Reactive Orange 143

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:

23 February 2012 to 11 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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 40854/A TE
- Physical state: Solid
- Storage condition of test material: At room temperature in the dark

Specific details on test material used for the study:

Identification: FAT 40854/A TE
Description: Reddish-brown powder
Batch: TZ 5719 / BOP 02-11
Test substance storage: At room temperature in the dark
Stability under storage conditions: Stable
Expiry date: 01 April 2016
Stability in vehicle: Water: Stability for at least 6 hours at room temperature
Solubility in vehicle: Water: More than 80 g/L

Test animals

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

NMRI BR mice (SPF) were used as the test system. These mice are recommended by international guidelines (e.g. OECD, EC). Females were nulliparous and non-pregnant. The animals were provided by Charles River, Sulzfeld, Germany.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6 weeks
- Weight at study initiation: 31 – 40 g
- Assigned to test groups randomly: yes
- Fasting period before study: yes
- Housing: In groups of 5 animals per sex per cage in polycarbonate cages containing sterilised sawdust as bedding material. Paper bedding was provided as cage-enrichment
- Diet: free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water: free access to tap-water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.9 – 21.7
- Humidity (%): 44 - 67
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: physiological saline
- Justification for choice of solvent/vehicle: Test compound was stable in water and a solution could be obtained in physiological saline. Physiological saline has been accepted and approved by authorities and international guidelines
- Concentration of test material in vehicle: 43.8, 87.5, 150 and 175 mg/ml
- Amount of vehicle (if gavage or dermal): The dosing volume was 10 ml/kg body weight

Details on exposure:

The mice received an oral intubation of a maximum tolerated (high), an intermediate and a low dose of FAT 40854/A. The route of administration was selected taking into account the possible route of human exposure during manufacture, handling and use.

Duration of treatment / exposure:

Treatment:
Solvent, positive control, low and mid dose level: 24 hours
Highest dose level: 24 and 48 hours

Frequency of treatment:

Once

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw (total dose)

Remarks:

Vehicle

Doses / concentrations 2

Dose / conc.:

2 000 mg/kg bw (total dose)

Remarks:

Group B,C

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw (total dose)

Remarks:

Group D

Doses / concentrations 4

Dose / conc.:

500 mg/kg bw (total dose)

Remarks:

Group E

Doses / concentrations 5

Dose / conc.:

40 mg/kg bw (total dose)

Remarks:

Group F- Cyclophosphamide

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s):
- Route of administration: Oral
- Doses / concentrations: 40 mg/kg body weight

Examinations

Tissues and cell types examined:

Bone marrow smears

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
-The dose level selected should be ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg.

DETAILS OF SLIDE PREPARATION:
- The smears are air-dried, fixed in methanol and stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer, allowed to air-dry and vover-slipped using mounting medium.

METHOD OF ANALYSIS:
- The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes.

Evaluation criteria:

A test substance is considered positive in the micronucleus test if:
-It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test, one-sided, p <0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) and the number of micronucleated polychromatic erythrocytes in the animals are above the historical control data range.

A test substance is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant (Wilcoxon Rank Sum Test, one-sided, p <0.05) increase in the incidence of micronucleated polychromatic erythrocytes and the number of micronucleated polychromatic erythrocytes in the animals are within the historical control data range.

Statistics:

Wilcoxon Rank Sum Test, one-sided, p <0.05

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg BW
- Clinical signs of toxicity in test animals:
The animals showed no treatment related clinical signs or mortality after dosing.

RESULTS OF DEFINITIVE STUDY

- Clinical signs of toxicity in test animals:
The animals showed no treatment related clinical signs or mortality after dosing.
- Induction of micronuclei (for Micronucleus assay):
No biologically relevant increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with FAT 40854/A.
- Ratio of PCE/NCE (for Micronucleus assay):
No decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test substance on erythropoiesis.

Any other information on results incl. tables

Dose range finding study

The animals showed no treatment related clinical signs or mortality after dosing.

 

Micronucleus main test

Mortality and toxic signs

Before use, animal numbers 27 to 30 (group F) had fighting marks. The animals of the groups treated with 2000, 1000 and 500 mg FAT 40854/A /kg body weight and the animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality.

 

Micronucleated polychromatic erythrocytes

The mean number of micronucleated polychromatic erythrocytes scored in FAT 40854/A treated groups were compared with the corresponding vehicle control group. No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of FAT 40854/A treated animals compared to the vehicle treated animals. The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals were within the historical vehicle control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, the acceptability criteria of the test were met.

 

Ratio polychromatic to normochromatic erythrocytes

The animals of the groups, which were treated with FAT 40854/A and the positive control showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which indicated a lack of toxic effects of this test substance on the erythropoiesis.

Applicant's summary and conclusion

Conclusions:

FAT 40854/A is not clastogenic or aneugenic in the bone marrow micronucleus test in male mice up to a dose of 2000 mg/kg.

Executive summary:

A GLP-compliant micronucleus test in bone marrow cells of the mouse with FAT 40854/A was carried out according to OECD guideline 474 and EU method B.12 to evaluate its genotoxic effect in developing erythrocytes (polychromatic erythrocytes) in the bone marrow. In the dose range finding study 3 males and 3 females were dosed via oral gavage with 2000 mg FAT 40854/A per kg body weight. The animals showed no treatment related clinical signs or mortality after dosing. Since there were no substantial differences in toxicity between sexes only males were used in the main study. In the main study male animals were dosed via oral gavage with vehicle or with 2000, 1000 and 500 mg FAT 40854/A per kg body weight. A positive control group was dosed via oral gavage with 40 mg cyclophosphamide (CP) per kg body weight. In total 6 treatment groups were used, each consisting of 5 animals. No treatment related clinical signs or mortality were noted in any animal treated with FAT 40854/A or control animals receiving vehicle or cyclophosphamide. Bone marrow of the groups treated with FAT 40854/A was sampled 24 or 48 (highest dose only) hours after dosing. Bone marrow of the negative and positive control groups was harvested 24 and 48 hours after dosing, respectively. No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with FAT 40854/A. The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals were within the historical vehicle control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, both criteria for an acceptable assay were met. The groups that were treated with FAT 40854/A and the group treated with cyclophosphamide showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test substance on erythropoiesis. It is concluded that FAT 40854/A is not clastogenic or aneugenic in the bone marrow micronucleus test when sampled at 24 and 48 hours post dosing of male mice up to a dose of 2000 mg/kg under the experimental conditions.