[No public or meaningful name is available]

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experiment start date - 29 July 2010; Experiment completion date - 25 August 2010; Study completion date - 29 September 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): FAT 40851/A TE
- Physical state: orange powder
- Analytical purity: 69.87% (all coloured components) (Dose calculation was not adjusted to purity.)
- Purity test date: not reported
- Lot/batch No.: TZ 5891 / BOP 02-09
- Expiration date of the lot/batch: July 31, 2014
- Stability under test conditions: not reported
- Storage condition of test material: at room temperature

Specific details on test material used for the study:

Identification: FAT 40851/A TE
Batch Number: TZ 5891 / BOP 02-09
Purity: 69.9 % all coloured components
Appearance: Orange powder
Expiry Date: July 31, 2014
Storage Conditions: At room temperature at about 20 °C

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 = 36.5 g (SD ± 1.7 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: 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 ± 3
- Humidity (%): 45 - 100
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water
- Source: B. Braun Melsungen AG 34212 Melsungen, Germany
- Catalogue no.: 6724092.00.00
- 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

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was formulated in in the vehicle (sterile water).

Duration of treatment / exposure:

single oral gavage

Frequency of treatment:

single oral gavage

Post exposure period:

no

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Vehicle control - 24 hr exposure

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

Low dose group - 24 hr exposure

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Mid dose group - 24 hr exposure

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

High dose group - - 24 and 48 hr exposure

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):

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 oral 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:

Non-parametric Mann-Whitney test

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

The animals treated with 2000 mg/kg b.w. did not express any toxic reactions. However, discoloured urine was observed (orange) in animals treated with 2000 mg/kg bw both in pre-test and main experiment.

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw
- Solubility: no problems with solubility reported
- Clinical signs of toxicity in test animals:
- no adverse effects
- orange discoloration of the urine, indicating bioavailability of the test item which is a orange 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:
- orange discoloration of the urine in the highest dose group, indicating bioavailability of the test item which is a orange dye
- testing up to the limit dose

Any other information on results incl. tables

- Table 2: Summary of Micronucleus Test Results

Test group	Dose mg/kg bw	Sampling time (h)	PCEs with micronuclei (%)	Range	PCE per 2000 erythocytes
vehicle	0	24	0.114	1 - 4	1275
test item	500	24	0.064	1 - 2	1240
test item	1000	24	0.100	0 - 5	1211
test item	2000	24	0.086	0 - 6	1166
positive control	40	24	2.736	39 -75	1047
test item	2000	48	0.107	1 - 3	1215

 

- 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 40851/A/kg b.w.; 24 h	-	n.t.
1000 mg FAT 40851/A/kg b.w.; 24 h	-	n.t.
2000 mg FAT 40851/A/kg b.w.; 24 h	-	n.t.
40 mg CPA/kg b.w.; 24 h	+	0.0003
2000 mg FAT 40851/A/kg b.w.; 48 h	-	n.t.

-         =         not significant

+         =         significant

n.t. = not tested

 

- Table 4: Historical controls from 2003 - 2009

	Negative Controls Males	Positive Controls (CPA) Males
Mean* ± SD	0.096 ± 0.040	2.332 ± 0.696
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:

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 40851/A 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 in a GLP-certified laboratory. The test item was formulated in sterile water, which was also used as vehicle control. The volume administered orally was 10 mL/kg bw. 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 a pre-experiment 2000 mg FAT 40851/A TE per kg bw (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 40851/A did not have any cytotoxic properties in the bone marrow. However, the animals showed discoloured urine after treatment with 2000 mg/kg b.w. 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 40851/A were near to the value of the vehicle control group. Additionally all values were within the historical vehicle control database. 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.