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:
from 30 January 2009 to 17 June 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: the study was performed according to internationally recognised guidelines and GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories GmbH, Borchen, Germany
- Age at study initiation: 6 - 10 weeks
- Weight at study initiation:
> Main experiment: 166.7g ± 4.6g (males) and 169.1g ± 4.2g (females)
> Experiment repeat: 184.6g ± 5.8g (males) and 163.2g ± 7.8g (females)
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: individually housed in Makrolon Type II cages, with wire mesh top and granulated soft wood bedding
- Diet: pelleted standard diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 25-70%
- Air changes (per hr): no data
- Photoperiod: artificial light 6:00 am - 6:00 pm

IN-LIFE DATES: From 3 February 2009 to 6 March 2009

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: chosen for its relative non-toxicity
- Concentration of test material in vehicle:
- Amount of vehicle: 10 mL/kg
- Purity: deionised water (main experiment) and sterile water (experiment repeat)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test item was formulated in water. Before preparation, the vehicle was degassed by sonication for 15 minutes and then saturated with inert gas, and kept under inert gas atmosphere for 15 minutes prior dosing.
Duration of treatment / exposure:
unique exposure
Frequency of treatment:
once
Post exposure period:
24 or 48 hours
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 375, 750, 1500 mg/kg bw (main experiment)
Basis:
other: nominal
Remarks:
Doses / Concentrations:
0, 1500 mg/kg (experiment repeat)
Basis:
other: nominal
No. of animals per sex per dose:
6 animals/sex/group and per sampling time (except in control groups: 5 animals/sex/group)
Control animals:
yes, concurrent vehicle
Positive control(s):
none; no data; cyclophosphamide
- Route of administration: orally
- Doses / concentrations: 20 mg/kg

Examinations

Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: based on a preliminary study on acute toxicity performed with 2 animals per sex under identical conditions as in the mutagenicity study

TREATMENT AND SAMPLING TIMES:
At the beginning of the treatment the animals were weighed and individual volume to be administered was adjusted to the animal body weight. The animals received the test item, the vehicle or the positive control once. The animals of all groups were examined for acute toxic symptoms at intervals of approx.. 1h, 2-4h, 6h, 24h and 48h after administration of the test item.
Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.

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 nucleated cells were separated from the erythrocytes using the method of Romagna. Four slides were made from each bone marrow sample, two of which were stained with May-Grunwald. The remaining two slides per animal were kept as reserve slides in case a confirmation of result using acridine orange staining becomes necessary.

METHOD OF ANALYSIS:
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.
Evaluation criteria:
A test item is classified as 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. Statistical methods (nonparametric Mann-Whitney test) will be used as an aid in evaluating the result. However, the primary point of consideration is the biological relevance of the results.
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:
nonparametric Mann-Whitney test

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
In the pre-experiment tests, groups of 4 animals (2 males and 2 females) received orally a single dose of test item formulated in deionised water at the following concentrations: 500, 1000, 2000, 1500, and 1750 mg/kg bw. They were then observed during 48 hours.
At 500, 1000 and 1500 mg/kg bw, the animals expressed reduction of spontaneous activity, ruffled fur and urine colored in orange/red. At 1750 and 2000 mg/kg, additional observation were abdominal position and death (1 male and A female at 1750 and 1 male and 2 females at 2000).
On the basis of these data 1500 mg/kg bw were estimated to be suitable.

RESULTS OF DEFINITIVE STUDY
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 the test item were below or near to the value of the vehicle control group.
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 the test item did not exert any cytotoxic effect in the bone marrow. The urine of all treated animals has taken the colour of the test item, indicating the systemic distribution of the test item and thus its bioavailability.
20 mg/kg bw cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency.
- Ratio of PCE/NCE: see table
- Toxic symptoms: in the first main experiment 8 animals (4 males and 4 females) died after treatment with 1500 mg/kg bw. A further experiment was, thus performed treating further animals (12 animals per sex). In the study repeat 1 male and 2 females died.

Any other information on results incl. tables

Table 1:Results of in vivo micronucleus test (Main experiment, sampling time 24h)

  

Vehicle

375 mg/kg

750 mg/kg

1500 mg/kg

CPA 20 mg/kg

Number of cells evaluated

2000

2000

2000

2000

2000

Sampling time (h)

24

24

24

24

24

Number of animals

11

12

12

12

11

Micronucleated cells per 2000 PCEs per animals (mean)

3.0

3.1

1.8

3.0

21.9

Percent cells with micronuclei (mean)

0.150

0.154

0.092

0.150

1.095

PCE per 2000 erythrocytes (mean)

1074

981

1117

1080

909

 

Table 2:Results of in vivo micronucleus test (Main experiment, sampling time 48h)

  

Vehicle

1500 mg/kg

Number of cells evaluated

2000 

 2000

Sampling time (h)

48

48

Number of animals

11

4

Micronucleated cells per 2000 PCEs per animals (mean)

3.0

2.3

Percent cells with micronuclei (mean)

0.150

0.113

PCE per 2000 erythrocytes (mean)

1127

1076

 

Table 3:Results of in vivo micronucleus test (Experiment repeat, sampling time 24h)

  

Vehicle

1500 mg/kg

CPA 20 mg/kg

Number of cells evaluated

2000 

 2000

2000

Sampling time (h)

24

24

24

Number of animals

12

10

12

Micronucleated cells per 2000 PCEs per animals (mean)

3.7

1.4

31.8

Percent cells with micronuclei (mean)

0.183

0.070

1.592

PCE per 2000 erythrocytes (mean)

1075

1099

851

 

Table 4:Results of in vivo micronucleus test (Experiment repeat, sampling time 48h)

  

Vehicle

1500 mg/kg

Number of cells evaluated

2000 

 2000

Sampling time (h)

48

48

Number of animals

12

11

Micronucleated cells per 2000 PCEs per animals (mean)

4.5

4.2

Percent cells with micronuclei (mean)

0.225

0.209

PCE per 2000 erythrocytes (mean)

1102

1109

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of the test, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the rat, after a unique oral administration up to 1500 mg/kg bw.
Executive summary:

In a Wistar rat bone marrow micronucleus assay performed according to OECD testing guideline 474 (GLP study, scored as validity 1 according to Klimisch criteria), 6 animals/sex/group and per sampling time were treated by a unique oral gavage withthe test itemat doses of 0, 375, 750 and 1500 mg/kg bw.  Bone marrow cells were harvested at 24 and/or 48 hours post-treatment.  The vehicle was deionised water (main experiment) or sterile water (repeat experiment).

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 the test item were below or near to the value of the vehicle control group.

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 the test item did not exert any cytotoxic effect in the bone marrow. The urine of all treated animals has taken the colour of the test item, indicating the systemic distribution of the test item and thus its bioavailability.

The positive control induced the appropriate response.

Under the conditions of the test, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the rat, after a unique oral administration up to 1500 mg/kg bw.