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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
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04-Feb.-2003 - 08-Apr.- 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
9 th addendum to OECD Section 4, No. 474 adopted July 21, 1997
Deviations:
no
Principles of method if other than guideline:
Pre-Experiment for Toxicity
A preliminary study on acute toxicity was performed with two 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 (200, 350, 500, 1000 and 1800 mg/kg bw in 1% CMC) with the test item and examined for acute toxic symptoms at
intervals of around 1 h, 2-4 h, 6 h, 24 h, 30 h, and 48 h after administration of the test item.

Deviations from the study plan:
In the main experiment not all the animals treated with the high and mid dose (350 and 175 mg/kg bw) were observed for clinical signs of toxicity at
the 6h post treatment interval.
This deviation, however, does not affect the validity of the experiment.
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Supplier: Evonik Röhm GmbH
- Purity: 99.3% (HPLC analysis)
- Lot/batch No.: 11121211 of December 20, 2002
- Expiration date of the lot/batch: December 2003
- Stability under test conditions: at least 15 hours in aqua deionised
- Storage condition of test material: Refrigerator (+4°C), light protected

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd., 4414 Füllinsdorf, Switzerland
- Number of animals. 72 (36 males/36 females)
- Age at study initiation: Males: 5 - 7 weeks; Females: 7 - 9 weeks
- Weight at study initiation: males mean value 33.9 g (SD ± 2.2 g)
females mean value 25.6 g (SD ± 1.7 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): 22 ±3°C
- Humidity (%): 30 - 70 %
- 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 sigle 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, Methacrylamide 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 and 48h
Frequency of treatment:
single dose
Doses / concentrationsopen allclose all
Dose / conc.:
87.5 mg/kg bw/day (nominal)
Remarks:
24 h
Dose / conc.:
175 mg/kg bw/day (nominal)
Remarks:
24 h
Dose / conc.:
350 mg/kg bw/day (nominal)
Remarks:
24 h
Dose / conc.:
350 mg/kg bw/day (nominal)
Remarks:
48 h
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
CPA; cyclophosphamide
- Supplier: Fluka; SIGMA-Aldrich Vertiebs-GmbH, 82041 Deisenhofen, Germany
- Purity: > 98%
- Dissolved in: deionised water
- 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 25 °C only 3.5% of its potency is lost after 24 hours.

Historical Controls
------------------
1999 - 2001
Negative controls:
Percent micronucleated polychrornatic erythrocytes
Range:0.010 to 0.150
Mean: 0.066* ± 0.032
Positive controls:
Cyclophosphamide; 40 mglkg b.w. [CPA]
Percent micronucleated polychromatic erythrocytes
Range:0.910 to 2.975
Mean: 1.644*± 0.446
*= mean of 95 experiments

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
2000 mglkg as the upper limit for non-toxic test items.
The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48
hours.
The volume to be administered should be compatible with physiological space available.
Three adequate spaced dose levels spaced by a factor of 2 were applied at the central sampling interval 24 h after treatment. For the highest dose
level an additional sample was taken at 48 h after treatment.

The highest dose (350 mg/kg) was estimated by a pre-experiment to be suitable.


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. The
animals of the highest dose group were examined for acute toxic symptoms at intervals of around 1 h, 2-4h, 6 h and 24 h after administration of the
test item.
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 (390 x g) 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-Griinwald (MERCK, D-64293
Darmstadt, Germany)/Giemsa (Gurr, BDH Limited Poole, Great Britain). 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 2000 polychromatic erythrocytes (PCE)
were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and total erythrocytes was determined in thesame sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.
Ten animals (5 males, 5 females) per test group were evaluated as described.
Evaluation criteria:
The study was considered vailid as the following criteria are met:
- the negative controls are in the range of our historical control data (0.01 - 0.15 %; mean= 0.066 ± 0.032 PCEs with micronuclei).
- the positive controls are in the range of our historical control data (0.91 - 2.975 %; mean = 1.644 ± 0.446 PCEs with micronuclei).
- at least 80 % of animals are evaluable
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
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
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)
- Dose range: 200 - 1800 mg/kg bw

Any other information on results incl. tables

Results:  

Pre-Experiment for Toxicity In the pre-experiment 4 animals (2 males, 2 females) per dose group received orally a single dose of 1800, 1000, 500, 350 and 200 mg/kg b.w. Methacrylamide formulated in 1% CMC. The volume administered was 10 ml/kg b.w.. The animals treated with Methacrylamide expressed toxic reactions as shown in the table:

toxic

reactions

hours post-treatment (male/female) 1800 mg/kg bw

hours post-treatment (male/female) 1000 mg/kg bw

hours

1

2-4

6

24

30

48

1

2-4

6

24

30

48

Reduction of spontaneous activity

2/2

2/2

2/2

0/0

-

-

2/2

2/2

2/2

0/0

-

-

Abdominal position

1/1

2/2

2/2

0/0

-

-

0/0

1/1

1/1

0/0

-

-

Eyelid closure

2/1

2/2

2/2

0/0

-

-

2/1

2/1

2/1

0/0

-

-

Ruffled fur

2/2

2/2

2/2

0/0

-

-

2/2

2/2

2/2

0/0

-

-

Apathy

1/0

1/1

1/1

0/0

-

-

0/0

1/1

1/1

0/0

-

-

death

0/0

0/0

0/0

2/2

-

-

0/0

0/0

0/0

2/2

-

-

toxic

reactions

hours post-treatment (male/female) 500 mg/kg bw

hours post-treatment (male/female) 350 mg/kg bw

hours

1

2-4

6

24

30

48

1

2-4

6

24

30

48

Reduction of spontaneous activity

2/2

2/2

2/2

2/0

0/-

0/-

1/1

2/2

2/2

0/0

0/0

0/0

Eyelid closure

0/0

2/2

2/2

0/0

0/-

0/-

1/0

0/0

0/0

0/0

0/0

0/0

Ruffled fur

2/0

2/2

2/2

2/0

0/-

0/-

1/1

1/1

1/1

0/0

0/0

0/0

death

0/0

0/0

0/0

0/2

0/-

0/-

n.d.

n.d.

n.d.

n.d

n.d.

n.d.

toxic

reactions

hours post-treatment (male/female)

200 mg/kg bw

hours

1

2-4

6

24

30

48

Reduction of spontaneous activity

2/1

0/0

0/0

0/0

0/0

0/0

Ruffled fur

1/1

1/0

0/0

0/0

0/0

0/0

Toxic symptoms in the Main Experiment

In the main experiment for the highest dose group 24 animals (12 males, 12 females) received orally a single dose of 350 mg /kg b.w.

Methacrylamide formulated in 1% CMC. The volume administered was 10 ml/kg b.w.. The animals treated with 350 mg /kg b.w. expressed

toxic reactions as shown in the table:

Toxic reactions

hours post treatment

male/female

Hours [hr]

1

2-4

6*

24

Reduction of spontaneous activity

9/8

9/10

3/5

0/1

Abdominal position

1/0

0/0

0/0

0/0

Eyelid closure

4/4

5/7

3/5

2/1

Ruffled fur

6/1

7/5

3/3

7/1

*: at the 6h post treatment interval only 6 of the 12 animals were observed.

For the mid dose group 12 animals (6 males, 6 females) received orally a single dose of 175 mg /kg b.w. Methacrylamide formulated in

1% CMC. The volume administered was 10 mllkg b.w..

The animals treated with 175 mg /kg b.w. expressed toxic reactions as shown in the table:

Toxic reactions

hours post treatment

male/female

Hours [hr]

1

2-4

6*

24

Reduction of spontaneous activity

2/2

4/2

-*/1

0/0

Eyelid closure

2/0

2/2

-*/2

0/0

Ruffled fur

2/0

4/0

-*/0

0/0

*: at the 6h post treatment interval the 6 males were not observed.

For the low dose group 12 animals (6 males, 6 females) received orally a single dose of 87.5 mg /kg b.w. Methacrylamide formulated in

1% CMC. The volume administered was 10 mllkg b.w..

The animals treated with 87.5 mg /kg b.w. did not express any toxic reactions.

Summary of Micronucleus Test Results

Test group

Dose

mg/kg bw.

Sampling

Time [hr]

PCEs with micro-nuclei [%]

Range

PCE per 2000

erythocytes

Vehicle control versus test group

Signifi-cance

p

Vehicle

0

24

0.030

0-2

1025

n.d.

n.d.

Methacrylamide

87.5

24

0.035

0-2

1037

-

0.5000

Methacrylamide

175

24

0.040

0-2

1041

-

0.4000

Methacrylamide

350

24

0.035

0-3

1023

-

0.5000

Positive control CPA

40

24

1.220

9-46

1015

+

0.0001

Methacrylamide

350

48

0.055

0-2

1014

-

0.1356

n.d.: not determined

-: not significant; +: significant

Statistical significance at the five per cent level (p 0.05) was evaluated by means of the non-parametric Mann-Whitney test.


The mean number of polychromatic erythrocytes was not increased after treatment with the test item as compared to the mean value of polychromatic erythotrcytes (PCEs) of the vehicle control indicating that Methacrylamide had no 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 Methacrylamide 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.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative
During the study decribed and under the experimental conditions reported, Methacrylamide did not induce micronuclei. This was determined by the
micronucleus test with bone marrow cells of the mouse (OECD guideline 474).
Therefore, Methacrylamide 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 Methacrylamide (99.3%) at doses of 0, 87.5, 175 and 350 mg/kg bw. Bone marrow cells were harvested at 24 and 48 hours post-treatment. The vehicle was 1% CMC (carboxymethyl cellulose). The animals received orally a single dose of Methacrylamide.  

There were no signs of toxicity during the study. Methacrylamide was tested at an adequate dose up to 350 mg/kg bw. The positive control induced the appropriate response.  There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.

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