type 3), on softwood granulate in groups of 5 animals
Room temperature: 22 ± 2 °C
Relative humidity: 55 ± 10 %
Lighting time: 12 hours daily
Animal identification: fur-marking with KMn04 and cage numbering

Route of administration:

oral: gavage

Vehicle:

Deionised water

Details on exposure:

The test compound dilutions were prepared fresh each day. 6250 mg substance was weighed in a beaker, mixed with deionized water, washed out in a 25 ml flask and topped up to the calibration mark. A solution was formed.

For the Endoxan® stock solution, 5 ml distilled water were added to 100 mg Endoxan® in an injection phial and shaken to form a clear solution. The solutions for administration were prepared from this stock solution. For this purpose, 2 ml of the 2 % stock solution were mixed with 6 ml distilled water.

The test compound was administered orally by gavage to male and female mice. The following doses were tested: 0 and 5000 mg/kg bodyweight. The 5000 mg/kg bodyweight dose level was chosen since a preliminary study had shown it to be the maximum applicable dose. The animals were treated once with the test compound and according to the test procedure the animals were killed 24, 48 or 72 hours after administration of the test compound.

Duration of treatment / exposure:

0 and 5000 mg/kg divided into 2 administrations of 10 ml/kg
Killing of animals (5 males + 5 females per group) and evaluation of micronuclei 24, 48, and 72 hours after administration

Frequency of treatment:

2 administrations within 2 hours on Day 1

Post exposure period:

24, 48, or 72 hours

Dose / conc.:

5 000 mg/kg bw (total dose)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (Endoxan) 50 mg/kg bw

Tissues and cell types examined:

Bone marrow smears:
- Polychromatic erythrocytes
- Normochromatic erythrocytes

Details of tissue and slide preparation:

Extraction of the bone marrow
In conformity with the test procedure the animals were killed by carbon dioxide asphyxiation 24, 48 or 72 hours after application. For each animal, about 3 ml foetal 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 formed. The mixture was then centrifuged for 5 minutes at 1200 rpm and almost all the supernatant discarded. One drop of the thoroughly mixed sediment was smeared on a cleaned slide, identified by project code and animal number and air-dried for about 24 hours.

Staining procedure
5 minutes in methanol
3 minutes in May-Gruenwald's solution
2 minutes in May-Gruenwald's solution diluted 1:1 with distilled water
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:

1000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. As a control measure 1000 mature erythrocytes were also counted and examined for micronuclei. In addition, the ratio of polychromatic to normochromatic erythrocytes was determined. All bone marrow smears for evaluation are coded to ensure that the group to which they belonged remains unknown to the investigator. The number of polychromatic erythrocytes with micronuclei occurring in the 1000 polychromatic erythrocytes counted, and the number of normocytes with micro-nuclei occurring in the 1000 normocytes counted, were evaluated statistically; comparison of dose groups with the simultaneous control group was performed according to Wilcoxon (paired, one-sided, increase), (4).

The results of the treatment groups (test substance) in the micronucleus test at each dose and killing time were compared with corresponding control values. The ratio of polychromatic to normochromatic erythrocytes was also evaluated statistically by the method of Wilcoxon (paired, two sided) (4).

Statistics:

The statistical evaluations were performed using the "Diamant" computer program Version 2.0, supplied by the Department of Information and Communication Hoechst AG. All statistical results are based on a 95 % level of significance. Actual data were also compared with historical controls.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

tested up to highest applicable dose

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Animals were treated with 0 and 5000 mg/kg body-weight to study the induction of micronuclei in bone marrow cells of mice. All animals survived after application of 5000 mg/kg bodyweight. No signs of toxicity were observed.
The bone marrow smears were examined for the occurrence of micronuclei in red blood cells.
The incidence of micronucleated polychromatic erythrocytes in the dose groups of the substance was within the normal range of the negative control groups. No statistically significant increase of micronucleated polychromatic erythrocytes has been observed. The number of normochromatic erythrocytes with micronuclei did not differ significantly from the values of the simultaneous control animals for each of the three killing times investigated. The ratio of polychromatic erythrocytes to normocytes remained essentially unaffected by the test compound. A small reduction in the ratio of polychromatic to normochromatic erythrocytes was within the normal range of the historical controls and was considered as of no toxicological significance.
Cyclophosphamide (Endoxan(R)) induced a marked and statistically significant increase of the number of polychromatic erythrocytes with micronuclei in both males and females indicating the sensitivity to the test system.

Conclusions:

The substance did not lead to a substantial increase of micro-nucleated polychromatic erythrocytes. Hence, it is not cytogenic in the micronucleus test.

Executive summary:

This test was performed according to OECD guideline for testing of chemicals 474, 1983; Genetic Toxicology, Micronucleus test. No unforeseen cir­cumstances were observed, which may have affected the quality and integrity of this study. The study was conducted in compliance with the principles of Good Laboratory Practice.

The test substance was tested in the micronucleus test. The test compound was administered orally by gavage to male and female mice. The following doses were tested: 0 and 5000 mg/kg bodyweight. 

The 5000 mg per kg bodyweight dose level was chosen since a preliminary study had shown it to be the maximum applicable dose.

The animals were treated once with the test compound and according to the test procedure the animals were killed 24, 48 or 72 hours after administration of the test compound.

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

The incidence of micronucleated polychromatic erythrocytes of the animals treated with the test substance was within the normal range of the negative control. The number of normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes remained essentially unaffected by the test compound. A small reduction in the ratio of polychromatic to normochromatic erythrocytes was noted for male animals at 24 h killing time only. However this value was within the normal range of the historical controls and was considered as of no toxicologocal significance.

Endoxan® induced in both males and females a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating the sensitivity of the system. The ratio of polychromatic erythrocytes to normocytes showed a significant difference to the negative control values.

The results indicate that, under the conditions of the present study, the test substanceis not mutagenic in the micronucleus test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Reactive Blue 250 was tested for mutagenicity with the strains Salmonella typhimurium TA100, TA1535, TA1537, TA1538, TA98 and Escherichia coli WP2uvrA. The mutagenicity studies were conducted in the absence and in the presence of a metabolizing system derived from rat liver homogenate. A dose range of six different doses from 20 µg/plate to 10000 µg/plate was used. Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies.

The test item proved to be not toxic to the bacterial strains. On the basis of the preliminary test results, 10000 µg/plate was chosen as the top dose level in the main assay. In the absence and presence of the metabolic activation system the test item did not show a dose dependent increase in the number of revertants in any of the bacterial strains. 

Summarising, it can be stated that Reactive Blue 250 is not mutagenic in these bacterial test systems either with or without exogenous metabolic activation at the dose levels investigated.

 

The bacterial mutation test for the structural analogue Reactive Black 5 was tested in the strains TA100, TA1535, TA1537, TA98 of Salmonella typhimurium. The mutagenicity studies were conducted in the standard plate test (Ames Test) and in a modified preincubation test (Prival Test). The studies were performed in the absence and in the presence of a metabolizing system derived from rat or hamster liver homogenate. A dose range of six different doses from 4 µg/plate to 5000 µg/plate was used. Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies.

The test item proved to be not toxic to the bacterial strains. Consequently, 5000 µg/plate was chosen as top dose level for the mutagenicity study.

In the standard Ames test, the test substance did not show a dose dependent increase in the number of revertants in any of the bacterial strains in the absence or presence of the rat liver metabolic activation system. Also in the presence of hamster liver S9 using the preincubation method according to Prival, Reactive Black 5 did not induce a significant increase in the number of revertant colonies, with any of the tester strains.

Summarizing, it can be stated that Reactive Black 5 is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Prival. Due to the similarity in structure and activity of the substance the same is considered ttrue for Reactive Blue 250.

 

The test structural analogues 01, 02 and 03 were investigated for their potential to induce gene mutations in two independent mutagenicity assays.  One assay was conducted with the plate incorporation method and the other as a modified preincubation test (Prival test). The studies were performed in the absence and in the presence of a metabolizing system derived from rat or hamster liver homogenate. The respective substance was assessed up to 5000 µg/plate in the standard plate test in the modified preincubation test. The substances did not cause a significant increase in the number of revertant colonies at any dose level in the absence or presence of metabolic activation system in either of the tests. Under the study conditions, the substances were found to be non-mutagenic in the bacterial reverse mutation assay including the Prival modification.

 

Structural analogue 04 was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. The study was performed in two independent experiments, using identical experimental procedures. In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

The main experiments were evaluated at the following concentrations:

exposure period	S9 mix	concentrations in µg/mL
		Experiment I
4 hours	-	525.0	1050	2100	3150	4200
4 hours	+	1050	2100	4200	6300	8400
		Experiment II
24 hours	-	134.2	268.4	536.9	805.3	1073.8
4 hours	+	268.4	536.9	1073.6	2147.5	4295.0

 

No precipitation of the test item was observed up to the maximum concentration in any of the experiments. Relevant cytotoxic effects, indicated by a relative cloning efficiency I or a relative cell density at first subcultivation of less than 50% in both parallel cultures, occurred in the first experiment at 1050 µg/mL and above without metabolic activation. In the second experiment relevant cytotoxic effects as described above were noted at 805.3 µg/mL and above without metabolic activation and at 4295 µg/mL with metabolic activation. The recommended cytotoxic range of approximately 10%-20% relative cloning efficiency or relative cell density was covered with and without metabolic activation. The difference in cytotoxicity noted in the first and the second experiment with metabolic activation is based on the variability of the cell density during treatment. According to the OECD 476 guideline proliferating cells should be treated so, the actual cell density varies from experiment to experiment.

No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration. The mutation frequency did not exceed the historical range of solvent controls, the induction factor did not reach or exceed the threshold of 3.0. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

 

The structural analogue Reactive Black 5 was tested for clastogenicity/aneugenicity in a mouse micronucleus test and a chromosome aberration test in vivo.

In the micronucleus test in mice, Reactive Black 5 was administered once orally by gavage as single doses of 0 (vehicle control), 2000 and 10000 mg/kg bodyweight to male and female NMRI mice (5 mice per sex and killing time point). 10000 mg/kg bw had been shown in a preliminary study to be the maximum feasible dose. A positive control group received Endoxan at an oral dose of 50 mg/kg body weight. Animals were killed 12, 24, or 48 hours after treatment by carbon dioxide asphyxiation. The bone marrow obtained from femora of the animals was prepared, placed on microscopic slides and stained. 1000 poly- and 1000 normochromatic erythrocytes were screened for micronuclei. In addition, the ratio of polychromatic to normochromatic erythrocytes was determined.

Under the conditions of the present study, Reactive Black 5 caused no significant increase in the number of cells with micronuclei in the bone marrow cells of treated animals as compared with the control group. Endoxan however produced a marked increase in number of cells with micronuclei.

The results indicate that, under the conditions of the present study, Reactive Black 5 is not clastogenic or aneugenic in the in vivo micronucleus test.

In the chromosome aberration test in the Chinese hamster, Reactive Black 5 was administered once orally by gavage in a single dose of 5000 mg/kg bodyweight to male and female Chinese hamsters. This dose had been shown in a preliminary study to be the maximum tolerated dose. A positive control group, induced exactly 24 hours later to run parallel with the negative control and the dose group, received Endoxan in an oral dose of 50 mg/kg bodyweight. Animals from each group were killed 12, 24 and 48 hours after treatment by carbon dioxide asphyxiation. Five males and five females from each group were killed at each of these times. The bone marrow obtained from femora of the animals was prepared, placed on microscopic slides and stained, after which 50 metaphases per animal were evaluated. The completeness in the number of chromosomes and the various chromatic and chromosomal aberrations were assessed.

Under the conditions of the present study, Reactive Black 5 caused no significant increase in the aberration rate in the bone marrow cells of the treated animals as compared with the control group. Endoxan however produced a marked increase in the aberration rate in the test animals.

The results indicate that, under the conditions of the present study, Reactive Black 5 is not clastogenic in the in vivo chromosome aberration test in bone marrow cells of the Chinese hamster.

 

Structural analogues 01 and 03 were assessed for their potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of mice according to OECD Guideline 474. The substances were tested at the maxiumum dose levels 0f 5000 and 2000 mg/kg bw, respectively. A vehicle control group treated with distilled water and a positive control group treated with cyclophosphamide (60 mg/kg bw intraperitoneally) were also included. The oral administration of the test substances did not induce any biologically relevant increase in the frequency of micronucleated polychromatic erythrocytes (MPCEs) in male or female mice compared to the vehicle control. No biologically important alteration in the ratio of polychromatic to normochromatic erythrocytes occurred in the treated groups when compared to the vehicle control. Treatment with the positive control, cyclophosphamide, caused significant increase in the number of micronucleated polychromatic erythrocytes (MPCEs), thus validating the test. Under the study conditions, the test substances did not induce micronuclei in bone marrow cells of the treated mice. Therefore, the test substances were not considered to be clastogenic in this micronucleus assay.

 

Structural analogue 02 was tested for the potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the rat according to OECD Guideline 474, EPA OPPTS 870.5395 and EU Method B.12, in compliance with GLP. The test substance was suspended in sesame oil and given twice at an interval of 24 h as a oral dose of 2000 mg/kg bw/d to male and female rats. The dose was selected based on the results of a previous rat acute oral toxicity study. The animals were sacrificed 24 h after the last administration and bone marrow cells were collected for micronuclei analysis. After treatment with the test substance, 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 and differed less than 20% from the control value.

Under the study conditions, the test substance did not induce micronuclei in bone marrow cells of the rat. Therefore, the test substance was not considered to be clastogenic in this micronucleus assay.

 

Based on the similarity of source and target substances with respect to structure, physico-chemical properties and mechanism of action, the results of the studies with the structural analogues can be used for read across to Reactive Blue 250.

Justification for classification or non-classification

Mutation and clastogenicity tests negative - No classification necessary