2,2'-[[3-acetamido-4-[(2-chloro-4-nitrophenyl)azo]phenyl]imino]diethyl diacetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Only bacteria-specific effects were noted in the bacteria reverse mutation assay. The test substance or its structural analogue was negative in all other genotoxicity studies.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

09 December 2014 to 04 May 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

The test item Disperse Blue ANT was examined for mutagenic activity by assaying for the induction of 6-thioguanine resistant mutants in Chinese hamster V79 cells after in vitro treatment.
6-thioguanine can be metabolised by the enzyme hypoxanthine-guaninphosphoribosyl-transferase (HPRT) into nucleotides, which are used in nucleic acid synthesis resulting in the death of HPRT-competent cells. HPRT-deficient cells, which are presumed to arise through mutations in the HPRT gene, cannot metabolise 6-thioguanine and thus survive and grow in its presence.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: EMEM medium supplemented with 10% Foetal Calf Serum (EMEM complete)
- Properly maintained: yes; permanent stock of V79 cells are stored in liquid nitrogen and subcoltures are prepared from the frozen stocks for experimental use.
- Periodically checked for Mycoplasma contamination: yes
- The karyotype, generation time, plating efficiency and mutation rates (spontaneous and induced) have been checked in this laboratory.
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 tissue fraction: Species: Rat Strain: Sprague Dawley Tissue: Liver Inducing Agents: Phenobarbital – 5,6-Benzoflavone Producer: MOLTOX, Molecular Toxicology, Inc. Batch Numbers. 3332, 3263 and 3417

Test concentrations with justification for top dose:

A preliminary cytotoxicity assay was performed at the following dose levels: 1250, 625, 313, 156, 78.1, 39.1, 19.5, 9.77 and 4.88 µg/mL.
A second toxicity test was performed in the absence of S9 metabolism using the following lower and closer concentrations: 10.0, 7.14, 5.10, 3.64 and 2.60 µg/mL.
Since no adequate toxicity for dose selection was obtained, a third toxicity test was performed in the absence of S9 metabolism, using the following concentrations: 40.0, 20.0, 10.0, 5.00 µg/mL.
Two independent assays for mutation to 6-thioguanine resistance were performed using dose levels:
Main Assay I (+S9): 600, 400, 267, 178, 119 and 79.0 µg/mL
Main Assay I (-S9): 40.0, 20.0, 10.0, 5.00, 2.50, 1.25 and 0.625 µg/mL
Main Assay II (+S9): 400, 308, 237, 182, 140 and 108 µg/mL
Main Assay II (+S9): 20.0, 15.4, 11.8, 9.10, 7.00 and 5.39 µg/mL

Vehicle / solvent:

Test item solutions were prepared using dimethylsulfoxide (DMSO).

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Preliminary cytotoxicity tests were undertaken in order to select appropriate dose levels for the mutation assays: a single experiment was performed in the presence of S9 metabolism, while three tests were performed in the absence of S9 metabolic activation.A single culture was used at each test point and positive controls were not included.
Two Mutation Assays were performed including negative and positive controls, in the absence and presence of S9 metabolising system.
Duplicate cultures were prepared at each test point, with the exception of the positive controls which were prepared in a single culture. On the day before the experiment, sufficient numbers of 75 cm2 flasks were inoculated with 2 million freshly trypsinised V79 cells from a common pool. The cells were allowed to attach overnight prior to treatment. Following treatment, the cultures were incubated at 37°C for three hours. At the end of the incubation period, the treatment medium was removed and the cell monolayers were washed with PBS. Fresh complete medium was added to the flasks which were then returned to the incubator at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) to allow for expression of the mutant phenotype.
Determination of survival: The following day, the cultures were trypsinised and an aliquot was diluted and plated to estimate the viability of the cells.
Subculturing: On Day 3, the cell populations were subcultured in order to maintain them in exponential growth. When Day 8 was used as expression time, subculturing was performed on Day 4 and Day 6.
Determination of mutant frequency: A single expression time was used for each experiment: Day 8 in Main Assay I and Day 6 in Main Assay II. At the expression time, each culture was trypsinised, resuspended in complete medium and counted by microscopy. After dilution, an estimated 1 x 10^5 cells were plated in each of five 100 mm tissue culture petri dishes containing medium supplemented with 6-thioguanine.
These plates were subsequently stained with Giemsa solutions and scored for the presence of mutants. After dilution, an estimated 200 cells were plated in each of three 60 mm tissue culture petri dishes. These plates were used to estimate Plating Efficiency (P.E.).

Evaluation criteria:

EVALUATION CRITERIA
For a test item to be considered mutagenic in this assay, it is required that:
- There is a five-fold (or more) increase in mutation frequency compared with the solvent controls, over two consecutive doses of the test item. If only the highest practicable dose level (or the highest dose level not to cause unacceptable toxicity) gives such an increase, then a single treatment-level will suffice.
- There must be evidence for a dose-relation (i.e. statistically significant effect in the ANOVA analysis).

Statistics:

The results of these experiments were subjected to an Analysis of Variance in which the effect of replicate culture and dose level in explaining the observed variation was examined. For each experiment, the individual mutation frequency values at each test point were transformed to induce homogeneous variance and normal distribution. The appropriate transformation was estimated using the procedure of Snee and Irr (1981), and was found to be y = (x + a)b where a = 0 and b = 0.275. A two way analysis of variance was performed (without interaction) fitting to two factors:
- Replicate culture: to identify differences between the replicate cultures treated.
- Dose level: to identify dose-related increases (or decreases) in response, after allowing for the effects of replicate cultures and expression time.
The analysis was performed separately with the sets of data obtained in the absence and presence of S9 metabolism.

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:

Survival after treatment:
In Main Assay I, following treatment in the absence of S9 metabolism, a severe toxic effect was observed at the highest dose level (40.0 µg/mL) reducing survival to 2% of the concurrent negative control value. At the next lower concentration of 20.0 µg/mL, survival was reduced to 13%. Mild toxicity was observed at 10.0 µg/mL, while no relevant toxicity was noted at the remaining concentrations tested. In the presence of S9 metabolic activation, severe toxicity (relative survival < 10%) was observed at the two highest dose levels (600 and 400 µg/mL), while test item treatment at 267 µg/mL yielded 41% relative survival. At lower dose levels, no relevant toxicity was noted. At the highest dose level tested, no cells were recovered on Day 6 both in the absence and presence of S9 metabolic activation. Since at low survival levels, mutation results are prone to a number of artefacts (selection effects, sampling error, founder effects) and mechanisms other than direct genotoxicity per se can lead to positive results (e.g. events associated with apoptosis, endonuclease release from lysosomes, etc.), the dose level of 400 µg/mL was excluded from analysis.
In Main Assay II, in the absence of S9 metabolism, survival was reduced to 15% at the highest dose level of 20.0 µg/mL; moderate toxicity (approximately 30% of RS) was noted at the two next lower concentrations; mild toxicity (RS=58%) was observed at 9.10 µg/mL, while no relevant toxicity was observed at the remaining dose levels. It should be noted that on Day 6 a lower number of cells, compared to the negative control value, was recovered at the highest dose level. In the presence of S9 metabolism, test item treatment at 400 µg/mL yielded 31% relative survival; mild toxicity (RS = 63%) was noted at the next lower dose level, while no relevant toxicity was observed over the remaining concentrations.
Mutation results:
In the presence of S9 metabolism, no increase over the spontaneous mutation frequency was observed in any experiment, at any treatment level. In the absence of S9 metabolic activation, no increase over the spontaneous mutation frequency was observed in Main assay I (Day 8), while a dose related increase in mutation frequency, which reached five-fold the concurrent negative control value at 15.4 µg/mL, was observed in Main Assay II (Day 6). Analysis of variance indicated that replicate culture was not a significant factor in explaining the observed variation in the data, in the absence and presence of S9 metabolism, in Main Assay I and II. Dose level was a significant factor (p < 0.05%) in explaining the observed variation in the data of Main Assay II in the absence of S9 metabolism.

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that Disperse Blue ANT does not induce mutation in Chinese hamster V79 cells after in vitro treatment, in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Executive summary:

The test item Disperse Blue ANT was examined for mutagenic activity by assaying for the induction of 6-thioguanine resistant mutants in Chinese hamster V79 cells after in vitro treatment. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. Test item solutions were prepared using dimethylsulfoxide (DMSO). A preliminary cytotoxicity assay was performed, in the absence and presence of S9 metabolic activation. Based on solubility features, the test item was assayed at a maximum dose level of 1250 µg/mL and at a wide range of lower dose levels: 625, 313, 156, 78.1, 39.1, 19.5, 9.77 and 4.88 µg/mL. In the absence of S9 metabolism, no cells survived at almost all the concentrations tested; survival was reduced to 50% of the concurrent negative control value at the lowest dose level (4.88 µg/mL). In the presence of S9 metabolism, severe toxicity was noted at the two highest concentrations, while survival was reduced to 44% of the concurrent negative control value at 313 µg/mL. No relevant toxicity was noted over the remaining concentrations tested. By the end of treatment time, precipitation of test item and/or a coloured film, adhering to the flask surface, was observed at all concentrations tested in the absence of S9 metabolism and at the three highest dose levels in its presence. In order to select the appropriate range of concentrations for the mutation assay, an additional toxicity test was performed in the absence of S9 metabolism using the following lower and closer concentrations: 10.0, 7.14, 5.10, 3.64 and 2.60 µg/mL. Since no adequate toxicity for dose selection was obtained, a third toxicity test was performed in the absence of S9 metabolism, using the following concentrations: 40.0, 20.0, 10.0, 5.00 µg/mL. Severe toxicity was noted at the highest concentration; survival was reduced to 12% and 37% of the concurrent negative control value at 20.0 and 10.0 µg/mL, respectively; while no relevant toxicity was observed at 5.00 µg/mL. Two independent assays for mutation to 6-thioguanine resistance were performed using the following dose levels:

Main Assay I (+S9): 600, 400, 267, 178, 119 and 79.0 µg/mL

Main Assay I (-S9): 40.0, 20.0, 10.0, 5.00, 2.50, 1.25 and 0.625 µg/mL

Main Assay II (+S9): 400, 308, 237, 182, 140 and 108 µg/mL

Main Assay II (+S9): 20.0, 15.4, 11.8, 9.10, 7.00 and 5.39 µg/mL

In the second experiment the concentration range was slightly modified on the basis of the toxicity results obtained in Main Assay I. No reproducible five-fold increases in mutant numbers or mutant frequency were observed following treatment with the test item at any dose level, in the absence or presence of S9 metabolism. Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system.

It is concluded that Disperse Blue ANT does not induce gene mutation in Chinese hamster V79 cells after in vitro treatment in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

October 01, 1997 to October 20, 1997

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:

First Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 471, "Salmonella typhimurium, Reverse Mutation Assay", adopted May 26, 1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EEC Directive 92/69, L 383 A, Annexe V, B 14, dated December 29, 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Based upon the results of the pre-experiment the concentrations applied in the main experiments were chosen.
The maximum concentration was 5000 μg/plate (active ingredient). The concentration range included two logarithmic decades. In this study six adequately spaced concentrations were tested. Two independent experiments were performed.
To evaluate the toxicity of the test article a pre-study was performed with strains TA 98 and TA 100. The plates with the test article showed normal background growth up to 5000 ug/plate in strain TA 98 and TA 100.
According to the dose selection criteria the test article was tested at the following concentrations:
33; 100; 333; 1000; 2500; and 5000 μg/plate (active ingredient)

Vehicle / solvent:

On the day of the experiment, the test article was dissolved in DMSO. The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-o-phenylene-diamine (4-NOPD); 2-aminoanthracene (2-AA)

Details on test system and experimental conditions:

Test System
Characterisation of the Salmonella typhimurium Strains
The histidine dependent strains are derived from S. typhimurium strain LT2 through a mutation in the histidine locus. Additionally due to the "deep rough" (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus". In the strains TA 98 and TA 100 the R-factor plasmid pKM 101 carries the ampicillin resistance marker.
In summary, the mutations of the TA strains used in this study can be described as follows:
Salmonella typhimurium
TA 1537: his C 3076; rfa·; uvrB·: frame shift mutations
TA 98: his D 3052; rfa·; uvrB-;R-factor: frame shift mutations
TA 1535: his G 46; rfa-; uvrB·: base-pair substitutions
TA 100: his G 46; rfa-; uvrB-;R-factor: base-pair substitutions
Regular checking of the properties of the strains regarding the membrane permeability, ampicillin- and tetracycline-resistance as well as spontaneous mutation rates is performed in the laboratory of CCR according to Ames et al. In this way it was ensured that the experimental conditions set down by Ames were fulfilled.
The bacterial strains TA 1535, TA 98, and TA 100 were obtained from Ames (University of California, 94720 Berkeley, U.S.A.). The bacterial strain TA 1537 was obtained from BASF (D-67063 Ludwigshafen).

Storage
The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO (MERCK, D-64293 Darmstadt) in liquid nitrogen.

Precultures
From the thawed ampoules of the strains 0.5 ml bacterial suspension was transferred into 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. A solution of 20 μl ampicillin (25 μg/ml) was added to the strains TA 98 and TA 100. This nutrient medium contains per litre:
8 g Merck Nutrient Broth (MERCK, D-64293 Darmstadt)
5 g NaCl (MERCK, D-64293 Darmstadt)
The bacterial culture was incubated in a shaking water bath for 8 hours at 37° C.

Selective Agar
The plates with the minimal agar were obtained from E. Merck, D-64293 Darmstadt.

Overlay Agar
The overlay agar contains per litre:
6.0 g MERCK Agar Agar*
6.0 g NaCl*
10.5 mg L-Histidine x HCl x Hp*
12.2 mg Biotin*
* (MERCK, D-64293 Darmstadt)
Sterilisations were performed at 121 °C in an autoclave.

Mammalian Microsomal Fraction S9 Mix
The bacteria used in these assays do not possess the enzyme systems, which, in mammals, are known to convert promutagens into active DNA damaging metabolites. In order to overcome this major drawback an exogenous metabolic system is added in form of mammalian microsome enzyme activation mixture.

Pre-Experiment for Toxicity
To evaluate the toxicity of the test article a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described for the experiment I below (plate incorporation test).
Toxicity of the test article can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

Experimental Performance
For each strain and dose level, including the controls three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 μl: Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
500 μl: S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 μl: Bacteria suspension (cf test system, pre-culture of the strains),
2000 μl: Overlay agar

After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark.

Data Recording
The colonies were counted using the AUTOCOUNT (Artek Systems Corporation, BIOSYS GmbH, D-61184 Karben). The counter was connected to an IBM AT compatible PC with printer which printed out both, the individual and mean values of the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates. Due to precipitation of the test article the colonies were counted manually from 333 μg/plate up to 5000 μg/plate.

Rationale for test conditions:

The most widely used assays for detecting gene mutations are those using bacteria. They are relatively simple and rapid to perform, and give reliable data on the ability of an agent to interact with DNA and produce mutations.
Reverse mutation assays determine the frequency with which an agent reverses or suppresses the effect of the forward mutation. The genetic target presented to an agent is therefore small, specific and selective. Several bacterial strains, or a single strain with multiple markers are necessary to overcome the effects of mutagen specificity. The reversion of bacteria from growth-dependence on a particular amino acid to growth in the absence of that amino acid (reversion from auxotrophy to prototrophy) is the most widely used marker.
The Salmonella typhimurium histidine (his) reversion system measures his- -> his+ reversions. The S. typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100) and frameshift (TA 1537, TA 98) mutations.
According to the direct plate incorporation method the bacteria are exposed to the test article with and without metabolic activation and plated on selective medium. After a suitable period of incubation, revertant colonies are counted.
To establish a dose response effect six dose levels with adequately spaced concentrations were tested. The maximum dose level was 5000 μg/plate (active ingredient).
To validate the test, reference mutagens are tested in parallel to the test article.

Evaluation criteria:

The generally accepted conditions for the evaluation of the results are:
- corresponding background growth on both negative control and test plates
- normal range of spontaneous reversion rates
A test article is considered positive if either a biologically relevant and reproducible dose related increase in the number of revertants or a biologically relevant and reproducible increase for at least one test concentration is induced.
A test article producing neither a biologically relevant and reproducible dose related increase in the number of revertants nor a biologically relevant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
A biologically relevant response is described as follows:
A test article is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98 and TA 100 or thrice on TA 1535 and TA 1537. Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the criteria described above or not.

Statistics:

A statistical analysis of the data is not required.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.
The plates incubated with the test article showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.
In experiment I, substantial and dose dependent increases in revertant colony numbers were observed following treatment with FAT 41'021/A in strain TA 1535 with metabolic activation and in strains TA 1537 and T A 98 with and without metabolic activation. The number of colonies reached or exceeded the threshold of twice (strain TA 98) and thrice (strain TA 1535 and TA 1537) the number of the corresponding solvent control at concentrations of 100 μg/plate and above. In experiment II a dose dependent increase in revertant colony numbers was observed in strains TA 1535 and TA 100 with S9 mix and in strains TA 1537 and TA 98 with and without S9 mix. The threshold was reached or exceeded at concentrations as low as 33 μg/plate and above.

Pre-Experiments for Toxicity

To evaluate the toxicity of the test article a pre-study was performed with strains TA 98 and TA 100. The results are given in the following table:

 

Substance	Concentration per plate μg	Revertants per plate
		TA 98		TA 100
		-	+	-	+
Negative control	-	21	28	105	83
Solvent control	-	21	28	105	91
4-NOPD	10.0	539	/	/	/
Sodium azide	10.0	/	/	910	/
2-aminoanthracene	2.5	-	634	-	602
Test article	3 10 33 100 333 1000 2500 5000	18 22 34 43 72 95 156 141	28 35 35 65 90 81 128 124	101 88 63 67 64 67 80 72	96 87 92 68 89 93 87 56

- = without S9 mix

+ = with S9 mix

/ = not performed

 

The plates with the test article showed normal background growth up to 5000 μg/plate in strain TA 98 and TA 100.

According to the dose selection criteria, the test article was tested at the following concentrations:

33; 100; 333; 1000; 2500 and 5000 μg/plate (active ingredient).

 

Experiment I: Plate Incorporation Test

Test article: FAT 41’021/A

S9 mix from: Rat liver (Batch R 030797)

Test strain: TA 1535

Without S9 mix

Concentration μg/plate	Plate			Revertants/plate
	1	2	3	Mean	s.d.	Factor*
Negative Control Solvent Control Positive Control# 33 100 333 1000 2500 5000	17 15 858 12 11 8 18 12 19	22 18 837 15 10 10 14 16 19	15 19 695 11 12 16 14 12 12	18 17 797 13 11 11 15 13 17	3.6 2.1 88.7 2.1 1.0 4.2 2.3 2.3 4.0	1.0 46.0 0.7 0.6 0.7 0.9 0.8 1.0

With S9 mix

Concentration μg/plate	Plate			Revertants/plate
	1	2	3	Mean	s.d.	Factor*
Negative Control Solvent Control Positive Control## 33 100 333 1000 2500 5000	10 23 216 13 15 19 23 39 79	10 22 202 10 13 15 47 52 84	13 17 169 16 9 20 32 45 69	11 21 196 13 12 18 34 45 77	1.7 3.2 24.1 3.0 3.1 2.6 12.1 6.5 7.6	1.0 9.5 0.6 0.6 0.9 1.6 2.2 3.7

                            Σ revertants /concentr. test article

*enhanced factor: = ------------------------------------------------------

                              Σ revertants / solvent control

# sodium azide 10 μg/plate

## 2-aminoanthracene 2.5 μg/plate

 

Experiment I: Plate Incorporation Test

Test article: FAT 41’021/A

S9 mix from: Rat liver (Batch R 030797)

Test strain: TA 1537

Without S9 mix

Concentration μg/plate	Plate			Revertants/plate
	1	2	3	Mean	s.d.	Factor*
Negative Control Solvent Control Positive Control# 33 100 333 1000 2500 5000	10 10 148 13 14 39 49 95 105	11 5 148 20 5 26 47 78 108	9 5 138 21 18 32 54 101 99	10 7 145 18 12 32 50 91 104	1.0 2.9 5.8 4.4 6.7 6.5 3.6 11.9 4.6	1.0 21.7 2.7 1.9 4.9 7.5 13.7 15.6

With S9 mix

Concentration μg/plate	Plate			Revertants/plate
	1	2	3	Mean	s.d.	Factor*
Negative Control Solvent Control Positive Control## 33 100 333 1000 2500 5000	9 11 102 7 5 33 118 104 166	14 9 90 9 11 52 130 150 204	11 10 156 11 5 40 95 147 192	11 10 116 9 7 42 114 134 187	2.5 1.0 35.2 2.0 3.5 9.6 17.8 25.7 19.4	1.0 11.6 0.9 0.7 4.2 11.4 13.4 18.7

                     Σ revertants /concentr. test article

*enhanced factor: = ------------------------------------------------------

                               Σ revertants / solvent control

# 4-nitro-o-phenylene-diamine 50 μg/plate

## 2-aminoanthracene 2.5 μg/plate

 

Experiment I: Plate Incorporation Test

Test article: FAT 41’021/A

S9 mix from: Rat liver (Batch R 030797)

Test strain: TA 98

Without S9 mix

Concentration μg/plate	Plate			Revertants/plate
	1	2	3	Mean	s.d.	Factor*
Negative Control Solvent Control Positive Control# 33 100 333 1000 2500 5000	22 20 564 33 43 77 82 169 125	18 15 567 33 41 67 107 142 153	22 27 487 35 45 71 96 156 144	21 21 539 34 43 72 95 156 141	2.3 6.0 45.3 1.2 2.0 5.0 12.5 13.5 14.3	1.0 26.1 1.6 2.1 3.5 4.6 7.5 6.8

With S9 mix

Concentration μg/plate	Plate			Revertants/plate
	1	2	3	Mean	s.d.	Factor*
Negative Control Solvent Control Positive Control## 33 100 333 1000 2500 5000	38 23 579 39 74 102 77 117 115	19 33 584 32 61 95 87 132 133	26 28 738 33 61 72 79 136 125	28 28 634 35 65 90 81 128 124	9.6 5.0 90.4 3.8 7.5 15.7 5.3 10.0 9.0	1.0 22.6 1.2 2.3 3.2 2.9 4.6 4.4

                              Σ revertants /concentr. test article

*enhanced factor: = ------------------------------------------------------

                                 Σ revertants / solvent control

# 4-nitro-o-phenylene-diamine 10 μg/plate

## 2-aminoanthracene 2.5 μg/plate

 

Experiment I: Plate Incorporation Test

Test article: FAT 41’021/A

S9 mix from: Rat liver (Batch R 030797)

Test strain: TA 100

Without S9 mix

Concentration μg/plate	Plate			Revertants/plate
	1	2	3	Mean	s.d.	Factor*
Negative Control Solvent Control Positive Control# 33 100 333 1000 2500 5000	117 99 854 62 68 64 63 88 74	94 105 968 62 64 68 70 73 72	103 110 909 65 70 60 67 78 69	105 105 910 63 67 64 67 80 72	11.6 5.5 57.0 1.7 3.1 4.0 3.5 7.6 2.5	1.0 8.7 0.6 0.6 0.6 0.6 0.8 0.7

With S9 mix

Concentration μg/plate	Plate			Revertants/plate
	1	2	3	Mean	s.d.	Factor*
Negative Control Solvent Control Positive Control## 33 100 333 1000 2500 5000	81 90 560 96 67 91 105 89 58	88 93 620 92 66 89 76 80 50	80 89 626 89 70 86 98 93 61	83 91 602 92 68 89 93 87 56	4.4 2.1 36.5 3.5 2.1 2.5 15.1 6.7 5.7	1.0 6.6 1.0 0.7 1.0 1.0 1.0 0.6

                               Σ revertants /concentr. test article

*enhanced factor: = ------------------------------------------------------

                                   Σ revertants / solvent control

# sodium azide 10 μg/plate

## 2-aminoanthracene 2.5 μg/plate

 

Experiment II: Plate Incorporation Test

Test article: FAT 41’021/A

S9 mix from: Rat liver (Batch R 030797)

Test strain: TA 1535

Without S9 mix

Concentration μg/plate	Plate			Revertants/plate
	1	2	3	Mean	s.d.	Factor*
Negative Control Solvent Control Positive Control# 33 100 333 1000 2500 5000	8 12 850 14 15 13 17 24 15	22 14 898 19 19 24 13 16 18	16 14 897 19 16 16 13 22 16	15 13 882 17 17 18 14 21 16	7.0 1.2 27.4 2.9 2.1 5.7 2.3 4.2 1.5	1.0 66.1 1.3 1.3 1.3 1.1 1.6 1.2

With S9 mix

Concentration μg/plate	Plate			Revertants/plate
	1	2	3	Mean	s.d.	Factor*
Negative Control Solvent Control Positive Control## 33 100 333 1000 2500 5000	17 14 195 11 11 20 24 61 171	9 16 213 14 10 20 32 57 112	12 17 217 11 15 23 31 48 135	13 16 208 12 12 21 29 55 139	4.0 1.5 11.7 1.7 2.6 1.7 4.4 6.7 29.7	1.0 13.3 0.8 0.8 1.3 1.9 3.5 8.9

                                Σ revertants /concentr. test article

*enhanced factor: = ------------------------------------------------------

                                   Σ revertants / solvent control

# sodium azide 10 μg/plate

## 2-aminoanthracene 2.5 μg/plate

 

Experiment II: Plate Incorporation Test

Test article: FAT 41’021/A

S9 mix from: Rat liver (Batch R 030797)

Test strain: TA 1537

Without S9 mix

Concentration μg/plate	Plate			Revertants/plate
	1	2	3	Mean	s.d.	Factor*
Negative Control Solvent Control Positive Control# 33 100 333 1000 2500 5000	17 16 93 29 30 43 23 43 99	14 17 85 29 19 41 38 44 86	9 14 89 20 20 34 35 52 93	13 16 89 26 23 39 32 46 93	4.0 1.5 4.0 5.2 6.1 4.7 7.9 4.9 6.5	1.0 5.7 1.7 1.5 2.5 2.0 3.0 5.9

With S9 mix

Concentration μg/plate	Plate			Revertants/plate
	1	2	3	Mean	s.d.	Factor*
Negative Control Solvent Control Positive Control## 33 100 333 1000 2500 5000	19 17 253 33 36 55 71 144 166	12 11 228 45 34 78 72 151 159	20 24 268 34 38 76 50 138 167	17 17 250 37 36 70 64 144 164	4.4 6.5 20.2 6.7 2.0 12.7 12.4 6.5 4.4	1.0 14.4 2.2 2.1 4.0 3.7 8.3 9.5

                               Σ revertants /concentr. test article

*enhanced factor: = ------------------------------------------------------

                                    Σ revertants / solvent control

# 4-nitro-o-phenylene-diamine 50 μg/plate

## 2-aminoanthracene 2.5 μg/plate

 

Experiment II: Plate Incorporation Test

Test article: FAT 41’021/A

S9 mix from: Rat liver (Batch R 030797)

Test strain: TA 98

Without S9 mix

Concentration μg/plate	Plate			Revertants/plate
	1	2	3	Mean	s.d.	Factor*
Negative Control Solvent Control Positive Control# 33 100 333 1000 2500 5000	20 20 1367 91 115 331 276 463 683	20 20 1315 101 117 343 357 501 629	14 15 1383 93 103 343 421 438 704	18 18 1355 95 112 339 351 467 672	3.5 2.9 35.6 5.3 7.6 6.9 72.7 31.7 36.7	1.0 73.9 5.2 6.1 18.5 19.2 25.5 36.7

With S9 mix

Concentration μg/plate	Plate			Revertants/plate
	1	2	3	Mean	s.d.	Factor*
Negative Control Solvent Control Positive Control## 33 100 333 1000 2500 5000	24 25 505 147 159 708 584 848 1510	34 44 633 173 149 593 721 925 1374	29 28 516 157 171 622 591 918 1299	29 32 551 159 160 641 632 897 1394	5.0 10.2 70.9 13.1 11.0 59.8 77.2 42.6 107.0	1.0 17.1 4.9 4.9 19.8 19.5 27.7 43.1

                                 Σ revertants /concentr. test article

*enhanced factor: = ------------------------------------------------------

                                    Σ revertants / solvent control

# 4-nitro-o-phenylene-diamine 10 μg/plate

## 2-aminoanthracene 2.5 μg/plate

 

Experiment II: Plate Incorporation Test

Test article: FAT 41’021/A

S9 mix from: Rat liver (Batch R 030797)

Test strain: TA 100

Without S9 mix

Concentration μg/plate	Plate			Revertants/plate
	1	2	3	Mean	s.d.	Factor*
Negative Control Solvent Control Positive Control# 33 100 333 1000 2500 5000	127 113 800 133 120 110 87 125 155	137 101 732 111 103 148 103 151 178	109 78 751 98 92 136 99 118 183	124 97 761 114 105 131 96 131 172	14.2 17.8 35.1 17.7 14.1 19.4 8.3 17.4 14.9	1.0 7.8 1.2 1.1 1.3 1.0 1.3 1.8

With S9 mix

Concentration μg/plate	Plate			Revertants/plate
	1	2	3	Mean	s.d.	Factor*
Negative Control Solvent Control Positive Control## 33 100 333 1000 2500 5000	128 117 788 128 147 188 185 237 371	131 115 658 115 151 206 192 264 325	119 112 625 134 136 241 198 248 365	126 115 690 126 145 212 192 250 354	6.2 2.5 86.2 9.7 7.8 27.0 6.5 13.6 25.0	1.0 6.0 1.1 1.3 1.8 1.8 2.2 3.1

                              Σ revertants /concentr. test article

*enhanced factor: = ------------------------------------------------------

                                Σ revertants / solvent control

# sodium azide 10 μg/plate

## 2-aminoanthracene 2.5 μg/plate

 

 

Summary of Results

Test article: FAT 41’021/A

S9 mix from: Rat Liver (Batch R 030797)

Without S9 mix

Concentration μg/plate	Revertants/plate mean from three plates
	TA 1535		TA 1537		TA 98		TA 100
	I	II	I	II	I	II	I	II
Negative control Solvent control Positive control# 33 100 333 1000 2500 5000	18 17 797 13 11 11 15 13 17	15 13 882 17 17 18 14 21 16	10 7 145 18 12 32 50 91 104	13 16 89 26 23 39 32 46 93	21 21 539 34 43 72 95 156 141	18 18 1355 95 112 339 351 467 672	105 105 910 63 67 64 67 80 72	124 97 761 114 105 131 96 131 172

With S9 mix

Concentration μg/plate	Revertants/plate mean from three plates
	TA 1535		TA 1537		TA 98		TA 100
	I	II	I	II	I	II	I	II
Negative control Solvent control Positive control## 33 100 333 1000 2500 5000	11 21 196 13 12 18 34 45 77	13 16 208 12 12 21 29 55 139	11 10 116 9 7 42 114 134 187	17 17 250 37 36 70 64 144 164	28 29 634 35 65 90 81 128 124	29 32 551 159 160 641 632 897 1394	83 91 602 92 68 89 93 87 56	126 115 690 126 145 212 192 250 354

# Sodium azide (10.0 μg/plate) strains TA 1535 and TA 100; 4-nitro-o-phenylene-diamine strains TA 1537 (50 μg/plate) and TA 98 (10.0 μg/plate)

## 2-aminoanthracene (2.5 μg/plate) strains TA 1535, TA 1537, TA 98 and TA 100

Conclusions:

During the described mutagenicity test and under the experimental conditions reported, the test article induced gene mutations by base pair changes and frameshifts in the genome of the strains used. Disperse Red 167:1 is considered mutagenic in bacteria in presence and absence of an exogenuous metabolic activation system.

Executive summary:

This study was performed to investigate the potential of the substance to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100.

 

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.

The test article was tested at the following concentrations:

33; 100; 333; 1000; 2500; and 5000 μg/plate (active ingredient)

 

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.

 

The plates incubated with the test article showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.

 

In both experiments, substantial and dose dependent increases in revertant colony numbers were observed following treatment with FAT 41'021/A with and without metabolic activation.

 

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

 

Conclusion

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article induced gene mutations by base pair changes and frameshifts in the genome of the strains used.

 

Therefore, Disperse Red 167:1 is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test substance did not induce micronuclei in the polychromatic erythrocytes of treated mice.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

October 26th 1998 to November 18th 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

OECD Guideline For Testing of Chemicals, 474 Genetic Toxicology, Mammalian Erythrocyte Micronucleus Test, Adopted 21st, July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EEC Directive 92/69, L 383 A, Annex B. 12., p. 154-156

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

U.S. EPA: OPPTS 870.5395 Health Effects Test Guidelines; Mammalian Erythrocyte Micronucleus Test, August 1998

Deviations:

no

GLP compliance:

yes

Type of assay:

other: Mammalian Erythrocyte Micronucleus Test

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

Species of animals: mouse
Strain of animals: HsdWin:NMRI
The mouse has been chosen for this study since it provides a convenient in vivo mammalian model.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species of animals: mouse
Strain of animals: HsdWin:NMRI
Origin (supplier) of animals: Harlan Winkelmann GmbH, Gartenstrasse 27, 33178 Borchen
Animal identification: fur marking with KMn04 and cage numbering
Body weight at start of study
male animals:
mean = 33.9 g (=100%)
min = 32.0 g (- 5.6 %)
max = 38.0 g (+ 12.1 %)
n = 15
female animals:
mean = 27.3 g (=100%)
min = 26.0 g (- 4.8 %)
max = 29.0 g (+ 6,2 %)
n = 15

Age at the start of study: male/female animals approximately 7 weeks
Randomization procedure: randomization schemes 98.0890 and 98.0891
Animal maintenance: in fully air-conditioned rooms in makrolon cages type 3 (five animals per cage) on soft wood granulate
Room temperature: 22 ± 3°C
Relative humidity: 50 ± 20%
Lighting times: 12 hours daily
Acclimatization: 5 days under study conditions
Food: rat/mice diet ssniff® R/M-H (V 1534), ad libitum ssniff® GmbH, Postbox 2039, 59480 Soest
Water: tap water in plastic bottles, ad libitum

Route of administration:

oral: gavage

Vehicle:

Tylose HEC 4000

Details on exposure:

On the days of administration the test substance was suspended in Tylose HEC 4000 (0.5% w/v) at the appropriate concentration. A magnetic stirrer was used to keep the preparation homogeneous until dosing had been completed.

Duration of treatment / exposure:

48 hours

Frequency of treatment:

twice at an interval of 24 hours

Post exposure period:

24 hours

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males/5 females per dose group (test, vehicle/negative control & positive control)

Control animals:

yes

yes, concurrent vehicle

Positive control(s):

Name or number of compound (I.N.N. or U.S.A.N): cyclophosphamide
Synonyms: Endoxan®
Formula of the compound: C7H15Cl2N2P – H2O
CAS-Register number: 50-18-0
Supplier of reference compound: ASTA Medica AG
Batch number: 6035758
Certificate of analysis: certified by the supplier

Formulation of reference compound: CPA dissolved in distilled water on the second day of experiment; final concentration: 0.5 % (w/v)

Tissues and cell types examined:

2000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. In addition, the ratio of polychromatic erythrocytes to 200 total erythrocytes was determined.

Details of tissue and slide preparation:

Animals were killed by carbon dioxide asphyxiation 24 hours after dosing. For each animal, about 3 ml fetal bovine serum was poured into a centrifuge tube. Both femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. The mixture was then centrifuged for 5 minutes at approx. 1200 rpm, after which almost all the supernatant was discarded. One drop of the thoroughly mixed sediment was smeared onto a cleaned slide, identified by project code and animal number and air-dried for about 12 hours.

Staining was performed as follows:
- 5 minutes in methanol
- 5 minutes in May-Grűnwald's solution
- brief rinsing twice in distilled water
- 10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2 (Weise)
- rinsing in distilled water
- drying
- coating with Entellan®

Evaluation criteria:

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

Statistics:

Main parameter for the statistical analysis, i.e. validity assessment of the study and mutagenicity of the test substance, was the proportion of polychromatic erythrocytes with micronuclei out of the 2000 counted erythrocytes. All bone marrow smears for evaluation were coded to ensure that the group from which they were taken remained unknown to the investigator.
A one-sided Wilcoxon-Test was evaluated to check the validity of the study. The study was considered as valid in case the proportion of polychromatic erythrocytes with micronuclei in the positive control was significantly higher than in the negative control (p=0.05).
If the validity of the study had been shown the following sequential test procedure for the examination of the mutagenicity was applied: Based on a monotone-dose relationship one-sided Wilcoxon tests were performed starting with the highest dose group. These test were performed with a multiple level of significance of 5%

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

All animals survived after treatment. Red colored urine but 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 group of C.l. Disperse Red 167 was within the normal range of the negative control groups. No statistically significant increase of micronucleated polychromatic erythrocytes was observed. The ratio of polychromatic erythrocytes to total erythrocytes remained essentially unaffected by the test compound and was not less than 20% of the control values.
Cyclophosphamide (Endoxan) induced a marked and statistically significant increase in the number of polychromatic erythrocytes with micronuclei, thus indicating the sensitivity of the test system.

Summary tables and statistics

Test compound:C.I. Disperse Red 167

Sex	Dose mg/kg bwt.	Killing time	Number		Poly/Ery   Mean	Poly/Ery SD Mean	Poly with MN   Mean	Poly with MN [%] Mean	Poly with MN SD Mean
			of animals	Poly counted
Male Male Male	0 – Control 2000 50 – Endoxan	24 h 24 h 24 h	5 5 5	2000 2000 2000	0.48 0.58 0.42	0.07 0.06 0.04	1.6 1.0 60.6	0.08 0.05 3.03	0.03 0.04 0.69
Female Female Female	0 – Control 2000 50 – Endoxan	24 h 24 h 24 h	5 5 5	2000 2000 2000	0.54 0.52 0.44	0.05 0.08 0.05	1.2 3.2 53.8	0.06 0.16 2.69	0.07 0.10 0.59

 

Sex	Dose mg/kg bwt.	Killing time	Number		Poly/Ery   Mean	Poly/Ery SD Mean	Poly with MN   Mean	Poly with MN [%] Mean	Poly with MN SD Mean	Mut. I.
			of animals	Poly counted
Pooled Pooled Pooled	0 – Control 2000 50 – Endoxan	24 h 24 h 24 h	10 10 10	2000 2000 2000	0.51 0.55 0.43	0.07 0.07 0.04	1.40 2.10 57.20*	0.1 0.1 2.9	0.05 0.09 0.63	1.0 1.5 40.9

Mut. I. = Mutagenic Index

Control = Vehilce Tylose HEC 4000 (0.5% (w/v))

* = significantly different from control (p<0.05)

 

A cross comparison of individual data and pooled data may show discrepancies since the values are rounded.

 

Table of individual data

Test compound:C.I. Disperse Red 167

Group	Animal number	Sex	Dose mg/kg b.w.	Poly/200Ery	Poly with MN	Poly/Ery	Poly with MN [%]
1 1 1 1 1 1 1 1 1 1	1 2 3 4 5 6 7 8 9 10	Male Male Male Male Male Female Female Female Female Female	0 – Control 0 – Control 0 – Control 0 – Control 0 – Control 0 – Control 0 – Control 0 – Control 0 – Control 0 – Control	114 78 97 102 91 111 99 123 97 113	2 2 1 2 1 0 2 1 0 3	0.57 0.39 0.49 0.51 0.46 0.56 0.50 0.62 0.49 0.57	0.10 0.10 0.05 0.10 0.05 0.00 0.10 0.05 0.00 0.15
2 2 2 2 2 2 2 2 2 2	31 32 33 34 35 36 37 38 39 40	Male Male Male Male Male Female Female Female Female Female	2000 2000 2000 2000 2000 2000 2000 2000 2000 2000	119 131 97 113 119 115 90 102 90 125	0 1 2 1 1 2 3 6 4 1	0.60 0.66 0.49 0.57 0.60 0.58 0.45 0.51 0.45 0.63	0.00 0.05 0.10 0.05 0.05 0.10 0.15 0.30 0.20 0.05
3 3 3 3 3 3 3 3 3 3	21 22 23 24 25 26 27 28 29 30	Male Male Male Male Male Female Female Female Female Female	50 – Endoxan 50 – Endoxan 50 – Endoxan 50 – Endoxan 50 – Endoxan 50 – Endoxan 50 – Endoxan 50 – Endoxan 50 – Endoxan 50 – Endoxan	73 96 83 79 84 80 82 79 100 95	79 71 56 49 48 58 61 33 60 57	0.37 0.48 0.42 0.40 0.42 0.40 0.41 0.40 0.50 0.48	3.95 3.55 2.80 2.45 2.40 2.90 3.05 1.65 3.00 2.85

 

 

Historical control values

 

Based on OECD 474, adopted 1997:

		Micronucleated PCE / 2000 PCE						PCE / Ery (200 Ery counted)
Vehicle	Sex	N	Mean	STD	Min	Med	Max	N	Mean	STD	Min	Med	Max
DMA / PEG / citrate buffer	Female	5	2.4	0.89	1	3	3	5	0.43	0.06	0.37	0.42	0.51
	Male	5	3.8	2.49	2	2	7	5	0.45	0.07	0.35	0.45	0.52
Deionized water	Female	25	1.88	1.36	0	2	6	25	0.50	0.06	0.41	0.51	0.66
	Male	25	1.64	1.19	0	1	5	25	0.45	0.07	0.33	0.46	0.59
Sesame oil	Female	5	2	1.58	0	2	4	5	0.50	0.08	0.40	0.51	0.60
	Male	5	3	1.58	1	3	5	5	0.48	0.10	0.34	0.47	0.59
Total	Female	35	1.97	1.32	0	2	6	35	0.49	0.07	0.37	0.50	0.66
	Male	35	2.14	1.65	0	2	7	35	0.46	0.07	0.33	0.46	0.59

PCE: Polychromatic Erythrocytes

NCE: Normochromatic Erythrocytes

Ery: Total Erythrocytes (PCE + NCE)

Conclusions:

The results lead to the conclusion that Disperse Red 167:1 did not lead to a substantial increase of micronucleated polychromatic erythrocytes and is not mutagenic in the micronucleus test under the conditions described in this report. The substance is not classifiable according to CLP criteria.

Executive summary:

The micronucleus test was carried out with the test item. The test compound was suspended in Tylose HEC 4000 (0.5% w/v) and was given twice at an interval of 24 hours as an orally dose of 2000 mg per kg body weight to male and female mice, based on the results of a previous dose range finding assay (see preliminary study).

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

 

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

 

The number of polychromatic erythrocytes containing micronuclei was not increased.

The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment with the test item and was not less than 20% of the control value.

 

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

 

Under the conditions of the present study the results indicate that Disperse Red 167:1 is not mutagenic in the micronucleus test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

The test itemDisperse Red 167:1, was tested positive in the Bacteria Reverse Mutation Assay (Ames test) in Salmonella strains, but negative in the in-vivo mouse micronucleus test. A close structural analogue was also tested positive in an Ames test, but negative in the mutation assay in mammalian cells and an in-vivo micronucleus test in rats. This positive effect in the bacterial mutation assay is a bacteria-specific effect due to bacterial nitro-reductases, which are highly effective in these bacterial strains, but not in mammalian cells.

This positive effect in the bacterial mutation assay is a bacteria-specific effect due to bacterial nitro-reductases, which are highly effective in these bacterial strains, but not in mammalian cells.

This effect was also investigated in-depths in two other nitro-dyes.

The first nitro dye was tested positive in an Ames test, but negative in the following tests:

Study Type	Metabolic activation	Result
Mitotic recombination assay withSaccharomyces cerevisae	Rat liver S9-mix	Negative
Point mutation assay withSaccharomyces cerevisae	Rat liver S9-mix	Negative
HPRT assay with V79 hamster cells	Rat liver S9-mix	Negative
UDS assay with rat hepatocytes	Metabolic competent hepatocytes were used	Negative
Micronucleus assay in vivo (mouse)	In vivo assay	Negative

The seconnd nitro-dye, Disperse Blue 291, was tested positive in an Ames test with nitroreductase and O‑acetyltransferase positive Salmonella typhimurium strains, but negative with nitroreductase and O-acetyltransferase negative strains and in a test for unscheduled DNA synthesis.

The nitroreductase family comprises a group of flavin mononucleotide (FMN)- or flavin adenine dinucleotide (FAD) -dependent enzymes that are able to metabolize nitroaromatic and nitroheterocyclic derivatives (nitrosubstituted compounds) using the reducing power of nicotinamide adenine dinucleotide (NAD(P)H). These enzymes can be found in bacterial species and, to a lesser extent, in eukaryotes. The nitroreductase proteins play a central role in the activation of nitro-compounds. Type I nitroreductases can transfer two electrons from NAD(P)H to form the nitroso and hydroxylamino intermediates and finally the amino group. Type II nitroreductases transfer a single electron to the nitro group, forming a nitro anion radical, which in the presence of oxygen generates the superoxide anion in a futile redox cycle, regenerating the nitro group [de Oliveira et al. 2010].

The positive effect in the bacterial reverse mutation test (Ames) was clearly related to a bacteria-specific metabolism of the test substance, as it is well-known for aromatic nitro compounds to be positive in the Ames assay resulting from metabolism by the bacteria-specific enzyme nitro-reductase [Tweats et al. 2012]. This could be also be proved to be true in studies with Disperse Blue 291, which was tested for mutagenic activity in the Salmonella assay with strains with different levels of nitroreductase and O-acetyltransferase[Umbuzeiro et al. 2005]. In this study,Disperse Blue 291 showed mutagenic activity with all standard strains of Salmonella typhimurium tested (TA1537, TA1538, TA98 and TA100), except for TA1535.In nitroreductase and O-acetyltransferasenegative strains (TA98NR, TA98DNP6) not mutagenic activity was observed in the absence of S9, whereas themutagenic activity was increased with the nitroreductaseand/or O‑acetyltransferaseoverproducing strains, (YG1021, YG1024 and YG1041) This shows the importance of the bacterial acetyltransferase enzyme in the activation of Disperse Blue 291. Because of the remarkable increase in the response with the nitroreductase and O‑acetyltransferase overproducing strain (YG1041), it is assumed that the product of the nitroreductaseis a substrate for the O-acetyltransferase. As there was a very slight increase in mutagenicity with TA98NR, TA98, YG1021, TA98DNP6, and YG1024 in the presence of S9, it was assumed that P450 enzymes have also a role in the activation of Disperse Blue 291, besides the bacterial enzymes. This could however not proven true in in-vitro and/or in-vivo studies with the test substance or the structural analogues.

It has also been demonstrated in various other publications that this mutagenic activity is a bacteria-specific effect and that these Ames positive nitro-substances are not mutagenic in mammalian assays.

That the reduction of these nitro-compounds to mutagenic metabolites is a bacteria-specific effect is demonstrated in the following by means of the two compounds AMP397 and fexinidazole.

• AMP397 is a drug candidate developed for the oral treatment of epilepsy. The molecule contains an aromatic nitro group, which obviously is a structural alert for mutagenicity. The chemical was mutagenic in Salmonellastrains TA97a, TA98 and TA100, all without S9, but negative in the nitroreductase-deficient strains TA98NR and TA100NR. Accordingly, the ICH standard battery mouse lymphomatkand mouse bone marrow micronucleus tests were negative, although a weak high toxicity-associated genotoxic activity was seen in a micronucleus test inV79 cells [Suter et al. 2002].The amino derivative of AMP397 was not mutagenic in wild type TA98 and TA100. To exclude that a potentially mutagenic metaboliteis released by intestinal bacteria, a Muta^TMMouse study was done in colon and liver with five daily treatments at the MTD, and sampling of 3, 7 and 21 days post-treatment. No evidence of a mutagenic potential was found in colon and liver. Likewise, a comet assay did not detect any genotoxic activity in jejunum and liver of rats, after single treatment with a roughly six times higher dose than the transgenic study, which reflects the higher exposure observed in mice. In addition, a radioactive DNA binding assay in the liver of mice and rats did not find any evidence for DNA binding. Based on these results, it was concluded that AMP397 has no genotoxic potential in vivo. It was hypothesized that the positive Ames test was due to activation by bacterial nitro-reductase, as practically all mammalian assays including fourin vivoassays were negative, and no evidence for activation by mammalian nitro-reductase or other enzymes were seen. Furthermore, no evidence for excretion of metabolites mutagenic for intestinal cells by intestinal bacteria was found.
• Fexinidazolewas in pre-clinical development as a broad-spectrum antiprotozoal drug by the Hoechst AG in the 1970s-1980s, but its clinical development was not pursued. Fexinidazole was rediscovered by the Drugs for Neglected Diseases initiative (DNDi) as drug candidate to cure the parasitic disease human African trypanomiasis (HAT), also known as sleeping sickness. The genotoxicity profile of fexinidazole, a 2-substituted 5-nitroimidazole, and its two active metabolites, the sulfoxide and sulfone derivatives were investigated [Tweats et al. 2012]. All the three compounds are mutagenic in the Salmonella/Ames test; however, mutagenicity is either attenuated or lost in Ames Salmonella strains that lack one or more nitroreductase(s). It is known that these enzymes can nitroreduce compounds with low redox potentials, whereas their mammalian cell counterparts cannot, under normal conditions. Fexinidazole and its metabolites have low redox potentials and all mammalian cell assays to detect genetic toxicity, conducted for this study either in vitro (micronucleus test in human lymphocytes) or in vivo (ex vivo unscheduled DNA synthesis in rats; bone marrow micronucleus test in mice), were negative.

Based on these data and the common mechanism between the reduction of these nitro-compounds, which is widely explored in literature [de Oliveira et al. 2010], it is concluded, that the mutagenic effects observed in the Ames test with Disperse Red 167:1 is a bacteria specific effect and not relevant to mammalians.

References

De Oliveira IM, Bonatto D, Pega Henriques JA. Nitroreductases: Enzymes with Environmental Biotechnological and Clinical Importance. InCurrent Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology; Mendez-Vilas, A., Ed.; Formatex: Badajoz, Spain, 2010:1008–1019.

Suter W, Hartmann A, Poetter F, Sagelsdorff P, Hoffmann P, Martus HJ. Genotoxicity assessment of the antiepileptic drug AMP397, an Ames-positive aromatic nitro compound. Mutat Res. 2002 Jul 25;518(2):181-94.

Umbuzeiro GA, Freeman H, Warren SH, Kummrow F, Claxton LD. Mutagenicity evaluation of the commercial product CI Disperse Blue 291 using different protocols of the Salmonella assay. Food and Chemical Toxicology 2005;43:49–56.

Tweats D, Bourdin Trunz B, Torreele E. Genotoxicity profile of fexinidazole--a drug candidate in clinical development for human African trypanomiasis (sleeping sickness). Mutagenesis. 2012 Sep;27(5):523-32.

Additional information

Bacteria reverse mutation

This study was performed to investigate the potential of the test substance to induce gene mutations according to the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.

The test article was tested at the following concentrations: 33; 100; 333; 1000; 2500; and 5000 μg/plate (active ingredient)

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

 In both experiments, substantial and dose dependent increases in revertant colony numbers were observed following treatment with the test substance with and without metabolic activation.

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

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article induced gene mutations by base pair changes and frameshifts in the genome of the strains used.

Therefore, Disperse Red 167:1 is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.

Mutagenicity in mammalian cells (HPRT) - Structual Analogue

The test item was examined for mutagenic activity by assaying for the induction of 6-thioguanine resistant mutants in Chinese hamster V79 cells after in vitro treatment. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. Test item solutions were prepared using dimethylsulfoxide (DMSO). A preliminary cytotoxicity assay was performed, in the absence and presence of S9 metabolic activation. Based on solubility features, the test item was assayed at a maximum dose level of 1250 µg/mL and at a wide range of lower dose levels: 625, 313, 156, 78.1, 39.1, 19.5, 9.77 and 4.88 µg/mL. In the absence of S9 metabolism, no cells survived at almost all the concentrations tested; survival was reduced to 50% of the concurrent negative control value at the lowest dose level (4.88 µg/mL). In the presence of S9 metabolism, severe toxicity was noted at the two highest concentrations, while survival was reduced to 44% of the concurrent negative control value at 313 µg/mL. No relevant toxicity was noted over the remaining concentrations tested. By the end of treatment time, precipitation of test item and/or a coloured film, adhering to the flask surface, was observed at all concentrations tested in the absence of S9 metabolism and at the three highest dose levels in its presence. In order to select the appropriate range of concentrations for the mutation assay, an additional toxicity test was performed in the absence of S9 metabolism using the following lower and closer concentrations: 10.0, 7.14, 5.10, 3.64 and 2.60 µg/mL. Since no adequate toxicity for dose selection was obtained, a third toxicity test was performed in the absence of S9 metabolism, using the following concentrations: 40.0, 20.0, 10.0, 5.00 µg/mL. Severe toxicity was noted at the highest concentration; survival was reduced to 12% and 37% of the concurrent negative control value at 20.0 and 10.0 µg/mL, respectively; while no relevant toxicity was observed at 5.00 µg/mL. Two independent assays for mutation to 6-thioguanine resistance were performed using the following dose levels:

Main Assay I (+S9): 600, 400, 267, 178, 119 and 79.0 µg/mL

Main Assay I (-S9): 40.0, 20.0, 10.0, 5.00, 2.50, 1.25 and 0.625 µg/mL

Main Assay II (+S9): 400, 308, 237, 182, 140 and 108 µg/mL

Main Assay II (+S9): 20.0, 15.4, 11.8, 9.10, 7.00 and 5.39 µg/mL

In the second experiment the concentration range was slightly modified on the basis of the toxicity results obtained in Main Assay I. No reproducible five-fold increases in mutant numbers or mutant frequency were observed following treatment with the test item at any dose level, in the absence or presence of S9 metabolism. Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system.

It is concluded that the test item does not induce gene mutation in Chinese hamster V79 cells after in vitro treatment in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

MNT in-vivo

The ability of the test item to induce cytogenetic damage and/or disruption of the mitotic apparatus in mouse bone marrow was investigated measuring the induction of micronuclei in polychromatic erythrocytes. The test compound was suspended in Tylose HEC 4000 (0.5% w/v) and was given twice at an interval of 24 hours as an orally dose of 2000 mg per kg body weight to male and female mice, based on the results of a previous dose range finding assay (see preliminary study).

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

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

The number of polychromatic erythrocytes containing micronuclei was not increased.

The ratio of polychromatic erythrocytes to total erythrocytes in both male and female animals remained unaffected by the treatment with the test item and was not less than 20% of the control value.

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

Under the conditions of the present study the results indicate that Disperse Red 167:1 is not mutagenic in the micronucleus test.

Justification for classification or non-classification

Based on the results of in vivo testing, no classification for genotoxicity is required for the test substance according to CLP (EC 1272/2008) criteria.