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:
1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted similarly to OECD Guideline 474 with minor deviations: no data on the housing conditions and individual body weight at the start of the test; number of micronucleated immature erythrocytes for individual animals not reported. 1000 polychromatic erythrocytes (PCE) were analysed per animal, which was a requirement of the old version of the OECD 474 (1983). The new version requires the analysis of at least 2000 PCE.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1988

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
no data on the housing conditions and body weight at the start of the test; number of micronucleated immature erythrocytes for individual animals not reported
Principles of method if other than guideline:
Not applicable
GLP compliance:
not specified
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Undecalactone
- Source: Japan Food Additives Association, Tokyo, Japan
- Purity: > 98.0 %

Test animals

Species:
mouse
Strain:
other: ddY mice
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Shizuoka Agricultural Cooperative Association for Laboratory Animals, Shizuoka, Japan
- Age at study initiation: 8 weeks
- Diet: Food pellets CE-2 (Japan Clea, Tokyo, Japan), ad libitum
- Water: ad libitum

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
Olive oil
Details on exposure:
None
Duration of treatment / exposure:
- 250, 500, 1000 and 2000 mg/kg bw: One day
- 500 mg/kg bw: 4 days
Frequency of treatment:
- 250, 500, 1000 and 2000 mg/kg bw: Single injection
- 500 mg/kg bw: 4 injections with 24 h intervals between the injections
Post exposure period:
24 h
Doses / concentrationsopen allclose all
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
Basis: actual injected
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
Basis: actual injected
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Basis: actual injected
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
Remarks:
Basis: actual injected
No. of animals per sex per dose:
6 males/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Mitomycin-C
- Source: Kyowa Hakko Ltd, Tokyo, Japan
- Route of administration: Intraperitoneal
- Doses: 2 mg/kg bw

Examinations

Tissues and cell types examined:
- Femora bones were removed for marrow extraction and the prepared slides were examined for polychromatic erythrocytes (PCEs) and total erythrocytes.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The maximum doses of the test material were set at the supposed maximum tolerated dose referring to LD50.

DETAILS OF SLIDE PREPARATION: Mice were killed by cervical dislocation at 24 h after exposure. Femoral marrow cells were flushed out with fetal bovine serum and smeared on clean glass slides. Cells were fixed with methanol for 5 minutes and stained with Giemsa stain.

METHOD OF ANALYSIS:
- 1000 polychromatic erythrocytes (PCEs) per mouse were scored using a light microscope with a high power objective (X 100) and the no. of micronucleated polychromatic erythrocytes (MNPCEs) were recorded.
- Proportion of polychromatic erythrocytes (PCEs) among the total erythrocytes was evaluated by observing 1000 erythrocytes on the same slide.
Evaluation criteria:
Result was considered as positive if one or more treatment group(s) showed a statistically significant difference (P < 0.01) from the spontaneous level of MNPCEs and the trend test indicated a positive dose response (P < 0.05).
Statistics:
Two-stage statistical procedure:
- In the first step of the procedure, the frequency of MNPCEs in each treatment group was compared with the binomial distribution specified by historical control data (Hayashi et al. 1985).
- In the second step, the dose-response relationship was tested by the Cochran- Armitage trend test (Armitage, 1955; Cochran, 1954; Margolin and Risko, 1988).

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
- See table 7.6.2/1 for MNPCEs (%) and PCEs (%)

Any other information on results incl. tables

Table 7.6.2/1: Results of micronucleus test

Groups

Dose (mg/kg bw)

No. of doses

Time between doses (24 h)

Sampling time (h)

MNPCEs (%)

PCEs (%)

Mortality

Vehicle (Olive oil)

0

1

-

24

0.10 ± 0.06

54.6 ± 6.4

0/6

Undecalactone

250

0.25 ± 0.12

51.6 ± 7.7

500

0.08 ± 0.08

43.9 ± 7.4

1000

0.18 ± 0.08

43.7 ± 9.2

2000

0.25 ± 0.07

52.3 ± 3.0

500

4

24

0.08 ± 0.10

48.7 ± 8.1

Mitomycin-C

2

1

-

4.78 ± 1.40*

43.5 ± 4.6

 

MNPCEs - Micronucleated polychromatic erythrocytes; PCEs - Polychromatic erythrocytes;

* value of MNPCE differ significantly from the historical control (p < 0.01)

Applicant's summary and conclusion

Conclusions:
Under the test conditions, Undecalactone is not considered as clastogenic in the mouse bone marrow micronucleus assay according to the criteria of the Annex I of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.
Executive summary:

In an in vivo mouse bone marrow micronucleus assay performed similarly to OECD Guideline 474, groups of ddY male mice (6/dose) were injected with Undecalactone in olive oil at 250, 500, 1000 and 2000 mg/kg bw (single treatment) and 500 mg/kg bw (4 injections with 24 h intervals between the injections) by intraperitoneal route. The positive control group was injected with mitomycin C at 2 mg/kg bw. Mice were killed by cervical dislocation 24 h after an administration. Femoral marrow cells were flushed out with fetal bovine serum and smeared on clean glass slides. Cells were fixed with methanol for 5 minutes and stained with Giemsa stain. 1000 polychromatic erythrocytes (PCEs) per mouse were scored for recording the incidence of micronucleated polychromatic erythrocytes (MNPCEs). The proportion of PCEs among the total erythrocytes was also evaluated by observing 1000 erythrocytes on the same slide.

Mice treated with Undecalactone did not show any significant increase in the frequency of MNPCEs. Positive control (mitomycin C) induced a statistically significant increase in MNPCEs indicating the validity of the study.

Under the test conditions, Undecalactone is not considered as clastogenic in the mouse bone marrow micronucleus assay according to the criteria of the Annex I of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.