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:
09-July-1989 - 31-Aug.- 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD 474, GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

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:
Pre-Experiment for Toxicity
A preliminary study on acute toxicity was performed with 2 animals per sex under identical conditions as in the mutagenicity study concerning:
starvation period, animal strain; vehicle; route, frequency, and volume of administration.
The animals were treated orally with a single dose of 5000 mg/kg bw in 1% CMC with the test item and examined for acute toxic symptoms.
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
- Supplier: Evonik Röhm GmbH, D-64293 Darmstadt, Germany

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL Tierfarm, Füllinsdorf, Switzerland
- Number of animals. 84 (42 males/42 females)
- Age at study initiation: minimum 11 weeks
- Weight at study initiation: approximately 30 g
- Assigned to test groups randomly: yes
- Fasting period before study: 18 hours before treatment
- Housing: single
- Diet: pelleted standard diet,ad libitum
- Water: tap water, ad libitum
- Acclimation period: minimum 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ±3°C
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs artifical light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: 1% CMC (carboxymethyl cellulose)
- Justification for choice of solvent/vehicle: vehicle was chosen to its relative non-toxicity for animals. All animals received a single standard volume
of 10 mL/kg body weight orally.
- Concentration of test material in vehicle:
- Supplier: Fluka; SIGMA-Aldrich Vertiebs-GmbH, 82041 Deisenhofen, Germany
- Lot/batch no.: no data
- Catalogue no.: 21902
- Purity: no data
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test substance was formulated in 1% CMC. All animals received a single standard volume of 10 ml/kg body weight
orally.

DIET PREPARATION
- Rate of preparation of diet (frequency): no data
- Mixing appropriate amounts with (Type of food): no data
- Storage temperature of food: no data
Duration of treatment / exposure:
24h, 48h and 72 hours
Frequency of treatment:
single dose
Doses / concentrations
Remarks:
Doses / Concentrations:
5000 mg/kg suspended in Carboxymethylcellulose (1 %)
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
CPA; cyclophosphamide
- Supplier: SERVA, Heidelberg, Germany
- Purity: commercial grade
- Dissolved in: physiological saline
- Route of administration: orally, once
- Doses / concentrations: 40 mg/kg bw
Volume administered: 10 mL/kg bw

The stability of CPA at room temperature is sufficient. At 20 °C only 1% of CPA is hydrolysed per day in aqueous solution.

Examinations

Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or
the highest dose that can be formulated and administered reproducibly.
The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 72
hours.
The volume to be administered should be compatible with physiological space available.

TREATMENT AND SAMPLING TIMES:

Treatment:
Approximately 18 hours before treatment the animals received no food but water ad libitum. At the beginning of the treatment the animals (including
the controls) were weighed and the individual volume to be administered was adjusted to the animals body weight. The animals received the test item, the vehicle or the positive control substance once. Twelve animals, six males and six females, were treated per dose group and sampling time.
Sampling of bone marrow was done 24, 48 and 72 hours after treatment, respectively.


DETAILS OF SLIDE PREPARATION:

Preparation of the Animals:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal
calf serum, using a syringe. The cell suspension was centrifuged at 1000 rpm for 5 minutes and the supernatant was discarded. A small
drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (Kindler, D-79110 Freiburg,
Germany/Giemsa. Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg, Germany). At least one slide was made from each bone
marrow sample.

METHOD OF ANALYSIS:
Analysis of Cells:
Evaluation of the slides was performed using NlKON microscopes with 100x oil immersion objectives. At least 1000 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 normochromatic erythrocytes per 1000 erythrocytes. The analysis was performed with coded
slides.
Ten animals (5 males, 5 females) per test group were evaluated as described. The remaining animal of each test group was evaluated in case an
animal had died in its test group spontaneously or due to gavage error.
Evaluation criteria:
A test article is classified as mutagenic if it induces either a statistically significant dose-related increase in the number of micronucleated
polychromatic erythrocytes or a reproducible statistically significant positive response for at least one of the test points.
A test article producing neither a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes nor a
statistically significant and reproducible positive response at anyone of the test points is considered nonmutagenic in this system.
This can be confirmed by means of the nonparametric Mann-Whitney test.
However, both biological and statistical significance should be considered together.
mntire
Statistics:
Statistical methods (nonparametric Mann-Whitney test) will be used as an aid in evaluating the results. However, the primary point of consideration
is the biological relevance of the results.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY (see Remarks on results including tables and figures)

In a pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 5000 mg/kg b.w., Dodecylmethacrylate
suspended in CMC (1%). The volume administered was 10 ml/kg b.w..
All treated animals expressed toxic reactions: reduction of spontaneous activity, eyelid closure and apathy. One female expressed pilo erection.

Any other information on results incl. tables

Summary of results test group                     dose mg/kg bw              sampling              PCEs with              range                            PCE/NCE                                                                                time (hours)        micronuclei        ------------------------------------------------------------------------------------------------------------------------------ suspending agent                     0                            24                     0.09%                 0 - 2                           1000 / 404                        test article                            5000                         24                    0.06%                  0 - 3                           1000 / 511 cyclophosphamide                40                           24                    1.06%                 3 - 27                           1000 /935         suspending agent                     0                            48                     0.14%                  1 - 4                           1000 / 688                      test article                            5000                          48                     0.03%                  0 - 2                          1000 / 900     suspending agent                     0                            72                      0.09%                 0 - 3                           1000 / 676                      test article                            5000                          72                      0.07%               0 - 2                           1000 / 670 -------------------------------------------------------------------------------------------------------------------------------

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative
During the study decribed and under the experimental conditions reported, Dodecylmethacrylate did not induce micronuclei. This was determined by the micronucleus test with bone marrow cells of the mouse (OECD guideline 474).
Therefore, Dodecylmethacrylate is considered to be non-mutagenic in this micronucleus assay.
Executive summary:

In a NMRI mouse bone marrow micronucleus assay, 5 animals/male/female/dose were treated orally with Dodecylmethacrylate (98%, stabilizied) at a dose of 0, 5000 mg/kg bw. The test article was suspended in Carboxymethylcellulose (1%). This suspending agent was used as negative control. The volume administered orally was 10 ml/kg b.w.. 24 h, 48 h and 72 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE.

The following dose level of the test article was investigated:

24 h, 48 h, and 72 h preparation interval: 5000 mg/kg b.w..

In a pre-experiment this dose level was estimated to be the maximum attainable dose. The animals expressed toxic reactions. After treatment with the test article the ratio between PCEs and NCEs was not affected as compared to the corresponding negative controls thus indicating no cytotoxic effects.

In comparison with the corresponding negative controls there was no enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article.

An appropriate reference mutagen was used as positive control which showed a distinct increase of induced micronucleus frequency. 

This study is classified as accceptable. This study satisfies the requirement for Test Guideline OECD 474 for in vivo cytogenetic mutagenicity data.

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