5-[[4-chloro-6-[(3-sulphophenyl)amino]-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2,7-disulphonic acid, sodium salt

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No genotoxicity observed

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 20th, 1988 to June 23th, 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Cytokinesis block (if used):

Colchicine

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

200, 1100, 2000 µg/ml

Vehicle / solvent:

water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium;

Evaluation criteria:

-The test substance is classified as mutagenic if it induces a significantly increased aberration rate as compared with the negative controls with one of the concentrations tested . The significance is obvious either by an enhancement of the rate clearly exceeding the control range or it is proven by adequate biometry (Binomial statistic with Fisher's exact test).
-The test substance is classified as mutagenic if there is a reproducible concentration related increase in the aberration rate.
-The test substance is classified as not mutagenic when it tests negatively both with and without metabolic activation.

Statistics:

Fisher's exact test

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

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:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

ambiguous

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
In a preliminary experiment Reactive Red 198 was assayed with respect to its solubility in cell culture medium. The highest concentration at which no visible precipitation was observed, was found to be 2000 µg/mL.
The cytotoxicity experiment proved that Reactive Red 198 was not toxic to the V79 cells in the absence of metabolic activation (S9-mix). Also in the presence of metabolic activation (S9-mix) no indication of toxicity was observed up to the limit of solubility.
On the basis of these results the preparation of chromosomes was done after 2 h-treatment with 2000 µg/ml at 6, 18 and 28 h, and 18 hours after treatment additionally with 1100 and 200 µg/mL, both with and without S9-mix.

Mutagenicity
The test substance Reactive Red 198 was assessed for its mutagenic potential in vitro in the chromosome aberration test with two independent cell cultures without metabolic activation and two independent cell cultures with metabolic activation.
A reproducible enhancement in the number of phases with aberrations, but only inclusive gaps over the range of the solvent control was found at the concentration of 2000 µg/mL at 28h after treatment without metabolic activation (S9-mix). A statistical increase of aberrations inclusive and exclusive gaps was observed at the same dose level and preparation time. The significant enhancement of aberrations exclusive gaps is of no genotoxic relevance, because of the low level (=0%) in the solvent control. The types of aberrations in the 28h interval induced preliminary consisted of iso-breaks, iso-fragments, minutes and exchanges.

Conclusions:

In conclusion Reactive Red 198 does not significantly induce clastogenic effects in V79 Chinese hamster cells in the presence and in the absence of a metabolic activation system, under the experimental conditions described.

Executive summary:

The test substance Reactive Red 198 was examined for mutagenic activity in V79 Chinese hamster cells. The induction of chromosome aberrations after in vitro treatment was investigated in the presence and absence of a fraction of liver homogenate for metabolic activation (S9-mix).

A preliminary cytotoxicity experiment was performed in order to select appropriate dose levels for the mutagenicity study. The test substance produced no significant cytotoxic effect (reduction of plating efficiency) without metabolic activation up to the limit of solubility (2000 µg/mL). No cytotoxic effect was also observed with metabolic activation up to the limit of solubility.

For mutagenicity testing two independent cell cultures with and without metabolic activation (S9-mix) up to the limit of solubility (2000 µg/mL) were used.

For main experiment dose levels of 200, 1100, 2000 µg/mL were used, in the absence and in the presence of S9-mix metabolic activation.

The test compound induced a significant increase in the number of phases with aberrations inclusive gaps, and in the number of aberrations inclusive and exclusive gaps 28h after treatment with 2000 µg/mL without S9-mix. At the other dose levels the test compound did not induce a significant increase of phases with aberrations. No relevant cytotoxic effect (reduction of mitotic index) of the compound was observed in the main experiments.

Marked increases in the rate of chromosome aberrations were obtained with the positive control substances indicating the sensitivity of the assay.

In conclusion Reactive Red 198 does indicate a slight mutagenic effect (=aberrations) in V79 Chinese hamster cell s in the absence of a metabolic activation system. This effect was considered as not sufficient to evaluate the compound as distinct genotoxic agent. In the presence of S9-mix no chromosome mutations were observed under the experimental conditions described.

 

Reference 2

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:

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

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.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

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:

Conclusion:
In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, Reactive Red 198 is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The test item Reactive Red 198 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
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 3

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
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.

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

The 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-1997 and U.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 of Good 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 substance was dissolved in deionised water, and each bacterial strain was exposed to 5 dose levels. Doses for both studies ranged from 50 to 5000 μg/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.

The substance did not precipitate on the plates up to the highest investigated dose of 5000 μg/plate. In both studies toxicity was not observed either with or without metabolic activation.

 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 or presence of the rat liver activation system (10 % (v/v)). Also in the absence and in the presence of hamster liver S9-mix (30 % (v/v)) using the preincubation method according to Prival, the 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 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1982

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

AMES, B.N. et al. (1973) "Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection."
Proceedings of the National Academy of Sciences 70, 2281 - 2285.
GREEN, M.H.L. and MURIEL, W.J. (1976) "Mutagen testing using TRP+ reversion of Escherichia coli."
Mutation Research 38, 3 - 32.

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
TA1538 hisD3052 Frameshift

Escherichia coli:
WP2uvrA trpE Base pair substitution

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix from rat liver

Test concentrations with justification for top dose:

4 to 10000 µg/plate

Vehicle / solvent:

deionised water, DMSO

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

other: 2-aminoanthracene, methylhydrazone derivative, streptocotocine, ENNG

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

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

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

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

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

Vehicle 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

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

The results lead to the conclusion that Reactive Red 198 is not mutagenic in the absence and presence of metabolic activation.

Executive summary:

Compound 51 / 82 A was examined in the mutagenicity screening test in bacteria first described by Ames and co-workers.

Under the conditions we employed 51 / 82 A in concentrations of 4 µg to 10,000 µg showed no mutagenic activity.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 April to 05 May 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

84/449/EWG, B.14, Prival Modification

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

TA98 hisD3052 Frameshift
TA100 hisG46 Base pair substitution
TA1535 hisG46 Base pair substitution
TA1537 hisC3076 Frameshift

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: histidine dependent

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix from rat and hamster liver

Test concentrations with justification for top dose:

Preliminary test: 4 to 10000 µg/plate
Main test: 20 to 10000 µg/plate

Vehicle / solvent:

At the day of the experiment the test substance was dissolved in Aqua bidest at appropriate concentrations. Two independent experiments were performed for each protocol (Ames, Prival).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

Remarks:

without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

congo red

other: 2-aminoanthracene, benzidine

Remarks:

with S9

Details on test system and experimental conditions:

Preparation and storage of a liver homogenate fraction ("S-9")
Liver preparations were performed from liver of Aroclor induced Sprague Dawley rats and from non pretreated Syrien hamsters. Male Sprague Dawley rats (200 -300 g) receive a single intraperitoneal injection of Aroclor 1254 (500 mg/kg bodyweight) 5 days before sacrifice. Preparation is performed at 0 to 4 °C using cold sterile solution and glassware. The livers from at least 5-6 Sprague Dawley rats or from 5-6 male Syrian golden hamsters (7-8 weeks old) are removed and pooled, washed in 150 mM KCl (approximately 1 ml/g wet livers). The washed livers are cut into small pieces and homogenized in three volumes of KC1. The homogenate is centrifuged at 9000 g for 10 minutes. The supernatant is the S-9 fraction. It is divided into small portions, rapidly frozen and stored at -80 °C for not longer than three months.

Preparation of S-9 Mix
Sufficient S-9 fraction is thawed immediately before each test at room tempera¬ture. One or three volumes of S-9 fraction is mixed with nine or seven volumes of the S-9 cofactor solution and kept on ice until used. This preparation is termed S-9 Mix. The concentrations of the different compounds in the S-9 Mix of the rat liver are:

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

According to the modification proposed by Prival (5) using 30 minutes preincubation in the presence of 30 % Syrian golden hamster S-9 Mix. The S-9 Mix consists of:

8 mM MgCl2
33 mM KCI
20 mM glucose-6-phosphate
2.8 units/ml glucose-6-phosphate dehydrogenase
4 mM NAPD+
2 mM NADH
2 mM FMN (Riboflavin-5’-phosphate-Na-salz)
100 mM phosphate buffer pH 7.4

Bacteria
Bacteria are grown overnight in nutrient broth (25 g Oxoid Nutrient Broth No 2 /liter) at 37 °C. The suitable amount of bacteria in the cell suspension is checked by nephelometry. For inoculation, stock cultures which are stored at -80 °C, are used. The compound is tested with the strains Salmonella typhimurium TA 100, TA 1535, TA 1537, and TA 98.

Toxicity experiments and dose range finding
Preliminary toxicity tests were performed with five or four tester strains using three plates per dose to get information on mutagenicity and toxicity for calculation of an appropriate dose range. A reduced rate of spontaneously occuring colonies as well as visible thinning of the bacterial lawn were used as indicator for toxicity. Thinning of the bacterial lawn was controlled microscopically. In combination with the main experiments, toxicity testing was performed as follows: 0.1 ml of the different dilutions of the test compound were thoroughly mixed with 0.1 ml of 10-6 dilution of the overnight culture of TA 100 and plated with histidine and biotin rich top agar (3 plates per dose). The solvent control is compared with the number of colonies per plate in the presence of the test compound. Results are given as a ratio of these values (= surviving fraction).

Mutagenicity test
Two independent experiments which each of the two protocols (Ames, Prival) were performed.

a) - with 10 % rat liver S-9 Mix or buffer and the strains TA 100, TA 1535, TA 1537, and TA 98 - with 30 % rat liver S-9 Mix and the stains TA 100, TA 1535, TA 1537 and TA 98

Top agar is prepared for the Salmonella strains by mixing 100 ml agar (0.6 % agar, 0.6 % NaCl) with 10 ml of a 0.5 mM histidine-biotin solution. The follow¬ing 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 10 % or 30 % rat liver S-9 Mix or buffer

After mixing, the liquid is poured into a petridish with minimal agar (1.2 % 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.

b) with 30 % Syrian golden hamster S-9 Mix and preincubation

0.1 ml test solution, 0.1 ml bacterial suspension and 0.5 ml S-9 Mix are incubated at 30 °C for the duration of 30 minutes. Subsequently, 2 ml of soft agar which consists of 100 ml agar (0.6 % agar + 0.6 % NaCl) and 10 ml amino-acid solution (minimal amino-acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) is added. After mixing, the samples are poured on to the Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds. After incubation for 48 to 72 hours at 37 °C in the dark, colonies (his+ revertants) are counted.

Positive controls
Positive control plates were included for each strain. The following substances were used as positive controls.

a) without metabolic activation:
Na-azide: TA 100, TA 1535
9-Aminoacridine: TA 1537
2-Nitrofluorene: TA 98

b) with rat liver S-9 Mix (10 %):
Benzo[a]pyrene: TA 98, TA 100, TA 1535, TA 1537
2-Aminoanthracene: TA 98, TA 100, TA 1535, TA 1537

c) with rat liver S-9 Mix (30 %):
Benzo[a]pyrene: TA 98, TA 100, TA 1535, TA 1537
2-Aminoanthracene: TA 98, TA 100, TA 1535, TA 1537

d) with hamster liver S-9 Mix (30 %):
2-Aminoanthracene: TA 100, TA 1535, TA 1537
Benzidine: TA 98
Congored: TA 98

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.

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 µg/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 µg/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 µg/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 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Reactive Red 198 was tested for mutagenicity with Salmonella typhimurium strains TA 98, TA 100, TA 1535, and TA 1537 in the absence and presence of a metabolic activation systems. S-9 Mix from Sprague Dawley rats and from Syrien golden hamsters were used. The number of colonies per plate with each strain as well as mean values of 3 plates, corrected to the next whole number are given.

Sterility checks and control plates
Sterility of S-9 Mix and the test compound were indicated by the absence of contamination on the test material and S-9 Mix sterility check plates. Control plates (background control and positive controls) gave the expected number of colonies.

Toxicity test
The test compound was tested at doses of 4 to 10000 microgram/plate and proved to be not toxic to the bacterial strains. For mutagenicity testing 10000 microgram/plate was chosen as the highest dose in the main experiments.

Mutagenicity test with Reactive Red 198
Ames-Test:
The test compound did not cause a significant increase in the number of revertant colonies with any of the tester strains either in the absence or in the presence of rat S-9 Mix (10 %). No dose dependent effect was obtained.

Prival-Test:
In the presence of rat liver S-9 Mix (30 %) and hamster liver S-9 Mix (30 %) using the preincubation method according to Prival the test compound did not show any relevant increases in the number of revertant colonies under the experimental conditions described.

Conclusions:

Reactive Red 198 is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Prival.

Executive summary:

Reactive Red 198 was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 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 20 microgram/ plate to 10000 microgram/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. 10000 microgram/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 bacteria1 strains. Also in the presence of a metabo1ic activation system, treatment of the cells with Reactive Red 198did not result in relevant increases in the number of revertant colonies.

b) Prival Test:

In the presence of hamster liver S-9 using the preincubation method according to Priva1 Reactive Red 198 di d not induce a significant increase in the number of revertant colonies, with any of the tester strains.

Summarizing, it can be stated that Reactive Red 198is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Prival.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The substance has no clastogenic properties

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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:

mammalian erythrocyte micronucleus test

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 KMnO4 and cage numbering

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Deionised water
- Justification for choice of solvent/vehicle: good water solubility
- Concentration of test material in vehicle: 25% (w/v)
- Amount of vehicle (if gavage or dermal): 20 mL/kg bw divided in two portions of 10 mL/kg bw two ours appart

Details on exposure:

The test compound dilutions were prepared fresh each day. 6250 mg was weighed in a beaker, mixed with deionisized 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 Reaktiv-Rot F-66 813 FW per 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.

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

Remarks:

Doses / Concentrations:
5000 mg/kg bw
Basis:

Remarks:

Doses / Concentrations:
250 mg/mL
Basis:
nominal conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Endoxan
- Route of administration: oral
- Doses / concentrations: 50 mg/kg

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-Grünwalds solution
2 minutes in May-Grünwalds 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 test substance per kg bodyweight 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 occurance of micronuclei in red blood cells.
The incidence of micronucleated polychromatic erythrocytes in the dose groups of the test 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 polycromatic to normochromatic erythrocytes was within the normal range of the historical controls and was considered as of no toxicological significance.
Cyclophosphamid (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:

Administration of the test substance did not lead to a substantial increase of micronucleated polychromatic erythrocytes. It is concluded that the test substance 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 test compound was given in two equal parts within two hours 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 substance is not mutagenic in the micronucleus test.

Additional information

The test substance was negative in two Ames tests with four different Salmonella strains, one additional with Prival modification and the other with one additional E. coli strain. In addition, Reactive Red 198 was negative in the mutagenicity test in mammalian cells (HPRT test). Reactive Red 198 did not show any clastogenic effects in an in-vitro chromosome aberration test with metabolic activation system. However, it showed a slight clastogenic effect in this test in the absence of the metabolic activation system. This is in line with the result of many in-vitro clastogenicity tests for vinyl-sulphone dyes.

It is well known that vinyl-sulphone compounds result in false positive test results in in-vitro tests for clastogenicity (Dearfield KL et al. (1991); Warra TJ et al. (1990)). This is due to the fact that these chemical agents react via the Michael addition reaction. Chemical reactivity via Michael addition is essential for many of the uses for which these compounds are important. As in the currently assessed dye, Reactive Red 198, vinyl sulphone moieties are used in fiber-reactive dyes (MacGregor et at. (1980)). These compounds are known to deplete glutathione in in‑vitro test systems, in which the concentration of phase II enzymes is very low. Glutathione plays a role in the detoxification of many compounds. Conjugation with glutathione via Michael addition and subsequent excretion is the most common bio-elimination route for these compounds. Since in-vitro systems have low levels of glutathione, the glutathione depletion leads to a positive result in the in-vitro test system, which is not the case in the in-vivo test system, where glutathione is present in adequate amount, as could be shown in the negative in-vivo micronucleus study with the structural analogue which showed also a positive in-vitro result.

 

 

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

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

Warra TJ et al. (1990). Methyl vinyl sulphone: A new class of Michael-type genotoxin. Mutat Res. 1990;245:191-199

Justification for classification or non-classification

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

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