3-aminomethyl-3,5,5-trimethylcyclohexylamine

Repeated dose toxicity: oral

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:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Reference 3

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

1981

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

3-aminomethyl-3,5,5-trimethylcyclohexylamine

EC Number:

220-666-8

EC Name:

3-aminomethyl-3,5,5-trimethylcyclohexylamine

Cas Number:

2855-13-2

Molecular formula:

C10H22N2

IUPAC Name:

3-aminomethyl-3,5,5-trimethylcyclohexylamine

Details on test material:

Isophorone diamine, purity 99 %

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

This test system is recognized by the international regulatory agencies as a suitable rodent model for studies of this type.

Sex:

male/female

Details on test animals or test system 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: Tap water at pretest and 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

Details on route of administration:

Oral, mixed with the drinking water. The water was available ad libitum. The control animals received drinking water without the test article.

Vehicle:

water

Details on oral exposure:

TEST ARTICLE PREPARATION
The test article was weighed into a glass beaker on a tared Mettler PK 300 balance and distilled water was added (g/g). The test article/drinking water mixtures were prepared daily during the first eleven days and twice weekly thereafter.

TEST ARTICLE ADMINISTRATION
METHOD
Oral, mixed with the drinking water. The water was available ad libitum. The control animals received drinking water without the test article.
Rationale: Proven efficacy for absorption of test article.

DOSE LEVELS (nominal)
Group 1, 0 mg/kg body weight/day
Group 2, 20 mg/kg body weight/day
Group 3, 60 mg/kg body weight/day
Group 4, 160 mg/kg body weight/day
Rationale: Based upon oral LD50 performed by the sponsor.

DURATION OF ACCLIMATION PERIOD
7 days

DURATION OF TREATMENT
13 weeks

VEHICLE
Tap water at pretest, distilled water during the treatment period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test article in the drinking water was performed during the first week of the study. Intercurrent sampling for concentration analyses were performed monthly. The analyses were performed in the analytical laboratories of RCC. The analyses were performed by capillary gas chromatography.
The mean concentrations in % of nominal and their standard deviations was found to be:
Group 2: 99.6 % (+/- 14.5 %)
Group 3: 94.2 % (+/- 5.6 %)
Group 4: 104.5 % (+/- 2.8 %)
The results of the stability test at room temperatureshowed that the test article was stable in drinking water for 96 hat least.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily (continuously)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control group

Doses / concentrations 2

Dose / conc.:

20 mg/kg bw/day (nominal)

Remarks:

low dose group
actual dose: 21.5 mg/kg bw/d for males; 22,6 mg/kg bw/d for females

Doses / concentrations 3

Dose / conc.:

60 mg/kg bw/day (nominal)

Remarks:

intermediate group
actual dose: 59 mg/kg bw/d for males; 62 mg/kg bw/d for females

Doses / concentrations 4

Dose / conc.:

160 mg/kg bw/day (nominal)

Remarks:

high dose group
actual dose: 150 mg/kg bw/d for males; 147 mg/kg bw/d for females

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:

OBSERVATIONS:
VIABILITY/ MORTALITY
Each animal was observed twice daily for viability.

SIGNS AND SYMPTOMS
Observations for clinical signs and symptoms of toxicity were performed twice daily. A description of any abnormality observed at any examination was recorded.

FOOD CONSUMPTION
The food consumption was recorded for 7-day periods and the mean daily food consumption calculated. These data were recorded weekly, using an on-line electronic recording system composed of a Mettler PK 4800 balance connected to a HewlettPackard 9835 desk computer.

WATER CONSUMPTION
The water consumption was recorded once weekly for a 24-hour period, using an on-line electronic recording system composed of a Mettler PK 4800 balance connected to a Hewlett-Packard 9835 desk computer.

BODY WEIGHTS
The body weight of each animal was recorded weekly, using an online electronic recording system composed of a Mettler PK 4800 balance connected to a Hewlett-Packard 9835 desk computer.

OPHTHALMOSCOPIC EXAMINATIONS
Ten animals per sex and group with the lowest identification numbers were examined for ocular abnormalities using a HeineBifocal ophthalmoscope (miraflex type) A description of any abnormality was recorded. Examinations were performed at pretest and at the end of study duration.

CLINICAL LABORATORY INVESTIGATIONS:
GENERAL
Blood samples for hematology and clinical biochemistry were collected from ten animals per group and sex with the highest identification numbers under light ether anesthesia. The animals were fasted for 18 hours before blood sampling but water was provided. Blood samples were collected from each animal between the hours of 7.00 and 8.30 a.m. to reduce biologic variation caused by circadian rhythm. Blood samples were drawn from the retro-orbital plexus.
Blood sampling, after 13 weeks November 11-12, 1985
The assays of blood parameters were performed under quality control conditions to assure reliable test results. The summary and individual tables were generated by a computer whose program limits the width of each column to 10 characters. Therefore, the names of some parameters have been abbreviated. Any nonstandard abbreviation has been defined in this section under "Parameter" in upper-case letters enclosed by parentheses. Clinical laboratory data are expressed in general accordance with the International System of Units (SI)
Remark code identification:
NV = no value
HEM = hemolytic sample

HEMATOLOGY
The following anticoagulants were used during blood collection, EDTA-K2 (hematology)
Sodium Citrate, 3.8 %. (coagulation, L10)
The following commercial reference controls were used to monitor the performance of the method:
Hematology:
Control Blood Plus - Level I (low range)
Control Blood Plus - Level II (normal range)
Control Blood Plus - Level III (high range)
(Merz & Dade AG, Duedingen/Switzerland)

Coagulation:
Ci-Trol-1 (normal range)
Ci-Trol-2 (high range)
(Merz & Dade AG, Duedingen/Switzerland)

The following methods were used to determine the values of the parameters listed:
Parameter // Method/ Instrument // Unit of measure
- Erythrocyte count (RBC) // Sysmex (TOA) CC-720 Multi-Parameter Automated Hematology Analyzer // T/ L
- Hemoglobin (HB) // Cyanmethemoglobin, Sysmex (TOA) CC-720 Multi-Parameter Automated Hematology Analyzer // mmol/ L
- Hematocrit (HCT) // Sysmex (TOA) CC-720 Multi-Parameter Automated Hematology Analyzer //L/ L
- Mean corpuscular volume (MCV) // Calculated value: HCT/RBCSysmex (TOA) CC-720 Multi Parameter Automated Hematology Analyzer // fl
- Mean corpuscular hemoglobin (MCH) // Calculated value: HB/RBC - Sysmex (TOA) CC-720 Multi Parameter Automated Hematology Analyzer // fmol
- Mean corpuscular hemoglobin concentration (MCHC) // Calculated value, HB/HCT - Sysmex (TOA) CC-720 Multi Parameter Automated Hematology Analyzer // mmol/ L
- Platelet count (PLATELETS) // Clay Adams Ultra Flo-100 Whole Blood Platelet Counter // G/ L
- Reticulocyte count (RETIC.) // Supravital staining with Brecher's New Methylene Blue solution - Manual count of 1000 erythrocytes of stained film, using a Leitz Laborlux 12 light microscope // 1
- Nucleated erythrocytes - normoblasts (NEN) // Reported as number of nucleated erythrocytes per 100 leukocytes in the differential count // NEN/100 WBC
- Total leukocyte count (WBC) // Sysmex /TOA) CC-720 MultiParameter Automated Hematology Analyzer // G/ L
- Differential leukocyte count (Diff. WBC Count) // Monocellular layer blood smears produced with a Perkin-Elmer Model 90 Coleman Uni-Smear Spinner Blood smear stained with a modified Wright's Eosin Methylene Blue solution (OMRON) using an Omron Microx AutoStainer Manual count of 100 leukocytes using a Leitz Laborlux 12 light microscope // 1 (rel.)
Cell classification:
BAND. = Band Neutrophil
SEG. = Segmented Neutrophil
ED. = Eosinophil
BASO. = Basophil
LYMPH. = Lymphocyte
MONO. = Monocyte
PLAS. = Plasma Cell
OTHER = Blast Cell (undifferentiated)
- Red cell morphology // By microscopic examination of stained blood smear. Erythrocytes that vary from the normal in size, shape and hemoglobin content, or contain greater amounts of nuclear remnants are indicated as abnormal erythrocytes, and are characterized as such // normal/ abnormal

COAGULATION:
Parameter // Method/ Instrument // Unit of measure
- Thromboplastin time (PT) // Quick's one stage method using Thromboplastin C (Merz & Dade) and a Amelung Digital Ball Coagulometer KC 10 // sec.
- Partial thromboplastin time (PTT) // Method using Actin Activated Cephaloplastin Reagent (Merz
& Dade) and a Amelung Digital Ball Coagulometer KC 10 // sec.

CLINICAL BIOCHEMISTRY
The following anticoagulant was used during blood collection, Lithium Heparin (140 U.S.P. Units). The following commercial reference controls were used to monitor the performance of the method:
Clinical Biochemistry
Seronorm (normal range)
Pathonorm L (low range)
Pathonorm H (high range)
(Nyegaard & Co., Oslo/Norway)
Moni-Trol I-E (normal range)
Moni-Trol II-E (abnormal range)
(Merz & Dade AG, Duedingen/Switzerland)
Protein Electrophoresis:
Seronorm Protein
(Nyegaard & Co., Oslo/Norway)

The following methods were used to determine the values of the
parameters listed:
Parameter // Method/ Instrument // Unit of measure
- Glucose // GOD/PAP - Greiner Selective Analyzer G-300 // mmol/ L
- Urea // Urease/Berthelot - Greiner Selective Analyzer G-300 // mmol/ L
- Creatinine // Jaffe-kinetic reaction - Greiner Selective Analyzer G-300 // µmol/ L
- Bilirubin, total (BILI. T.) // Diazotized 2,4-dichloroaniline - Greiner Selective Analyzer G-300 // µmol/ L
- Cholesterol, total (CHOLEST. T. ) // CHOO/ PAP - Greiner Selective Analyzer G-300 // mmol/ L
- Aspartate aminotransferase (ASAT (GOT)) // MOH/ NAOH coupled reaction, optimized method based on Scand. Soc. for Clin. Chem. recommendations - Greiner Selective Analyzer G-300 // µkat/ L (37 °C)
- Alanine aminotransferase (ALAT (GPT)) // LDH / NADH coupled reaction, optimized method based on Scand. Soc. for Clin. Chem. recommendations - Greiner Selective Analyzer G-300 // µkat/ L (37 °C)
- Lactate dehydrogenase (LDH) // Direct NADH coupled reaction using pyruvate as substrate (PYR --> LAC reaction), optimized method based on German Soc. for Clin. Chem. recommendations - Greiner Selective Analyzer G-300 // µkat/ L (37 °C)
- Alkaline phosphatase (ALP) // p-nitrophenylphosphate./ diethanolamine (PNPP/DEA),
optimized method based on Scand. Soc. for Clin. Chem. recommmendations - Greiner Selective Analyzer G-300 // µkat/ L (37 °C)
- Calcium // Reaction with o-cresolphthalein complexone - Gilford Analyzer, System 203 // mmol/ L
- Phosphorus // Unreduced phosphomolybdate complex without deproteinization (360 nm) - Gilford Analyzer, System 203 // mmol/ L
- Sodium // Flame photometry with internal Lithium standard - Greiner Selective Analyzer G-300 (IL-743, OEM) // mmol/ L
- Potassium // Flame photometry with internal Lithium standard - Greiner Selective Analyzer G-300 (IL-743, OEM) // mmol/ L
- Chloride // Mercuric thiocyanate - Greiner Selective Analyzer G-300 // mmol/ L
- Protein, total // Biuret end point - Greiner Selective Analyzer G-300 // g/ L
- Protein electrophoresis (PROT. ELECTROPH.) // Horizontal electrophoresis, using agarose gel film and a Bio-Rad (s) horizontal electrophoresis system (Model 1415 Horizontal Electrophoresis Cell and Model 500 Constant Power Supply), and a Beckman CDS-200 Computing Densitometer // 1 (rel.), g/ L (abs.)
Electrophoretic fractions:
ALBUMIN = Albumin
A1-GLOB. = Alpha 1-globulin
A2-GLOB. = Alpha 2-globulin
SB-GLOB. = Summ of beta globulins
G-GLOB. = Gamma globulin
Calculated value based on peak area: A/G RATIO= Albumin to Globulin ratio.

Sacrifice and pathology:

ORGAN WEIGHTS
The following organ weights of all animals necropsied after 13 weeks were recorded, Adrenals, kidneys, liver and testes.

NECROPSY AND HISTOPATHOLOGY
Necropsy of all rats was performed by RCC. Animals were killed by exsanguination following the induction of anesthesia by intraperitoneal injection of sodium pentobarbitone. Tissue samples were fixed in neutral phosphate buffered 4 % formalin, processed and embedded in paraffin wax. Sections were cut at an approximate thickness of 4 micrometers and stained with hematoxylin and eosin. Sections of the following organs and tissues were examined microscopically from all rats of groups 1 and 4 and from those which had gross lesions (number of sections per organ), Adrenal glands (2), aorta (thoracic; 1), bone (sternumi 1), bone marrow (sternum1 1), brain (3), cecum (1), colon (1), duodenum (1), epididymides (2), esophagus (1), eyes (2), Harderian glands (2), heart (1), ileum (1), jejunum (1), kidneys (2), liver (2), lungs with mainstem bronchi (2), lymph nodes (mandibular, mesenteric; 1), mammary gland (1), ovaries (2), pancreas (1), pituitary gland (1), prostate (1), rectum (1), salivary gland (mandibular, sublinguali 1), sciatic nerve (1), seminal vesicles (2), skeletal muscle (1), skin (1), spinal cord (1), spleen (1), stomach (1), testes (2), thymus (1), thyroid gland (2), tongue (1), trachea (1), urinary bladder (1), uterus (3), and all macroscopic abnormalities.

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:

The following statistical methods were used to analyze the body weights, water and food consumption, organ weights, mortality, and clinical laboratory data:
Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied for the comparison between the treated groups and the control groups.
The Steel-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
For the overall spontaneous mortality data, the Fisher's exact test for 2x2 tables was applied.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.
Individual values, means, standard deviations and statistics were rounded off before printing. For example, test statistics were calculated on the basis of exact values for means and pooled variances and then rounded off to two decimal places. Therefore, two groups may display the same printed means fot a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related sign of toxicity or clinical symptom was noted in any animal of any group.

Mortality:

no mortality observed

Description (incidence):

No animal died during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain: Lower in group 4 than in other groups (statistically significant: males -16%; females -21%)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

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).

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

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%

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No treatment-related finding was observed in any animal.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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%

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

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
- alpha-2 globulin fraction (relative -18.6% and abolute-21.4%) for group 4 males

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

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 pathological findings:

no effects observed

Description (incidence and severity):

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.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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; group 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)

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

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.

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

1

Dose descriptor:

LOAEL

Effect level:

160 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

other: histopathological alterations of kidneys

2

Dose descriptor:

NOAEL

Effect level:

59 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no treatment related adverse effects were seen in the intermediate dose group

3

Dose descriptor:

NOAEL

Effect level:

62 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no treatment related adverse effects were seen in the intermediate dose group

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 the test item 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 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 the test item 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.