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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:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and OECD guideline compliant study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015
Reference Type:
study report
Title:
Unnamed
Year:
2015

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis(1,2,2,6,6-pentamethyl-4-piperidyl) [[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methyl]butylmalonate
EC Number:
264-513-3
EC Name:
Bis(1,2,2,6,6-pentamethyl-4-piperidyl) [[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methyl]butylmalonate
Cas Number:
63843-89-0
Molecular formula:
C42H72N2O5
IUPAC Name:
bis(1,2,2,6,6-pentamethylpiperidin-4-yl) butyl(3,5-di-tert-butyl-4-hydroxybenzyl)malonate
Details on test material:
- Name of test material (as cited in study report): Propanedioic acid, [[3,5-bis(1,1-dimethylethyl)-4- hydroxyphenyl]methyl]butyl-, bis(1,2,2,6,6-pentamethyl-4- piperidinyl) ester
- Physical state: solid
- Analytical purity: 99.8%
- Lot/batch No.: 0005039980
- Expiration date of the lot/batch: 21 August 2015
- Stability under test conditions: stable
- Storage condition of test material: At room temperature

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, 97633 Sulzfeld, Germany
- Age at study initiation: 6 - 10 weeks (beginning of treatment)
- Weight at study initiation: mean value (before treatment) 34.5 g (SD +/- 1.9 g) mean value (before sacrifice) 33.7 g (SD +/- 2.6 g)
- Assigned to test groups randomly: yes
- Fasting period before study: none
- Housing: single
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days prior to the start of dosing

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

IN-LIFE DATES: From: 2015-01-28 To: 2015-03-03

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Grinding of the test item in a mortar was used to formulate the test item. Homogeneity of the test item in vehicle was maintained during treatment using a magnetic stirrer. The preparations were made freshly before each dosing occasion.
Duration of treatment / exposure:
one gavage dose
Frequency of treatment:
once
Post exposure period:
24 and 48 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000 and 2000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
7
Control animals:
yes, concurrent vehicle
Positive control(s):
40 mg CPA/kg b.w.; 24 h sacrifice

Examinations

Tissues and cell types examined:
polychromatic erythrocytes (PCE) of bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Two dose-range finding study with each 2 males and 2 females

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 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 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/Giemsa. Cover slips were mounted with EUKITT. 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 4000 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 total erythrocytes. The analysis was performed with coded slides.
Seven males per test group were evaluated for the occurrence of micronuclei except for the negative and positive control groups with five animals each. Per animal 4000 polychromatic erythrocytes (PCEs) were scored for micronuclei.

OTHER: The animals of all dose groups, except the positive control, were examined for acute toxic symptoms at intervals of around 0-1 h, 2-4 h, 5-6 h, 24 h, and/or 48 h after administration of the test item.

For determination of the substance in plasma, blood samples (~0.5 mL each) of additional satellite animals were taken. For this purpose, blood sampling was performed 1 h and 4 h after a single oral application of the highest test item dose. 3 animals for each sampling time were used, resulting in 6 samples. Additionally, 3 animals were dosed with the negative control item once and their blood were withdrawn 1 h after application.
These satellite animals were allocated as follows: vehicle group: animal numbers 44 – 46, high dose group 1 h sampling: animal numbers 47 – 49, high dose group 4 h sampling: animal numbers 50 – 52.
The blood of the animals was collected in tubes containing K3-EDTA. The blood samples were centrifuged at 10’000 rpm for about 5 minutes to obtain plasma samples. The plasma samples were stored at - 65°C and were sent to the sponsor for further analysis .
Evaluation criteria:
The study is considered valid as the following criteria are met:
- at least 5 animals per group could be evaluated.
- PCE to erythrocyte ratio was not less than 20 % of the negative control.
- the positive control shows a statistically significant and biological relevant increase of micronucleated PCEs compared to the vehicle control.

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 above the laboratory’s historical solvent control data range. Statistical methods (nonparametric Mann-Whitney test) were used as an aid in evaluating the results, if necessary. 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
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw
- Clinical signs of toxicity in test animals: yes
- Rationale for exposure: Acute oral toxicity data in rats

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): see table 2
- Ratio of PCE/NCE (for Micronucleus assay): not affected
- Appropriateness of dose levels and route: appropriate
- Statistical evaluation: yes

The observed systemic toxicity at the tested doses is indicative for 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 the plasma.

Any other information on results incl. tables

Table 1: Clinical symptoms observed during the study

 

hours post-treatment (males)

Clinical symptoms

0-1

2-4

5-6

24

48

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

Eyelid closure (partially)

1

3

4

4

2

Ruffled fur

7

10

14

14

7

Reduction of spontaneous activity

2

7

7

6

4

Diarrhoea

0

0

0

3

4

Medium dose: 1000 mg/kg b.w. (7 males)

Eyelid closure (partially)

1

1

0

0

-

Ruffled fur

3

5

6

5

-

Reduction of spontaneous activity

2

3

3

2

-

Diarrhoea

0

0

0

1

-

Low dose: 500 mg/kg b.w.(7 males)

Ruffled fur

3

3

3

1

-

The animals treated with the vehicle control (corn oil) did not express any clinical symptoms.

Table 2: Summary of the micronucleus test results

Test
Group

Dose
mg/kg
b.w.

Sampling
time

Mean MN/4000 PCE

SD MN/4000 PCE

Mean
%
MN

Range

Ratio
 PCE/ total Ery

%

ratio
Vehicle

min

max

Vehicle

0

24

6.8

3.8

0.2

4

13

0.590

100.00

Dose 1

500

24

5.4

2.4

0.1

3

10

0.591

100.17

Dose 2

1000

24

4.6

1.1

0.1

3

6

0.585

99.15

Dose 3

2000

24

6.4

2.4

0.2

3

11

0.585

99.15

CPA

40

24

66.0

28.9

1.7

46

116

0.583

98.81

Vehicle

0

48

3.8

1.6

0.1

2

6

0.594

100.00

Dose 3

2000

48

4.6

4.0

0.1

0

11

0.515

86.70

Table 3: Biomentry

Negative control versus test group

Significance

p

Dose 1 - 500 mg Tinuvin 144/kg b.w.; 24 h

no

0.617

Dose 2 - 1000 mg Tinuvin 144/kg b.w.; 24 h

no

0.353

Dose 3 - 2000 mg Tinuvin 144/kg b.w.; 24 h

no

0.935

Positive Control - 40 mg CPA/kg b.w.; 24 h

yes

0.012

Dose 3 - 2000 mg Tinuvin 144/kg b.w.; 48 h

no

1.000


Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Executive summary:

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 substance were below or near to the value of the vehicle control group. 40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

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