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Description of key information

In repeated dose studies, decreased body weights and water and feed consumption has been observed, and are probably related to the irritating nature of EDA and it’s high pH. Hepatocellular pleomorphism has been observed and also increased AST and ALS values.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was performed pre-GLP. No guideline was available.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
no
Remarks:
pre-GLP
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Microbiological Associates, Inc., Walkersville, MD
- Age at study initiation: 41 days
- Weight at study initiation: 101-152 g (males) 91-110 g (females)
- Fasting period before study: no info
- Housing: suspended wire-bottom-and-front stainless steel cages. Three males or 5 females were housed in each cage.
- Water (e.g. ad libitum): ad libitum
Route of administration:
oral: feed
Details on oral exposure:
New concentrations of feed were prepared weekly, with the percentage of EDA.2HCl in the diet adjusted to maintain a constant dosage level in mg/kg for each sex according to the average body weight gain and diet consumption.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A spectrophotometric method was employed for the analysis of EDA.2HCI in the diet.
Duration of treatment / exposure:
3 months
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
50 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
260 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1040 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10 animals per sex
Control animals:
yes, plain diet
Observations and examinations performed and frequency:

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION : Yes
- Time schedule for examinations: Monthly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Immediately before sacrifice
- Parameters c examined: Red and white blood cell counts, measurement of hemoglobin and mean corpuscular volume, calculation of hematocrit, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, and determination of differential white blood cell counts

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Immediately before sacrifice
- Animals fasted: No data
- How many animals:
- Parameters examined: Measurement of serum concentrations of glucose, urea nitrogen, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, total protein, albumin and creatinine.

URINALYSIS: Yes
- Time schedule for collection of urine: One week before sacrifice

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
All rats were given a complete gross necropsy examination and organ weights were recorded for the brain, liver, kidneys, spleen, heart, adrenals and
testes. Tissues were taken and fixed in 10% neutral buffered formalin. All tissues were processed for paraffin embedding, sectioned at 5 microns and stained with hematoxylin and eosin. Microscopic lesions were graded as to severity, where possible, into 4 categories (mild, moderate, marked or severe)
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No deaths or clinical signs of toxicity.

BODY WEIGHT AND WEIGHT GAIN
Body weight gain was depressed markedly for both sexes at the high dosage level

FOOD AND WATER CONSUMPTION
There was marked reduction in diet consumption in the high level dose females and a significant increase in the low level dose females. While water
consumption rates were equivalent in the male test and control animals, there was a dose-related reduction for the female test animals.

CLINICAL CHEMISTRY
Changes in clinical chemistry values included a reduction in serum glucose level and an elevation of alkaline phosphatase activity, AST and ALT activities

HAEMATOLOGY
In male rats, depression of the red blood cell counts and increased mean corpuscular volumes were seen. In the female rats, in addition to the above changes, there were also depression of hematocrit and hemoglobin values and an increase in mean corpuscular hemoglobin.

URINALYSIS
The median urinary pH at the high dosage level was lowered significantly in both sexes

ORGAN WEIGHTS
In the male rats, there was a significant reduction in liver, kidney, spleen and heart weight and a concomitant decrease in some of the relative organ weights. Similarly, in the females, there was a reduction in liver, heart, adrenal and brain weights with an decrease of relative liver weight.

PATHOLOGY
There was no dose-related gross lesions in any animal on the study. The most significant histologic changes were present in the livers of the high dosage level animals, particularly in the females. The liver changes, termed "hepatocellular pleomorphism", consisted of an increase in the size of hepatocytes and hepatocyte nuclei, increased variation in nuclear size and shape, and an increase in the number of multinucleate hepatocytes. Occasional degenerating hepatocytes were also seen.
Dose descriptor:
NOAEL
Effect level:
22 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Recalculated to EDA base
Dose descriptor:
LOAEL
Effect level:
114 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Clinical chemistry: elevation of alanine aminotransferase activity in males;  Haematology :Increased mean corpuscular volumes in females. Recalculated to EDA base.
Critical effects observed:
not specified

High dose group: Diet and water consumption significantly reduced in the high dose female rats. Significant reduction in body weight gain of both sexes in the high dose group; significant reduction in absolute weights of liver and heart (both sexes), adrenal and brain (female), kidney and spleen (male) in the high dose group; increase of relative weight of brain (both sexes), spleen and heart (female) and testis in the high dose group. Significant elevation of alkaline phosphatase activity in males and females. Significant elevation of alanine aminotransferase activity in males and females of high dose groups. Increased mean corpuscular volumes in males and females. Significant increase of mean corpuscular hemoglobin and significant depression of hematocrit and hemoglobin values; significant depression of red blood cell counts, serum glucose level and urinary pH (from 6.0 to 5.0) and significant elevation of aspartate aminotransferase activity in both sexes of the high dose group; hepatocellular pleomorphism in 7/10 female and 2/10 male (control: 0/10 of each sex) in high dose group, hepatocellular degeneration and significant increased
prevalence of tracheitis in male of the high dose group.


Intermediate dose group:  Water consumption significantly reduced in female rats. Significant elevation of alanine aminotransferase activity in males of intermediate dose groups. Increased mean corpuscular volumes in females.

Low dose group: Water consumption significantly reduced in female rats. Significant elevation of alkaline phosphatase activity in males, this effect was not noted in the intermediate dose group.

Conclusions:
The LOAEL was 250 mg/kg/day for a 13 week study in rats. Since the water consumption was only slightly decreased at 50 mg/kg/day, the No-Observed-Adverse-Effect-Level (NOAEL) was considered to be approximately 50 mg/kg/day of EDA*2HCl. Re-calculated to EDA this corresponds to 22 mg/kg/day. The LOAEL level corresponds to 114 mg/kg/day EDA.
Executive summary:

In a three month dietary study, male and female rats were fed targeted doses of 0, 50, 250 or 1000 mg/kg/day of EDA-2HCl . There were no deaths and no abnormal clinical signs noted during the study. Body weight gains were significantly decreased in the high dose group which affected a number of absolute and relative organ weights in both males and females. Water consumption was comparable to control values at all dose levels in males but was decreased in a dose-response manner in female rats at all 3 dose levels.

Slight reductions in serum glucose levels and an elevation of alkaline phosphatase, AST and ALT activities were observed in the high dose group. An elevation of ALT activity was also observed in the intermediate dose male rats. Urinary pH in the high dose group was decreased in both males and females. There were no dose-related gross lesions in any animal on the study. The most significant histopathologic lesion, hepatocellular pleomorphism, was observed primarily in the high dose female and, to a lesser extent, male rats.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
22 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Well report good quality study of its era. Klimisch 2.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: pre-GLP, no guideline
Principles of method if other than guideline:
Four exposure groups of 30 rats (15 female/15 male) were exposed to EDA vapour. Each exposure group had a control group.
GLP compliance:
no
Species:
rat
Strain:
Sherman
Sex:
male/female
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber air was passed through an acidified glassbead adsorption tube. The captured Ethylenediamine was quantified by titration.
Duration of treatment / exposure:
6 weeks
Frequency of treatment:
7 h/day, 5 days/week
No. of animals per sex per dose:
15 male and 15 female
Control animals:
yes
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Dose descriptor:
NOAEC
Effect level:
59 ppm (analytical)
Sex:
male/female
Basis for effect level:
other: clinical signs; body weight; organ weights;
Dose descriptor:
NOAEC
Effect level:
144 mg/m³ air (analytical)
Sex:
male/female
Dose descriptor:
NOAEL
Effect level:
48 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Recalculated using rat weight 0.425 kg (mean male/female) and inhalation volume 0.33 m3/7h (values from guidance document)
Dose descriptor:
LOAEC
Effect level:
132 ppm
Based on:
test mat.
Sex:
male/female
Dose descriptor:
LOAEC
Effect level:
323 mg/m³ air (analytical)
Sex:
male/female
Dose descriptor:
LOAEL
Effect level:
107 mg/kg bw/day (actual dose received)
Sex:
male/female
Critical effects observed:
not specified

132 ppm: The death of 4/30 animals was attributed to lung infection (not substance-related); slight depilation; body weight gain and relative weights of liver and kidney were not affected; no substance-related macroscopic or histologic changes.

225 ppm: The death of 16/30 was substance-related and another 10 death were considered not to be substance-related; the 4 rats which survived showed significantly lower weight gain and higher relative weights of liver and kidney; cloudy swelling of the liver and of the loop and convoluted tubules of the kidney; lung congestion was observed in exposed as well as in control rats in similar proportions.
484 ppm: All rats died within 20 days of initial exposure; depilation was first observed on the 6th day of exposure; cloudy swelling of the liver (in 23/28 animals), cloudy swelling and degeneration of convoluted tubules (in 7/28 animals); congestion of the lung (in 17/28 animals) and of the adrenal cortex (in 5/28 animals).
59 ppm: No effect on weight gain or organ weights. No depilation. No significant damage to examined tissues; lung, liver and kidney.

Conclusions:
NOAEC in a subacute inhalation study was 144 mg/m³ air .
Executive summary:

In a 6 week study four exposure groups of 30 Sherman rats (15 female/15 male) were exposed to EDA vapour 7 h /day for 5 days per week. Each exposure group had a control group. The NOAEC was 144 mg/m³ air

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
144 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
Some what limited study of 6 week duration, full details are not available. in the publication.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
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.
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
Species:
mouse
Strain:
C3H
Sex:
male
Details on test animals or test system 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
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
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
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.
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
Critical effects observed:
not specified

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.

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

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
8 mg/kg bw/day
Study duration:
chronic
Species:
mouse
Quality of whole database:
Study limited by single dose level but did include positive control. Study was aimed at comparing two samples of ethylene diamine with different impurity profiles. Didi demonstrate no adverse effecst at highest none irritant dose.

Additional information

There are three oral repeat dose studies, the key study being a reasonable modern 90 day dietary study, with Ethylenediamine dihydrochloride. A NOAEL was determined at 22mg/kg/day after adjustment to the dose level as Ethylenediamine base. There is also a 2 year cancer bioassay which had a NOAEL of 9mg/kg/day but this may have been higher as the next dose level was 45mg/kg/day as Ethylenediamine base. The third oral study was investigated specific effects on the eyes at significantly higher dose with no NOAEL. There is a very old 6 week inhalation study which established a NOAEC of 144 mg/m3 and dermal study which had a NOAEL at the only dose tested of 8mg/kg/day.

 

Based on this the key value is the 22mg/kg day NOAEL from the 90 day dietary study.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
90 day study equivalent to OECD408 but predates the guideline.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Only available repeat exposure inhalation study, old stdy be sufficient details reported. Klimisch 2.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver

Repeated dose toxicity: inhalation - systemic effects (target organ) respiratory: lung

Justification for classification or non-classification

The classification for repeat dose toxicity for a 90 day study is based of serious adverse systemic effects seen at dose levels of 100mg/kg or less. In the 90 day study the only serious probably none reversible effects were seen at the highest dose level of 456mg/kg/day with relatively minor effects at 114mg/kg/day as ethylendiamine base. As the top dose where the serious adverse effects were seen was well above the 100mg/kg upper limit for a classification of STOT (Specific Target Organ Toxicity) at Category 2. Based on this, no classification for repeat dose toxicity if required under the EU CLP and GHS criteria.