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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:
12 January 2016 - 3 March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline study and performed according to GLP requirements.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
Envigo CRS GmbH
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Identification: LZ 649
Appearance: Clear colourless to yellowish liquid
Retest Date: 2 July 2016
Storage Conditions
(provided by the Sponsor): Keep refrigerated (2-8°C) and in the dark

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
Number of animals for
the pre-test: 2 males and 2 females for each pre-test
Number of animals for
the main study: 42 males for the main study
9 additional males for determination of bioavailability
Age (beginning of treatment): 6 – 10 weeks
Body weight: between 32.1 g and 40.0 g
Acclimation: At least 5 days prior to the start of dosing under test conditions after health examination.
Only animals without any visible signs of illness were used for the study.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
70 % Polyethylene glycol
Details on exposure:
Application volume: 10 mL/kg b.w.
Duration of treatment / exposure:
The animals of all dose groups, except the positive control group, were examined for acute toxic symptoms at intervals of around 0-1 h, 2-4 h, 5-6 h, 24 h, and 48 h after administration of the test item. Sampling of the bone marrow was done 24 and 48 h after treatment, respectively.
Frequency of treatment:
1
Post exposure period:
Pre-Experiment on Toxicity: 0-1 h, 2-4 h, 5-6 h, 24 h, and 48 h after administration of the test item.
Main experiment: 0-1 h, 2-4 h, 5-6 h, 24 h, and 48 h after administration of the test item.


24h + 48h
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
500 mg/kg b.w.
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg b.w.
Basis:
actual ingested
Remarks:
Doses / Concentrations:
2000 mg/kg b.w.
Basis:
actual ingested
Remarks:
Doses / Concentrations:
Positive control 40 mg/kg b.w. Cyclophosphamide
Basis:
actual ingested
No. of animals per sex per dose:
Number of animals for the pre-test: 2 males and 2 females for each pre-test
Number of animals for the main study: 42 males for the main study
Main study: 7 males per test group
24 h preparation interval: 500, 1000, and 2000 mg/kg b.w.
48 h preparation interval: 2000 mg/kg b.w.
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide 40 mg/kg b.w.

Examinations

Tissues and cell types examined:
Tissue: bone marrow
Cell type: polychromatic erythrocytes (PCE)
Details of tissue and slide preparation:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives.
4000 polychromatic erythrocytes (PCE) per animal were analysed for micronuclei.
Evaluation criteria:
To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per total erythrocytes. The analysis was performed with coded slides.


Statistics:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test using the validated statistical program RScript Wilcoxon_2.Rnw.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
clinical signs
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
As estimated by a pre-experiment 2000 mg LZ 649 per kg b.w. (the maximum guideline-recommended dose) was suitable as highest treatment dose.Clinical symptoms in the main experiment included ruffled fur, reduced spontaneous activity and eyelid closure in some of the animals treated with the high dose of test item. The animals treated with the mid and low dose did not exhibit any clinical symptoms.The observed systemic toxicity suggests a systemic distribution of the test item. Thus, bioavailability of the test item under the tested conditions is assumed. This was additionally confirmed by analytical detection of the test item in plasma (Study Number 41502651).
The mean number of polychromatic erythrocytes was not substantially decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that the test item 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 test item were below or near to the value of the vehicle control group.

Any other information on results incl. tables

 

hours post-treatment (males)

Clinical symptoms

1

2-4

6

24

48

High dose: 2000 mg/kg b.w. (14 males at 1 to 24 h; 7 males at 48 h)

reduction of spontaneous activity

1

2

0

1

0

eyelid closure

0

 

2

0

0

0

ruffled fur

1

 

2

1

2

0

The animals treated with the vehicle control (PEG 400), or with the low and mid dose of the test item did not express any clinical symptoms.

Summary of Micronucleus Test Results:

Test Group

Dose mg/kg b.w.

Sampling time

Mean MN/4000 PCE

SD MN/4000 PCE

Range

Ratio PCE / total Ery

% ratio Vehicle

min

max

Vehicle

0

24

4.1

2.6

1

9

0.572

100.00

Dose 1

500

24

4.7

2.6

2

8

0.577

100.87

Dose 2

1000

24

3.7

1.9

1

5

0.591

103.32

Dose 3

2000

24

2.6

1.6

0

4

0.621

108.57

Positive

40

24

90.4

33.8

51

144

0.654

114.34

Dose 3

2000

48

4.0

2.2

2

8

0.546

95.45

MN= micronuclei

PCE= polychromatic erythrocytes

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test substance does not produce relevant increases of the numbers of micronuclei in polychromatic erythrocytes after in vivo treatment of mice of either sex of the test strain at doses of 500, 1000 and 2000 mg/kg b.w. Therefore, the test item is considered to be non-mutagenic in this in vivo micronucleus assay.
Executive summary:

The in vivo study was peformed to investigate the potential of the tests item to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The study was performed in year 2016 according to OECD Guideline 474 and GLP.

The observed systemic toxicity suggests a systemic distribution of the test item. Thus, bioavailability of the test item under the tested conditions is assumed. This was additionally confirmed by analytical detection of the test item in plasma (supporting phase report 41502651).

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 effects in the bone marrow.

In conclusion, in 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, the test item is considered to be non-mutagenic in this in vivo micronucleus assay.