1,4-Benzenediamine, N,N'-mixed Ph and tolyl derivs.

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 study; study run comparable to regulatory guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1994

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

1,4-Benzenediamine, N,N'-mixed Ph and tolyl derivs.

EC Number:

273-227-8

EC Name:

1,4-Benzenediamine, N,N'-mixed Ph and tolyl derivs.

Cas Number:

68953-84-4

Molecular formula:

C18-20H16-20N2

IUPAC Name:

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

Species:

mouse

Strain:

CD-1

Sex:

male/female

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:

intraperitoneal

Vehicle:

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.

Examinations

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.

Statistics:

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

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

only at 2500 mg/kg bw dose group at 48h time point

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

Conclusions:

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.