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Toxicological information

Genetic toxicity: in vivo

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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 July 21, 2009 to October 16, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted GLP guideline study.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories GmbH, 33178 Borchen
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: males mean value 33.8 g (SD ± 3.0 g)
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: single, Makrolon Type II, with wire mesh top (EHRET GmbH, 79302 Emmendingen, Germany)
- Diet: pelleted standard diet, ad libitum (Harlan Laboratories GmbH, 33178 Borchen, Germany)
- Water: tap water, ad libitum (Gemeindewerke, 64380 Rossdorf, Germany)
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2 °C
- Humidity (%): 45 - 65 %
- Air changes (per hr): no data
- Photoperiod: 12 hrs dark / 12 hrs light (artificial light 6.00 a.m. - 6.00 p.m.)

IN-LIFE DATES: no data

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil (Sigma-Aldrich Vertriebs GmbH, 82041 Deisenhofen)
- Justification for choice of solvent/vehicle: no data
- Concentration of test material in vehicle: no data
- Amount of vehicle: 10 mL/kg bw
- Lot/batch no. (if required): no data
- Purity: no data
Details on exposure:
At the beginning of the treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animal’s body weight. The animals received the test item, the vehicle or the positive control substance once.
Frequency of treatment:
once
Post exposure period:
24 or 48 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 75, 150, 300 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
7 males/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide (Sigma-Aldrich Vertriebs GmbH, 82041 Deisenhofen, Germany. Purity: > 98 %)
- Justification for choice of positive control(s): no data
- Route of administration: orally
- Doses / concentrations: 40 mg/kg bw (10 mL/kg bw)

Examinations

Tissues and cell types examined:
bone marrow cells from femora
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: based on the results of the pre-experiments on toxicity.
A preliminary study on acute toxicity was performed with two animals per sex under identical conditions as in the mutagenicity study concerning: animal strain, vehicle, route, frequency, and volume of administration. 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, 8 h (except for the fourth pre-experiment), 10 h (except for the third and fourth pre-experiment), 24 h, 30 h, and 48 h after administration of the test item.

TREATMENT AND SAMPLING TIMES: 24 and 48 hours after treatment

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 Freiburg, Germany). At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS: 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. The analysis was performed with
coded slides.

OTHER: The animals of all dose groups (excepted the positive control) were examined for acute toxic symptoms at intervals of approximately 1 h, 2 - 4 h, 6 h, 24 h and 48 h after administration of the test item. In case of problems occurred during the treatment of the animals connected to the corrosive characteristics of the test item it was considered to have two additional early observation intervals (8 h and 10 h after treatment). However, these were skipped since the animals did not show any abnormal clinical signs in the pre-experiments demonstrating the redundancy of further observation intervals.
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 (8)) will be used as an aid in evaluating the results. 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:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
At 300 mg/kg bw, reduction of spontaneous activity in all the animals 1 h after treatment. At 150 mg/kg bw, ruffled fur in 1 animal 1 h after treatment.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 300 - 1000 mg/kg bw
- Clinical signs of toxicity in test animals:
1st test (100 mg/kg bw): The animals treated with 100 mg/kg b.w. did not express any toxic reactions.
2nd test (1000 mg/kg bw): reduction of spontaneous activity, abdominal position, apathy. The two males and one female died approximately 2 hours after application of the test item. The second female animal died 3 hours after application of the test item.
3rd test (500 mg/kg bw): reduction of spontaneous activity, abdominal position, ruffled fur
4th test (300 mg/kg bw): reduction of spontaneous activity, ruffled fur

Any other information on results incl. tables

Table 7.6.2/1: Summary of micronucleus test results

Test group

Dose (mg/kg bw)

Sampling time (h)

PCEs with micronuclei (%)

Range

PCE per 2000 erythrocytes

Significance

p

Vehicle

0

24

0.129

0-5

1376

-

 

Test item

75

24

0.179

1-6

1355

-

0.1949

Test item

150

24

0.143

1-6

1340

-

0.3569

Test item

300

24

0.171

1-7

1356

-

0.2756

Positive control

40

24

3.107

34-99

1175

+

0.0003

Test item

300

48

0.129

0-4

1237

n.t.

-

- = not significant

+ = significant

n.t = not tested, as the mean micronucleus frequency was not above the vehicle control value

 

Table 7.6.2/2: Toxic symptoms in the main experiment

Toxic reactions

Hours post-treatment

1 h

2-4 h

6 h

24 h

48 h

300 mg/kg bw : reduction of spontaneous activity

14

0

0

0

0*

150 mg/kg bw : ruffled fur

1

0

0

0

-

*: data only from 7 males.

 

Table 7.6.2/3: Pre-experiments results for toxicity

Toxic reactions

Dose (mg/kg bw)

Hours post-treatment (male / female)

1 h

2-4 h

6 h

8 h

10 h

24 h

30 h

48 h

Reduction of spontaneous activity

100

0/0

0/0

0/0

0/0

0/0

0/0

0/0

0/0

1000

2/2

0/1

-

-

-

-

-

-

500

2/2

2/2

0/2

0/2

-/-

0/2

0/0

0/0

300

2/2

0/1

0/0

-/-

-/-

0/0

0/0

0/0

Abdominal position

100

0/0

0/0

0/0

0/0

0/0

0/0

0/0

0/0

1000

2/2

0/1

-

-

-

-

-

-

500

2/2

1/2

0/0

0/0

-/-

0/0

0/0

0/0

300

0/0

0/0

0/0

-/-

-/-

0/0

0/0

0/0

Apathy

100

0/0

0/0

0/0

0/0

0/0

0/0

0/0

0/0

1000

0/0

0/1

-

-

-

-

-

-

500

0/0

0/0

0/0

0/0

-/-

0/0

0/0

0/0

300

0/0

0/0

0/0

-/-

-/-

0/0

0/0

0/0

Ruffled fur

100

0/0

0/0

0/0

0/0

0/0

0/0

0/0

0/0

1000

-

-

-

-

-

-

-

-

500

0/0

1/2

1/2

1/2

-/-

1/2

1/0

1/1

300

0/0

0/1

0/1

-/-

-/-

0/0

0/0

0/0

Death

100

0/0

0/0

0/0

0/0

0/0

0/0

0/0

0/0

1000

0/0

2/2

-

-

-

-

-

-

500

0/0

0/0

0/0

0/0

-/-

0/0

0/0

0/0

300

0/0

0/0

0/0

-/-

-/-

0/0

0/0

0/0

-/- no observation made

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the reported experimental conditions, the substance did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.
Executive summary:

In an in vivo micronucleus assay, performed according to the OECD guideline No. 474 and in compliance with GLP, male NMRI mice (7/dose) were exposed to the substance (98 % pure) diluted in corn oil by a single gavage dose.

On the basis of pre-experiments data, 300 mg/kg bw was estimated to be suitable as top dose. No gender specific differences were observed, therefore, the main experiment wasperformed using males only.The following dose levels of the test item were investigated:

- 24 h preparation interval: 75, 150, and 300 mg/kg bw

- 48 h preparation interval: 300 mg/kg bw.

The volume administered orally was 10 mL/kg bw. 24 h and 48 h after a single administrationof the test item the bone marrow cells were collected for micronuclei analysis.Per animal 2000polychromatic erythrocytes (PCEs) were scored for micronuclei.

The same assay was done with the vehicle control and the positive control (Cyclophosphamide at 40 mg/kg bw).

The animals treated with 300 mg/kg bw expressed reduction of spontaneous activity 1 hour after treatment. One animal treated with 150 mg/kg bw showed ruffled fur 1 hour after treatment. All the observed effects were reversible within 2 -4 hours.

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 the substance did not have any cytotoxic properties in the bone marrow.

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 substance were below or near to the value of the vehicle control group. The positive control showed a statistically significant increase of induced micronucleus frequency.

Under the experimental conditions, the test item did not induce a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.

This study is acceptable and satisfies the requirement of the OECD 474 guideline for the cytogenicity.