Registration Dossier

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted in accordance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid, melt
Details on test material:
Please refer to the section "confidential details on test material" below.

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: 8 - 11 weeks
- Weight at study initiation: male mean value 36.0 g (SD +/- 1.7 g), female mean value 28.6 g (SD +/- 1.4 g)
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: single
Cage Type: Makrolon Type II/III, with wire mesh top (EHRET GmbH, 79302 Emmendingen, Germany)
Bedding: 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: tap water, ad libitum (Gemeindewerke, 64380 Rossdorf, Germany)
- Acclimation period: minimum of five days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 2 °C
- Humidity: 45 - 65 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6.00 p.m.

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: corn oil
- Justification for choice of vehicle: relative non-toxicity for the animals
- Concentration of test material in vehicle:
Main experiment:
# Males: 187.5, 375 and 750 mg/kg b.w. as low, mid and high dose
# Females: 100, 200 and 400 mg/kg b.w. as low, mid and high dose
- Amount of vehicle (if gavage or dermal): 10 mL/kg b.w.
- Lot/batch no.: MKBF8603V (Catalogue no.: C8267; Sigma-Aldrich Vertriebs GmbH, 82041 Deisenhofen, Germany)
Frequency of treatment:
Vehicle control, positive control and dose groups were treated once.
Post exposure period:
Samples of bone marrow were taken 24 hours after the treatment and for the highest dose level an additional sample was taken at 48 h after treatment.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
Males: 187.5, 375 and 750 mg/kg b.w. as low, mid and high dose
Basis:
actual ingested
based on the pre-experiment
Remarks:
Doses / Concentrations:
# Females: 100, 200 and 400 mg/kg b.w. as low, mid and high dose
Basis:
actual ingested
based on the pre-experiment
No. of animals per sex per dose:
six
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Justification for choice of positive control: standard according to OECD TG 474
- Route of administration: orally
- Doses / concentrations: 40 mg/kg b.w.; dissolved in sterile water; volume administered: 10 mL/kg b.w



Examinations

Tissues and cell types examined:
Bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The highest dose level represents in each gender the maximum tolerated dose as determined in the pre-experiment.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
Three adequately spaced dose levels spaced by a factor of 2 were administered, and samples were collected at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.

DETAILS OF SLIDE PREPARATION:
The femora of sacrificed animals (using CO2 followed by bleeding) were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1500 rpm (390x g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (Merck, 64293 Darmstadt, Germany) / Giemsa (Merck, 64293 Darmstadt, Germany). Cover slips were mounted with EUKITT (Kindler, 79110 Freiburg, Germany). At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Slides were evaluated using microscopes with 100x oil immersion objectives. Per animal (all animals per sex and test group) 2000 polychromatic erythrocytes (PCE) were analysed for micronuclei. To investigate a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.
Evaluation criteria:
A test item is classified as mutagenic if it 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) are used as an aid in evaluating the results, if necessary. However, the primary point of consideration is the biological relevance of the results.
A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.
Statistics:
See evaluation criteria above.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
other: vehicle control serves as negative control
Positive controls validity:
valid
Additional information on results:
On the basis of the pre-experiments 750 mg/kg b.w. for the males and 400 mg/kg b.w. for the females were estimated to be suitable as highest dose levels. Gender specific differences in toxicity were observed. In accordance with the guidelines both genders were used in the main experiment.


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: no - test item, positive control induced the appropriate response
- Ratio of PCE/NCE:
- Appropriateness of dose levels and route: yes, triggered by observed gender specific differences in toxicity
- Statistical evaluation: yes, non-parametric Mann-Whitney test
- All animals treated with the vehicle control (corn oil) did not express any clinical signs of toxicity.

Any other information on results incl. tables

Table 1: Rates of micronuclei (Males)

Test group

Dose
(mg/kg b.w.)

Sampling time (h)

PCEs with micronuclei
(%)

Range

PCE per 2000 erythrocytes

Vehicle control

          0

24

0.075

    1 -3

1292

Test item

   187.5

24

0.100

    0 -4

1294

Test item

      375

24

0.058

    0 -2

1307

Test item

      750

24

0.092

    0 -4

1276

Positive control

        40

24

2.075

  29 -54

1255

Test item

      750

48

0.142

    0 -8

1345

 

Table 2: Rates of micronuclei (Females)

Test group

Dose
(mg/kg b.w.)

Sampling time (h)

PCEs with micronuclei
(%)

Range

PCE per 2000 erythrocytes

Vehicle control

          0

24

0.108

    1 - 4

1295

Test item

      100

24

0.083

    0 - 3

1371

Test item

      200

24

0.175

    1 - 7

1381

Test item

      400

24

0.092

    0 - 4

1265

Positive control

        40

24

1.733

  18 -52

1260

Test item

      400

48

0.092

    1 - 3

1400

 

Applicant's summary and conclusion