2,2'-[[5-acetamido-4-[(2-bromo-4,6-dinitrophenyl)azo]-2-methoxyphenyl]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, whereas the mutagenicity study in mammalian cells was negative.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Not conducted under GLP, no information on test substance purity

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0, 10, 50, 100, 500, 1000 and 5000 µg/plate

Vehicle / solvent:

The substance was dissolved 50 mg/ml in dimethylsulfoxide and further diluited in the same solvent according the need.

Negative solvent / vehicle controls:

yes

Details on test system and experimental conditions:

Media: Vogel-Bonner E agar medium (VB) has the following compositlon:

MgSO4 x 7H20 0.2 g/l
citric acid x H2O 2 g/l
K2HPO4 10 g/l
NaNH4HPO4 x 4 H2O 3.5 g/l
glucose 2 g/l
Agar 15 g/l

It was prepared by mixing 750 ml of 2% molten autoclave sterillzed water agar wlth 250 ml of.4 times concentrated salt solution and 10 ml of 20 % glucose solution autoclaved separately.
For the antibacterlal test, histidine was also added to give a final concentratlon of 10 µg/ml.
10 cm plastic petri dishes were prepared containing 20 ml each of VB agar and used within 3 days from preparation.
Top agar was prepared by mlxing 100 ml of autoclave sterilised 0.6 % agar and 0.5 % NaCl in water with 10 ml of a millipore filter sterillzed 0.5 mM histidine, 0.5 mM blotln solutlon. Top agar was dlspensed in 2 ml amounts into 16x100 mm glass tubes kept at 42 °C.

TA1535, TA1537, TA1538 Ames strains were kept at 4 °C in deep soft nutrient agar, strains TA100 and TA98 were kept at -80 °C in nutrient broth plus ampicillin (20 µg/ml) and dimethysulfoxide (80 µg/ml).
The day before the test, fresh cultures were prepared by inoculating 10 ml of nurtrient broth with each strain, which were incubated overnight at 37 °C. Concurrently with each series of test, cultures were checked for relevant characters, namely:
-histidine requirement
-biotin requirement
- uvrB character
-rfa charcter
- presence or absence of the pKM101 prototrophy
-induced reversion rate by known mutagens

For testing the antibacterial activity :
0.1 ml of solvent containing the required amount of substance + 0.1 ml of a 10^-5 dilution of an overnight culture of TA1535 or TA98 containing between 100 and 500 colony forming units (CFU) + if requested S9 mix
were added in sequence to each top agar tube. Controls received 0.1 ml of the solvent mixture.
The contents of each tube were quickly mixed and poured on the surface of a histidine containing VB plate.
Colonies were counted after 24 hours of incubation at 37 °C. Tests were carried put inm triplicate.

For testing mutagenicity:
0.1 ml of solvent containing the required amounts of substance or 0.1 ml of sovlent alone + 0.1 ml of overnight culture of each strain containing 10^7 bacteria and if reuired 0.5 ml of S9 mix were added in sequence to each top agar tube. The contents of each tube were quickly mixed and pured on the surface of a VB plate.
Revertant colonies were counted after 2 days of incubation at 37 °C. Tests were carried out in triplicate.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

up to 5000 ug/plate

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Key result

Species / strain:

S. typhimurium, other: TA1535, TA1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Conclusions:

Under the study conditions, the substance was mutagenic in strains TA98 and TA100 and ambiguous in strain TA1538 with metabolic activation.

Executive summary:

A study was conducted to determine the mutagenic potential of the test substance (purity not given), according to OECD Guideline 471. Five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA 1538) were exposed to the test substance, dissolved in dimethylsulfoxide, at concentrations of 0, 10, 50, 100, 500, 1000 and 5000 µg/plate with or without metabolic activation (S-9 mix) for 48 h. Negative and positive control experiments were valid. Under the experimental conditions, the substance was mutagenic in strains TA98 and TA100, and ambiguous in TA1538 with metabolic activation. The mutagenicity is of frame shift type (Archroma, 1980).

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

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:

not specified

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Rat S-9 mix

Test concentrations with justification for top dose:

1st experiment
Strains: TA 1535, TA 100, TA 1537, TA 98
Doses: 0, 20, 100, 500, 2500 and 5000 µg/plate

2nd experiment:
Strain: TA 98
Doses: 0, 0.8, 4, 20, 100 and 500 µg/plate

Untreated negative controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

9-aminoacridine

other: 2-aminoanthracene for all four strains, N-methyl-N'-nitro-N-nitroso-guanidine for the strains TA 100 and TA 1535, 4-nitro--o-phenylenediamine for the strain TA 98.

Details on test system and experimental conditions:

Test tubes containing 2 mL portions of soft agar (0.6% agar +0.6% NaCl) and 10 mL amino acid solution are kept in a water bath at 45°C and the remaining components are added in the following order:
0.1 mL test solution, 0.1 mL bacterial suspension, 0.5 mL S-9 mix (in tests with metabolic activation) or 0.5 mL phosphate buffer (in tests without metabolic activation).
After mixing, the samples are poured onto the Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 sec. After incubation at 37°C for 48 h in the dark, the bacterial colonies (his+ revertants) are counted.

Evaluation criteria:

The test substance was evaluated as positive if it fulfilled the following requirements:
-doubling of the spontaneous mutation rate (compared to control)
-dose-response relationship
-reproducibility of the results

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation was obtained due to incomplete solubility of the test substance from 500 µg/plate onwards.

INFORMATION ON CYTOTOXICITY:
- Cytotoxicity: no cytotoxicity was observed at any of the tested concentrations

Conclusions:

Under the study conditions, the test substance was considered to be mutagenic.

Executive summary:

A study was conducted to determine the mutagenic potential of the test substance (purity 40%) according to OECD Guideline 471. Four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) were exposed to the test substance at concentrations of 0, 0.8, 4, 20, 100 and 500 µg/plate (for TA 98) and 0, 20, 100, 500, 2500 and 5000 µg/plate for (TA100, TA1537 and TA1535), with or without metabolic activation (S-9 mix). Negative and positive controls were valid. The test substance induced mutagenic activity with and without metabolic activation in all the four tested strains; in all cases of a positive response the number of revertants was greater than twice the number of spontaneous mutations. Under the study conditions, the test substance was considered to be mutagenic (Engelhardt, 1990).

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Summary of method and results available

Qualifier:

no guideline followed

Principles of method if other than guideline:

The test substance was tested for mutagenic activity in a reduced Ames screening test. S. typhimurium strains TA98 and TA100 were tested in a standard plate test without and with metabolic activation (rat S-9 mix) at test substance concentrations of 100-5000 µg/plate.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Metabolic activation:

with and without

Metabolic activation system:

Rat S-9 mix

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene and 4-nitro-o-phenylenediamine

Details on test system and experimental conditions:

S.typhimurium strains TA98 and TA100 were tested in a standard plate test without and with metabolic activation (Rat S-9 mix) at test substance concentrations of 100-5000 µg/plate.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation was observed from 500 µg/plate concentration

INFORMATION ON CYTOTOXICITY:
- Cytotoxicity: no cytotoxic effect was observed at any of the concentration

Without S-9 mixed weakly mutagenic effect from 100-500 mg /plate (factor 1,4-1,8) with an increase of the revertantenzahl by the factor 4,6 at 5000 mg / platte. According to a metabolising system, a positive 








































  
  
  
  
  
  
  
  
  
  
  
  
  
  

effect was demonstrated over the entire dose range.

Conclusions:

Under the study conditions, the test substance was considered to be mutagenic.

Executive summary:

A study was conducted to determine the mutagenic potential of the test substance according to a reduced version of the Ames test. Five strains of Salmonella typhimurium (TA98 and TA100) were exposed to the test substance at concentrations of 100 to 5000 μg/plate with or without metabolic activation (S-9 mix). Negative and positive controls were valid. The test substance induced mutagenic activity with and without metabolic activation in the TA98 strain, whereas in TA100 strain the test substance induced mutagenic activity only with metabolic activation; in all cases of a positive response the number of revertants was greater than twice the number of spontaneous mutations. Under the study conditions, the test substance was considered to be mutagenic (Engelhardt, 1993).

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Not conducted under GLP

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 micronuclei in the polychromatic erythrocytes of treated rats and did not induce sex-linked recessive lethal mutations in Drosophila melanogaster.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

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 study conditions, the test substance was not found to be genotoxic.

Executive summary:

A study was conducted to determine the in vivo genetic toxicity of the test substance (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 (Archroma, 1985).

Reference 2

Endpoint:

in vivo insect germ cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The test substance was tested for mutagenic activity (the induction of sex-linked recessive lethal mutations) in Drosophila melanogaster adult males exposed by injection. A standard genetic scheme (Base females crossed to Canton-s wild type males) was employed and germ cells which were post­meiotic at the time of exposure were tested for lethal mutations.

GLP compliance:

yes

Type of assay:

Drosophila SLRL assay

Species:

Drosophila melanogaster

Strain:

other: Canton S-wild type

Sex:

male/female

Details on test animals or test system and environmental conditions:

The Drosophila melanogaster stocks used are laboratory lines in existence for many years. The Canton-S wild type stock (from which males were taken for exposure) was a line in use in the University of Wisconsin Genetics laboratory for approximately 20 years and is being used in sex-linked recessive lethal assay since 1989. The "Basc" stock (which supplied the females for the parental matings) was obtained from Brown University in 1986. The stocks are transferred weekly and observed for genetic purity (intact phenotypic markers).

The culture medium for the stocks as well as for the test matings is a standard Drosophila culture medium. All stocks and Fi and Fa matings were kept at room temperature (at 22°C). The photoperiod was not controlled as it is not known to affect results.

Route of administration:

intraperitoneal

Vehicle:

1.9% DMSO and 0.1% Tween 80 in 0.7% NaCl

Details on exposure:

Please refer to "Any other information on materials and methods" for more details

Frequency of treatment:

The male Drosophila were injected only once and kept for recovery for 1-2 days before mating.

Post exposure period:

1-2 days

Dose / conc.:

50 ppm (nominal)

Control animals:

yes, concurrent vehicle

Positive control(s):

N-dimethylnitrosamine (500 ppm) in 0.7% aqueous NaCl.

Tissues and cell types examined:

99 chrmosomes from each tested male were tested for the induction of mutations.

Statistics:

When an individual P1 male produces more than 1 lethal among his progeny, the group of lethals can be called a "multiple". A multiple may result from many independent mutations in the treated post-meiotic germ cells (as is the case with a potent mutagen) or from a single spontaneous mutation in an untreated gonial cell, which then divides to produce a "cluster" of sperm cells carrying identical recessive lethal mutations. Thus each multiple must be subjected to statistical analysis to determine whether or not it could be considered a cluster. The statistical test is based on a cumulative Poisson distribution. One large multiple of 52 was recovered in the positive control group of experiment 474, and another multiple of 13 was recovered in the positive control group of experiment 476. These were the only multiples determined to be clusters. The data from the males which produced clusters were removed from the data before analysis.before analysis. The recessive lethal data were evaluated by comparing the overall mutation frequency of the treated group with the overall mutation frequency of concurrent negative controls, by applying the "Normal Test". For this experiment, P = 0.46, and is therefore assessed as negative. All values are within the range of recent historical controls in this laboratory.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Under the study conditions, the test substance did not induce sex-linked recessive lethal mutations in the post­meiotic germ cells of Drosophila melanogaster.

Executive summary:

A study was conducted to determine the in vivo genetic toxicity of the test substance (in the form a green powder of 97% purity) to induce sex-linked recessive lethal mutations in Drosophila melanogaster according to an internal protocol of the test facility. The potential of the test substance to induce sex-linked recessive lethal mutations was investigated in this assay. The test substance was injected to 2 -3 day old male Drosophila flies of the 'Canton S' wild-type and kept for 1 -2 days before mating. 3 -4 day old control and the treated males were mated singly to three virgin females (3 -10 day old) of the balancer stock 'Basc' which was termed Brood 1. Each male was transferred to three new virgin females to create Brood 2 and thereafter Brood 3. The heterozygous females of F1 generation were mated individually to their respective siblings in order to produce F2. The P1 males and females were later discarded. Sperm/spermatid/spermatocyte samples from the three broods were collected and approximately 99 chromosomes from each treated male were observed. Under the study conditions, the test substance did not induce sex-linked recessive lethal mutations in the post­meiotic germ cells of Drosophila melanogaster (Foureman, 1990).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

The test substance (and the structural analogue 1) was positive in the Ames assay, but negative in a Drosophila melanogaster Sex-Linked Recessive Lethal Assay, a mutation assay in mammalian cells and in anin vivomouse 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

A study was conducted to determine the mutagenic potential of the read across substance 'structural analogue 1' according to OECD Guideline 471. Five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA 1538) were exposed to the test substance, dissolved in dimethylsulfoxide, at concentrations of 0, 10, 50, 100, 500, 1000 and 5000 µg/plate with or without metabolic activation (S-9 mix) for 48 h. Negative and positive control experiments were valid. Under the experimental conditions, the substance was mutagenic in strains TA98 and TA100, and ambiguous in TA1538 with metabolic activation. The mutagenicity is of frame shift type (Archroma, 1980).

A study was conducted to determine the mutagenic potential of the test substance (purity 40%) according to OECD Guideline 471. Four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) were exposed to the test substance at concentrations of 0, 0.8, 4, 20, 100 and 500 µg/plate (for TA 98) and 0, 20, 100, 500, 2500 and 5000 µg/plate for (TA100, TA1537 and TA1535), with or without metabolic activation (S-9 mix). Negative and positive controls were valid. The test substance induced mutagenic activity with and without metabolic activation in all the four tested strains; in all cases of a positive response the number of revertants was greater than twice the number of spontaneous mutations. Under the study conditions, the test substance was considered to be mutagenic (Engelhardt, 1990).

A study was conducted to determine the mutagenic potential of the test substance according to a reduced version of the Ames test. Five strains of Salmonella typhimurium (TA98 and TA100) were exposed to the test substance at concentrations of 100 to 5000 μg/plate with or without metabolic activation (S-9 mix). Negative and positive controls were valid. The test substance induced mutagenic activity with and without metabolic activation in the TA98 strain, whereas in TA100 strain the test substance induced mutagenic activity only with metabolic activation; in all cases of a positive response the number of revertants was greater than twice the number of spontaneous mutations. Under the study conditions, the test substance was considered to be mutagenic (Engelhardt, 1993).

A study was conducted to determine the genotoxicity of the read across substance 'structural analogue 1' 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).

In vivo

A study was conducted to determine the in vivo genetic toxicity of the read across substance 'structural analogue 1' 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).

A study was conducted to determine the in vivo genetic toxicity of the test substance (in the form a green powder of 97% purity) to induce sex-linked recessive lethal mutations inDrosophila melanogasteraccording to an internal protocol of the test facility. The potential of the test substance to induce sex-linked recessive lethal mutations was investigated in this assay. The test substance was injected to 2 -3 day old male Drosophila flies of the 'Canton S' wild-type and kept for 1 -2 days before mating. 3 -4 day old control and the treated males were mated singly to three virgin females (3 -10 day old) of the balancer stock 'Basc' which was termed Brood 1. Each male was transferred to three new virgin females to create Brood 2 and thereafter Brood 3. The heterozygous females of F1 generation were mated individually to their respective siblings in order to produce F2. The P1 males and females were later discarded. Sperm/spermatid/spermatocyte samples from the three broods were collected and approximately 99 chromosomes from each treated male were observed. Under the study conditions, the test substance did not induce sex-linked recessive lethal mutations in the post­meiotic germ cells of Drosophila melanogaster (Foureman, 1990).

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.