2-[(8-amino-7-{[4-substituted-2-sulfonatophenyl]diazenyl}-1-hydroxy-3,6-disulfonaphthalen-2-yl)diazenyl]-4-[(4-chloro-6-{[3-(substituted)phenyl](ethyl)amino}-heteromonocycl-2-yl)amino]arylsulfonic acid, potassium and sodium salts

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-04-07 to 2010-07-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD guideline compliant GLP compliant

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

as at 1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

as at 2008

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test material (as cited in study report): FAT 40849/A TE
- Physical state: blue powder
- Analytical purity: 76.7 % all coloured components, (dose calculation not adjusted to purity)
- Purity test date: not reported
- Lot/batch No.: TZ 5463 / BOP 03-09
- Expiration date of the lot/batch: June 30, 2014
- Stability under test conditions: not reported
- Storage condition of test material: at room temperature

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: males mean value = 34.6 g (SD ± 3.0 g)
- Assigned to test groups randomly: not reported
- Fasting period before study: no
- Housing: singly in Makrolon Type II/III cages with wire mesh top (EHRET GmbH, 79302 Emmendingen, Germany) and granulated soft wood bedding
(Rettenmaier & Söhne GmbH + Co. KG,73494 Rosenberg, Germany)
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum(Harlan Laboratories B.V. Postbus 6174, 5960 AD Horst, The Netherlands)
- Water (e.g. ad libitum): tap water, ad libitum, (Gemeindewerke, 64380 Rossdorf, Germany)
- Acclimation period: >= 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 26 - 65
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: CMC (carboxymethyl cellulose) 1%
- Source: FLUKA Chemie AG, 9471 Buchs, Switzerland
- Catalogue no.: 21902
- Justification for choice of solvent/vehicle: chosen for its relative non-toxicity for animals
- Concentration of test material in vehicle: 50, 100, 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg
- Source: FLUKA Chemie AG, 9471 Buchs, Switzerland
- Catalogue no.: 21902

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in 1% carboxymethylcellulose (CMC) on the day of the experiment.

Duration of treatment / exposure:

single oral gavage

Frequency of treatment:

single oral gavage

Post exposure period:

no

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
main experiment: 500, 1000, 2000 mg/kg bw
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
toxicity pretest: 100, 1000, 2000 mg/kg bw
Basis:

No. of animals per sex per dose:

- main test: 7 males per dose (only males were used in the main study as the toxicity pretest showed comparable susceptibility of males and females)
- toxicity pre-test: 2 males and 2 females per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

none; no data; cyclophosphamide (CPA)
- Supplier: Fisher Scientific GmbH, 61130 Nidderau, Germany
- Justification for choice of positive control(s): recommended as positive control substance in the OECD guideline
- Route of administration: oral gavage
- Doses / concentrations: 40 mg/Kg bw (concentration in vehicle (water): 4 mg/mL)

Examinations

Tissues and cell types examined:

bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
2000 mg/kg bw is the limit dose according to the OECD guideline. Two lower doses with a spacing factor of 2 were selected.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
- single aoral treatment
- Sampling:
- pre-test: examination for acute toxic symptoms 1 h, 2-4 h, 6 h, 24 h, 30 h, and 48 h after administration
- main test: examination for acute toxic symptoms 1 h, 2-4 h, 6 h, 24 h (and 48 h) after administration; animals of all dose levels sacrificed at 24 h post dosing, an additional high dose group sacrificed 48 post treatment

DETAILS OF SLIDE PREPARATION:
- sacrifices of animals using CO2 followed by bleeding
- removal of the femora, cutting off of epiphyses, marrow flushed out with foetal calf serum using a syringe
- centrifugation of the cell suspension at 1500 rpm (390 x g) for 10 minutes
- discarding of supernatant, resuspesion of the remaining cell pellet and spreading of a small drop of the suspension on a slide
- air drying of the slide and staining with May-Grünwald (Merck, 64293 Darmstadt, Germany)/Giemsa (Merck, 64293 Darmstadt, Germany)
- mounting of cover slips with EUKITT (Kindler, 79110 Freiburg, Germany)
- preparation of at least one slide per each bone marrow sample

METHOD OF ANALYSIS:
- slide analysis using NIKON microscopes with 100x oil immersion objectives
- analysis for micronuclei of 2000 polychromatic erythrocytes (PCE) per animal
- determination of ratio between polychromatic and normochromatic erythrocytes in the same sample for detection of cytotoxicity
- cytotoxicity expressed in polychromatic erythrocytes per 2000 erythrocytes
- slides were coded
- use of samples from all animals per test group

OTHER:

Evaluation criteria:

The study was considered valid as the following criteria are met:
- at least 5 animals per group can be evaluated.
- PCE to erythrocyte ratio should not be less than 20 % of the negative control.
- the positive control shows a statistically significant and biological relevant increase of
micronucleated PCEs compared to the negative control.

Positive result:
If the test item induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. Statistical methods (nonparametric Mann-Whitney test) will be used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results.

Negative result
If the test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes it is considered non-mutagenic in this system.

Statistics:

see above under Evaluation criteria

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

(2 males of the lowest dose group in the pre-test showed ruffeled fur from 6 to 48 h post dosing; no other effects were found)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 100, 1000 and 2000 mg/kg bw
- Solubility: no problems with solubility reported
- Clinical signs of toxicity in test animals:
- ruffled fur in 2 males of the lowest dose group in the pre-test from 6 to 48 h post dosing; due to the absence of adversity and a dose relation effects are not regarded as treatment related
- blue discoloration of the urine in the two highest dose groups, indicating bioavailability of the test item which is a blue dye
- Evidence of cytotoxicity in tissue analyzed: no
- Rationale for exposure: 2000 mg/kg bw is the limit dose recommended in the OECD guideline.


RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): not applicable
- Induction of micronuclei (for Micronucleus assay):
- No micronuclei induced in any of the treatment groups
- Micronuclei induced in the positive control group at the expected rate, indicating the sensitivity of the test system
- for details see Tables 2 and 3
- Ratio of PCE/NCE (for Micronucleus assay):
- not significantly changed in any of the treatment groups
- for details see Tables 2 and 3
- Appropriateness of dose levels and route:
- blue discoloration of the urine in the two highest dose groups, indicating bioavailability of the test item which is a blue dye
- testing up to the limit dose
- Statistical evaluation: non-parametric Mann-Whitney test

Any other information on results incl. tables

- Table 2: Summary of Micronucleus Test Results

test group	Dose mg/kg b.w.	Sampling time (h)	PCEs with micronuclei (%)	range	PCE per 2000 erythocytes
vehicle	0	24	0.086	0 -4	1291
test item	500	24	0.136	2 - 4	1224
test item	1000	24	0.136	1 -4	1216
test item	2000	24	0.121	2 -4	1258
positive control	40	24	4.164	68 -117	1175
test item	2000	48	0.100	1 -3	1193

- Table 3: Statistical significance at the five per cent level (p < 0.05, evaluated by means of the non-parametric Mann-Whitney test).

Vehicle control versus test group	Significance	p
500 mg FAT 40849/A TE/kg b.w.; 24 h	-	0.1145
1000 mg FAT 40849/A TE/kg b.w.; 24 h	-	0.1235
2000 mg FAT 40849/A TE/kg b.w.; 24 h	-	0.2063
40 mg CPA/kg b.w.; 24 h	+	0.0003
2000 mg FAT 40849/A TE/kg b.w.; 48 h	-	0.4199

-         =         not significant

+         =         significant

- Table 4: Historical controls from 2003 - 2009

	Negative Controls Males	Positive Controls (CPA) Males
Mean* ± SD	0.10 ± 0.04	2.33 ± 0.70
Range**	0.01 - 0.22	0.70 - 4.52
No. of Experiments	318	316

*:        mean value (percent micronucleated cells)

**:      range of the mean group values (percent micronucleated cells)

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The test item FAT 40849/A TE was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse according to OECD TG 474 (single oral doses of 500, 1000 and 2000 mg/kg bw).
No micronuclei wereinduced in any of the treatment groups while micronuclei were induced in the positive control group at the expected rate, indicating the sensitivity of the test system. Blue discoloration of the urine in the two highest dose groups, indicating bioavailability of the test item which is a blue dye.
In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Executive summary:

The test item FAT 40849/A TE was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse according to OECD TG 474.

The test item was formulated in 1 % carboxymethylcellulose (CMC), which was also used as vehicle control. The volume administered orally was 10 mL/kg b.w.. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis.

Seven males per test group were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes. The following dose levels of the test item were investigated: 24 h preparation interval: 500, 1000, and 2000 mg/kg bw; 48 h preparation interval: 2000 mg/kg bw.

As estimated by pre-experiments 2000 mg FAT 40849/A TE per kg b.w. (the maximum guideline-recommended dose) was suitable.

The mean number of polychromatic erythrocytes was not decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that FAT 40849/A TE did not have any cytotoxic properties in the bone marrow. However, the animals showed discoloured urine after treatment with at least 1000 mg/kg bw indicating the bioavailability of the test item.

In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values

of micronuclei observed after treatment with FAT 40849/A TE were near to the value of the vehicle control group. Additionally the values of all test item treated animals were within the range of the historical vehicle control data.

40 mg/kg bw cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.