Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1985-08-05 - 1985-11-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1986
Report Date:
1986
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
1981
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Isophorone diamine, purity 99 %

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ORGANISMS
- Source: KFM Kleintierfarm Madoerin AG, CH
- Age: 6 weeks
- Weight at study initiation:
males 136-157 g, females 117-139 g
- Number of animals: Total 80 males, 80 females; 20 per sex and group
- Fasting period before study:
- Housing: groups of 5 in Makrolon type 4 cages
- Diet: ad libitum, Kliba 343 rat maintenance diet
- Water: Distilled water for the treatment period ad libitum
- Acclimation period: 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Humidity (%): 55 +/- 10 %
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 h/12h

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on oral exposure:
ADMINISTRATION / EXPOSURE
- Duration of test/exposure: 13 weeks
- Type of exposure: oral, drinking water
- Post exposure period: -
- Vehicle: drinking water
- Doses:
Group 1 = control;
group 2 = 20 mg/kg bw/day;
group 3 = 60 mg/kg bw/day;
group 4 = 160 mg/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration and stability analysis of isophorone diamine were performed by gas chromatography.
The results of the stability test at room temperature showed that isophorone diamine was stable in drinking water for at least 96 h.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily (continuously)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
20, 60, or 160 mg/kg bw d (nominal) = groups 2, 3, and 4
Basis:
nominal in water
Remarks:
Doses / Concentrations:
males: 0; 21.5; 59; 150 mg/kg bw per day / females: 0; 22.6; 62; 147 mg/kg bw per day
Basis:
other: mean daily dose received (calculated) over 13 weeks
No. of animals per sex per dose:
20
Control animals:
yes
Details on study design:
Post-exposure period: none
Positive control:
no positive control

Examinations

Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS AND FREQUENCY:
- Clinical signs: twice daily
- Mortality: twice daily
- Body weight: weekly
- Food consumption: weekly (7 day consumption)
- Water consumption: weekly (24 hours consumption)
- Ophthalmoscopic examination: at pretest and end of study;
10 animals per group (lowest ID numbers) and sex examined
- Haematology: end of study, 10 animals per group and sex
(highest ID numbers)
- Biochemistry: end of study, 10 animals per group and sex
(highest ID numbers)
Sacrifice and pathology:
ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC):
- Macroscopic: all organs listed below under "Microscopic";
weights of adrenals, kidneys, liver, testes
- Microscopic: (all rats of groups 1 and 4 plus those which had gross lesions): adrenal glands, aorta (thoracic), bone (sternum), bone marrow
(sternum), brain, cecum, colon, duodenum, epididymides, esophagus, eyes, Harderian glands, heart, ileum, jejunum, kidneys, liver, lungs with
mainstem bronchi, lymph nodes (mandibular, mesenteric), mammary gland, ovaries, pancreas, pituitary gland, prostate, rectum, salivary gland
(mandibular, sublingual), sciatic nerve, seminal vesicles, sekletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid gland, tongue,
trachea, urinary bladder, uterus
Other examinations:
see addendum to the pathology report:
In an attempt to further elucidate the toxicological significance of findings in the kidneys, a larger sample of renal tissue was re-examined from rats
of all groups. In addition to the already available sections, another set of histological slides was prepared from the wet tissue of rats of groups 1 and 4 which was matched by two sections from each rat of groups 2 and 3.
Statistics:
Univariate one-way analysis of variance for significance of intergroup differences;
Dunnett-test for comparison between treated groups and control group (if normal distribution assumed)
Steel-test (if normal distribution not assumed)
Fisher's exact test for spontaneous mortalities

Results and discussion

Results of examinations

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:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
NOAEL = 59 mg/kg bw d (males),
62 mg/kg bw d (females)
ACTUAL DOSE RECEIVED BY DOSE LEVEL BY SEX
males 21.5 / 59 / 150 mg/kg bw d
females 22.6 / 62 / 147 mg/kg bw d
TOXIC RESPONSE/EFFECTS BY DOSE LEVEL:
- Mortality and time to death:
No animal died during the study
- Clinical signs:
No treatment-related sign or symptom was noted.
- Body weight gain: Lower in group 4 than in other groups (statistically significant: males -16%; females -21%)
- Food/water consumption:
The mean food consumption of the group 4 animals was reduced when compared with that of the control rats (statistically significant for males at
week 9-13 and for females at week 8-10).
The mean water consumption was decreased when compared with the controls (statistically significant in group 4 animals):
Males group 2: -18.0%; group 3: -20.1%, group 4: -40.4%
Females group 2: -13.5%; group 3: -19.9%; group 4: -47.8%
- Ophthalmoscopic examination:
No treatment-related finding was observed in any animal.
- Clinical chemistry: The assessment of clinical biochemical data indicated no changes of toxicological significance. Some treatment-unrelated effects were noted and considered to be secondary. They were not supported by morphological findings. The following changes were statistically
significant:
urea level for group 4 males +40.4%
calcium level for group 3 females (-3.1%) and for group 4 males (-4.0%) and females (-7.1%)
phosphorus levels for group 3 males (-10.5%) and females (-15.4%) and for group 4 males (-18.7%) and females (-27.5%)
total protein levels for group 4 females -7.0%
albumin fraction (absolute) of the protein electrophoretic pattern for group 4 females -7.7%
alpha-1 globulin fraction (relative males -14.0%, females -10.4% and absolute males -18.1%, females -16.3%) of the protein electrophoretic pattern
for group 4 animals
alpha-2 globulin fraction (relative -18.6% and abolute-21.4%) for group 4 males
- Haematology: The assessment of hematological data indicated no changes of toxicological significance after 13 weeks of treatment. Some treatment unrelated effects were noted and considered to be secondary. They were not supported by morphological findings. The following changes were
statistically significant:
hemoglobin in group 4 females -6.2%
platelet count in group 4 males (+29.0%) and females (+12.5%)
reticulocyte count for group 4 females +46.7%
total leukocyte count for group 2 males (-20.2%), group 3 males (-20.2%) and females (-24.1%), and group 4 males (-22.9%) and females (-25.3%)
prolonged prothrombin time for group 4 females +4.7%
- Organ weights:
Kidney weights in group 4 males absolute +8.1% (not significant), relative +16.4% (statistically significant) and females absolute +13.5%, relative
+25.0% (both statistically significant)
Liver weights absolute +20.7%, relative +16.7% (both statistically significant) in group 3 males, absolute -13.8% (statistically significant), relative
-4.8 % (not significant) in group 4 females, absolute -3.3%, relative +3.6% (both insignificant) in group 4 males
Other absolute and relative organ weights were not affected significantly.
- Gross pathology: No treatment-related macroscopic findings were observed. A few spontaneous gross lesions were encountered in both control
and treated rats. Their incidence and severity are considered to be similar in all groups.
- Histopathology: In further examination of the kidneys (see addendum to the pathology report, Reference RCC 1989), isolated very small foci of
tubular atrophy were recorded in one organ only. The statistical analysis for positive trend with respect to dose rate yielded a significant result for males (Z=2.29, one-tailed P=0.01) and a negative one for females (Z=1.61, one-tailed P=0.55).
Under the conditions of the experiment, the test article produced morphological alterations in the kidneys of rats at 160 mg/kg bw/day (nominal; gr oup 4)
(see second addendum to the report, Reference RCC 2000). The findings consisted of an increased incidence in tubular basophilia (both sexes of
group 4), and tubular casts (both sexes of group 4) along with a higher incidence of lymphoid foci (both sexes of group 4). These changes are
indicative for tubular nephrosis. All findings were of minor severity degrees, but were statistically significant. The remainder of findings recorded
did not differ between controls and rats treated with the test article.
Frequency of findings treated (control)
-----------------------------------------------------------
Tubular basophilia males 17/20 (0/20), females 13/20 (6/20)
Tubular casts males 8/20 (1/20), females 11/20 (1/20)
Lymphoid foci males 16/20 (5/20), females 13/20 (4/20)
-----------------------------------------------------------
- Other: The remainder of findings recorded did not differ between controls and rats treated with the test article. They were considered to be within
the range of spontaneous background lesions which may be recorded in Wistar rats of this strain and age.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: histopathological alterations of kidneys
Dose descriptor:
LOAEL
Effect level:
160 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: histopathological alterations of kidneys
Dose descriptor:
NOAEL
Effect level:
59 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: histopathological alterations of kidneys
Dose descriptor:
NOAEL
Effect level:
62 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: histopathological alterations of kidneys
Dose descriptor:
NOAEL
Effect level:
> 160 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: effects on examined fertility organs

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

no further remarks

Applicant's summary and conclusion

Conclusions:
In this 13-week oral toxicity study according to OECD TG 408 isophorone diamine was administered in the drinking water to Wistar rats (20 animals/sex/dose) which received nominal daily doses of 0, 20, 60, and 160 mg/kg bw ( actual dose 21.5 / 59 / 150 mg/kg bw day for males, 22.6 / 62 / 147 mg/kg bw day for females). The NOAEL for isophorone diamine was 59 mg/kg bw/day for males and 62 mg/kg bw/day for females when administered orally in the drinking water to Wistar rats.
Executive summary:

In this 13-week oral toxicity study according to OECD TG 408 isophorone diamine was administered in the drinking water to Wistar rats (20 animals/sex/dose) which received nominal daily doses of 0, 20, 60, and 160 mg/kg bw ( actual dose 21.5 / 59 / 150 mg/kg bw day for males, 22.6 / 62 / 147 mg/kg bw day for females). No treatment-related clinical signs, symptoms or mortality were noted during the study. Food and water consumption and body weight gain were significantly reduced in high dose animals. In addition, animals of this group revealed higher absolute and relative liver and kidney weights. In the 60 mg/kg bw/day group there was a statistically significant decrease in total leukocyte count (-20.2 %) and increase in the absolute liver weights (+20.7 %) and the relative liver weights (+16.7 %) for males. Along with some other statistically significant hematological and clinical chemical findings in the higher dose groups, the decrease in total leukocyte count was considered to be secondary and not treatment-related, as these effects were in general not supported by morphological findings. The variations in liver weights were also considered to be not treatment related because of the lack of dose-dependency and supporting morphological findings.

However, under the conditions of the experiment, the test article produced morphological alterations in the kidneys of rats at 160 mg/kg bw/day (nominal). The findings consisted of an increased incidence in tubular basophilia (both sexes), and tubular casts (both sexes) along with a higher incidence of lymphoid foci (both sexes). These changes are indicative for tubular nephrosis and may correspond to some of the clinical chemical findings, particularly the increased urea level for high dose males (+40.4 %). All findings were of minor severity degrees, but were statistically significant. The remainder of findings recorded did not differ between controls and rats treated with the test article. They were considered to be within the range of spontaneous background lesions which may be recorded in Wistar rats of this strain and age. The NOAEL for isophorone diamine was 59 mg/kg bw/day for males and 62 mg/kg bw/day

for females when administered orally in the drinking water to Wistar rats.