N,N'-bis(1,3-dimethylbutylidene)-2,2'-iminobis(ethylamine)

Acute Toxicity: dermal

Administrative data

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Meets generally accepted scientific standards, well documented and acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Initial Submission: Letter submitting two enclosed irritation studies and one enclosed toxicity study on Epon Curing Agent H-1 with attatchments

Author:

US EPA

Year:

1991

Bibliographic source:

OTS0534688; New Doc ID: 88-920000220; Old Doc ID: 8EHQ-1191-1584

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

yes

Remarks:

some animals were abraded, occlusive coverage of the application site

GLP compliance:

not specified

Test type:

standard acute method

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-bis(1,3-dimethylbutylidene)-2,2'-iminobis(ethylamine)

EC Number:

234-205-3

EC Name:

N,N'-bis(1,3-dimethylbutylidene)-2,2'-iminobis(ethylamine)

Cas Number:

10595-60-5

Molecular formula:

C16H33N3

IUPAC Name:

N-(1,3-dimethylbutylidene)-N'-{2-[(1,3-dimethylbutylidene)amino]ethyl}ethane-1,2-diamine

Details on test material:

- Name of test material (as cited in study report): Epon Curring Agent H-1
- Physical state: liquid
- Analytical purity: 94 %
- Impurities (identity and concentrations):
CAS # 108-10-1 (Methyl isobutyl keton): 1.4 %
CAS # 111-40-0 [2,2'-iminodi(ethylamine)]: 4.4 %
water: 0.2 %
- Stability under test conditions: no data
- Storage condition of test material: room temperature

Test animals

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Ray Nichols Rabbitry, Lumberton Texas
- Age at study initiation: no data
- Weight at study initiation: 2.5 - 3.1 kg
- Housing: individually
- Diet: Purina Rabbit Chow, ad libitum
- Water: ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
no data

Administration / exposure

Type of coverage:

occlusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

TEST SITE
- Area of exposure: back of the trunck
- % coverage: 12 x 18 cm
- Type of wrap if used: occlusive

REMOVAL OF TEST SUBSTANCE
- Washing (if done): with warm water
- Time after start of exposure: 24 hours

Duration of exposure:

24 hours

Doses:

0, 1250 and 2500 mg/kg

No. of animals per sex per dose:

4

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: observations were made every hour for the first six hours after treatment and at least once daily thereafter for 14 days. Individual body weights were recorded just prior to treatment and daily thereafter for 14 days.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight,organ weights, histopathology, skin irritation

Statistics:

no data

Results and discussion

Effect levels

Sex:

male/female

Dose descriptor:

LD50

Effect level:

ca. 2 500 mg/kg bw

Based on:

test mat.

Mortality:

no animals died at 1250 mg/kg bw; 3/4 males and 1/4 females died at 2500 mg/kg bw.

Clinical signs:

Signs of erythema and edema were observed in both treatment groups until the end of the study. Prominent in-life observations for the control animals included diarrhea and few feces. Prominent in-life observations for animals treated with the test substance (both dose levels) included activity decrease, ataxia, diarrhea, few feces, hematuria, labored breathing, no feces, ptosis, rapid breathing, and small amount of no urine.

Body weight:

mean body weight control: day 0: 2.66 kg, day 7: 2.83 kg, day 14: 2.90 kg
mean body weight high dose group: day 0: 2.63 kg, day 7: 2.48 kg, day 14: 2.60 kg
mean body weight low dose group: day 0: 2.76 kg, day 7: 2.74 kg, day 14: 2.91 kg
Statistical analysis of the organ to body weight ratios revealed no statistically significant differences between any of the groups. Statistical analysis could not be conducted for high dose males versus control males or low dose males due to the high mortality which occurred on that group.

Gross pathology:

Gross necropsy of animals in the control group revealed diarrhea, cecum distended and filled with gas, and surface indentations on kidneys. All other tissues appeared normal.
Of the gross necropsy findings of animals in high and low dose group, those described as meaturia, discoloration of contents of intestines and urinary bladder, discoloration of kidneys and liver, discoloration of the exposure area, heart hard, spleen large, surface indentations on kidneys, testes drawn into the abdominal cavity, discoloration of the lungs, tickening or hardening of the exposure area, and red watery liquid in the abdominal cavity in the area of the kidneys, undulations of the surface of the gall bladder, and variations thereof were considered unusual findings and possibly related to treatment.

Other findings:

- Histopathology:
Results 2500 mg/kg vs. control:
Microscopic examination was performed on testicles from 4 rabbits exposed to 2500 mg/kg of the test substance applied to the skin. In two of these rabbits, the skin was abraded. A similar number of controls, two with abraded skin, were used as sham controls.
Testicles from control animals showed a somewhat lower level of spermatogenesis than expected in adult males. Also noted were a few degenerated spermatids and a greater number of giant cells than expected. Giant cell formation and degeneration of spermatids were seen only in the center of the seminiferous tubules in the area occupied by the daughter cells of secondary spermatogonia after the meiotic division. Epididymi were present on all testicles, but were of little value because most were empty.
In the exposed groups, testicles from the abraded and unabraded animals could be easily separated from the control animals and from each other. In those animals with the abraded skin, numerous giant cells were formed. These were not only more numerous than those on control testicles, they were also larger and appeared more viable. Some had in excess of twenty nuclei. Degeneration of spermatids and spermatogonia was not a prominent finding in these testicles. The testicles from the unabraded animals however, did not show giant cell formation in excess of the control animals, nor did they show a greater number of degenerated cells. But, the degenerate cells were in the area occupied by the primary spermatogonia - alon the basement membranes of the seminiferous tubules. The degenerated cells were characterized by eosinophilic cytoplasm and karyorrhexis. The difference between the abraded animals and the controls was easily discernible and unmistakeable. The difference between the unabraded and the control animals was subtle, but verified by two blind readings. In all exposed animals many normal appearing seminiferous tubules were present.
Conclusion 2500 mg/kg vs. controls:
Within the limits of the experiment, the test substance had a direct effect on spermatogenesis with dermal application at a dosage of 2500 mg/kg bw.
When placed on abraded skin, it inhibited maturation of secondary spermatogonia into maturing spermatids forming large giant cells instead. When placed on unabraded skin, it caused degeneration and cell death of a minimal number if cells along the basement membranes of the seminiferous tubules. These cells are most likely primary spermatogonia. In the unabraded animals, most areas of seminiferous tubules appear to be functioning as well as control animals.
Results 1250 mg/kg vs. control:
Microscopic examination was performed on testicles from 4 rabbits exposed to 1250 mg/kg of the test substance applied to the skin. In two of these rabbits, the skin was abraded. A similar number of controls, two with abraded skin, were used as sham controls.
Testicles from control animals showed a somewhat lower level of spermatogenesis than expected in adult males. Also noted were a few degenerated spermatids and a greater number of giant cells than expected. Giant cell formation and degeneration of spermatids were seen only in the center of the seminiferous tubules in the area occupied by the daughter cells of secondary spermatogonia after the meiotic division and the nuclei were pyknotic. Epididymi were present on all testicles, but were of little value because most were empty.
In the exposed group, the testicles from the animals with abraded skin could not be separated from the control animals on blind reading. This was attempted several times and using several criteria that are listed as findings in the appendix. The animals with unabraded skin however, could be separated from the cintrols, in animal number 1860, the number of giant cells was so much more than that found in the controls, that it could be identified. These giant cells occupied the center of the seminiferous tubules and contained many more nuclei and the nuclei appeared less pyknotic. The testicles from animal number 1779 could also be selected out by the presence of one of this type of giant cell. However, this single giant cell was not present in the other testicel and that testicle could not be separated from controls on blind reading. The morphology of these giant cells is the same that was found on the acute dermal toxicity of higher doses. However, in the higher doses previously examined, these giant cells were found only in the abraded skin animals. This is in direct contrast with this examination where they were found only the the unabraded skin animals. This difference is believed to be an indication that specificity of the findings in both the high and the low dose animals concerning giant cell formation could be artificial because of the small number of animals in the experiments. Animal number 1961 had minimal calcification of seminiferous tubules and this is considered an incidental finding.
Conclusion 1250 mg/kg vs. controls:
Testicles form animals exposed to 1250 mg/kg of the test substance, when applied to abraded skin, could not be separated from the controls on the basis of morphology of the testicles and epididymis. Those rabbits exposed to the same agent at the same dosage but on unabraded skin, could be separated from the control animals by the presence of either the number and/or the morphology of giant cells within the seminiferous tubules. It would appear that the test substance ahd an effect on the testicles of rabbits when applied to unabraded skin, but not to abraded skin. However, when this examination is correlated with previous examinations at much higher doses, this apparent effect is open to serious question.