Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Remarks:
Screening assay
Adequacy of study:
weight of evidence
Study period:
1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Qualifier:
equivalent or similar to
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 98
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S9
Test concentrations with justification for top dose:
up to 1000 µg/plate
the substance is toxic at concentrations of 1000 µg/plate and above
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Dimethylsulphoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
not specified
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
Conclusions:
Under the conditions of this assay, the test substance gave a positive response with S. typhimurium strain TA98 in the presence and absence of an auxiliary metabolising system (S9).
Executive summary:

The mutagenic activity of Disperse Blue 284 (82.4%purity),was investigated in a screening assay in the plate incorporation test using the Salmonella typhimurium strain TA 98. The test article was tested at the concentrations up to 1000 µg/plate up to 1000 µg/plate. The test substance was toxic at concentrations of 1000 µg/plate and above.

Reproducible, dose-dependent increases in revertant colony numbers were obtained with and without S9 mix. 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 frameshifts in the genome in the tested strain. As the screening test was positive, no further experiment was conducted.

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 nor did it induce DNA repair (as measured by unscheduled DNA synthesis) in rat liver as seen in studies with a close structural analogue.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
C57BL
Details on species / strain selection:
C57BL/6JfBL10/Alpk male and female mice
Sex:
male/female
Route of administration:
oral: gavage
Duration of treatment / exposure:
Bone marrow samples were taken 24 hours after dosing at 3130 mg/kg and 24, 48 and 72 hours after dosing at 5000 mg/kg.
Frequency of treatment:
Twice at an interval of 24 h

Post exposure period:
Bone marrow samples were taken 24 hours after dosing at 3130 mg/kg and 24, 48 and 72 hours after dosing at 5000 mg/kg.

Dose / conc.:
3 130 mg/kg bw (total dose)
Dose / conc.:
5 000 mg/kg bw (total dose)
No. of animals per sex per dose:
5/sex/dose and killing time

Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Route of administration: oral, gavage
- Doses / concentrations: 65 mg/kg bw
Tissues and cell types examined:
Bone marrow erythrocytes

Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Based on patterns of lethalities or severe toxicity observed over a 4-d observation period following a single oral dose in a maximum tolerated dose (MTD)-Phase I

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Bone marrow smears were prepared 24 and 48 h after dosing for the vehicle control and treated animals and 24 h after dosing for the cyclophosphamide treated animals.

DETAILS OF SLIDE PREPARATION: The preparations were stained with polychrome methylene blue and eosin to visualise the various cell types.

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:
Number of polychromatic erythrocytes (PCE) per slide: 1000 PCE
Percentage of polychromatic erythrocytes in the total erythrocyte population: 1000 Erythrocytes

Evaluation criteria:
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
Statistics:
- The incidence of micronucleated PCE and percentage PCE in the erythrocyte sample, were considered by ANOVA.
- All analyses were carried out using the GLM procedure in SAS.
- One-sided Student's t-test:
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
Under the test conditions, test substance is not clastogenic in the mouse micronucleus assay.
Executive summary:

Disperse Blue 284 was tested in C57BL/6JfBL10/Alpk male and female mice at dose levels of 3130 and 5000 mg/kg, the higher dose level being the limit dose for this assay. Bone marrow samples were taken 24 hours after dosing at 3130 mg/kg and 24, 48 and 72 hours after dosing at 5000 mg/kg.

No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes, over vehicle control values, were seen at either dose level in the females at any of the sampling times investigated. A small but statistically significant increase in the incidence of micronucleated polychromatic erythrocytes was noted in male mice 24 hours after being dosed at 5000 mg/kg. Extended analysis of a further 2000 polychromatic erythrocytes from all males at the 24 hour sampling time was conducted. No statistically or biologically significant increases were observed in the extended analyses or when the original andextended analyses were combined prior to statistical analysis. The original increase observed in the males is therefore considered not to be of any biological significance.

The test system positive control, cyclophosphamide, induced statistically significant and biologically meaningful increases in micronucleated polychromatic erythrocytes, compared to the vehicle control values, thus demonstrating the sensitivity of the test system to a known clastogen.

It is therefore concluded Disperse Blue 284, under the conditions of test, is not clastogenic in the mouse micronucleus test.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
GLP compliance:
yes
Type of assay:
unscheduled DNA synthesis
Species:
rat
Strain:
Fischer 344
Details on species / strain selection:
Fischer 344 (F344) rats were used because of the UDS data available for this strain within CTL and other laboratories.
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, Kent
- Age at study initiation: 6-8 weeks
- Weight at study initiation: 189-236 g
- Assigned to test groups randomly: the animals were allocated according to the order in which they were removed from the stock cage
- Housing: 5/cage
- Diet (e.g. ad libitum): Porton Combined Diet , ad libitum
- Water (e.g. ad libitum): Filtered tap water, ad libitum
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 40-60
- 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: Forms good suspension
- Concentration of test material in vehicle: 125 and 200 mg/mL
- Lot/batch no. (if required): CTL reference number Y00790/004


Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Dosing suspensions of the test substance were preparedas a suspension in corn oil by homogenisation. Concentrations prepared for the UDS assay were 125 and 200 mg/mL
All dosing preparations were administered at a volume of 10 mL/kg bw.


Duration of treatment / exposure:
Preparation of hepatocytes took place 2 or 16 hours after dosage. Actual achieved exposure times were in the range of lh 55min to 2h 30min for the 2 hour time point, and 15h 55min to 16h 45min for the 16 hour treatment. In experiment 2, 2-AAF-treated rats were sampled 4h after dosage.

Frequency of treatment:
Single dose

Post exposure period:
2 and 16 h after treatment

Dose / conc.:
1 250 mg/kg bw (total dose)
Dose / conc.:
2 000 mg/kg bw (total dose)
No. of animals per sex per dose:
preliminary study: 5 (treatment group only)
5 males/dose in treatment group
2 males/group in vehicle and positive control group

Control animals:
yes, concurrent vehicle
Positive control(s):
Positive control substances were 2-acetylaminofluorene (2-AAF, CTL reference number Y00892/050) in experiments 1, 2 and 3 and N-nitrosodimethylamine (NDMA, CTL referencenumber Y01468/004) in experiment 4
- Route of administration: oral, gavage
- Doses / concentrations: AAF: 25 mg/kg bw / 2.5 mg/mL suspension in 0.5% w/v hydroxypropylmethylcellulose in 0.1% w/v PS80 (HPMC).
NDMA: 10 mg/kg bw / 1 mg/mL dissolved in deionised water
Tissues and cell types examined:
Hepatocytes

Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Based on a preliminary study, in which the acute MTD for the test substance was >2000 mg/kg

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 2 or 16 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:
- 30 morphologically normal cells/slide and a total of 60 cells/animal.
- 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 (AMS 40-10) 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:
Criteria for a positive response:
- A mean animal net nuclear grain count [N-C] value of greater than zero represents a biologically significant departure from normal.
- The radiolabelling of the nucleus exceeds that of the cytoplasm, indicating of a real net synthesis of nuclear DNA
Criteria for a negative response:
- The mean net nuclear grain count of all test substance treated animals is less than 0.
Statistics:
No data
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
blue discolouration of the internal organs served as proof for exposure
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: >2000 mg/kg in acute oral study

RESULTS OF DEFINITIVE STUDY
- Animal Toxicity:Over the period of the main UDS experiments (2 or 16 hours post-treatment), no signs of acute toxic effects were observed in treated animals. Although applied at a limit test dose in the main UDS experiments, the observation of blue colouration of the internal organs of treated rats showed that absorbtion and distribution of the test substance or its metabolites had taken place.
- Cytotoxicity: Hepatocytes prepared from animals dosed with the test substance were examined microscopically. No apparent signs of excessive cytotoxicity were observed ie: few cells present, or a high proportion of cells of abnormal morphology or large numbers of pyknotic cells compared with the solvent controls.
- Appropriateness of dose levels and route: the blue colouration of the internal organs of treated rats showed that absorbtion and distribution of the test substance
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:

 

The test substance was tested for the ability to induce unscheduled DNA synthesis (UDS) in an in vivo rat hepatocyte assay incorporating an autoradiographic technique. Male Fischer 344 rats were given a single oral dose by gavage at 1250 or 2000 mg/kg body weight. The highest test treatment, 2000 mg/kg, was the limit dose for this assay. Two sampling times were used, 2 hours and 16 hours following administration of the chemical. Two independent experiments were carried out at each time point, validated by concurrent control treatments of rats with corn oil, the solvent for the test substance, and with the carcinogens 2-acetylaminofluorene (2-AAF) or N-nitrosodimethylamine (NDMA).

Hepatocytes from treated rats were assessed for the induction of UDS at both dose levels, 1250 and 2000 mg/kg. Examination of the mean net nuclear grain count and percentage of cells in repair showed that the test substance did not induce DNA repair, as measured by UDS, at either dose level or time point.

It is concluded that, when tested up to a limit dose of 2000 mg/kg, the test substance did not induce DNA repair in the liver of the rat in vivo.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

The test itemDisperse Blue 284 was tested positivein a screening Bacteria Reverse Mutation Assay (Ames test) in Salmonella strain TA98, but negative in an in-vivo micronucleus test in mice. A close structural analogue was also tested positive in an Ames test, but negative in an in-vitro cytogenicity test human lymphocytes, an in-vivo Unscheduled DNA Synthesis (UDS) assay in rat hepatocytes and an in-vivo micronucleus test in mice.

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

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

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

Study Type

Metabolic activation

Result

Mitotic recombination assay withSaccharomyces cerevisae

Rat liver S9-mix

Negative

Point mutation assay withSaccharomyces cerevisae

Rat liver S9-mix

Negative

HPRT assay with V79 hamster cells

Rat liver S9-mix

Negative

UDS assay with rat hepatocytes

Metabolic competent hepatocytes were used

Negative

Micronucleus assay in vivo (mouse)

In vivo assay

Negative

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

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

The positive effect in the bacterial reverse mutation test (Ames) was clearly related to a bacteria-specific metabolism of the test substance, as it is well-known for aromatic nitro compounds to be positive in the Ames assay resulting from metabolism by the bacteria-specific enzyme nitro-reductase [Tweats et al. 2012]. This could be also be proved to be true in studies with Disperse Blue 291, which was tested for mutagenic activity in the Salmonella assay with strains with different levels of nitroreductase and O-acetyltransferase[Umbuzeiro et al. 2005]. In this study,Disperse Blue 291 showed mutagenic activity with all standard strains of Salmonella typhimurium tested (TA1537, TA1538, TA98 and TA100), except for TA1535.In nitroreductase and O-acetyltransferasenegative strains (TA98NR, TA98DNP6) not mutagenic activity was observed in the absence of S9, whereas themutagenic activity was increased with the nitroreductaseand/or O‑acetyltransferaseoverproducing strains, (YG1021, YG1024 and YG1041) This shows the importance of the bacterial acetyltransferase enzyme in the activation of Disperse Blue 291. Because of the remarkable increase in the response with the nitroreductase and O‑acetyltransferase overproducing strain (YG1041), it is assumed that the product of the nitroreductaseis a substrate for the O-acetyltransferase. As there was a very slight increase in mutagenicity with TA98NR, TA98, YG1021, TA98DNP6, and YG1024 in the presence of S9, it was assumed that P450 enzymes have also a role in the activation of Disperse Blue 291, besides the bacterial enzymes. This could however not proven true in in-vivo studies with Disperse Blue 291 and the structural analogue (UDS) or Disperse Blue 284 and the structural analogue (MNT).

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

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

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

Based on these data and the common mechanism between the reduction of these nitro-compounds, which is widely explored in literature [de Oliveira et al. 2010], it is concluded, that the mutagenic effects observed in the Ames test with Disperse Blue 284 is a bacteria specific effect and not relevant to mammalians.

Disperse Blue 284 and its structural analogue were not genotoxic in the in-vivo UDS and MNT test. Therefore, a direct genotoxic effect as well as a metabolisation towards genotoxic structures by mammalian species can be excluded.

References

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

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

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

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

Additional information

Bacteria reverse mutation

The mutagenic activity of Disperse Blue 284 (82.4%purity),was investigated in a screening assay in the plate incorporation test using the Salmonella typhimurium strain TA 98. The test article was tested at the concentrations up to 1000 µg/plate up to 1000 µg/plate. The test substance was toxic at concentrations of 1000 µg/plate and above.

Reproducible, dose-dependent increases in revertant colony numbers were obtained with and without S9 mix. 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 frameshifts in the genome in the tested strain. As the screening test was positive, no further experiment was conducted.

MNT in-vivo

The ability of the test item to induce cytogenetic damage and/or disruption of the mitotic apparatus in mouse bone marrow was investigated measuring the induction of micronuclei in polychromatic erythrocytes. Disperse Blue 284 (82.4% purity), was tested in C57BL/6JfBL10/Alpk male and female mice at dose levels of 3130 and 5000 mg/kg body weight, the higher dose level being the limit dose for this assay. Bone marrow samples were taken 24 hours after dosing at 3130 mg/kg body weight and 24, 48 and 72 hours after dosing at 5000 mg/kg body weight. No statistically or biologically significant increases in the incidence of micronucleated polychromatic erythrocytes, over vehicle control values, were seen at either dose level in the females at any of the sampling times investigated. A small but statistically significant increase in the incidence of micronucleated polychromatic erythrocytes was noted in male mice 24 hours after being dosed at 5000 mg/kg body weight. Extended analysis of a further 2000 polychromatic erythrocytes from all males at the 24 hour sampling time was conducted. No statistically or biologically significant increases were observed in the extended analyses or when the original and extended analyses were combined prior to statistical analysis. The original increase observed in the males is therefore considered not to be of any biological significance. The test system positive control, cyclophosphamide, induced statistically significant and biologically meaningful increases in micronucleated polychromatic erythrocytes, compared to the vehicle control values, thus demonstrating the sensitivity of the test system to a known clastogen. It is therefore concluded Disperse Blue 284, under the conditions of test, is not clastogenic in the mouse micronucleus test.

Unscheduled DNA Synthesis

The structural analogue 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 the test substance by gavage at 1250, or 2000 mg/kg body weight. The highest test dose, 2000 mg/kg was the limit test dose for a non-toxic test agent in this assay. Animals were killed and hepatocytes prepared two hours and sixteen hours following administration of the chemical. Two independent experiments were carried out for each time point. Hepatocytes from treated rats were exposed to [4]-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 the test substance and with the carcinogens 2-acetylaminofluorene [2AAF] or N‑nitrosodimethylamine [NDMA]. 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 test animals were responsive to known carcinogens requiring metabolic activation for genotoxic activity. Hepatocytes from test substance treated animals were assessed for UDS at both dose levels. Treatments with the test substance 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 substance did not induce DNA repair (as measured by unscheduled DNA Synthesis) in rat liver.

Justification for classification or non-classification

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