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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item Bromo(hexahydro-2H-azepin-2-onato-N)magnesium is considered as "not mutagenic under the conditions of the test".

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01/2008 to 05/2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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: 26.07.2008

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature 20 ± 5 °C

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no

Species / strain / cell type:

other: TA 97a, TA 98, TA 100, TA 102 and TA 1535

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

4986 to 50 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-Nitro-1,2-phenylene diamine (NPD) in DMSO, 2-Amino anthracene (2-AA) in DMSO

Details on test system and experimental conditions:

Plate incorporation method

Per strain and dose, four plates with and four plates without S9 mix were used. 10 ml of
the test solution of the appropriate concentration were membrane filtrated into sterile vessels.
Top agar basis was melted in a microwave oven, after melting, 10 ml of histidinebiotin-
solution 0.5 mMol per 100 ml basis was added and the bottle was placed in the
water bath at 45 °C.
0.1 ml of the appropriate solution of the test item were given into a sterile tube. After mixing
with 0.1 ml overnight culture of the respective strain and 0.5 ml phosphate buffer
(only for treatments without S9) or 0.5 ml S9 mix, 2 ml Top-Agar were added. The mixture
was gently vortexed, then poured on a minimal glucose plate and distributed evenly,
using a Drigalski spatula. The plates were closed, covered with brown paper and left to
harden for a few minutes, then inverted and placed in the dark incubator at 37 °C.

Pre-incubation method

Per strain and dose, four plates with and four plates without S9 mix were used. 10 ml of
the test solution of the appropriate concentration were membrane filtrated into sterile vessels.
Top agar basis was melted in a microwave oven, after melting, 10 ml of histidinebiotin-
solution 0.5 mMol per 100 ml basis was added and the bottle was placed in the
water bath at 45 °C.
0.1 ml of the appropriate solution of the test item were given into a sterile tube. After mixing
with 0.1 ml overnight culture of the respective strain, 0.5 ml phosphate buffer (only
for treatments without S9) or 0.5 ml S9 mix were added. The mixture was incubated in an
incubation chamber at 37°C for 20 minutes. During this time the vessels were aerated
through careful shaking. Then 2 ml top agar were added. The mixture was vortexed gently,
then poured on a minimal glucose plate and distributed evenly, using a Drigalski spatula.
The plates were closed, covered with brown paper and left to harden for a few minutes,
then inverted and placed in the dark incubator at 37 °C.

Evaluation criteria:

The colonies were counted visually, the numbers were recorded.

Statistics:

A spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations as well as the increase factor of revertant induction.

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Conclusions:

Under the conditions of the test, the test item didn't show mutagenic effects towards Salmonella typhimurium, strains TA 97a, TA 98, TA 100, TA 102 and TA 1535. Therefore, no concentration-effect relationship could be determined.

Executive summary:

The test item Bromo(hexahydro-2H-azepin-2-onato-N)magnesium is considered as "not mutagenic under the conditions of the test".

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test item Bromo(hexahydro-2H-azepin-2-onato-N)magnesium did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mouse spot test

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

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.

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

Dose / conc.:

437.5 mg/kg bw/day (nominal)

Dose / conc.:

875 mg/kg bw/day (nominal)

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

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.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

No classification according to CLP-regulation required due to negative results in in-vitro OECD 471 study and in-vivo OECD 474 study.