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:
10-January-2000 - 22-March- 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study OECD 474, GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted July 21, 1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
N-(hydroxymethyl)methacrylamide
EC Number:
213-086-1
EC Name:
N-(hydroxymethyl)methacrylamide
Cas Number:
923-02-4
Molecular formula:
C5H9NO2
IUPAC Name:
N-(hydroxymethyl)-2-methylprop-2-enamide
Details on test material:
- Supplier: Evonik Röhm GmbH, D-64293 Darmstadt, Germany
- Expiration date of the lot/batch: no data
- Stability under test conditions: stable
- Storage condition of test material: at room temperature

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelman, D-33178, Borchen, Germany
- Age at study initiation: no data
- Weight at study initiation: approximately 21 - 32 g
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: 5 animals of identical sex per cage
- Diet: pelleted standard diet
- Water: tap water, ad libitum
- Acclimation period: minimum 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25 °C
- Humidity (%): 55 ± 10%
- 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: 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 33.3 mL/kg body weight orally.
- Concentration of test material in vehicle:
- Supplier: Fluka; SIGMA-Aldrich Vertiebs-GmbH, 82041 Deisenhofen, Germany
- Lot/batch no.: 36H0738
- Purity: no data
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test substance was formulated in CMC. All animals received a single standard volume of 33.3 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 and 48h hours
Frequency of treatment:
single dose
Doses / concentrations
Remarks:
Doses / Concentrations:
24 h: 83.9, 251.7, 839 mg/kg suspended in Carboxymethylcellulose 48 h: 839 mg/kg b.w. suspended in Carboxymethylcellulose
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
CPA; cyclophosphamide
- Supplier: SIGMA-Aldrich Vertiebs-GmbH, 82041 Deisenhofen, Germany
- Purity: > 98%
- Dissolved in: 0.9 % NaCl (physiological saline)
- Route of administration: i.p., once
- Doses / concentrations: 30 mg/kg bw
- Volume administered: 10 mL/kg bw
- Sampling time: 24 hours

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 that producing some indication of cytotoxicity (change in the ratio of polychromatic to normochromatic erythrocytes).
The volume to be administered should be compatible with physiological space available.

TREATMENT AND SAMPLING TIMES:

Treatment:
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. Ten animals, five males and five females, were treated per dose group and sampling time.
Sampling of bone marrow was done 24 and 48 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 1500 rpm for 10 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 (Merck, D-64293 Darmstadt,
Germany)/Giemsa (Merck, D-64293 Darmstadt, Germany). Cover slips were mounted with EUKITT (KINDLER, D-79108 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 Olympus microscopes with 100x oil immersion objectives. 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 normochromatic erythrocytes per 1000 erythrocytes. The analysis was performed with coded
slides.
5 animals per test group were evaluated as described.
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 PCE's
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.
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

Pre-Experiment for Toxicity:
A preliminary study on acute toxicity was performed with 3 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 1520 mg/kg bw of the test item and examined for acute toxic symptoms. At this The volume
administered was 33.3 ml/kg b.w.. At this dose one animal died within 24 h. A second dose was tested 1267 mg/kg b.w. were two animals died after 48 h. A third dose 839 mg/kg b.w. was administered where only slight toxic signs were found (reduced spontaneous activity) 1 hour after application. One animal died within 24 hours.

Any other information on results incl. tables

Summary of Results (tables please see attached document (attached background material).

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
During the study decribed and under the experimental conditions reported, N-Methylolmethacrylamide did not induce micronuclei. This was
determined by the micronucleus test with bone marrow cells of the mouse according to OECD guideline 474.
Therefore, N-Methylolmethacrylamide 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 N-Methylolmethacrylamide (purity: 52%) at a dose of 0, 83.9, 251.7 and 839 mg/kg bw. The test article was suspended in Carboxymethylcellulose. This suspending agent was used as negative control. The volume administered orally was 33.3 ml/kg b.w.. 24 h and 48 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. 2000 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.

In a pre-experiment 839 mg/kg b.w.was estimated to be the maximum attainable dose. At this dose level the animals expressed only slight toxic effects (reduced spontaneous reactivity) up to 6 hours after application of the test substance. One male animal died in the 24 h 839 mg/kg b.w. dose group of the main experiment. After treatment with the N-Methylolmethacrylamide at a dose of 839 mg/kg b.w. at 24 and 48 hours the ratio between PCEs and NCEs was affected. This indicated cytotoxic effects of N-Methylolmethacrylamide.

In comparison with the corresponding negative controls there was no enhancement in the frequency of the detected micronuclei at any preparation dose level after application of the test article. The mean values of micronuclei observed after treatment with N-Methylolmethacrylamide (0.11-0.23% all micronuclei and 0.11 - 0.21 % small micronuclei) were in the same range as

compared to the negative control groups (0.14 - 0.19% all micronuclei and 0.14 - 0.17% small micronuclei).

An appropriate reference mutagen Cyclophosphamide (30 mg/kg b.w., administered i.p).was used as positive control which showed a distinct increase of induced micronucleus frequency (percentage of cells with micronuclei was 1.04% for male and 1.29% for female mice and with small micronuclei 1.01% for male and 1.27% for female mice). 

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

Therefore, under the experimental conditions reported, N-Methylolmethacrylamide did not induce micronuclei as determined by micronucleus test in bone marrow cells of the mouse. It is considered to be non-mutagenic in micronucleus assay.