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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:
June-July 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD guideline No. 474 and under GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Pentaerythritol, ethoxylated, esters with acrylic acid
EC Number:
500-111-9
EC Name:
Pentaerythritol, ethoxylated, esters with acrylic acid
Cas Number:
51728-26-8
Molecular formula:
(C2 H4 O)n (C2 H4 O)n (C2 H4 O)n (C2 H4 O)n C17 H20 O8
IUPAC Name:
Pentaerythritol, ethoxylated, esters with acrylic acid
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): C-SAT 070087
- Substance type: UVCB
- Physical state: almost colourless liquid
- Analytical purity: 100% active ingredient
- Lot/batch No.: S772960026
- Expiration date of the lot/batch: October 23, 2008

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Germany
- Age at study initiation: 8 - 10 weeks
- Weight at study initiation: males, mean weight 39.3 gram; females, mean weight 30.7 gram
- Assigned to test groups randomly: yes
- Housing: Makrolon Type I cages with wire mesh top
- Diet (e.g. ad libitum): pellet standard diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 plus/minus 3 degr. Celsius
- Humidity (%): 30-85%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): artifical light from 6.00 a.m. - 6.00 p.m.

IN-LIFE DATES: From: To: June 16, 2008 to July 10, 2008

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: 40% ethanol + 60% polyethylene glycol 400
- Amount of vehicle (if gavage or dermal): 10 ml/kg b.w.
Details on exposure:
Route = oral

PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test substance was formulated in 40% ethanol + 60% polyethylene glycol 400. This vehicle was chosen because of its relative non-toxicity for the animals. All animals received a single standard volume of 10 ml/kg b.w.
Duration of treatment / exposure:
After having received a single, oral dose of the test substance, the animals were examined for acute toxic symptoms at intervals at around 1 h, 2-4 h, 6 h, 24 h, 30 h and 48 h after adminstration of the test substance. Sampling of the bone marrrow was done 24 and 48 hours after treatment.
Frequency of treatment:
Once
Doses / concentrations
Remarks:
Doses / Concentrations:
437.5, 875 and 1750 mg/kg b.w.
Basis:
nominal conc.
No. of animals per sex per dose:
Six males and six females were assigned to each test group. Five males and five females per test grouo were evaluated for the occurrence of micronuclei.
Control animals:
yes, concurrent vehicle
Positive control(s):
- Postiive control: cyclophosphamide
- Route of administration: orally, once
- Doses / concentrations: 40 mg/kg b.w.

Examinations

Tissues and cell types examined:
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.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:

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

DETAILS OF SLIDE PREPARATION:

METHOD OF ANALYSIS:

OTHER:
Evaluation criteria:
The study is considered valid as the following criteria are met:
- the negative controls are in the range of the historical control data of the laboratory
- the positive controls are in the range of the historical data of the laboratory
- at least 5 animals per group and sex can be evaluated
- PCE to erythrocyte ratio should not be less than 20% of the negative control

A tes substance is considered to be mutagenic is this assay if it induces a dose-related increase or a clear increase in the number of polychromatic erythrocytes in a single dose group.
Statistics:
The non-parametric Mann-Whitney test will be used as an aid in evaluating the results.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF FIRST RANGE-FINDING STUDY
- Dose range: 2000 mg/kg (oral, single dose)
- Number of animals used; 2 males and 2 females
- Clinical signs of toxicity in test animals:
* reduction of spontaneous activity up to 6 hours after dosing
* ruffled fur up to 30 h after dosing
* one male died at 24 h after dosing

RESULTS OF SECOND RANGE-FINDING STUDY
- Dose range: 1750 mg/kg (oral, single dose)
- Number of animals used; 2 males and 2 females
- Clinical signs of toxicity in test animals:
* reduction of spontaneous activity up to 6 hours after dosing
* ruffled fur up to 30 h after dosing
* no deaths within 48 hours after dosing

RESULTS OF MAIN STUDY
- Induction of micronuclei (for Micronucleus assay): no statisitically significant or biologically relevant increase in the frequency of micronuclei at any preparation interval and dose level after administration of the test substance.
- Ratio of PCE/NCE (for Micronucleus assay): The mean number of PCEs was not decreased after treatment with the test substance as compared to the vehicle control indicating that the test substance did not have cytotoxic properties in the bone marrow.
- Statistical evaluation: no statistically significant increase in the frequency of micronuclei

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the experimental conditions reported, the test substance did not induce micronuclei in the bone marrow of the mouse. Therefore, the test substance is considered to be non-mutagenic in the micronucleus assay.
Executive summary:

The study was performed to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test substance was formulated in 40% ethanol and 60% PEG 400 which was also used as vehicle control. The volume administerred orally was 10 ml/kg b.w.

24 and 48 h after a single administration of the test substance, the bone marrow cells were collected for micronuclei analyis. Ten animals (5 males, 5 females) per test group were evaluated for the occurence of micronuclei. At least 2000 polychlromatic erythrocytes (PCEs) per animal were scored for micronuclei. Cytotoxicity was determined by the ratio between polychromatic and normochromatic erythroycytes and was reported as number of PCES per 2000 erythrocytes.

The following dose levels of the test substance were investigated:

- 24 h interval: 437.5, 875 and 1750 mg/kg bw

- 48 h interval: 1750 mg/kg bw

The mean number of PCEs was not decreased after treatment with the test substance as compared to the vehicle control indicating that the test substance did not have cytotoxic properties in the bone marrow.

The test substance did not cause a statisitically significant or biologically relevant increase in the frequency of micronuclei at any preparation interval and dose level after administration of the test substance. The mean values of micronuclei observed after treatment with the test substance were below or near the value of the vehicle control group.

The positive control, cyclophosphamide, showed a statistically significant increase in the frequency of micronuclei.

In conclusion, under the experimental conditions reported, the test substance did not induce micronuclei in the bone marrow of the mouse. Therefore, the test substances is considered to be non-mutagenic in the micronucleus assay.