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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:
22 August 2006 to 20 October 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Directive 2000/32/EC, Annex 4C
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: in vivo mouse micronucleus

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethyl 4-dimethylaminobenzoate
EC Number:
233-634-3
EC Name:
Ethyl 4-dimethylaminobenzoate
Cas Number:
10287-53-3
Molecular formula:
C11H15NO2
IUPAC Name:
ethyl 4-(dimethylamino)benzoate
Test material form:
solid
Details on test material:
- Appearance: White solid
- Storage conditions of test material: Room temperature

Test animals

Species:
mouse
Strain:
NMRI
Details on species / strain selection:
The mouse has been used for many years as suitable experimental animal in cytogenetic investigations. There are many data available from such investigations which may be helpful in the interpretation of results from the micronucleus test. In addition, the mouse is an experimental animal in many physiological, pharmacological and toxicological studies.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 8 to 10 weeks prior to acclimatisation
- Mean weight at study initiation: Females: 28.6 ± 1.8 g, Males: 35.1 ± 1.8 g
- Assigned to test groups randomly: Yes
- Fasting period before study: No
- Housing: Singly housed in wire mesh top cages with granulated soft wood bedding
- Diet: Pelleted standard diet ad libitum
- Water: Tap water ad libitum
- Acclimation period: Minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 to 90 % (relative)
- Photoperiod: Artificial light from 6.00 am to 6.00 pm

IN-LIFE DATES: Not reported

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: 30 % DMSO, 70 % PEG 400
- Justification for choice of solvent/vehicle: The vehicle was chosen for its relative non-toxicity for the animals.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: On the day of the experiment the test material was formulated in the vehicle

- Dose volume: 10 mL/kg bodyweight
Duration of treatment / exposure:
24 h and 48 h after a single administration of the test material the bone marrow cells were collected for micronuclei analysis.
Frequency of treatment:
A single dose administration
Post exposure period:
Groups were sacrificed after 24 hours (500, 1000 and 2000 mg/kg bodyweight) or 48 hours (2000 mg/kg bodyweight only).
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
6 males and 6 females were assigned per group; 5 of each sex were evaluated for micronuclei
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Route of administration: Oral
- Doses / concentrations: 40 mg/kg bodyweight at a dose volume of 10 mL/kg bodyweight
- Stability: The stability of Cyclophosphamide at room temperature is sufficient. At 25 °C only 3.5 % of its potency is lost after 24 hours. The solution was prepared on the day of administration.

Examinations

Tissues and cell types examined:
Slides were prepared from the bone marrow of the femora and stained. The numbers of micronucleated polychromatic erythrocytes and normochromatic erythrocytes were recorded.
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 with respect to animal strain, vehicle, route, frequency and volume of administration. The animals were treated orally with the test material at doses of 100, 500, 1000 and 2000 mg/kg bw and examined for acute toxic symptoms at intervals of around 1, 2-4, 6, 24, 30 and 48 h after administration of the test material.
It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test materials. The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours. The volume to be administered should be compatible with physiological space available. Three adequately spaced dose levels spaced by a factor of 2 were applied at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment. On the basis of the data from the preliminary study, 2000 mg/kg bodyweight was estimated to be suitable.

TREATMENT AND SAMPLING TIMES: A single dose was administered with sacrifice after 24 (500, 1000 and 2000 mg/kg bodyweight) or 48 hours (2000 mg/kg bodyweight only).

DETAILS OF SLIDE PREPARATION: The animals were sacrificed using CO2 following by bleeding. The femora 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 (390 x 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/Giemsa. Cover slips were mounted with EUKITT . At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS: Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe 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. Ten animals (5 males, 5 females) per test group were evaluated as described. The remaining 6th animal of each sex in the respective test group test group is usually evaluated in case an animal dies in its test group spontaneously.
Evaluation criteria:
ACCEPTANCE CRITERIA
The study is considered valid if the following criteria are met:
- The negative controls are in the range of historical control data.
- The positive controls are in the range of historical control data.
- At least 4 animals per group and sex can be evaluated.
- PCE to erythrocyte ratio should not be less than 20 % of the negative control.

EVALUATION OF RESULTS
A test material 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.
A test material that fails to produce a biologically relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.
Statistics:
The nonparametric Mann-Whitney test was used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
minor clinical signs observed at 2000 mg/kg bw; no cytotoxicity
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
PRE-EXPERIMENT FOR TOXICITY
- Clinical signs of toxicity in test animals: Reduction of spontaneous activity and ruffled fur was observed in some or all animals at all concentrations but no symptoms were seen after 48 hours. Eyelid closure was observed at 2000 mg/kg bodyweight in two animals but this was not seen after 24 hours.
- Rationale for exposure: On the basis of the results from the pre-experiment for toxicity, 2000 mg/kg bodyweight was selected as the highest dose for the main study.

RESULTS OF DEFINITIVE STUDY
A summary of the micronucleus test results can be seen in Table 1. The mean number of polychromatic erythrocytes was not decreased after treatment with the test material as compared to the mean value of PCEs of the vehicle control indicating that the test material did not have any cytotoxic properties in the bone marrow.
Animals treated with the low (500 mg/kg) and mid (1000 mg/kg) dose levels of test material did not express any toxic reactions. Animals treated at the high dose level (2000 mg/kg) showed some minor signs. Two to 4 hours after treatment with the test material, 5 males and 6 females exhibited a reduction of spontaneous activity and ruffled fur. Six hours after treatment, the reduction of spontaneous activity was still evident in 3 males and 4 females. Ruffled fur was recorded in three animals of each sex. By the 24 h observation, all animals appeared normal.
40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

DISCUSSION
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 material. The mean values of micronuclei observed after treatment with the test material were below or near to the value of the vehicle control group. The mean frequency of micronucleated PCEs in the low dose group (0.180 %) is slightly above the upper historical control range (0.16 %). The number of micronucleated PCEs per 2000 PCEs (1-8 microncuelated PCEs per 2000 PCEs) of the individual animals within this group, however, does not show any relevant increase and is within the historical range (up to 8 per 2000 PCEs). This increase is not observed at higher doses and the observed increase is also not statistically significant. Therefore, it is concluded that the observed increase in the low dose group is biologically irrelevant.

Any other information on results incl. tables

Table 1: Summary of Micronucleus Test Results

Test group

Dose mg/kg b.w

Sampling time (h)

PCEs with micronuclei (%)

Range

PCE per 2000 erythocytes

Vehicle

0

24

0.150

0-7

1199

Test material

500

24

0.180

1-8

1215

Test material

1000

24

0.130

0-5

1212

Test material

2000

24

0.145

1-6

1212

Positive control

40

24

2.765

17-138

1109

Test material

2000

48

0.105

1-4

1222

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study, the test material is considered to be non-mutagenic.
Executive summary:

The potential of the test material to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse was investigated in accordance with the standardised guidelines OECD 474 and Commission Directive 2000/32/EC Annex 4C under GLP conditions.

The test material was formulated in 30 % DMSO/ 70 % PEG 400, which was also used as vehicle control. The volume administered orally was 10 mL/kg bodyweight. 24 and 48 hours after a single administration of the test material the bone marrow cells were collected for micronuclei analysis.

Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test material 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 material were investigated: 24 hour preparation interval: 500, 1000 and 2000 mg/kg bodyweight; 48 hour preparation interval: 2000 mg/kg bodyweight. The highest dose (2000 mg/kg, maximum guideline-recommended dose) was estimated by a pre-experiment to be suitable.

After treatment with the test material the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that the test material did not exert any cytotoxic effects in the bone marrow.

In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test material and with any dose level used. 40 mg/kg bodyweight cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency.

The test material did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse, therefore under the conditions of this study the test material is considered to be non-mutagenic.

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