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: GLP guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 8 - 11 weeks
- Weight at study initiation: mean value males: mean value 34.7 g (SD +- 2.0 g); females: mean value 28.4 g (SD +- 1.6 g)
- Assigned to test groups randomly: [yes]
- Housing: single
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: for a minimum of five days after their arrival

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 2 °C
- Humidity (%): 30 - 65 %
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil]
- Justification for choice of solvent/vehicle: The vehicle was chosen due to its relative non-toxicity for the animals.
The administered volume was 10 mL/kg b.w. including test substance.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test item was dissolved in corn oil. All animals received a single standard volume orally.
Duration of treatment / exposure:
single oral application
Frequency of treatment:
once
Post exposure period:
24 or 48 hours
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
500, 1000, 2000 mg/kg bw in males
Basis:
actual ingested
Remarks:
Doses / Concentrations:
375, 750, 1500 mg/kg bw in females
Basis:
actual ingested
No. of animals per sex per dose:
6 mice/sex at low and mid dose, 12 mice/sex at the high dose
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s):
- Route of administration potent inducer of micronuclei
- Doses / concentrations: 40 mg/kg bw.

Examinations

Tissues and cell types examined:
Per animal 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.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A preliminary study on acute toxicity was performed with two animals per sex under identical conditions as in the mutagenicity study concerning: animal strain, vehicle, route, frequency, and volume of administration.
The animals were treated orally with the test item and examined for acute toxic symptoms at intervals of approximately 1 h, 2-4 h, 6 h, 24 h, 30 h, and 48 h after administration of the test item.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
At the beginning of the treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animal’s body weight. The animals received the test item, the vehicle, or the positive control substance once orally. Six males and six females were treated per dose group and sampling time. Five males and five females each were treated for each vehicle and the positive control group. The animals of all dose groups, except the positive control were examined for clinical signs at intervals of around 1 h, 2 - 4 h, 6 h, 24 h, and/or 48 h after administration of the test item and vehicles.
Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.
Evaluation criteria:
The study was considered valid as the following criteria are met:
- at least 5 animals per test group can be evaluated.
- PCE to erythrocyte ratio should not be less than 20 % of the vehicle control.
- the positive control shows a statistically significant and biological relevant increase of micronucleated PCEs compared to the vehicle control.

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 (8)) 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:
Statistical methods (nonparametric Mann-Whitney test are used as an aid in evaluating the results, if necessary.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
Two animals of each sex treated in the pre-experiments received the test item 2-Butenoic acid, 4-oxo-4-(tridecylamino)-, (Z)-, branched dissolved in corn oil once orally. The volume administered was 10 mL/kg b.w.. A correction factor of 1.09 was applied. On the basis of these data 2000 mg/kg b.w. for the males and 1500 mg/kg b.w. for the females were estimated to be suitable as highest dose level.
Gender specific differences in toxicity were observed. In accordance with the test guidelines both sexes of animals were used in the main experiment.


RESULTS OF DEFINITIVE STUDY
In the main experiment for the highest dose group 12 males and 12 females received once orally administrations of 2-Butenoic acid, 4-oxo-4-(tridecylamino)-, (Z)-, branched dissolved in corn oil. The volume administered was 10 mL/kg b.w.. A correcton factor of 1.09 was applied. The symptoms of toxicity observed following treatment (affected males/females) are shown below for each dose group. The animals treated with the negative control (corn oil) did not express any toxic reaction.
High dose: Reduction of spontaneous activity (1 male, 1 female), Eyelid closure (3 males, 4 females), Ruffled fur (8 males, 9 females), Tumbling (1 male, 1 female), Tremor (1 female), Diarrhoea (11 males, 8 females), death (1 male)
Mid dose: Eylid closure (2 females)
Low dose: No clinical signs of toxicity were observed.

Any other information on results incl. tables

The test item 2-Butenoic acid, 4-oxo-4-(tridecylamino)-, (Z)-, branched was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test item was dissolved in corn oil, 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. A correction factor of 1.09 was applied.

Six males and six females per test group (except the vehicle and positive control groups with 5 males and 5 females each) were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei.

To investigate 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:

Males: 500, 1000 and 2000 mg/kg b.w. at the 24 h preparation interval
and 2000 mg/kg b.w. at the 48 h preparation interval,

Females: 375, 750 and 1500 mg/kg b.w. at the 24 h preparation interval
                and 1500 mg/kg b.w. at the 48 h preparation interval.

The highest dose levels were estimated by pre-experiments to be suitable.

The animals treated with the test item showed clinical signs such as reduced spontaneous activity, eyelid closure, tumbling, tremor, diarrhoea and/or ruffled fur in the high dose group (2000 and 1500 mg/kg b.w.) and few female mice showed eyelid closure at 375 mg/kg b.w. in the main experiment. One male of the high dose group (animal no. 67) died 24 hours after treatment.After treatment with the test item at 48h preparation interval the number of PCEs per 2000 erythrocytes was not substantially decreased as compared to the mean value of PCEs per 2000 erythrocytes of the vehicle control thus indicating that 2-Butenoic acid, 4-oxo-4-(tridecylamino)-, (Z)-, branched did not induce cytotoxic effects in the bone marrow.

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 (except mid dose females, discussed below) after administration of the test item. The mean values of micronuclei observed after treatment with all doses of 2-Butenoic acid, 4-oxo-4-(tridecylamino)-, (Z)-, branched were below or near to the value of the vehicle control group.

The micronucleus frequency found in the mid dose group for the females was statistically significantly higher compared to the vehicle control. However, all values observed in the test item treated dose groups at any preparation interval were very well within the laboratory’s historical vehicle control data. Additionally no dose dependence was observed in any gender. Thus, the observed significance was not biologically relevant and considered to be incidental.


40 mg/kg b.w. 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, 2-Butenoic acid, 4-oxo-4-(tridecylamino)-, (Z)-, branched did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Applicant's summary and conclusion