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
Study period:
no data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study is non-GLP, it does not use the maximum tolerated dose or 2000 mg/kg bw, nor the minimum number of animals required by the OECD guideline No 474. However, 1600 and 1400 mg/kg bw are not so far from 2000 mg/kg bw, and 7 to 8 animals were used per dose level. Also, the intraperitoneal dose route was used, which is likely to achieve a greater exposure to the bone marrow than the oral route, which is the normal route currently used. Finally, a study done according to OECD standards is unlikely to provide a different result, particularly as there is no concern for genotoxicity in the other available in vitro studies.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
publication
Title:
Macrocyclic musk compounds - an absence of genotoxicity in the Ames test and the in vivo Micronucleus assay
Author:
Abramsson-Zetterberg L, Slanina P
Year:
2002
Bibliographic source:
Toxicology Letter 135: 155-163

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
the maximum tolerated dose or 2000 mg/kg bw was not achieved, the minimum number of animals required was not used
Principles of method if other than guideline:
not applicable
GLP compliance:
not specified
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
- Name of test material: ethylene brassylate
- Analytical purity: 86 % (GC/MS)
- Source: Dr. G. Rimkus, Dept of Residue and Contamination Analysis, LVUA, Schleswig-Holstein, Neumünster, Germany

Test animals

Species:
mouse
Strain:
other: male CD-1 and female NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Uppsala
- Age at study initiation: 8-9 weeks
- Weight at study initiation: males: 30-40 g; females: 25-30 g
- Assigned to test groups randomly: yes, under following basis: - in Exp. 1 (males): 5 groups, 3 in each
- in Exp. 2 (females): 5 groups, 5 in each (except one with 4)
- Housing: no data
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
no data

IN-LIFE DATES: no data

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: rape oil
- Dose volume: 10 mL/ kg bw
Details on exposure:
none
Duration of treatment / exposure:
Exp. 1: 46 and 70 h; Exp. 2: 42 and 70 h
Frequency of treatment:
once
Post exposure period:
none
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
Exp. 1: 100, 1000 or 1600 mg/kg bw
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
Exp. 2: 350, 700 or 1400 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
Exp. 1: 3 males per dose
Exp. 2: 5 females per dose, except the highest dose-group which includes only 4 females (1 died under anaesthesia)
Control animals:
yes, concurrent vehicle
Positive control(s):
Colchicine
- Justification for choice of positive control(s): direct acting agent with an aneugenic effect resulting in an increased fMPCE in peripheral blood at about 45 hours after treatment
- Route of administration: intraperitoneal
- Doses / concentrations: 1 mg/kg bw

Examinations

Tissues and cell types examined:
Polychromatic erythrocytes (PCE) from peripheral blood
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
Blood samples were collected, under light anaesthesia with Fluothane, from the orbital plexus of each animal, at 46 and 70 h after injections in Exp. 1 and at 42 and 70 h after in,jections in Exp. 2. The choice of the first sampling time is based on the knowledge of the time between the appearance of polychromatic erythrocyte in bone marrow and peripheral blood. From each animal, about 50 µL blood was drawn at each sampling occasion. Two parallel aliquots of 5 µL blood were layered for purification on a 65 % Percoll gradient, as described by Grawé et al. (1993).

DETAILS OF SLIDE PREPARATION:
The cells in the pellet were fixed in a solution of glutaraldehyde according mainly to a method by Hayashi et al. (1992). The fixed cells were then stored during 1-2 days at 4 °C and the following staining was made in a buffer prepared by adding the fluorescent dyes Hoechst 33342 and thiazole orange to PBS. The samples from the two sampling occasions were stained and analysed at the same time.

METHOD OF ANALYSIS:
The samples were analysed on a dual laser FACStar Plus flow cytometer. The setting and equipment of the flow cytometer has been described before. From each of the peripheral blood samples about 100 000 PCE were analysed (from each animal at each occasion almost 200 000 PCE - two parallels - were analysed).
Evaluation criteria:
a statistically significant difference between the treated and control groups should be demonstrated
Statistics:
Student t-test

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
only one female died under the anaesthesia
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no significant increase difference in the mean fMPCE (frequency of micronucleated PCE) in the groups of the animals treated as compared to the controls in both experiments.
- Ratio of PCE/NCE (for Micronucleus assay): see Table 7.6.2/1
- Appropriateness of dose levels and route:
- Statistical evaluation: no statistical difference between treated and control groups
- Positive control: In Exp. 1, the mice injected with colchicine showed a significant increase in the mean fMPCE at 46 h after the treatment. In Exp. 2, similarly treated mice showed a significant increase at 42 h.

Any other information on results incl. tables

Table 7.6.2: Results from the micronucleus test

Strain and sex

N° of animals

Treatment (mg/kg bw)

Time after injection (h)

PCE (%)

PCE (No. of analysed)

MPCE (No. Of scored)

fMPCE (per mille)

SD

CD1, male

3

Control

46

2.7

594325

617

1.04

0.31

3

EB, 100

46

3.7

599023

648

1.08

0.15

3

EB, 1000

46

2.5

570505

524

0.92

0.22

3

EB, 1600

46

2.8

598598

592

0.99

0.13

3

Col, 10

46

0.7*

211822

2172

10.25*

0.17

3

Control

70

2.9

564638

623

1.10

0.23

3

EB, 100

70

3.0

548490

666

1.21

0.16

3

EB, 1000

70

2.2

513417

473

0.92

0.26

3

EB, 1600

70

2.5

536533

535

1.00

0.10

3

Col, 10

70

1.0*

293865

463

1.58

0.50

NMRI, female

5

Control

42

2.5

893089

1364

1.52

0.23

5

EB, 350

42

2.5

790404

1269

1.60

0.48

5

EB, 700

42

2.4

776752

1017

1.31

0.26

5

EB, 1400

42

2.7

903936

1271

1.40

0.19

3

Col, 10

42

1.7

375588

2334

6.18*

4.4

5

Control

70

2.7

753506

1304

1.73

0.21

5

EB, 350

70

1.9

665457

969

1.45

0.13

5

EB, 700

70

1.9

602829

806

1.34

0.22

4

EB, 1400

70

2.0

585127

855

1.46

0.39

4

Col, 10

70

1.9

405598

712

1.75

0.87

Col = Colchicine

* = Significant separated from the control

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time in both experiments.
Executive summary:

In an in vivo micronucleus assay conducted similarly to OECD 474 guideline, female NMRI and male CD-1 mice were intraperitoneally injected with Ethylene Brassylate diluted in rape oil. In experiment 1, males were dosed with 100, 1000 or 1600 mg/kg bw whereas in experiment 2, females were dosed with 350, 700 or 1400 mg/kg bw. Peripheral blood samples were collected at 46 and 70 h in experiment 1 and at 42 and 70 h in experiment 2.

All vehicle (solvent) controls had frequencies of micronucleated polychromatic erythrocytes (fMPCE) within the range expected for normal peripheral blood. The positive control material, Colchicine, induced statistically significant increases in the fMPCE at the first sampling time indicating the satisfactory performance of the test.

A female dosed with 1400 mg/kg bw died under anaesthesia. No other sign of toxicity was observed.

There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time in both experiments.

Under the test conditions, the test material is not classified according to the annex VI of the Regulation EC No. 1272/2008 (CLP) and of the Directive 67/548/EEC.

The study is non-GLP, it did not use the maximum tolerated dose or 2000 mg/kg bw, nor the minimum number of animals required by the OECD guideline No. 474. However, 1600 and 1400 mg/kg bw are considered to be close to the maximum recommended dose level of 2000 mg/kg bw, and 7 to 8 animals were used per dose level, which is only 2 or 3 less than recommended. Also, the intraperitoneal dose route was used, which is likely to achieve a greater exposure to the bone marrow than the oral route, which is the standard route currently used. Finally, a study done according to the current OECD standard is unlikely to provide a different result, particularly as there is no concern for genotoxicity in the other available in vitro studies.

This study is therefore considered as acceptable and satisfies the requirement for in vivo cytogenetic mutagenicity data.