Bromo(hexahydro-2H-azepin-2-onato-N)magnesium

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09/2008 to 11/2008

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 guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mouse spot test

Test material

Test material information

Constituent 1

Reference substance name:

Bromo(hexahydro-2H-azepin-2-onato-N)magnesium

EC Number:

241-158-2

EC Name:

Bromo(hexahydro-2H-azepin-2-onato-N)magnesium

Cas Number:

17091-31-5

Molecular formula:

C6H10BrMgNO

IUPAC Name:

magnesium(2+) ion 2-oxoazepan-1-ide bromide

additive 1

Reference substance name:

ε-caprolactam

EC Number:

203-313-2

EC Name:

ε-caprolactam

Cas Number:

105-60-2

Molecular formula:

C6H11NO

IUPAC Name:

azepan-2-one

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 07072601
- Expiration date of the lot/batch: July 2009

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

Housing: single

Cage Type: Makrolon Type I, with wire mesh top (EHRET GmbH, D-79302 Emmendingen)

Bedding: granulated soft wood bedding (Harlan Laboratories GmbH, D-33178 Borchen)

Feed: pelleted standard diet, ad libitum (Harlan Laboratories GmbH, D-33178 Borchen)

Water: tap water, ad libitum, (Gemeindewerke, D-64380 Roßdorf)

Environment: temperature 22 ± 3 °C

relative humidity 30 - 70 %

artificial light 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

deionised water

Duration of treatment / exposure:

single administration of the test item

Frequency of treatment:

once

Post exposure period:

24 h and 48 h, respectively

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

437.5 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

875 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 750 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

Yes, cyclophosphamide

Examinations

Tissues and cell types examined:

bone marrow cells

Details of tissue and slide preparation:

The animals were sacrificed using CO2 followed 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 (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.

Evaluation criteria:

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.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

test group dose mg/kg b.w.     sampling time (h)   PCEs with micronuclei (%)  range   PCE per 2000 erythrocytes  vehicle  0  24  0.054  0 - 3  1184  test item  437.5  24  0.083  0 - 4  1259  test item  875  24  0.104  0 - 4  1181  test item  1750  24  0.092  0 - 5  1246   positive control 40   24  2.417 21 -87  1175   test item  1750  48  0.150  1 - 7  1217

Applicant's summary and conclusion

Conclusions:

The test item Bromo(hexahydro-2H-azepin-2-onato-N)magnesium 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 deionised water, which was also used as vehicle control.
The volume administered orally was 10 mL/kg b.w.. 24 h and 48 h after a single
administration of the test item the bone marrow cells were collected for micronuclei
analysis.

Where possible twelve animals (6 males, 6 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: 437.5, 875 and 1750 mg/kg b.w..
48 h preparation interval: 1750 mg/kg b.w..
As estimated by a pre-experiment 1750 mg Bromo(hexahydro-2H-azepin-2-onato-
N)magnesium per kg b.w. was suitable. In the main study 1 female (animal no. 72) died
after treatment with this dose.

The mean number of polychromatic erythrocytes was not decreased after treatment with
the test item as compared to the mean value of PCEs of the vehicle control indicating that
Bromo(hexahydro-2H-azepin-2-onato-N)magnesium did not have any cytotoxic properties
in the bone marrow.

In comparison to the corresponding vehicle controls there was no biologically relevant
enhancement in the frequency of the detected micronuclei at any preparation interval and
dose level after administration of the test item. A statistically significant enhancement in
the frequency of micronucleated cells was observed in the groups treated with the mid
dose and high dose (prepared at the 48 h preparation interval). This increase was,
however, not dose related in the case of the mid dose. For the high dose there was not
any indication why the effect should turn up at the later interval without any prior signs
(such as organ toxicity) at the 24 h interval. Furthermore, all obtained values were within
the historical vehicle control range. Therefore, the significant increases are regarded as
biologically irrelevant.

40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which
showed a statistically significant increase of induced micronucleus frequency.

Executive summary:

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.