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Repeated dose toxicity: dermal

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Administrative data

chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1984 or before
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference Type:
Dermal Oncogenicity studies on ethylenediamine in male C3H mice.
DePass LR, Fowler EH, Yang RSH
Bibliographic source:
Fundam. Appl. Toxicol. 4:641-645

Materials and methods

Test guideline
no guideline followed
Principles of method if other than guideline:
The maximum tolerated dose (for local effects) was applied onto the back of 50 male mice three times weekly throughout their lifespan. Positive (3-methylcholanthrene) and negative (water) control groups (50 mice/group) were included.
GLP compliance:
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
Substance no 1: Purity 99,91%, 0,07% ammonia
Substance no 2: Purity 99.1%;
0.54 % pyrazine,
0.08 % ammonia,
0.03 % water,
0.02 % monomethylamine,
0.02 % ethylamine,
0.02 % N-methyl-piperazine,
0.02 % methylpyrazine,
trace dimethylamine,
trace ethanol,
trace N-ethylpiperazine,
trace ethylpyrazine ,
Specific details on test material used for the study:
Two samples of ethylenediamine from different manufacturers were selected for this study, they were high purity and a detailed analysis was performed. Although the samples were very similar, EDA No. 2 contained detectable concentrations of several amines not present in EDA No. I. These analyses indicated that the samples contained a minimum of 99.1% EDA and were stable throughout the study.

Test animals

Details on test animals or test system and environmental conditions:
- Source: C3H/HeJ mice from Jackson Laboratories, Bar Harbor, Maine
- Age at study initiation: 74 to 79 days of age
- Housing: Housed individually in stainless steel cages with wire mesh floors. All mice were housed in the same room with
controlled lighting
- Diet (e.g. ad libitum): Ziegler Bros. NIH 07 pellets (Gardners, Pa.)
- Water (e.g. ad libitum): water from an automatic watering system

Administration / exposure

Details on exposure:
Mice were treated three times weekly for their complete life span with 25 µl per application of each of the two EDA substances. Substances were applied with an Eppendorf pipet to the back of each mouse from which the fur was clipped once weekly.

Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
The stability of 1% aqueous dilutions was demonstrated, and the concentrations were verified monthly during the study.
Duration of treatment / exposure:
complete life span

Frequency of treatment:
Doses / concentrations
No. of animals per sex per dose:
Treatment group singly housed: 50 mice
Control animals:
yes, concurrent vehicle
Details on study design:
Control group singly housed: 50 mice Control group housed 5/cage: 40 mice in total
Deionized water from a MilliQ reagentgrade water system (2020 11574, Millipore Corp., Bedford, Mass.) was used as the solvent and negative control substance.
Positive control:
Positive control group housed 5/cage: 40 mice in total
3-Methylcholanthrene (MC, Eastman Kodak, Rochester, N.Y.) as a 0.1% dilution in acetone (Fisher Scientific Company, Pittsburgh, Pa.) was used as the positive control substance to demonstrate the susceptibility of the mice to induction of skin tumors.


Observations and examinations performed and frequency:
All mice were examined daily, and the onset and progress of tumor growth were recorded monthly.
Sacrifice and pathology:
Ten mice from the EDA and individually housed water control groups were scheduled for sacrifice at 18 months to evaluate their tissues for possible pathologic changes. Complete necropsies were performed on all mice. The dorsal skin from all animals plus all gross lesions were examined histologically after sectioning and staining with hematoxylin and eosin. In addition, all livers, kidneys and lungs from the 18 month sacrifice were fixed for histopathologic examination.
Mantel-Cox test and Breslow test

Results and discussion

Results of examinations

mortality observed, non-treatment-related
Description (incidence):
The mean survival time of the EDA No. 2 group (598 days) was significantly (p < 0.05) shorter than that of the water controls (626 days) by the Mantel-Cox test but not by the Breslow test. The difference in these statistical results is understandable since the Breslow test gives greater weight to earlier observations. In this case, the survival curves did not differ for the first 600 days of the study. The survival comparisons included the mice sacrificed at 18 months.
The mean survival time of the group-housed water controls (488 days) was significantly reduced compared to the singly housed controls by the Breslow but not the Mantel-Cox test. Since these curves differed primarily in the first 600 days of the study, the difference was significant by the test that gave greater weight to early deaths.
Description (incidence and severity):
No treatment-related macroscopic findings.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
In the exposure group for substance 1, animals (11) had mild to moderate dermal fibrosis, suggesting skin irritation.

Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related histopathological findings; one mouse of the exposure group had a mammary adenocarcinoma. One sebaceous adenoma of the skin of the thorax was noted in the control group individually housed.
In the exposure group for substance 1, one mouse had a myxosarcoma at the base of the tail.

In the 3-methylcholanthrene group, 39 of 40 mice had skin tumors including 37 with confirmed squamous cell carcinomas.

Effect levels

open allclose all
Dose descriptor:
Effect level:
other: 25 μl of 1% solution
Based on:
test mat.
Basis for effect level:
other: Mild fibrosis suggesting irritation to skin
Dose descriptor:
Effect level:
>= 8.3 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: based on BW of male mice of 30 g

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

The dermal repeated dose NOAEL for carcinogenicity was >=8.3 mg/kg bw; none of the two EDA samples was oncogenic (carcinogenic) under the conditions of this study.
Executive summary:

The dermal oncogenic potential of ethylenediamine (EDA) was assessed by applying 25 µL of a 1% solution in deionized water to the skin of 50 male C3H/HeJ mice. This was the highest concentration not producing irritation or weight changes in a preliminary 2-week study. Two EDA samples (Nos. 1 and 2) from different production sources were tested. Applications were made three times weekly until the death of the animals. A negative control group received deionized water only. This group and the EDA-treated groups were individually housed. A fourth group of 40 mice, housed 5 per cage, received 0.1% 3-methylcholanthrene (MC) in acetone as a positive control substance. A fifth group of 40 mice, housed 5 per cage, also received deionized water to determine the effect of group housing on survival. No skin tumors were observed in the EDA-treated groups. In the positive control group, however, 39 animals (98%) had skin tumors including 37 (92%) with confirmed squamous cell carcinomas. Eleven mice (22%) which received EDA No. 1 had dermal fibrosis indicative of probable skin irritation in this group; there was no such lesion in the controls. No skin tumors were observed in the EDA treated animals.