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:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Standard NTP protocol, according to international standad procedures.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: according to standard NTP test protocol for Micronucleus test
Principles of method if other than guideline:
Bone marrow assays

Rat and mouse bone marrow micronucleus tests typically employ 1 to 3 treatments of the chemical under study; treatments are administered at 24 hr.intervals, and there are normally 5 male animals per treatment group. Doses extend up to the maximum tolerated dose. The route of administration in these short-term tests is usually either intraperitoneal injection or oral gavage. Based on the cell cycle and maturation times of the erythrocytes,
harvesting of the bone marrow usually occurs 24 hours after the last dosing; this interval is indicated in the data tables as "sample collection time." At that time, about 50% of the erythrocytes in the bone marrow are immature, newly formed erythrocytes, and these are the cell types that are checked
for presence of micronuclei. The animals are euthanized by CO2 inhalation and the femurs are removed.

The bone marrow is flushed from the femurs and spread onto slides. The slides are air-dried, fixed, and stained with a fluorescent DNA-specific stain that easily illuminates any micronuclei that may be present. Typically, 2000 polychromatic erythrocytes (PCEs, reticulocytes; immature erythrocytes) are scored per animal for frequency of micronucleated cells in each of 5 animals per dose group. In addition, the percentage of PCEs among the total erythrocyte population in the bone marrow is scored for each dose group as an indicator of chemical-induced toxicity. In non-treated healthy mice
and rats, the %PCE in bone marrow is usually around 50-60%. If a chemical interferes with the production of erythrocytes in the bone marrow, then the %PCE in the bone marrow may decline from the typical normal level. Conversely, if erythrocyte production is stimulated by chemical exposure, then a higher percentage of immature erythrocytes may be observed.

As part of these bone marrow micronucleus tests measuring induction of chromosomal changes after short-term exposures to potentially mutagenic agents, peripheral blood samples are sometimes taken, usually about 48 hr after treatment. This sample collection time is based on cell cycle and
erythrocyte maturation data, as well as the timing of erythrocyte translocation from the bone marrow compartment to the peripheral circulation.
Thus, in these instances, although micronuclei are induced in erythrocytes in the bone marrow, it is the circulating erythrocyte population that is
analyzed. In these cases, 2000 PCE are analyzed for frequency of micronucleated cells, as described above, but 1000 erythrocytes are scored for
determination of %PCE, since the percentage of PCE in blood is only around 3-5% in a healthy animal.
GLP compliance:
not specified
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:
- Name of test material (as cited in study report): 2-Propenamide, N-(3-(dimethylamino)propyl)-2-methyl-
- Substance type: organic
- Physical state at room temperature: liquid
- Analytical purity: no data
- Isomers composition: not applicable
- Purity test date: no data
- Lot/batch No.: no data
- Expiration date of the lot/batch: no data
- Stability under test conditions: Stability in water: Several days at room temperature, refrigerated and in the freezer
- Storage condition of test material: no data

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
male
Details on test animals and environmental conditions:
No data.

Administration / exposure

Route of administration:
oral: gavage
Details on exposure:
PREPARATION OF DOSING: No data


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:
Sample collection time (time after the last dosing): 24 hours
Frequency of treatment:
GAV x 4, 4 Days
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 250.25, 500.5, 1000.1 mg/kg
Basis:
no data
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s):
- Route of administration: no data
- Doses / concentrations: 25.25 mg/kg

Examinations

Tissues and cell types examined:
Erythrocytes in the bone marrow of the femurs.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Doses extend up to the maximum tolerated dose.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Based on the cell cycle and maturation times of the erythrocytes, harvesting of the bone marrow usually occurs 24 hours after the last dosing; this
interval is indicated in the data tables as "sample collection time." At that time, about 50% of the erythrocytes in the bone marrow are immature,
newly formed erythrocytes, and these are the cell types that are checked for presence of micronuclei. The animals are euthanized by CO2
inhalation and the femurs are removed.

DETAILS OF SLIDE PREPARATION:
The bone marrow is flushed from the femurs and spread onto slides. The slides are air-dried, fixed, and stained with a fluorescent DNA-specific stain that easily illuminates any micronuclei that may be present.

METHOD OF ANALYSIS:
Typically, 2000 polychromatic erythrocytes (PCEs, reticulocytes; immature erythrocytes) are scored per animal for frequency of micronucleated cells
in each of 5 animals per dose group. In addition, the percentage of PCEs among the total erythrocyte population in the bone marrow is scored for
each dose group as an indicator of chemical-induced toxicity. In non-treated healthy mice and rats, the %PCE in bone marrow is usually around
50-60%. If a chemical interferes with the production of erythrocytes in the bone marrow, then the %PCE in the bone marrow may decline from the
typical normal level. Conversely, if erythrocyte production is stimulated by chemical exposure, then a higher percentage of immature erythrocytes
may be observed.
Evaluation criteria:
Data are typically presented as the mean number of micronucleated cells per 1,000 cells for each treatment group. A positive trend test is one in
which the P value is equal to or less than 0.025. For the micronucleus frequency in any dose group to be considered significantly elevated over the
control group, the P value must be equal to or less than 0.025 divided by the number of chemical-treatment groups. Thus, if the number of treated
groups is 3, then the required pairwise P value is 0.008. This adjustment in the pairwise P value is a correction for multiple comparisons of the same
data. In the short-term studies, tests that give positive results are repeated to confirm the response.

Factors that must be considered in analyzing micronucleus test data include number of animals per dose group (a minimum of 3 is required), dose
levels and number of doses administered, route of administration, tissue and cell type analyzed, sample time (interval between last dosing and
harvesting of cells for analysis), frequencies of micronucleated cells in the negative and positive controls, and the results of the statistical analyses.
The final conclusion for a micronucleus test is determined by considering the results of statistical analyses, the reproducibility of any observed
effects, and the magnitude and biological significance of those effects.
Statistics:
A formal statistical analysis of the data is performed that includes a trend test, to determine if there is an overall increase across all doses in the
frequency of cells containing micronuclei, and a pairwise comparison of each dose group to the corresponding control, to see if any one dose group
is statistically different from the control group in frequency of micronucleated cells.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
At 1000.1 mg/kg 2/5 male mice died.
Vehicle controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Peripheral Blood

Study ID Result Route
Male Female
F89655 Negative Not Tested Gavage

Start Date Sample Collection Time Sex Dosing Regimen
10/17/2006 24 Hours Male GAV x 4, 4 Days
  Dose
mg/kg
Animal
Number
Polychromatic Erythrocytes
Trend P = 0.098
Normochromatic Erythrocytes
Trend P = 0.008
No. Examined Total MN Cells Percent PCE MN Cells
per 1000
No. Examined Total MN Cells Percent NCE MN Cells
per 1000
Vehicle Control SALN
 0     
1 20000 66 2.0 3.3 997553 1288 98.0 1.3
 
 0     
2 20000 46 2.1 2.3 938193 1175 97.9 1.3
 
 0     
3 20000 46 2.3 2.3 844520 1139 97.7 1.3
 
 0     
4 20000 52 1.9 2.6 1031948 1488 98.1 1.4
 
 0     
5 20000 40 1.8 2.0 1089692 1413 98.2 1.3
Average ± SEM           2.0 ± 0.1 2.5 ± 0.2     98.0 ± 0.1 1.3 ± 0.0
 
Test Chemical
250.25     
10 20000 48 2.4 2.4 825749 1085 97.6 1.3
 
250.25     
6 20000 31 2.1 1.6 945000 1276 97.9 1.4
 
250.25     
7 20000 45 2.0 2.3 971457 1269 98.0 1.3
 
250.25     
8 20000 38 2.2 1.9 871415 1097 97.8 1.3
 
250.25     
9 20000 43 2.0 2.2 980310 1142 98.0 1.2
Average ± SEM           2.1 ± 0.1 2.1 ± 0.1     97.9 ± 0.1 1.3 ± 0.0
            Pairwise P: 0.927     Pairwise P: 0.880
 
Test Chemical
500.5     
11 20000 45 1.8 2.3 1104561 1417 98.2 1.3
 
500.5     
12 20000 47 2.0 2.4 964836 1269 98.0 1.3
 
500.5     
13 20000 54 2.3 2.7 838304 1122 97.7 1.3
 
500.5     
14 20000 60 2.3 3.0 860037 1114 97.7 1.3
 
500.5     
15 20000 54 2.3 2.7 852628 1107 97.7 1.3
Average ± SEM           2.1 ± 0.1 2.6 ± 0.1     97.9 ± 0.1 1.3 ± 0.0
            Pairwise P: 0.380     Pairwise P: 0.698
 
Test Chemical
1000.1     
16 20000 79 1.7 4.0 1142650 1746 98.3 1.5
 
1000.1     
17 20000 40 1.8 2.0 1071652 1481 98.2 1.4
 
1000.1     
18d 0 0 0 0.0 0 0 0 0.0
 
1000.1     
19d 0 0 0 0.0 0 0 0 0.0
 
1000.1     
20 20000 50 2.0 2.5 987243 1356 98.0 1.4
Average ± SEM           1.8 ± 0.1 2.8 ± 0.6     98.2 ± 0.1 1.4 ± 0.1
            Pairwise P: 0.205     Pairwise P: 0.013
 
Positive Control CPA
25.25    
21 20000 370 1.3 18.5 1542690 2236 98.7 1.4
 
25.25     
22 20000 488 0.2 24.4 10459899 17103 99.8 1.6
 
25.25     
23 20000 327 0.8 16.4 2547208 4126 99.2 1.6
 
25.25     
24 20000 425 0.8 21.3 2584665 4476 99.2 1.7
 
25.25     
25 20000 363 0.8 18.2 2469715 4302 99.2 1.7
Average ± SEM           0.8 ± 0.2 19.7 ± 1.4     99.2 ± 0.2 1.6 ± 0.1
 

d= dead

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative
In conclusion, it can be stated that during the decribed mutagenicity test and under the experimental conditions reported, the test article did not
induce gene mutations in the erythrocytes of the bone marrow tested.
Therefore, N-Dimethylaminopropyl methacrylamide has to be judged as nonmutagenic up to 500 mg/kg according to the Micronucleus test results.
Executive summary:

In a B6C3F1 mouse bone marrow micronucleus assay (standard NTP protocol), (number of anmials: 5 per dose group. male only) were treated by oral gavage with 2 -Propenamide, N-(3 -(dimethylamino)propyl)-2 -methyl- (Purity: no data) at doses of 0, 250.25, 500.5, 1000.1mg/kg bw.  Bone marrow cells were harvested at 24 hr after last dosing.  The vehicle was SALN. The mice were exposed by gavage x 4, 4 days, treatments were administed at 24 hr. intervals.

 

There were signs of toxicity at the highest dose group (1000.1 mg/kg) during the study. 2/5 test animals die

Dimethylaminopropyl methacrylate was tested at an adequate dose based on maximum tolerated dose. 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 acceptable.  This study satisfies the requirement for bone marrow micronucleus assay (standard NTP protocol) for in vivo cytogenetic mutagenicity data.