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:
2009-04-20 till 2009-05-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study under GLP without deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Directive 2000/32/EC, Annex 4C, dated May 19, 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- public name of test material:
Reaction mass of 6,13-dichloro-3,10-bis{[2-({[(2-chloroethyl)sulfonyl]alkanoyl}amino)ethyl]-amino}- polycarboheterocyclo 4,11-disulfonic acid, mono and/or disodium salt and 6,13-dichloro-3-{[2-({[(2-chloroethyl)sulfonyl]alkanoyl}amino) ethyl]amino}-10-[(2-{[4-(ethenylsulfonyl)alkanoyl]amino}ethyl)amino] polycarboheterocyclo -4,11-disulfonic acid, mono and/or di sodium salt
- Physical state: solid, dark blue powder
- Analytical purity: approx. 86.6%
- Lot/batch No.: VER 2108 BOP 02/07
- Expiration date of the lot/batch: November 30, 2012
- Storage condition of test material: At room temperature at about 20 °C

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Harlan Laboratories GmbH 33178 Borchen, Germany
- Age at study initiation: 8-12 weeks
- Weight at study initiation: males mean value 38.9 g (SD ± 2.8 g), females mean value 33.7 g (SD ± 3.5 g)
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: single, cage: Makrolon Type I, with wire mesh top Bedding: granulated soft wood bedding
- Diet (e.g. ad libitum): single pelleted standard diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum,
- Acclimation period: min 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 45 - 65%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light):artificial light 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: sterile water
- Justification for choice of solvent/vehicle: high solubility of the test item in water (60g/L
- Concentration of test material in vehicle: 50, 100 and 200mg/mL
- Amount of vehicle (if gavage or dermal): 10mL/kg bw
- Type and concentration of dispersant aid (if powder): not applicable
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: test item dissolved in vehicle (sterile water) at the respective concentration
- negative control: vehicle
- postive control : CPA; Cyclophosphamide, Supplier: Sigma-Aldrich Vertriebs GmbH, 82041 Deisenhofen, Germany
Dissolved in: deionised water, Dosing: 40 mg/kg b.w., Route and frequency of administration: orally, once, Volume administered: 10 mL/kg b.w., Solution prepared on day of administration.
Duration of treatment / exposure:
single oral dose
Frequency of treatment:
single oral dose
Post exposure period:
animals sacrified 24 or 48h post dosing
Doses / concentrations
Remarks:
Doses / Concentrations:
50, 100, 200 and 1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
6 males and 6 females
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide;
- Justification for choice of positive control(s):no data
- Route of administration: single oral dose
- Doses / concentrations: 40 mg/kg b.w., Volume administered: 10 mL/kg b.w.

Examinations

Tissues and cell types examined:
The animals were treated orally with the test item and examined for acute toxic symptoms at intervals of approximately 1 h, 2-4 h, 6 h, 24 h, 30 h, and 48 h after administration of the test item.

the animals(including controls) were weighed at the day of dosing, just prior to dosing

Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
based on dose range finding study (2 animals per sex) with 100, 1000 and 2000mg/kg bw (see table 1)

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): see specific fields

DETAILS OF 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, 64293 Darmstadt, Germany)/Giemsa (Gurr, BDH Limited Poole, Great Britain)
- Cover slips were mounted with EUKITT (Kindler, 79110)

METHOD OF ANALYSIS:
- The analysis was performed with coded slides
- Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives.
- Per animal 2000 polychromatic erythrocytes (PCE) were analysed 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.

OTHER:
Evaluation criteria:
MUTAGENIC:
- if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group.
NON-MUTAGENIC in this system.
- A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.


Statistics:
Statistical methods (nonparametric Mann-Whitney test (8)) will be used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
ruffled fur, reduction of spontaneous activity, see table 1
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 500 to 2000 mg/kg bw
- Solubility: very soluable, 60g/L
- Clinical signs of toxicity in test animals: ruffled fur and reduction of spontaneous activity (see table 1)
- Evidence of cytotoxicity in tissue analyzed: not analyzed
- Rationale for exposure: 2000mg/kg bw is the highest allowed dose for animal testing in the EU


RESULTS OF DEFINITIVE STUDY
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 test material did not have any cytotoxic properties in the bone marrow. (see table 2 for details)

In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with test material were near to the value of the vehicle control group.

Any other information on results incl. tables

Table 2 Summary of Micronucleus Test Results

test group

dose mg/kg b.w.

sampling time (h)

PCEs with micronuclei (%)

range

PCE per 2000 erythocytes

vehicle

0

24

0.088

0 -4

1206

test item

500

24

0.096

1 -4

1154

test item

1000

24

0.138

1 -6

1234

test item

2000

24

0.104

0 -5

1180

positive control

 40

24

2.142

16 -61

1124

test item

2000

48

0.121

1 -5

1207

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.
Therefore, test material is considered to be non-mutagenic in this micronucleus assay.
Executive summary:

This study was performed to investigate the potential of test material to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test item was formulated in sterile 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.

Twelve animals (6 males, 6 females) per test group were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) 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: 500, 1000, and 2000 mg/kg b.w..

48 h preparation interval: 2000 mg/kg b.w..

The highest dose (2000 mg/kg; maximum guideline-recommended dose) was estimated by pre-experiments to be suitable.

After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that test material did not exert any cytotoxic effects in the bone marrow.

In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used.

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

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Therefore, test material is considered to be non-mutagenic in this micronucleus assay.