Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007-08-15 to 2007-08-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

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, 19 May 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
3-methyl-5-phenylpent-2-enenitrile
EC Number:
299-682-2
EC Name:
3-methyl-5-phenylpent-2-enenitrile
Cas Number:
93893-89-1
Molecular formula:
C12 H13 N
IUPAC Name:
(2E)-3-methyl-5-phenylpent-2-enenitrile

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 8-10 weeks
- Weight at study initiation: males 37 g (mean), females 29.6 g (mean)
- Assigned to test groups randomly: yes
- Housing: single
- Diet: pelleted standard diet, ad libitum (Harlan Winkelmann GmbH, D-33178 Borchen)
- Water: tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-72
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle/solvent used: corn oil
- Justification for choice of solvent/vehicle: The vehicle was chosen to its relative non-toxicity for the animals.
- Amount of vehicle: total amount: 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test item was formulated in corn oil.

Duration of treatment / exposure:
single treatment, sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.
Frequency of treatment:
only one application
Post exposure period:
The animals of all dose groups were examined for acute toxic symptoms at intervals of around 1 h, 2 - 4 h, 6 h, 24 h and 48 h after administration of the test item. Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.
Doses / concentrationsopen allclose all
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Remarks:
24 h preparation interval
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
24 h preparation interval
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
24 h preparation interval
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
48 h preparation interval
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: orally
- Doses / concentrations: 40 mg/kg bw

Examinations

Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION:
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 (Merck, D-64293 Darmstadt)/Giemsa (Merck, D-64293 Darmstadt). Cover slips were mounted with EUKITT (Kindler, D-79110 Freiburg). 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.
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) will be used as an aid in evaluating the results. 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 significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 100, 500, 750 and 1000 mg/kg bw
- Clinical signs of toxicity in test animals: The animals treated with 750 and 1000 mg/kg bw expressed toxic reactions (ruffled fur, reduction of spontaneous activity, abdominal position, eyelid closure, apathy, tremor, death)

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: The mean values of micronuclei observed after treatment with the test substance were below or near to the value of the vehicle control group.
- Ratio of PCE/NCE: The ratio of PCE to NCE was not relevantly decreased after treatment with the test item as compared to the mean value of PCE/NCE of the vehicle control.

Any other information on results incl. tables

Table 1: Micronuclei in polychromatic erythrocytes (PCE) and relationship PCE per 2000 total erythrocytes

Test group

Dose mg/kg bw

Sampling time

PCEs with micronuclei [%]

range

PCE per 2000 erythrocytes

vehicle

0

24

0.12

0-6

1137

Test item

125

24

0.1

1-4

1132

Test item

250

24

0.12

0-6

1146

Test item

500

24

0.155

1-6

986

Positive control

40

24

2.385

12-68

1025

Test item

500

48

0.115

1-5

1020

 

Applicant's summary and conclusion

Conclusions:
The test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.
Executive summary:

The test item 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 formulated in corn oil, which was also used as vehicle control. The volume administered orally was 10 mL/kg bw. 24 h and 48 h after a single administration of the test item 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 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: 24 h preparation interval: 125, 250 and 500 mg/kg bw. 48 h preparation interval: 500 mg/kg bw. The analysis of the formulations used for dosing the animals showed, that the obtained results for the formulation concentrations correspond to their respective nominal values. The recovery values ranged between 93.7 to 100.7 % of the nominal values. As estimated by a pre-experiment 500 mg test substance per kg bw was suitable. In the main experiment one male (animal no. 66) died after treatment with this dose. The mean number of polychromatic erythrocytes was not relevantly decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that the test substance did not have any cytotoxic properties 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 after administration of the test item. The mean values of micronuclei observed after treatment with the test item were below or near to the value of the vehicle control group. 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, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.