Magnesium octadecylbenzenesulphonate

Genetic toxicity: in vivo

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Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11-Jun-2014 to 08-Oct-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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

Test material form:

solid: crystalline

Details on test material:

Batch No.: E00873-025-001
Physical Description: Black solid
Storage Conditions: Kept in a controlled room temperature area

Test animals

Species:

rat

Strain:

Wistar

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: 144 - 169 gram
- 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 (e.g. ad libitum): free access
- Water (e.g. ad libitum): free access
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 - 23.0°C
- Humidity (%): 45 - 81%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Propylene glycol
- Justification for choice of solvent/vehicle: Test compound was stable in Propylene glycol (Stability for at least 6 hours at room temperature is confirmed over the concentration range 20 to 200 mg/mL)and a suspension could be obtained in Propylene glycol. Propylene glycol has been accepted and approved by authorities and international guidelines. EXP1313100 concentrations were treated with ultra-sonic waves to obtain a homogeneous suspension


- Concentration of test material in vehicle: 100, 200 and 400 mg/ml
- Amount of vehicle (if gavage or dermal): The dosing volume was 5 ml/kg body weight

Details on exposure:

Chemical analysis of dose preparations
The concentrations analysed in the formulations of the dose levels groups 500, 1000 and 2000 mg/kg BW were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%).

A small response at the retention time of the test substance was observed in the chromatograms of the Group A formulation. It was considered not to derive from the formulation since a small response was obtained in the analytical blanks.

The formulations of the dose levels groups 500 and 2000 mg/kg BW were homogeneous (i.e. coefficient of variation = 10%).

Formulations at the entire range were stable at room temperature under normal laboratory light conditions for at least 4 hours

Duration of treatment / exposure:

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

Frequency of treatment:

Once

Doses / concentrations

Remarks:

Doses / Concentrations:
500, 1000 and 2000 mg/kg/bw
Basis:
analytical conc.

No. of animals per sex per dose:

Five animals per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s):
- Route of administration: Oral
- Doses / concentrations: 20 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: No treatment related clinical signs of toxicity or mortality.


RESULTS OF DEFINITIVE STUDY

- Dose range: 500, 1000 and 2000 mg/kg BW
- Clinical signs of toxicity in test animals: No treatment related clinical signs of toxicity or mortality
- 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 EXP1313100.
- 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.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
It is concluded that EXP1313100 is not clastogenic or aneugenic in the bone marrow micronucleus test when sampled at 24 and 48 hours post dosing of male rats up to a dose of 2000 mg/kg the recommended dose in accordance with current regulatory guidelines under the experimental conditions described in the report.

Executive summary:

No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with EXP1313100 compared to the vehicle treated animals.

 

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent 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 EXP1313100 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. The group that was treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle control, demonstrating toxic effects on erythropoiesis.