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:
February - September 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: "the Guidelines for the Testing of Chemicals (2004)" in China 474
Deviations:
not specified
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-iminobis[4,6-diamino-1,3,5-triazine]
EC Number:
222-695-1
EC Name:
2,2'-iminobis[4,6-diamino-1,3,5-triazine]
Cas Number:
3576-88-3
Molecular formula:
C6H9N11
IUPAC Name:
2-N-(diamino-1,3,5-triazin-2-yl)-1,3,5-triazine-2,4,6-triamine
Details on test material:
- Name of test material (as cited in study report): 2,2'-iminobis[4,6-diamino-1,3,5-triazine]
CAS No.: 3576-88-3
Supplier: DSM Engineering Plastics
Physical state: Off-white Powder
Sample storage: At normal room temperature and in a dry place

Test animals

Species:
mouse
Strain:
not specified
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Shanghai SILAIKE Experimental Animal Co. Ltd.
- Weight at study initiation: 25-30g
- Assigned to test groups randomly: yes, under following basis: by body weight, half of each sex.
- Housing: plastic feeding cages (L46 xW31 xH20cm) and Cages were replaced twice weekly.
- Diet: ad libitum; Commercial diet was provided by Suzhou Shuangshi Laboratory Animal Feed Science and Technology LTD.
- Water: ad libitum; Commercial water


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25°C
- Humidity (%): 40-70%
- Photoperiod (hrs dark / hrs light): Lighting was artificial, and a 12h light/dark cycle.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: 1 % CMC (carboxymethyl cellulose)

Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Sample preparation was following the dosage design to weigh the test sample of all groups. Then
5000mg, 2500mg and 1250mg of the test sample were weighed and 1% sodium carboxymethylcellulose
were added to 20ml and mixed extensively as the test sample.
Duration of treatment / exposure:
Double oral gavages (24h's interval).
Dose volume 0.4mL/20g bw.
20h after the 2nd gavage, mice were executed by cervical dislocation.
Frequency of treatment:
double oral gavages
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
5000 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
2500 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1250 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0 mg/kg
Basis:

No. of animals per sex per dose:
5 groups by body weight, 10 for each group, half of each sex
Control animals:
yes
Positive control(s):
cyclophosphamide

Examinations

Tissues and cell types examined:
Femur marrow
polychromatic erythrocytes (PCE)
normal chromatic erythrocytes (NCE)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: acute oral LD50 of male and female mice

DETAILS OF SLIDE PREPARATION: Femur marrow was fetched out and mixed with bovine serum.
Slides were smeared, fixed and stained by Giemsa's solution.

METHOD OF ANALYSIS: rate of cell containing micronucleus of the dosage groups was
analysed by U-test of Poisson distribution
Evaluation criteria:
Observed under the microscope, 2000 polychromatic erythrocytes (PCE) of each mouse were counted and number
of PCE containing micronucleus was recorded, and then rate of cell containing micronucleus
was calculated as per mil. As 200 polychromatic erythrocytes (PCE) were counted, normal
chromatic erythrocytes (NCE) were also counted to obtain the value of PCE/RBC.
Statistics:
U test of Poisson distribution

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

For the Table "Result of the Mice Erythrocyte Micronucleus Test of 2,2' -iminobis[4,6-diamino-1,3,5-triazine] (CAS No.:3576-88-3)" see attachment.

Historical Data:

Historical data of this lab: the ratio of negative control was 0 per mil - 4per mil, the ratio of positive control was 21 per mil - 34 per mil.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Mammalian Erythrocyte Micronucleus Test of 2,2'-iminobis[4,6-diamino-1,3,5-triazine] is negative.
Executive summary:

Summary

According to the test protocol of the Guidelines for the Testing of Chemicals (2004) 474 of National

Environment Protection Agency, Mammalian Erythrocyte Micronucleus Test of

2,2'-iminobis[4,6-diamino-1,3,5-triazine] (CAS No.: 3576-88-3) has been performed.

Mice were divided randomly into 5 groups by body weights, 10 for each group, half of each sex.

The acute oral LD50 of male and female mice were all greater than 5000mg/kg. Then 5000mg/kg was set as

the highest dose group for this test, 1/2 LD50 and 1/4 LD50 were middle dosage group and low dosage

group respectively, namely 5000mg/kg, 2500 mg/kg, 1250mg/kg, a negative control group (1% sodium

carboxymethylcellulose), and a positive control group of cyclophosphamide.

The test sample of various doses, the positive control and the negative control was administered to the mice by oral gavages twice

daily (24h's interval). All mice were weighed and gavage volume was 0.4ml/20g bw. 20h after the 2nd

gavage, mice were executed by cervical dislocation.

Femur marrow was fetched out and mixed with bovine serum.

Slides were smeared, fixed and stained by Giemsa's solution. Observed under the

microscope, 2000 polychromatic erythrocytes (PCE) of each mouse were counted and the number of

PCE containing micronucleus was recorded, and then rate of cell containing micronucleus was

calculated as per mil. As 200 polychromatic erythrocytes (PCE) were counted, normal chromatic

erythrocytes (NCE) were also counted to obtain the value of PCE/RBC. Compared with the negative

group, rate of cell containing micronucleus of the dosage groups was analyzed by U test of Poisson

distribution.

Result and conclusion:

Mammalian Erythrocyte Micronucleus Test of 2,2'-iminobis[4,6-diamino-1,3,5-triazine] (CAS No.: 3576-88-3) is negative.