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Repeated dose toxicity: oral

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Administrative data

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:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1983

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethylenediamine
EC Number:
203-468-6
EC Name:
Ethylenediamine
Cas Number:
107-15-3
Molecular formula:
C2H8N2
IUPAC Name:
ethane-1,2-diamine
Details on test material:
- Name of test material (as cited in study report): Ethylendiamine
- Analytical purity: 99 %
Specific details on test material used for the study:
Due to corrosivity of EDA, ethylene diammonium dichloride (ECDA.2HCl) was used (CAS no. 333-18-6; EC no. 206-369-6).

Test animals

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

Administration / exposure

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.
Actual concentrations were: 0, 50, 260 and 1040 mg/kg bw/day.
Duration of treatment / exposure:
3 months
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 animals per sex
Control animals:
yes, plain diet

Examinations

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
- 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)

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs of toxicity.
Mortality:
no mortality observed
Description (incidence):
No deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weight gain was depressed markedly for both sexes at the high dosage level.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was a marked reduction in diet consumption in the high dose females and a significant increase in the low dose females.
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
While water consumption rates were equivalent in the male test and control animals, there was a dose-related reduction for the female test animals.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
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.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Changes in clinical chemistry values included a reduction in serum glucose level and an elevation of alkaline phosphatase activity, AST and ALT activities.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
The median urinary pH at the high dosage level was significantly lower in both sexes.
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In the high dose group there was a significant reduction in absolute weight of liver and heart (both sexes), adrenal and brain (female), kidney and spleen (male) and an increase in relative weight of brain (both sexes), spleen and heart (female) and testis.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no dose-related gross lesions in any animal on the study.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The most significant histopathological changes were present in the livers of the high dose 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. Degenerating hepatocytes were also seen occasionally. Also, a significant increased prevalence of tracheitis was observed in males of the high dose group.
Histopathological findings: neoplastic:
no effects observed
Details on results:
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 males 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. Siginificant elevation of ALP activity in males; this effect was not noted in males of the intermediate dose group, as such no dose-response relationship was observed.

Effect levels

open allclose all
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: elevation of ALAT in males; increased MCV in females. Recalculated to EDA base.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The No-Observed-Adverse-Effect-Level (NOAEL) was considered to be 50 mg/kg/day of EDA*2HCl. Re-calculated to EDA this corresponds to 22 mg/kg/day.
Executive summary:

In a three month dietary study, male and female rats were fed targeted doses of 0, 50, 250 or 1000 mg/kg bw/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, ASAT and ALAT activities were observed in the high dose group. An elevation of ALAT 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 histopathological lesion, hepatocellular pleomorphism, was observed primarily in the high dose females and to a lesser extent in males.

The LOAEL was 250 mg/kg bw/day in 13 -week study in rats. Since the water consumption was only slightly decreased in females at 50 mg/kg/day, the No-Observed-Adverse-Effect-Level (NOAEL) was considered to be 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.