1,4-dioxacycloheptadecane-5,17-dione

Genetic toxicity: in vivo

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-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

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

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

Test material information

Constituent 1

Reference substance name:

1,4-dioxacycloheptadecane-5,17-dione

EC Number:

203-347-8

EC Name:

1,4-dioxacycloheptadecane-5,17-dione

Cas Number:

105-95-3

Molecular formula:

C15H26O4

IUPAC Name:

1,4-dioxacycloheptadecane-5,17-dione

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 or test system 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 / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
Exp. 1: 100, 1000 or 1600 mg/kg bw
Basis:
nominal conc.

Doses / concentrations 2

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.