Chromate(2-), [2,4-dihydro-4-[(2-hydroxy-4-nitrophenyl)azo]-5-methyl-3H-pyrazol-3-onato(2-)][3-[[4,5-dihydro-3-methyl-1-(4-methylphenyl)-5-oxo-1H-pyrazol-4-yl]azo]-4-hydroxy-5-nitrobenzenesulfonato(3-)]-, disodium

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a reverse gene mutation assay in bacteria, strains TA 100, TA 1537, TA 1538 and TA 98 of S. typhimurium were exposed to two batches of Acid Red 414 at concentrations of up to 10000 µg/plate.

Concentrations of up to and including 10000 µg/plate did not cause any bacteriotoxic effects. The total numbers of bacteria remained unchanged. No inhibition of growth was observed.

Evidence of a mutagenic activity of Acid Red 414 was found for both batches. In all Salmonella typhimurium strains used a biologically relevant increase in the mutant count of more than double of the corresponding negative control was found. The lowest effective concentrations for batch Ök.Nr. 1190 and Ök.Nr. 2027 were 10 and 50 µg/plate for Salmonella typhimurium TA 1538, 1000 and 5000 µg/per plate for TA 100, 20 µg/plate for TA 1537 and 500 µg/plate for TA 98, respectively. Both batches of Acid Red 414 showed hence mutagenic effects in the Salmonella/microsome test with and without a metabolic activation.

The present study was conducted in compliance with OECD Guideline For Testing Of Chemicals, 476 "Genetic Toxicology: In vitro Mammalian Cell Gene Mutation Test" in compliance with the Principles of Good Laboratory Practice (GLP).

The study was performed to investigate the potential of Acid Red 414 to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster in vitro.

Two independent experiments were conducted both with and without an exogenous rat liver microsomal activation system (S9-mix).

The compound was suspended in cell culture medium and tested at the following concentrations:

Main experiment with and without metabolic activation: 100, 250, 500, 750, 1000#, 1375*, 1750* µg/ml

Repeat main experiment with and without metabolic activation: 100, 250, 500, 750, 1000* µg/ml

*= because of high toxicity no mutant selection was performed.

#= in the absence of S9-mix no mutant selection was performed

 

The concentration ranges were based on the results of preliminary tests for solubility and toxicity.

In the absence of S9 metabolic activation in both mutation experiments a dose-related decrease in survival was observed reaching 16.8 and 3.8 % of the solvent control value in the microtiter plates at a dose level of 1000 µg/ml. In the presence of S9-mix survival was reduced to 31.2 and 8.2 % of the solvent control value at the same concentration.

With the concentrations of 250 and 750 µg/ml the test compound induced a statistically significant enhancement of the mutation rate over the range of the solvent controls in the presence of a metabolic activation in the second main experiment. But these results were not reproducible, not three fold higher than the solvent control group and not dose-dependent and therefore of no biological relevance.

The sensitivity of the test system was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control compounds.

In conclusion, Acid Red 414 did not induce gene mutation, i.e. was not mutagenic, in this HPRT-test with V79 Chinese hamster cells, in either the presence or absence of metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

No E. coli strain tested

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine locus

Species / strain / cell type:

S. typhimurium, other: TA 1538, TA 1537, TA 100, TA 98

Metabolic activation:

with and without

Metabolic activation system:

rat S9-mix

Test concentrations with justification for top dose:

Concentrations used: 10000, 5000, 1000, 500, 100, 50, 10 and 5 µg/plate
No bacteriotoxic effect up to 10000 µg/plate

Vehicle / solvent:

demineralised water

Untreated negative controls:

no

Remarks:

because the vehicle was water

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Cyclophosphamide, trypaflavine, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation: 0.1 mL TS+0.1 mL bacteria+0.5 mL S9+2 mL soft agar: 30 sec at 45 °C
- Incubation period: 48 hours at 37°C

NUMBER OF REPLICATIONS: 4 plates/strain/concentration

DETERMINATION OF CYTOTOXICITY
- Method: - background growth
- marked and dose-dependent reduction in mutant count compared to negative controls
- titer determination

Acceptance criteria:
a) The negative controls had to be within the expected range, as defined by published data (i.e. Maron and Ames, 1983) and the laboratory's own historical data
b) The positive controls had to show sufficient effects, as defined by the laboratory's experience
c) Titer determinations had to demonstrate sufficient bacterial density in the suspension.

An assay which did not comply with at least one of the above criteria was not used for assessment. Furthermore, the data generated in this assay needed to be confirmed by two additional independent experiments. Even if the criteria for points (a), (b) and (c) were not met, an assay was accepted if it showed mutagenic activity of the test compound.

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result; this increase should be about twice the amount of negative controls.

Statistics:

N.A.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

TA 100: 1.6-fold increase at cytotoxic concentrations

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

There was no indication of a bacteriotoxic effect of the test item at up to 10000 µg per plate. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. Nor was any inhibition of growth noted.

All four strains concerned showed a dose related and biologically relevant increase in mutant counts over those of the negative controls. This applied both to both batches with and without S9 mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Both batches of Acid Red 414 showed mutagenic effects in the Salmonella/microsome test with and without a metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria, strains TA 100, TA 1537, TA 1538 and TA 98 of S. typhimurium were exposed to two batches of Acid Red 414 at concentrations of up to 10000 µg/plate.

Concentrations of up to and including 10000 µg/plate did not cause any bacteriotoxic effects. The total numbers of bacteria remained unchanged. No inhibition of growth was observed.

Evidence of a mutagenic activity of Acid Red 414 was found for both batches. In all Salmonella typhimurium strains used a biologically relevant increase in the mutant count of more than double of the corresponding negative control was found. The lowest effective concentrations for batch Ök.Nr. 1190 and Ök.Nr. 2027 were 10 and 50 µg/plate for Salmonella typhimurium TA 1538, 1000 and 5000 µg/per plate for TA 100, 20 µg/plate for TA 1537 and 500 µg/plate for TA 98, respectively. Both batches of Acid Red 414 showed hence mutagenic effects in the Salmonella/microsome test with and without a metabolic activation.

 

The positive controls Endoxan, trypaflavine and 2-amino-anthracene acted markedly mutagenic, as can be seen from the biologically relevant increase of mutant colonies compared with the corresponding negative control.

 

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

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998

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)

Version / remarks:

1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell transformation assay

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase) locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Test organism: cell line W9 of Chinese hamster lung fibroblasts
- Source of cells: cell bank of "Genetic Toxicology", HMR Germany
- Cell culture medium: MEM (minimal essential medium) with Hanks-salts and 25 mM Hepes-buffer
- Experimental conditions in vitro: approx. 37 °C and approx. 4 % CO, in plastic flasks

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Rationale for dose selection
Evaluation of the solubility of Isolan Rot S-RL in cell culture medium showed that 5000 µg/mI was the highest practicable concentration and produced only a slight microscopic visible precipitation of the test substance at concentrations of 2000 µg/ml and above. 5000 µg/ml is the highest dose level tolerated by the test system. Accordingly, the preliminary toxicity study was carried out using a maximum concentration of 5000 µg/ml and a range of lower dose levels down to 100 µg/ml.
In the absence and presence of S9 metabolic activation high cytotoxicity was observed. Without metabolic activation survival declined in a dose-related manner reaching 2.1 % of the solvent control value at a dose level of 2000 µg/ml. With
metabolic activation survival was reduced to 3.3 % of the solvent control value at the same concentration.
Based on these results 1750 µg/ml was selected as the maximum dose level for the first main mutation experiment in both the absence and in the presence of S9-mix. Six lower concentrations down to 100 µg/ml were also included.

The following dose levels were used:
Main experiment with and without metabolic activation: 100, 250, 500, 750, 1000#, 1375*, 1750* µg/ml
Repeat main experiment with and without metabolic activation: 100, 250, 500, 750, 1000* µg/ml
*= because of high toxicity no mutant selection was performed.
#= in the absence of S9-mix no mutant selection was performed

Vehicle / solvent:

cell culture medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Reference Compounds

Without metabolic activation
Name or number of compound (I.N.N. orU.SAN): ethyl methane sulfonate
Synonyms: EMS
Formula of the compound: C3Ha03S
CAS-Register number: 62-50-0
Product number / Code: 820774
Supplier of reference compound: Dr. Theodor Schuchardt & Co. Chemische Fabrik, Germany
Batch number: 40606721
Certificate of analysis: certificated by the supplier, Analytical Department, Dr. Bolkart dated December 18th, 1995

With metabolic activation
Name or number of compound (I.N.N. orU.S.A.N): 9,10-dimethyl-1,2-benzanthracene
Synonyms: DMBA
Chemical name: 7,12-dimethylbenz[a]anthracene
CAS-Register number: 57-97-6
Product number / Code: 21,626-7
Supplier of reference compound: Fa. Aldhch – Chemie, Germany
Batch number: TL 1825LL
Certificate of analysis: certificated by the supplier, Aldrich chemical company, David Swessel, dated April 3rd, 1996

Test system

Test groups
with and without metabolic activation: 5.0, 7.5, 12.5, 25.0, 50.0, 75.0, 125.0, 250.0, 500.0, 750.0 and 1285.0 μg/ml (main mutation experiment)
50.0, 75.0, 125.0, 250.0, 500.0, 750.0, 1000.0 and 1285.0 μg/ml (repeat mutation experiment)

Control groups
negative controls: untreated control & cultures treated with the solvent

positive controls:
without metabolic activation: EMS (Ethyl methane sulfonate)
with metabolic activation: DMBA (9,10-dimethyl-1,2-benzanthracene)

Formulation of test compounds: suspended in DMSO at appropriate concentrations immediately before use.

Formulation of reference compounds: EMS dissolved in cell culture medium on the day of treatment, final concentration: 1.0 mg/ml = 8 mM.
DMBA dissolved in DMSO and frozen in small portions. Aliquot thawed on the day of treatment, final concentration in cell culture medium: 7.7 μg/ml = 30 μM

Source of biological material: cell bank of "Genetic Toxicology", HMR Deutschland GmbH, ProTox

Test organism: cell line V79 of Chinese hamster lung fibroblasts

Cell culture medium: MEM (minimal essential medium) with Hanks-salts and 25 mM Hepes-buffer

Experimental conditions in vitro: approx. 37 °C and approx. 4 % CO2 in plastic flasks
Observations and Measurements

Preparation and storage of a liver homogenate fraction (S9)
The S9 fraction was prepared by the testing facility according to Ames et. al (1975). Male Sprague Dawley rats (200-300 g), supplied by Harlan Winkelmann, Gartenstrasse 27, 33178 Borchen, Germany, received a single intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight) 5 days before killing. The livers were removed from at least 5-6 animals at approx. 0 to 4 °C using cold sterile solutions and glassware, and were then pooled and washed in approx. 150 mM KCI (approximately 1 ml/g wet liver). The washed livers were cut into small pieces and homogenized in three volumes of KCI. The homogenate was cenfrifuged at approx. 9000g for 10 minutes. The supernatant, the S9 fraction, was divided into small portions, rapidly frozen and stored at approx. - 80 °C for not longer than six months. 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 dimethylbenzanthracene to confirm metabolic activation by microsomal enzymes.

Preparation of S9-mix
Sufficient S9 fraction was thawed to room temperature immediately before each test. An appropriate quantity of S9 fraction (batch no. 98/1 for both mutation experiments, protein concentration 51.8 g/l) was mixed with S9 cofactor solution to yield a final protein concentration of 0.3 mg/ml in the cultures which was kept on ice until used. This preparation is termed S9-mix. The concentrations of the different components of the S9-mix were:
8 mM MgCI3
33 mM KCI
5 mM glucose-6-phosphate
5 mM NADP
100 mM phosphate buffer pH 7.4

Cell culture
Large stocks of the mycoplasma-free V79 cell line are stored in liquid nitrogen in the cell bank of "Genetic Toxicology", thus permitting repeated use of the same cell culture batch for numerous experiments. The identical characteristics of the cells ensure comparability of the experimental parameters.
Thawed stock cultures were kept at approx. 37 °C and approx. 4 % C03 in 175 cm2 plastic flasks. About 5 x 105 to 1 x 106 cells were seeded into each flask in 30 ml of MEM-medium supplement with approx. 10 % (v/v) FCS (fetal calf serum) containing approx. 2 mM L-glutamine and approx. 0.1 % (w/v) neomycinsulfate. The cells were subcultured twice a week.
For the selection of mutants the medium was supplemented with approx. 11 μg/ml thioguanine.

Toxicity experiments and dose range finding
A preliminary toxicity test was undertaken in order to select appropriate dose levels for the mutation assay. In this test a wide range of dose levels of test compound was used. Cell cultures were subjected to the same treatment conditions as in mutation assays, and the survival of the cells was subsequently determined.

The test included the following treatments:
Solvent control : the maximum final concentration of organic solvents will not exceed approx. 1 % (v/v).
Test compound : the highest dose level for the preliminary toxicity test was determined by the solubility of the test compound up to the maximum of 10 mM or 5000 μg/ml.
Treatments were performed both in the presence and absence of S9 metabolic activation system using a single cell culture at each test point.

Test procedure
In preliminary toxicity experiments approximately 4500 cells were seeded in each well of a microliter plate, allowed to attach overnight and then exposed to the test and control compound for four hours.
For each concentration at least 6 wells were used. Approx. 24 hours after treatment, the cells were fixed and stained with crystal violet.
Survival was determined by measurement of the crystal violet extinction.

In the main mutation experiments the cultures for assessing toxicity were prepared and treated with the test compound in the same way as for the preliminary experiment. 24 hours after seeding of approx. 4500 cells per well in a microtiter plate, the medium was replaced with serum-reduced (5 % v/v) medium containing the test compound to which either buffer or S9-mix was added as appropriate. After 4 hours the treatment medium was replaced with normal medium after rinsing twice with this. The cultures were stained with crystal violet and survival was determined after an incubation period of approx. 24 hours.

Rationale for dose selection
For non-toxic, freely soluble test compounds, the top dose should be 10 mM or 5000 ug/ml according to international testing guidelines.
For non-toxic, poorly soluble test compounds, the top dose should be the highest evaluable dose.
For toxic compounds the percentage survival relative to the solvent control should be calculated for each treatment. The dose level which resulted in a predicted survival of less than 30 % should be chosen as the highest dose level. At least eight respectively seven lower dose levels should be also included in each experiment.

Mutagenicity test
Two independent mutation tests were performed.
Two-day old, exponentially growing cultures which were more than 50 % confluent were trypsinated and a single cell suspension was prepared. The trypsin concentration was approx. 0.25 % (v/v) in Ca-Mg-free salt solution. The Ca-Mg-free salt solution was prepared as follows (per liter): NaCI 6.8 g; Ka 0.4 g; glucose 1 g; NaHC03 2.2 g; phenol red 5 mg; trypsin 2.5 g.
Subsequently the cells were replated to determine the mutation frequency and plating efficiency.

The treatment schedule of the mutagenicity test is described below:

Day 1: Subculturing of an exponentially growing culture
a) Approx. 4500 cells in each well of a microtiter plate for determination of the plating efficiency.
b) 6x 105 - 1 x 106 cells in 175 cm2 flasks with 30 ml medium for the mutagenicity test, one flask per experimental point.

Day 2: Treatment of a) and b) with the test compound in the presence and absence of S9-mix (final protein concentration: approx. 0.3 mg/ml) for 4 hours.

Day 3: Fixation and staining of the cells in a) for the determination of the plating efficiency.

Day 5: Subculturing of b) in 175 cm2 flasks

Day 9: Subculturing of b) in five 75 cm1 flasks with culture medium containing 6-thioguanine:
Mutant selection (about 300 000 cells/flask);
subculturing of b) in two 25 cm2 flasks for plating efficiency (about 400 cells per flask)

Day 16: Fixation and staining of colonies of b) - from subcultures seeded on day 9.

All incubations were carried out at approx. 37 °C and 4 % CO2.
Staining was performed with approx. 10 % (v/v) methylene blue in approx. 0.01 % (w/v) KOH solution.
Only colonies with more than 50 cells were counted.

Evaluation criteria:

Evaluation of data

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 induced increases in the mutation frequency which were both statistically significant and within the laboratory's normal range
the plating efficacy for the solvent control was greater than 50 %

Criteria for a positive response
The test compound is classified as mutagenic if:
it reproducibly induces with one of the test compound concentrations a mutation frequency that is three times higher than the spontaneous mutant frequency in this experiment
there is a reproducible dose-related increase in the mutation frequency. Such an evaluation may be considered independently from the number induced mutants
survival of the responding dose group is at least 30 %
However, in a case by case evaluation both decisions depend on the level of the corresponding negative control data.

Statistics:

The biometry of the results for the test compound is performed off-line with the MANN-WHITNEY-U-TEST

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: Before treatment, the pH values and osmolality of the treatment media were determined. The addition of test compound solutions did not have any effect on these parameters.
- Water solubility: solubility of Isolan Rot S-RL in cell culture medium showed that 5000 µg/mI was the highest practicable concentration and produced only a slight microscopic visible precipitation of the test substance at concentrations of 2000 µg/ml and above.
- Precipitation: slight microscopic visible precipitation of the test substance at concentrations of 2000 µg/ml and above

Conclusions:

The test substance did not induce gene mutation, i.e. was not mutagenic, in this HPRT-test with V79 Chinese hamster cells, either in the presence or in the absence of metabolic activation.

Executive summary:

The present study was conducted in compliance with OECD Guideline For Testing Of Chemicals, 476 "Genetic Toxicology: In vitro Mammalian Cell Gene Mutation Test" in compliance with the Principles of Good Laboratory Practice (GLP).

The study was performed to investigate the potential of Acid Red 414 to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster in vitro.

Two independent experiments were conducted both with and without an exogenous rat liver microsomal activation system (S9-mix).

The compound was suspended in cell culture medium and tested at the following concentrations:

Main experiment with and without metabolic activation: 100, 250, 500, 750, 1000#, 1375*, 1750* µg/ml

Repeat main experiment with and without metabolic activation: 100, 250, 500, 750, 1000* µg/ml

*= because of high toxicity no mutant selection was performed.

#= in the absence of S9-mix no mutant selection was performed

 

The concentration ranges were based on the results of preliminary tests for solubility and toxicity.

In the absence of S9 metabolic activation in both mutation experiments a dose-related decrease in survival was observed reaching 16.8 and 3.8 % of the solvent control value in the microtiter plates at a dose level of 1000 µg/ml. In the presence of S9-mix survival was reduced to 31.2 and 8.2 % of the solvent control value at the same concentration.

With the concentrations of 250 and 750 µg/ml the test compound induced a statistically significant enhancement of the mutation rate over the range of the solvent controls in the presence of a metabolic activation in the second main experiment. But these results were not reproducible, not three fold higher than the solvent control group and not dose-dependent and therefore of no biological relevance.

The sensitivity of the test system was demonstrated by the enhanced mutation frequency in the cell cultures treated with the positive control compounds.

In conclusion, Acid Red 414 did not induce gene mutation, i.e. was not mutagenic, in this HPRT-test with V79 Chinese hamster cells, in either the presence or absence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The Mammalian Erythrocyte Micronucleus Test according to OECD Test Guideline No 474 was carried out with Acid Red 414 in male and female NMRI mice. The test compound was suspended in deionized water 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. According to the test procedure the animals were killed 24 hours after test substance administration.

Cyclophosphamide 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 Acid Red 414 and was not less than 20% of the control value.

Cyclophosphamide induced a marked statistically significant increase in the number of poly-chromatic 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 Acid Red 414 is not mutagenic in the micronucleus test.

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:

1998-09-07 to 1998-11-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann Gartenstr. 27; D-33178 Borchen
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: M=38.3 g; F=30.5 g
- Assigned to test groups randomly: yes, randomization schemes 98.0818 and 98.0819
- Fasting period before study: -
- Housing: in fully air-conditioned rooms in makrolon cages type 3 (five animals per cage) on soft wood granulate
- Diet (e.g. ad libitum): rat/mice diet ssniff RIM-H (V 1534), ad libitum ssniff GmbH, Postbox 2039, 59480 Soest
- Water (e.g. ad libitum): tap water in plastic bottles, ad libitum
- Acclimation period: 5 days

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

Route of administration:

oral: gavage

Vehicle:

deionized water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
On the days of administration the test substance was suspended in deionized water at the appropriate concentration. A magnetic stirrer was used to keep the preparation homogeneous until dosing had been completed.
The test substance preparation was administered twice at an interval of 24 hours orally by gavage

Duration of treatment / exposure:

The test substance was administered twice at an interval of 24 hours orally by gavage to the test animals at a dose of 2000 mg per kg body weight; animals were killed 24 hours after dosing

Frequency of treatment:

twice at an interval of 24 hours

Post exposure period:

24 h

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide: 50 mg per kg body weight. orally per gavage

Tissues and cell types examined:

bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: In a preliminary dose range finding study, oral administration of 2000 mg Acid Red 414 per kg body weight did not cause any toxic effects in male and female mice over an observation period of 7 days. This dose level was therefore the regularly limit dose, selected as the highest dose for the main study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The test substance was administered twice at an interval of 24 hours orally by gavage to the test animals at a dose of 2000 mg per kg body weight. The vehicle, deionized water, was administered in the same way to the negative control groups. The study included a concurrent positive control using Cyclophosphamide, which was administered once orally by gavage at a dose of 50 mg per kg body weight.
All animals were killed by carbon dioxide asphyxiation 24 hours after dosing and bone marrow samples were taken from both femora.

DETAILS OF SLIDE PREPARATION:
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-Grünwald's solution
- brief rinsing twice in distilled water
- 10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2 (Weise)
- rinsing in distilled water
- drying
- coating with Entellan

METHOD OF ANALYSIS:
2000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. In addition, the ratio of polychromatic erythrocytes to 200 total erythrocytes was determined. 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-relation-ship one-sided Wilcoxon tests were performed starting with the highest dose group. These test were performed with a multiple level of significance of 5%.

OTHER:

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:

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-relation-ship 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

Remarks:

but reddish urine was observed

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

All animals survived after treatment. Red discolored feces and reddish urine but no signs of toxicity were observed during the duration of the whole study.
The dissection of the animals revealed red coloured contents of the gastro-intestinal tract as macroscopic finding.
The bone marrow smears were examined for the occurrence of micronuclei in red blood cells. The incidence of micronucleated polychromatic erythrocytes in the test substance treated group 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.

Conclusions:

Acid Red 414 did not lead to a substantial increase of micronucleated polychromatic erythrocytes and is not genotoxic in the micronucleus test

Executive summary:

The Mammalian Erythrocyte Micronucleus Test according to OECD Test Guideline No 474 was carried out with Acid Red 414 in male and female NMRI mice. The test compound was suspended in deionized water 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. According to the test procedure the animals were killed 24 hours after test substance administration.

Cyclophosphamide 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 Acid Red 414 and was not less than 20% of the control value.

Cyclophosphamide induced a marked statistically significant increase in the number of poly-chromatic 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 Acid Red 414 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 item Acid Red 414 was tested positive in the Bacteria Reverse Mutation Assay (Ames test), but was negative in the mutation assay in mammalian cells (HPRT) testing and the in-vivo Micronucleus Test in mice, testing for clastogenicity and aneugenicity. 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.

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]. However, it has been demonstrated in various publications that this is a bacteria-specific effect and that these Ames positive substances are not mutagenic in mammalian assays.

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 nitrocompounds [de Oliveira et al. 2010].

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.

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.

AMP397is 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 inSalmonellastrains 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.

As a conclusion, it could be said that 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 Acid Red 414 is a bacteria specific effect and not relevant to mammalians.

Acid Red 414 was not genotoxic in the mammalian in-vitro cell mutagenicity test (HPRT assay) and the in-vivo MNT test. Therefore, a direct genotoxic effect as well as a metabolisation towards genotoxic structures by mammalian species can be excluded.

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.

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.

Justification for classification or non-classification

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