N-[2-[(2-bromo-4,6-dinitrophenyl)azo]-5-(diethylamino)phenyl]acetamide

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, whereas the mutagenicity study in mammalian cells with the structural analogue was negative.

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:

supporting study

Study period:

1990

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only screening with one strain

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 98

Metabolic activation:

with and without

Metabolic activation system:

S9

Vehicle / solvent:

DMSO - test substance is not water soluble

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2AA

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Compount is significantly more toxic in the absence of S9

Conclusions:

The test substance is positive in Salmonella typhimurium TA 98

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Jan to 21 Apr 1999

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:

adopted July 21, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine locus

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction

Test concentrations with justification for top dose:

According to the results of the pre-experiment the concentrations applied in the main experiments were chosen:
33, 100, 333, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

On the day of the experiment, the test article was dissolved in DMSO (purity > 99 %, MERCK, D-64293 Darmstadt). The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
The test article precipitated at the higher concentrations in the overlay agar. The nondissolved particles of the test article had no influence on the data recording.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9, TA 1535 and TA 100

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-NOPD

Remarks:

without S9, TA 1537 and TA 98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9, all strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours at 37 °C

NUMBER OF REPLICATIONS: 3 plates/strain/dose level

DETERMINATION OF CYTOTOXICITY: Toxicity of the test article can be evidenced by a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

ACCEPTED CONDITIONS FOR EVALUATION:
The Salmonella typhimurium reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of historical data
- the positive control substances should produce a significant increase in mutant colony frequencies

Rationale for test conditions:

N.A.

Evaluation criteria:

A test article is considered positive if either a dose related increase in the number of revertants or a biologically relevant increase for at least one test concentration is induced.
A test article producing neither a dose related increase in the number of revertants nor a biologically relevant positive response at any one of the test points is considered nonmutagenic 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 in strains 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.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test article precipitated at the higher concentrations in the overlay agar. The nondissolved particles of the test article had no influence on the data recording.

Toxic effects, evident as a reduction in the number of revenants, were observed with and without S9 mix in strains TA 1535 and TA 1537 at 2500 and 5000 µg/plate, and in strain TA 100 with S9 mix in experiment II.

Conclusions:

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 TA 1535, TA 1537, TA 98, and TA 100. Therefore, the test item is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

 In a reverse gene mutation assay in bacteria, strains TA 1535, TA 100, TA 1537, TA 98 of S. typhimurium were exposed to the test item (60% purity) at concentrations of 33, 100, 333, 1000, 2500 and 5000 µg/plate in the presence and absence of mammalian metabolic activation. The test item was tested up to the limit concentration (5000 µg/plate).

Toxic effects, evident as a reduction in the number of revenants, were observed with and without S9 mix in strains TA 1535 and TA 1537 at 2500 and 5000 µg/plate, and in strain TA 100 with S9 mix in experiment II.

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, a substantial and dose dependent increase in revertant colony numbers was observed following treatment with the test substance in strains TA 1535, TA 1537, TA 98, and TA 100.

However, overlapping toxic effects reduced the number of revertant colonies in strains TA 1535, TA 1537 (with and without metabolic activation), and TA 100 from 1000 up to 5000 µg/plate.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase in 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 TA 1535, TA 1537, TA 98, and TA 100.

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 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Not conducted under GLP

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read across is based on the same physico-chemical properties, a close structural similarity and the same mechanism of action during use processes.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
- Source: Disperse Blue 79, EC 222-813-1
- Target: Disperse Violet 93:1
Substance Name: N-[2-[(2-bromo-4,6-dinitrophenyl)azo]-5-(diethylamino)phenyl]acetamide
EC Number: 258-110-1
CAS Number: 52697-38-8

3. ANALOGUE APPROACH JUSTIFICATION
see attachment section 13

4. DATA MATRIX
see attachment section 13

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

no

Type of assay:

mammalian cell gene mutation assay

Target gene:

hgprt

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0, 0.05, 0.1, 0.25, 0.5 and 1.0 µg/ml

Vehicle / solvent:

Dimethylsulfoxide

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

7,12-dimethylbenzanthracene mutagenic with S9 mix

Details on test system and experimental conditions:

On Day 1 of the experiment, 1.500.000 cells were seeded per I50 rnn Ø dish. On the following day, the cells were exposed to the test compound with the exception that 6-fold larger volumes of media and test solution were used. After removal of the test compound and washing of the plates with PBS, the cultures were maintained untíl Day 8 with 30 ml normal DME-FCS with one subculture on Day 5. This period is required for expression of the new genotype i.e., for sufficient dllution and catabolism of the previously formed hypoxanthíne guanine phosphoribosyl transferase. Afterwards the cells were harvested by trypsinisation and replated at a density of 1.000.000 per 150 mm Ø dish in DME-FCS containing 6-thioguanine (7 µg/ml) for selection of mutants (6 replicate plates), or at 100 cells per 60 mm Ø dish in medium without 6-thioguanine for the estimation of cloning efficiencies (3 replicate plates). The cultures were fixed and stained after 8 days (cloning efficiency plates) or 10 to 11 days (6-thioguanine plates).

Evaluation criteria:

Negative: if solvent and positive controls show results within the norm and if the test compound does not increase the mutation frequency 2-fold above the mean of the solvent controls under any condition, or if the mutation frequency is always lower than 10 x 10-6 and if at least 1.000.000 cells per condition have
been evaluated, the compound is considered as negative in the test and no further experlments are performed.

Positive: In case of a dose-dependent increase of the mutation frequency to at least 5-fold solvent control and at least 40 x 10-6 both in the presence and
absence of S-9 mix, the compound is considered as positlve in the test without additional experiments.

In all other cases, the test at the suspected optimal concentration range is repeated. Then, the result' is considered as positive if in both experiments
(at similar concentrations) the mutation frequency is at least 2-fold above the solvent control and at least 10 x 10-6. Otherwise the result is considered
as negative.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Under the experimental conditions, the substance did not show mutagenic potential to V79 cells.

Executive summary:

A study was conducted to determine the genotoxicity of the test substance (93% purity) according to OECD Guideline 476. Chinese hamster lung fibroblasts (V79) were exposed at concentrations of 0, 0.05, 0.1, 0.25, 0.5 and 1.0 µg/mL with or without S-9 metabolic activation. Cell survival and mutations were assessed. In the experiment for direct mutagenicity, N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) served as a positive control. In the S9-mix mediated assay, 7,12-dimethylbenz(a)anthracene (DMBA) was used. Negative and positive control experiments were valid. The substance showed acute toxicity to the V79 cells in the direct assay. However, there were no signs of mutagenicity in the presence or absence of S9 mix at various concentrations up to the limits of toxicity and solubility. Under the study conditions, the test substance was not considered to be mutagenic in Chinese hamster lung fibroblasts (V79) (Archroma, 1984).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test substance did not induce DNA repair (as measured by unscheduled DNA synthesis) in rat liver nor did the structural analogue induce micronuclei in the polychromatic erythrocytes of treated rats.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Read-across

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read across is based on the same physico-chemical properties, a close structural similarity and the same mechanism of action during use processes.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
- Source: Disperse Blue 79, EC 222-813-1
- Target: Disperse Violet 93:1
Substance Name: N-[2-[(2-bromo-4,6-dinitrophenyl)azo]-5-(diethylamino)phenyl]acetamide
EC Number: 258-110-1
CAS Number: 52697-38-8

3. ANALOGUE APPROACH JUSTIFICATION
see attachment section 13

4. DATA MATRIX
see attachment section 13

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

no

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

Mice are recommended for micronucleus assay as international standard.
Source: Kleintierfarm Madoerin AG, Switzerland
Age at the beginning of the test: 6 weeks (m), 7 weeks (f)
Initial bodyweight: 26-36 g (m), 23-34 g (f)
number of animals: 54 males and 54 females
Acclimation: 7 days at the test conditions under veterinary examination
Animals were randominzed
Method of identification: cages were labeled with project code, sex and dose. Animal identification by indelible inert color spots on the tail
Housing: groups of six animals
Cage: Makrolon Type 3 with wire mesh top and granulated softwood bedding
Environment: air conditioned temperature 22 ± 2 °C
Relative humidity: 55 ± 10 %
12 hours light/dark per day
Feed: pelleted standard Kliba 343-A, mouse diet ad libitum
Drinking: tap water ad libitum
and granulated :oftr¡ood bedding

Route of administration:

oral: gavage

Vehicle:

2% carboxymethylcellulose and distilled water (suspension prepated just before the application). Homogeneity of the suspension was mantained during application by magnetic stirrer

Details on exposure:

The maximum tolerated dose was based on acute oral toxicity data. A preliminary acute oral LD50 (limit tes, two doses, 3 males and 3 females per dose) performed with the same mouse species as was used in this study showed the following results after 14 days of observation:
1000 mg/kg bw : 0 mortality in 6 animals
5000 mg/kg bw: 0 mortality in 6 animals
The 5000 mg/kg bw was used in this study as maximum tolerated dose.

the negative control received the test article vehicle, i.e. 2 % CMC in distilled water
the test group received 5000 mg/kg bw of test article (volume applied 20 ml/kg)
the positive control group received 50 mg/kg bw of cyclophosphamide (reference mutagen) dissolved in 0.9 % saline solution immediately before application

At 24, 48 and 72h after treatment, six mice per sex and group were sacrificed for examination. The first five animals of each sex were evaluated. The remaining animal of each sex was evaluated if macroscopic examination of the slides revelaed technical imperfections that may have prevented accurate microscopic analysis or if a test animal died even spontaneously or from gavage error.

Frequency of treatment:

Once

Post exposure period:

72 h

Remarks:

Doses / Concentrations:
5000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

18 males + 18 females per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide

Details of tissue and slide preparation:

AII mice were sacrificed by cervical dislocation. The femora were removed from each rmouse and freed of adherent tissue.
The proximal end of the femur was cut with scissors. The needle of a plastic syringe containing 0.2 ml calf serum* was inserted into the proximal part of the marrow canal which was closed at the distal end. The femur was submerged in 1.5 ml calf serum in a Iabeled centrifuge tube.
The bone marrow celIs were dispersed in the calf serum as a homogeneous suspension. The tube containing the bone marrow ceIIs of both femora was centrifuged at 1000 r.p.m. fon 5 minutes.
The supernatant was removed, Ieaving a thin layer of serum. The cells of the sediment were suspended by aspiration in a siliconized pasteur pipette. Asmall drop of the marrow serum suspension was smeared on the sIide, which was identified by project code and the animal number, and allowed to dry overnight. Two slides per animal were prepared. The following day, the smears were stained using the panoptic stain method developed by Pappenheim.
The slides were coded before microscopic analysis. If rnacrocopic evaluation revealed technical imperfections, the first slide was replaced by the second slide prepared. From each animal, one thousand polychromatic erythrocytes (PCE) were scored under the microscope (magnification 1000x)*, for the incidence of micronuclei.
Additional information could be obtained by scoring nonmochromatic erythnocytes for micronuclei.
The calculated ratio polychromatic to normochromatic erythrocytes (PCE/NCE), based on 1000 erythrocytes scored per sIide, measured the toxic efficacy of the test article.

Evaluation criteria:

The frequencies of micronuclei of the treated male and female groups were compared with those of the negative control groups at each sampling time. A regression model assuming a Poisson distribution was applied. Estimation and testing were performed by maximum Iikelihood method.
If a test article produced no statistically significant and reproducible positive response at any one of the test points, it was considered non-mutagenic in this system.

Key result

Sex:

male/female

Genotoxicity:

negative

Remarks:

no significant test article related increase of micronucleated polychromatic erythrocytes

Toxicity:

no effects

Additional information on results:

No deaths occured. Sedation was observed in all test artcile treated animals for at least 6 hours after application.

Conclusions:

Under the experimental conditions the potential to induce the formation of micronuclei is negative.

Executive summary:

A study was conducted to determine the in vivo genetic toxicity of the structural analogue 2 according to OECD Guidelines 474. The ability of the test substance to induce cytogenetic damage and/or disruption of the mitotic apparatus in rat bone marrow was investigated measuring the induction of micronuclei in polychromatic erythrocytes. Male and female rats (15/sex/group) were exposed to the test substance at concentrations of 0 or 5000 mg/kg bw/day by gavage in a single application of a 2% carboxymethylcellulose (CMC) distilled water suspension. A positive control group (mitomycin-C, 2.0 mg/kg) was also tested. The examinations were performed at 24, 48 and 72 h by sacrifice of 6 animals per sex. The substance did not show an increase of micronuclei from bone marrow compared to the vehicle control. The values for the positive and negative controls were within the expectation ranges. The experiment was therefore considered valid. Under the study conditions, the test substance was not found to be genotoxic (Archroma, 1985).

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Deviations:

yes

Remarks:

see below

Principles of method if other than guideline:

Two doses only of the test compound were applied: Only two test compound concentrations were required for scoring as the test substance dosed at a limit concentration is not cytotoxic in the liver. A third dose was therefore considered unnecessary.

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Fischer 344

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: ICI Barriered Animal Breeding Uni (BABU), Alderley Park, Macclesfield
- Age at study initiation:
- Weight at study initiation: 180-280 g
- Assigned to test groups randomly: yes - according to the order in which they are removed from the stock cage
- Fasting period before study:
- Housing: - pre dosing: up to 5 per cage
- for dosing: up to three per cage in a fume cupboard for a total period not exceeding 24 hours
- Diet (e.g. ad libitum): Porton Combined Diet, Special Diets Services Ltd, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: Since the animals were eventually moved in a fume cupboard, acclimatisation was not considered appropriate


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 21
- Humidity (%): 40 - 60
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: test substance in insoluble in water
- Concentration of test material in vehicle: 125 and 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg body weight

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Dosing suspensions of the test substance were prepared in corn oil.

Duration of treatment / exposure:

single dose

Frequency of treatment:

once

Post exposure period:

4 and 12 h after treatment

Dose / conc.:

1 250 mg/kg bw/day (nominal)

Remarks:

in corn oil

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

in corn oil

No. of animals per sex per dose:

5 males/dose/time point in treatment group
4 males/group/time point in vehicle and positive control group - only 1 animal/group/time point scored

Control animals:

yes, concurrent vehicle

Positive control(s):

6-p-dimethylaniinophenylazobenzthiazole [6BT]: CTL Reference No. Y01226/016 [12h Experiments].
N-nitroso-dimethylamine [NDMA, DMN] : CTL Reference No. Y01468/004 [4h Experiments].

Tissues and cell types examined:

Hepatocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on acute oral toxicity study in rats, in which the acute MTD for the test substance was >5000 mg/kg bw and a renge finding study in 5 males at 2000 mg/kg bw

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Single oral dose by oral gavage. Slides from the animals were subsequently analysed. Of the two positive and two vehicle control animals in each experiment, only one of each was scored for induction of UDS

DETAILS OF SLIDE PREPARATION: Preparation of hepatocytes were made 4 or 12 h after dosage. Hepatocytes were prepared from treated animals by a two stage collagenase perfusion technique. Hepatocyte cultures were prepared by allowing cells to attach to plastic cover slips. Medium was removed from the dishes and replaced with fresh medium containing [3H] thymidine. After 4 h incubation at 37 °C within a 5% CO2/95% air (v/v) atmosphere, the medium was removed, the cells washed three times with medium containing unlabelled thymidine and the cultures incubated overnight with the same medium. Cultures were fixed and coverslips mounted onto microscope slides. Slides were coated with photographic emulsion and left for 14 d at 4 °C in the dark. The emulsion was developed, fixed and the cell nuclei and cytoplasm stained with Meyers haemalum and eosin Y phloxine.
Slides were examined microscopically for signs of undue cytotoxicity to enable selection of those to be examined for UDS.


METHOD OF ANALYSIS: Prior to microscopic assessment, all slides were furnished with code numbers, so that the counting was blind. The following counts were made:
- at least 25 but normally 50 cells/slide of two slides for each animal. The third slide was not normaly read, unles a total of 10 cells could not be obtained from the
first two slides examined.
- The nuclear count (the number of silver grains over the nucleus) and the cytoplasmic count (the number of grains in an adjacent, nuclear sized, most heavily labelled area of cytoplasm) were measured using an automated image analyser and the data captured directly into a computer.
- The mean net grain count (nuclear count - cytoplasmic count), the mean nuclear count and cytoplasmic counts and the percent of cells in repair (net grain count of 5 or greater) to be calculated.

Evaluation criteria:

EVALUATION CRITERIA
Criteria for a positive response:
- The criteria outlined in the ASTM Guideline are adopted, based on the mean net group grain count and the percentage of cells in repair. A treated group showing mean net nuclear grain counts of 5 or greater and 20% or greater of the cells in repair is considered to be a positive response. A compound can only be assigned as an unequivocal genotoxin in this assay if such a response is reproduced.

Criteria for a negative response:
- A negative response is obtained where the mean net nuclear grain count of the treated cultures is less than 0, and the percentage of cells in repair is also less than 20.


VALIDATION CRITERIA
Negative Controls
Cytoplasmic counts of less than 40 are considered acceptable. Mean net nuclear grain counts for the negative controls should be less than zero.

Positive Controls
Mean net grain counts for the positive control in each experiment shall be sufficient to be regarded as an unequivocal positive response; mean net nuclear grain value of 5 or greater, with at least 20% of the cells examined in repair (ie with 5 or more net grains).

Statistics:

difference between treated and control cultures

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

No significant adverse reactions to treatment

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: No adverse effects at 5000 mg/kg bw in acute oral or 2000 mg/kg body weight for four days in range finding study

RESULTS OF DEFINITIVE STUDY
- Animal Toxicity: No significant signs of acute toxic effects were observed.

Others:
Test substance caused no significant increases over the vehicle control in mean net nuclear grain count, nor in percentage of cells in repair, at either dose level (1250 or 2000 mg/kg bw) or time point (4 or 14 h) investigated. Hepatocytes from test substance treated animals had mean net nuclear grain values of less than zero. These data therefore provided no evidence for induction of UDS by test substance.

None

Conclusions:

Under the test conditions, the test substance did not induce DNA repair in rat liver in vivo up to a limit dose of 2000 mg/kg bw

Executive summary:

Disperse Violet 93.1 was tested for the ability to induce unscheduled DNA synthesis (UDS) in an in vivo rat hepatocyte assay. Male Fischer 344 rats were treated with a single oral dose of Disperse Violet 93.1 by gavage at 1250 and 2000mg/kg bodyweight. The highest test dose, 2000 mg/kg bw was the limit test dose for a non-toxic test agent in this assay. Animals were killed and hepatocytes prepared four hours and twelve hours following administration of the chemical. Two independent experiments were carried out for each time point.

Hepatocytes from treated rats were exposed to [³H]-thymidine and the amount of radioactivity incorporated into the nucleus and an equal area of cytoplasm determined by autoradiography. The cytoplasmic grain count was subtracted from that of the nucleus. The value obtained, the mean net nuclear grain count [N-C], is an Index of UDS activity. In this laboratory no negative control animal has shown a mean net nuclear grain count of greater than zero. An [N-C] value of greater than zero is therefore considered indicative of a UDS response.

Each experiment was validated by concurrent control treatments of rats with corn oil, the solvent for Disperse Violet 93.1 and with the carcinogens 2-acetylaminofluorene [2AAF] at twelve hours or N‑nitrosodimethylamine [NDMA] at four hours. Solvent treated rats gave rise to mean net nuclear grain counts of less than zero, whilst hepatocytes from 2AAF or NDMA treated animals had mean net nuclear grain counts of greater than +5. These data showed that background levels of UDS were normal and that the tester animals were responsive to known carcinogens requiring metabolic activation for genotoxic activity.

Hepatocytes from Disperse Violet 93.1 treated animals were assessed for UDS at both dose levels. Treatments with Disperse Violet 93.1 in no case resulted in a mean net nuclear grain count greater than zero, at either time point.

It is concluded that, when tested up to 2000 mg/kg body weight, the test sample of Disperse Violet 93.1 did not induce DNA repair (as measured by unscheduled DNA synthesis) in rat liver.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

The test substance was positive in the Ames assay, but negative in a test for unscheduled DNA synthesis; furthermore, a structural analogue was negative in a mutation assay in mammalian cells and in an in vivo mouse micronucleus assay. The positive effect in the Ames test is a bacteria-specific effect due to bacterial nitro-reductases, which are highly effective in these bacterial strains, but not in mammalian cells.

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).

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 Salmonella strains 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 metabolite is released by intestinal bacteria, a Muta™Mouse 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.

Fexinidazole was 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 (Tweatset 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.

Conclusion

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 the test and read-across substances is a bacteria specific effect and not relevant to mammalians.

References

De Oliveira IM, Bonatto D, Pega Henriques JA (2010). Nitroreductases: Enzymes with environmental biotechnological and clinical importance. In: Current Research, Technology and Education Topics in Applied Microbiology and Microbial Biotechnology. Mendez-Vilas, A., Ed.; Formatex: Badajoz, Spain. 1008–1019.

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

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

Additional information

In vitro

In a reverse gene mutation assay in bacteria, strains TA 1535, TA 100, TA 1537, TA 98 of S. typhimurium were exposed to the test item (60% purity) at concentrations of 33, 100, 333, 1000, 2500 and 5000 µg/plate in the presence and absence of mammalian metabolic activation. The test item was tested up to the limit concentration (5000 µg/plate). Toxic effects, evident as a reduction in the number of revenants, were observed with and without S9 mix in strains TA 1535 and TA 1537 at 2500 and 5000 µg/plate, and in strain TA 100 with S9 mix in experiment II. 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, a substantial and dose dependent increase in revertant colony numbers was observed following treatment with the test substance in strains TA 1535, TA 1537, TA 98, and TA 100. However, overlapping toxic effects reduced the number of revertant colonies in strains TA 1535, TA 1537 (with and without metabolic activation), and TA 100 from 1000 up to 5000 µg/plate. Appropriate reference mutagens were used as positive controls. They showed a distinct increase in 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 TA 1535, TA 1537, TA 98, and TA 100.

A study was conducted to determine the genotoxicity of the structural analogue 01 (93% purity) according to OECD Guideline 476. Chinese hamster lung fibroblasts (V79) were exposed at concentrations of 0, 0.05, 0.1, 0.25, 0.5 and 1.0 µg/mL with or without S-9 metabolic activation. Cell survival and mutations were assessed. In the experiment for direct mutagenicity, N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) served as a positive control. In the S9-mix mediated assay, 7,12-dimethylbenz(a)anthracene (DMBA) was used. Negative and positive control experiments were valid. The substance showed acute toxicity to the V79 cells in the direct assay. However, there were no signs of mutagenicity in the presence or absence of S9 mix at various concentrations up to the limits of toxicity and solubility. Under the study conditions, the test substance was not considered to be mutagenic in Chinese hamster lung fibroblasts (V79).

In vivo

Disperse Violet 93.1 was tested for the ability to induce unscheduled DNA synthesis (UDS) in an in vivo rat hepatocyte assay. Male Fischer 344 rats were treated with a single oral dose of Disperse Violet 93.1 by gavage at 1250 and 2000mg/kg bodyweight. The highest test dose, 2000 mg/kg bw was the limit test dose for a non-toxic test agent in this assay. Animals were killed and hepatocytes prepared four hours and twelve hours following administration of the chemical. Two independent experiments were carried out for each time point. Hepatocytes from treated rats were exposed to [3H]-thymidine and the amount of radioactivity incorporated into the nucleus and an equal area of cytoplasm determined by autoradiography. The cytoplasmic grain count was subtracted from that of the nucleus. The value obtained, the mean net nuclear grain count [N-C], is an Index of UDS activity. In this laboratory no negative control animal has shown a mean net nuclear grain count of greater than zero. An [N-C] value of greater than zero is therefore considered indicative of a UDS response. Each experiment was validated by concurrent control treatments of rats with corn oil, the solvent for Disperse Violet 93.1 and with the carcinogens 2-acetylaminofluorene [2AAF] at twelve hours or N‑nitrosodimethylamine [NDMA] at four hours. Solvent treated rats gave rise to mean net nuclear grain counts of less than zero, whilst hepatocytes from 2AAF or NDMA treated animals had mean net nuclear grain counts of greater than +5. These data showed that background levels of UDS were normal and that the tester animals were responsive to known carcinogens requiring metabolic activation for genotoxic activity. Hepatocytes from Disperse Violet 93.1 treated animals were assessed for UDS at both dose levels. Treatments with Disperse Violet 93.1 in no case resulted in a mean net nuclear grain count greater than zero, at either time point. It is concluded that, when tested up to 2000 mg/kg body weight, the test sample of Disperse Violet 93.1 did not induce DNA repair (as measured by unscheduled DNA synthesis) in rat liver.

A study was conducted to determine the in vivo genetic toxicity of structural analogue 01 (93% purity) according to OECD Guideline 474. The ability of the test substance to induce cytogenetic damage and/or disruption of the mitotic apparatus in rat bone marrow was investigated measuring the induction of micronuclei in polychromatic erythrocytes. Male and female rats (15/sex/group) were exposed to the test substance at concentration of 0 or 5000 mg/kg bw by gavage in a single application of a 2% carboxymethylcellulose (CMC) distilled water suspension. A positive control group (mitomycin-C, 2.0 mg/kg) was also tested. The examinations were performed at 24, 48 and 72 h by sacrifice of 6 animals per sex. The substance did not show an increase of micronuclei from bone marrow compared to the vehicle control. The values for the positive and negative controls were within the expectation ranges. The experiment was therefore considered valid. Under the study conditions, the test substance was not found to be genotoxic.

Justification for classification or non-classification

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