Registration Dossier

Administrative data

Endpoint:
repeated dose toxicity: dermal
Remarks:
other: carcinogenicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No data on guideline and GLP.

Data source

Reference
Reference Type:
publication
Title:
Dermal Oncogenicity studies on ethylenediamine in male C3H mice.
Author:
DePass, L.R., Fowler, E.H. and Yang, R.S.H
Year:
1984
Bibliographic source:
Fundam. Appl. Toxicol. 4:641-645.

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The maximum tolerated dose (for local effects) was applied to 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:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Substance no 1: Purity 99,91%, 0,07% ammonia
Substance no 2: Purity 99.1%;
Impurities:
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 ,

Test animals

Species:
mouse
Strain:
C3H
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- 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

Vehicle:
water
Details on exposure:
Mice were treated three times weekly for their complete life span with 25 µl per application of each substance. 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:
yes
Details on analytical verification of doses or concentrations:
Concentrations verified monthly during the study
Duration of treatment / exposure:
complete life span

Frequency of treatment:
3x/wk
Doses / concentrations
Remarks:
Doses / Concentrations:
25 μl of 1% aqueous solution/application
Basis:

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 received distilled water. Control group housed 5/cage: 40 mice received water
Positive control:
Positive control group housed 5/cage: 40 mice received 0.1% 3-methylcholanthrene in acetone

Examinations

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.
Statistics:
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.

Results and discussion

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
other: 25μl 1% solution
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Mild fibrosis suggesting irritation to skin was noted
Dose descriptor:
NOAEL
Effect level:
8.3 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Weight of male mice 0.03 kg

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Mean survival time of the exposure group for substance 2 (598 days) was shorter than that of the singly housed control group (626 days); no treatment-related macroscopic or histopathologic findings; one mouse of the exposure group had a dermal fibrosis at application site and another one 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, 1 mouse had a myxosarcoma at the base of the tail, and 11 animals had mild to moderate dermal fibrosis, suggesting skin irritation. Survival time did not differ from negative control groups.

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

Applicant's summary and conclusion

Conclusions:
The dermal repeated dose NOAEL was 8.3 mg/kg bw.
Executive summary:

The dermal oncogenic potential of ethylenediamine (EDA) was assessed by applying 25 µ1 of a I% 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 thrice 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. In the positive control group 98% of the animals had skin tumors.

Neither Ethylenediamine sample was oncogenic (carcinogenic) to the skin or systemically under the conditions of this study.

In a dermal carcinogenicity study, the maximum tolerated dose for local effects was applied to the back of 50 male mice three times weekly throughout their lifespan. There were no treatment-related systemic macroscopic or histopathologic findings