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Toxicological information

Genetic toxicity: in vivo

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in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study; study run comparable to regulatory guidelines.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
equivalent or similar to guideline
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1,4-Benzenediamine, N,N'-mixed Ph and tolyl derivs.
EC Number:
EC Name:
1,4-Benzenediamine, N,N'-mixed Ph and tolyl derivs.
Cas Number:
Molecular formula:
Reaction mass of N-phenyl,N'-o-tolyl-phenylene diamine, N,N'-diphenyl-p-phenylene diamine and N,N'-di-o-tolyl-phenylene diamine

Test animals

Details on test animals or test system and environmental conditions:
Mice were obtained from Charles River Labs, Massachusetts. Mice were held in quarantine for 25 days prior to study initiation at which time, the mice were approximately 10 wks of age. Males weighed 27-44 g, and females 27-35 g. Mice were randomly assigned to one of five study groups, 5 males & 5 females per group. Holding rooms were maintained at 22 +/-degrees C, and 12 hr light/12 hr dark cycle. Relative humidity was 55 +/- 15%. Water & diet (Harlan Teklad Rodent Diet) was provided ad libitum. A maximum of 5 mice were housed in wire mesh cages throughout study.

The in-life phase was conducted from 1993-05-11 to 1993-06-17.

Administration / exposure

Route of administration:
The dosing solvent was mixture of DMSO & corn oil, approximately 1:6 ratio. This produced dosing suspensions of DAPD with concentrations of test material of 25, 125, & 250 mg/10 ml. Delivered dosing volumes was 10 ml/kg.
Details on exposure:
A single intraperitoneal dose was administered at a volume of 10 ml/kg BW. Delivered doses were 250, 1250 & 2500 mg/kg for test animals. Negative controls received solvent only
Duration of treatment / exposure:
Single exposures administered.
Frequency of treatment:
One dose of control and test substance was administered.
Post exposure period:
At each dose level, mice were grouped to provide 5 males, 5 females for sampling at 3 time periods - 24, 48, & 72 hrs post dosing.
No. of animals per sex per dose:
Five mice per sex per dose.
Positive control(s):
Triethylenemelamine (TEM) was administered at dose of 0.5 mg/kg in saline solution.


Tissues and cell types examined:
Bone marrow:
Erythropoietic cells were removed from the femur marrow canal of mice at sacrifice. Cells with micronuclei were graded as polychromatic MPCEs) or normochromatic (MNCEs) erythrocytes.
Details of tissue and slide preparation:
At sacrifice, marrow cells were removed from femurs by perfusing bone channel with fetal bovine serum. Collected cells were concentrated with centrifugation followed by splacing a drop of the cell suspension onto a glass slide. Smears were dried, dipped in methanol, and air-dried. Staining was performed with a Modified Wrights Stain Pak containing polychrome methylene blue-eosin. Scoring of slides were performed in a blind design.

Cytogenetic analysis: an erythrocyte containing one or more micronuclei was consider a micronucleated erythrocyte. 1000 PCEs per mouse were scored for presence of MPCEs as well as # of MNCEs contained within the 1000-PCE field. Data were then expressed as number & % of MPCEs for 10,000 PCEs per group.
Evaluation criteria:
An acceptable study resulted when (a) frequency of MPCEs for negative control is <0.25%, (b) this value for positive control group is statistically increased (p<0.05) compared to negative control values, and (c) a minimum of 7 mice per dose level survive to be scored. A depression in the PCE/NCE ratio indicates a toxic response by a test chemical.
A positive response is when there is increase in incidence of MPCEs in the test chemical group(s) compared to negative controls as determined by a one-tailed t-test.

Results and discussion

Additional information on results:
A rangefinding study was conducted under same conditions as described for definitive study. Dose levels were administered to 4 mice per group at doses from 250 to 5000 mg/kg BW. One of 4 mice died at the 5000 mg/kg dose. Mice also exhibited writhing, piloerection, ptosis & decreased activity at this dose level over a 72 hr observation period. Based upon this finding, doses used in definitive study were 250, 1250 & 2500 mg/kg.

In the definitive study, 2 deaths were observed in the 1250 mg/kg group, and 3 deaths in the 2500 mg/kg group. Piloerection, abnormal stance, and writhing were noted in all dose groups, but in a dose-related trend. Results of the erythrocytes evaluations for micronuclei are presented in table below.

Any other information on results incl. tables

DAPD - Induction of Micronuclei in Erythrocytes in vivo
Compound Dose - mg/kg Sac. Time (hr) # Mice MPCEs-%(S.D.)* t-Test PCE/NCE Ratio
Solvent 10 mL/kg 24 10 0.040 (0.052)  - 0.972
DAPD 250 24 10 0.050 (0.053) NS 0.977
" 1250 24 10 0.060 (0.052) NS 0.948
" 2500 24 10 0.060 (0.070) NS 1.024
TEM 0.5 24 10 2.540 (1.213) p<0.01 1.035
Solvent 10 mL/kg 48 10 0.040 (0.052)  - 1.293
DAPD 250 48 10 0.050 (0.070) NS 1.323
" 1250 48 10 0.075 (0.046) NS 1.141
" 2500 48 10 0.050 (0.071) NS 0.906 (p<0.01)
Solvent 10 mL/kg 72 10 0.090 (0.074)  - 0.91
DAPD 250 72 10 0.060 (0.070) NS 0.97
" 1250 72 10 0.040 (0.052) NS 0.843
" 2500 72 10 0.057 (0.079) NS 1.256

* Micronucleated polychromatic erythrocytes - percentage compared to total

polychromatic erythrocytes

Applicant's summary and conclusion

Interpretation of results (migrated information): negative
DAPD was not found to possess clastogenic activity in this mouse erythropoietic cell in vivo assay for micronuclei.
Executive summary:

DAPD was administered to mice by intraperitoneal injections at doses that included toxic responses (lethality, behavioural effects, and decreased PCE/NCE ratio). Erythrocytes were isolated from the femur marrow at periods up to 72 hr post dosing. These cells were assessed for evidence of micronuclei induction in PCEs with an increase seen only for the positive control TEM. DAPD was not found to possess clastogenic activity in this mouse erythropoietic cell in vivo assay for micronuclei.