N,N-dimethylacrylamide

Genetic toxicity: in vivo

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

GLP compliance:

yes (incl. QA statement)

Remarks:

Harlan Cytotest Cell Research GmbH (Harlan CCR), In den Leppsteinswiesen 19, 64380 Rossdorf, Germany

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

N,N-dimethylacrylamide

EC Number:

220-237-5

EC Name:

N,N-dimethylacrylamide

Cas Number:

2680-03-7

Molecular formula:

C5H9NO

IUPAC Name:

N,N-dimethylacrylamide

Details on test material:

- Name of test material (as cited in study report): N,N-Dimethylacrylamide
- Test Item No.: 11/0599 - 2
- Physical state: colorless to yellowish, liquid
- Analytical purity: 99.04%
- Lot/batch No.: 20120615
- Storage condition of test material: At room temperature, protected from light

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: 8 - 11 weeks
- Weight at study initiation: 35.2 g (males); 28.6 g (females)
- Assigned to test groups randomly: yes
- Housing: single in Makrolon Type II/III cages, with wire mesh top (EHRET GmbH, 79302 Emmendingen, Germany) and granulated soft wood bedding (Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg, Germany)
- Diet: pelleted standard diet, ad libitum (Harlan Laboratories B.V.; Postbus 6174; 5960 AD Horst; The Netherlands)
- Water: tap water, ad libitum, (Gemeindewerke, 64380 Rossdorf, Germany)
- Acclimation period: minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 30 - 65
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle/solvent used: sterile water
- Justification for choice of solvent/vehicle: The vehicle was chosen due to its relative non-toxicity for the animals. Moreover, stability of the test item (different batch) in drinking water over a period of 7 days at room temperature was shown in a separate analytical study prior to start of the current study.
- Amount of vehicle: 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test item was dissolved in sterile water.

DOSING:
At the beginning of the treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animal’s body weight (10 ml/kg body weight). The animals received the test item, the vehicle, or the positive control substance once orally.

Duration of treatment / exposure:

single administration

Frequency of treatment:

once

Post exposure period:

24 or 48 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
Males: 100, 200, 400 mg/kg bw at 24 h, 400 mg/kg bw at 48 h; Females: 50, 100, 200 mg/kg bw at 24 h, 200 mg/kg bw atr 48 h
Basis:
actual ingested

No. of animals per sex per dose:

6 (test groups)
5 (negative and positive control groups)

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: orally
- Doses / concentrations: 40 mg/kg bw (dissolved in sterile water)

Examinations

Tissues and cell types examined:

bone marrow cells

Details of tissue and slide preparation:

Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.
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 (Merck, 64293 Darmstadt, Germany)/Giemsa (Merck, 64293 Darmstadt, Germany). Cover slips were mounted with EUKITT (Kindler, 79110 Freiburg, Germany). At least one slide was made from each bone marrow sample.
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. Per animal 2000 polychromatic erythrocytes (PCE) were analysed for micronuclei. To investigate a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.

Evaluation criteria:

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.
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 methods (nonparametric Mann-Whitney test) are used as an aid in evaluating the results, if necessary. However, the primary point of consideration is the biological relevance of the results.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

reduced spontaneous activity, eyelid closure and/or ruffled fur

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

PRELIMINARY EXPERIMENT:
Two animals/dose were treated orally (males 200, 400 or 800 mg/kg bw; females 200 or 400 mg/kg bw) and examined for acute toxic symptoms up to about 48 h after dosing. Based on the results (clinical signs of toxicity and mortality: both males given 800 mg/kg died, one female given 400 mg/kg died) dose levels of 400 and 200 mg/kg bw were estimated to be suitable for males and females, respecively. As gender specific differences in toxicity were observed, both sexes were used in the main study.

MAIN STUDY:
Cytotoxicity:
After treatment with the test item at the 24h and 48h preparation interval the number of PCEs per 2000 erythrocytes was not substantially decreased as compared to the mean value of PCEs per 2000 erythrocytes of the vehicle control thus indicating that the substance did not induce cytotoxic effects in the bone marrow.

Micronuclei:
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 substance were below or very close to the value of the vehicle control group and all values in dose groups were very well within the historical vehicle control data range of the laboratory.

Positive control:
40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

Any other information on results incl. tables

Males

 

Test group	Dose (mg/kg b.w.)	Sampling time (h)	PCEs with micronuclei (%)	Range	PCE per 2000 erythrocytes
Vehicle control	0	24	0.140	1 -       5	1129
Test item	100	24	0.075	0 -       5	1113
Test item	200	24	0.075	0 -       3	1188
Test item	400	24	0.058	0 -       3	1161
Positive control	40	24	1.960	26 -     52	1140
Vehicle	0	48	0.130	0 -       6	1184
Test item	400	48	0.133	1 -       5	1132

 

Females

 

Test group	Dose (mg/kg b.w.)	Sampling time (h)	PCEs with micronuclei (%)	Range	PCE per 2000 erythrocytes
Vehicle control	0	24	0.070	0 -       3	1214
Test item	50	24	0.092	0 -       4	1199
Test item	100	24	0.125	0 -       6	1188
Test item	200	24	0.083	0 -       3	1183
Positive control	40	24	1.600	24 -     42	1146
Vehicle	0	48	0.120	1 -       5	1175
Test item	200	48	0.117	0 -       4	1126