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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 1991-04-26 to 1991-05-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1994

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): diisopropylamine
- Physical state: liquid
- Analytical purity: 99.61%
- Purity test date: 1991-02-15
- Lot/batch No.: F9010
- Expiration date of the lot/batch: 1year (until march 1992)
- Stability under test conditions: 2hours
- Storage condition of test material: stable under standard conditions
- Other: origin: La Chambre

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles river France (Saint Aubin lès Elbeuf, 76410 Cléon, France)
- Age at study initiation: 7 weeks
- Weight at study initiation: 250g (males) and 200g (females)
- Fasting period before study: no
- Housing: individually inwire mesh bottomed, stainless steel cages (Iffa Crédo, 69210 l'Arbresle, France): 369cm²x18cm
- Diet (e.g. ad libitum): ad libitumA 04 C (U.A.R., Villemoisson, 91360 Epinay sur Orge, France)
- Water (e.g. ad libitum): ad libitum tap water
- Acclimation period: 16 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-2
- Humidity (%): 40-70
- Air changes (per hr): approx. 10-11
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 10-Apr-1991 (Arrival) To: 29-May-1991 (Necropsy)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: daily

ADMINISTRATION VOLUME=5mL/kg
4doses: 0, 15, 50 and 150 mg/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of the test compound under the conditions of administration was determined prior to the initiation of the study. The identity and concentration of the compound in the vehicle were verified once while the study was in progress.
Duration of treatment / exposure:
33 days
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
15, 50 and 150 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
15 per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: range-finding study n° DDO 432
Positive control:
not appropriate

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: twice weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes twice weekly

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Days 9 and 29
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: all

- Parameters:
Red blood cells: Erythrocyte count (RBC), Hemoglobin (Hb), Mean cell volume (MCV), Packed cell volume (PCV), Mean cell. Hb conc. (MCHC), Reticulocytes
White blood cells: Platelets (PLAT), Leucocytes (WBC), Total leukocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: days 7 and 32
- Animals fasted: Yes
- How many animals: all

- Parameters: Electrolytes: Sodium, Potassium, Chloride, Calcium
Metabolites and Proteins: Albumin, A/G ratio, Cholesterol (total), Creatinine, Globulins, Glucose, Protein (total), Triglycerides, Urea
Enzymes: Alanine aminotransferase: ALT /GPT, Aspartate aminotransferase: AST /GOT, Alkaline phosphatase (ALP)

URINALYSIS: Yes
- Time schedule for collection of urine: days 2 and 29
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data

- Parameters:
Quantitative parameters: Specific gravity, pH-value
Semiquantitative parameters : Proteins, Glucose, Ketones, Bilirubin, Nitrites, Blood, Urobilinogen, Leucocytes

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Parathyroid glands, Mammary tissue, Uterine cervix, Stomach: glandular area, Intestinal contents, Cecum, Colon, Larynx, Trachea, Skin and subcutaneous tissues, Spleen, Costochondral joint, Sternum, Thymus, Heart, Tracheobronchial lymph nodes, Urinary bladder, Vagina, Prostate, Aorta, Sciatic nerve, Femur and bone marrow, Crural muscle, Pancreas, Esophagus, Stomach contents, Jejunum, Ileum, Rectum, Mesenteric lymph nodes, Salivary glands, Tongue, Skull cavity, Pituitary, Inner ear, Kidneys, Adrenals, Lungs, Ovaries, Uterine tubes, Testes, Epididymides, Seminal vesicles, Popliteal lymph nodes,
Thyroid glands, Eyes, Harderian glands.

Weight: Uterine cervix, Spleen, Thymus, Heart, Prostate, Skull cavity, Kidneys, Adrenals, Lungs, Ovaries, Uterine tubes, Testes, Seminal vesicles, Eyes, Liver, Brain.

HISTOPATHOLOGY: Yes
Electronic microscopy on liver and kidneys
Light microscopy on parathyroid glands, Mammary tissue, Stomach: glandular area, Intestinal contents, Larynx, Kidneys, Liver, Uterine cervix, Spleen, Thymus, Heart, Prostate, Lungs, Testes, Seminal vesicles, Eyes, Cecum, Colon, Trachea, Skin and subcutaneous tissues, Tracheobronchial lymph nodes, Urinary bladder, Vagina, Aorta, Sciatic nerve, Crural muscle, Pancreas, Esophagus, Stomach contents, Jejunum, Ileum, Rectum, Mesenteric lymph nodes, Salivary glands, Pituitary, Epididymides, Popliteal lymph nodes, Harderian glands, Adrenals, Ovaries, Uterine tubes, Thyroid glands, Skull cavity
Statistics:
1-way analysis, pair wise comparisons of variance (Snedecor) and student t-test

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
not examined
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:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
at 150 mg/kg 1 male (accidental death), 4 females (days 7, 23, 34 and 36)
at 150 mg/kg ptyalism, slight somnolence and piloerection (all animals); loud breathing and bloody nasal discharge (some animals)

BODY WEIGHT AND WEIGHT GAIN
At 150 mg/kg slightly to moderately decreased growth rate in males.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No treatment related effects.

HAEMATOLOGY
The following variations were imputed to treatment:
- Slight decrease in hemoglobin levels in high dose (150 mg,'kg/d) females, associated with a decrease in mean corpuscular hemoglobin concentration (MCHC), but without modification in total red blood tell count (RBC) and packed tell volume (PCV);
- Moderate incrcase in the neutrophil count from the mid-dose (50 mg/kg/d) upwards;
- Slight increase in the platelet count in high dose (150 mg/kg/d) males.
These slight changes have no toxicological relevante and are probably related to the digestive lesions observed.

CLINICAL CHEMISTRY
An increase in sodium and chloride concentration was noted from the mid-dose (50 mg/kg/d) upwards in both sexes, with a dose-related effect.
Further modifications were noted at the high dose (150 mg/kg/d): moderate incrcase in triglyceride levels in females, slight increase in potassium levels in males, and slight decrease in albumin levels as well as slight increase in choiesterol levels in both sexes.

The other variations observed were not imputed to treatment because they were either too slight (incrcased triglyceride levcls in Group 2 (50 mg/kg/d) animals, incrcased ASAT activity in Group 3 (150 mg/kg/d) males) or without any toxicological relevante (decreased urea and globulin levels and decreased alkaiine phosphatase activity in Group 3 (150 mg/kg/d) females).

URINALYSIS
A moderate decrease in sodium and chloride urinary concentration and excretion was noted in males at the high dose (150 mg/kg).
ORGAN WEIGHTS
The following modifications were observed at the high dose (150 mg/kg): increase in liver absolute and relative weight in females, decrease in kdney and heart absolute weight in males and slight decrease in thymus absolute and relative weight in both sexes.

The other variations observed were imputed to the weight loss noted in Group 3 (150 mg/kg) males (increase in the relative weight of the lungs, eyes, brain and testes).

GROSS PATHOLOGY
Macroscopic examinations performed at necropsy on the animais sacrificed at the end of the study revealed digestive lesions at the high dose (150 mg/kg/d), from the esophagus to the ileum: red spots or red areas on the gastric mucosa and irregular aspect of this mucosa, and dilatation of the esophagus and small intestine.

Macroscopic examinations performed on the five high dose (150 mg/kg/d) animals round dead during the study revealed a few digestive lesions closely similar to those observed in the animais sacrificed at the end of the study, as well as pulmonary lesions (lungs not collapsed, crackling when squeered and congested).

Microscopic:
All the organs and tissues scheduled in the protocol were examined in Group 0 and 3 animals. In view of the modifications observed and for technical reasons, the esophagus, forestomach, glandular stomach, duodenum and jejunum were examined in ail Group 1 and 2 animals.

The following changes were imputed to diisopropylamine :
Glandular stomach and forestomach:
Very slight to marked subacute or chronic gastritis, mostly fibrosing, erosive or ulcerative.
Inflammation was mainly localized in the submucosa and often associated with edema in the forestomach; it involved the mucosa and submucosa with, in a few cases, edema in the glandular area.
Necrotic and hemorrhagic foci or areas were observed also in the glandular mucosa, as well as hyperkeratose epithelial hyperplasia in the forestomach mucosa.
Gastritis (considered as significant from the slight degree of severity) and epithelial hyperpiasia were noted with a dose-related effect from 50 mg/kg, the other changes at the dose of 150 mg/kg only (epithelial hyperplasia observed isolated in one animal given 15 mg/kg/d is hardly attributable to diisopropylamine).
These changes give evidence of gastric intolerance to the test compound.

Larynx - trachea :
Erosive or ulcerative purulent subacute laryngo-tracheitis in 7 animals given 150 mg kg, (Group 3), which could also be secondary to a local intolerance.

Health deterioration changes :
- subacute or chronic thymic involution,
- subacute splenic lymphoid atrophy,
- subacute lymphoid atrophy of the popliteal or mesenteric lymph nodes,
- adreno-cortical hyperplasia with acidophilic homogeneization of the cytoplasm of the cortical cells in one case,
- decreased quantity of subcutaneous fat tissue,
- atrophy of the uterine tubes,
- muscular atrophy,
- pancreatic serous dedifferentiation.


HISTOPATHOLOGY: NON-NEOPLASTIC
at 150 mg/kg laryngotracheitis, erosive and/or ulcerative gastritis in animals found dead; erosions or ulcerations and necrotic and hemorrhagic foci in the glandular and forestomachal mucosae, erosive or ulcerative purulent subacute laryngotracheitis and changes indicative of deterioration of health in surviving animals
from 50 mg/kg on: very slight to marked subacute or chronic gastritis


Animals found dead during the study:

Five animals from Group 3 (150 mg/kg) were found dead during the study:
no. 102 M on D9,
no. 110 F on D36,
no. 111 F on D23,
no. 115 F on D7,
no. 120 F on D34.
Besides the changes previously described and particularly laryngo-tracheitis and health deterioration changes, which could account for death, these animals presented:
Changes probably of agonal nature:
- Marked hepatic congestion, centrilobular (no. 115) or panlobular (no. 120),
- Marked pulmonary congestion with alveolar edema (and sometimes perivascular) in all animals.

Other changes of littie significance, mainly:
- congestivo-hemorrhagic changes of various organs in ail animals,
- splenic extramedullary erythropoiesis, possibly related to gastritis (no. 110),
- macrophage infiltration of the popliteai (no. 102) and mesenteric (nos. 110, 111, 115) lymph nodes,
- non-steatosic panlobular hepatocyte vacuolation, probably autolytic (nos. 111, 120).

Other findings:

They are physiological or belong to the spontancous pathology of the species.
In summary, light microscopic examinations revealed:
- From 50 mg/kg upwards: very slight to marked subacute or chronic gastritis, mostly fibrosing, with hyperkeratose epithelial hyperplasia of the forestomach mucosa,
- at 150 mg kg/d: worsening of gastric changes with erosions or ulcerations and necrotic and hemorrhagic foci in the glandular and forestomach mucosae,
- erosive or ulcerative purulent subacute lary•ngo-tracheitis (not investigated at mid doses),
- changes indicating health deterioration changes,

and in animals found dead at the dose of 150 mg/kg/d, which death is imputable to disopropylamine: changes of agonal nature (hepatic congestion and pulmonary edema) and worsening of health deterioration changes.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Remarks:
Gastric irritation
Effect level:
15 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: Moderate gastric irritation
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
50 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: mortality, body weight, haematological and clinical chemistry changes

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
NOAEL(local) 15 mg/kg based on moderate gastric inflammation at 50 mg/kg.
NOAEL(systemic) 50 mg/kg based on mortality, body weight, haematological and clinical chemistry changes at 150 mg/kg
Executive summary:

The Repeated oral toxicity of Diisopropylamine (DIPA) was evaluated in male and female rats according to OECD N°407 guideline (Repeated Dose 28-Day Oral Toxicity in Rodents). DIPA was administered orally once daily to Sprague Dawley rats for 4 weeks at doses of 0, 15, 50, and 150mg/kg/d. Clinical examinations were realized twice daily. Animal weights and food intake were recorded twice weekly. Hematological analysis was realized on days -9 and 29 and biochemistry on days -7 and 32. Unrinalysis was realized on five animals per sex per group. All animals were necropsied and subjected to complete macroscopic examination.

At the high dose (150 mg/kg), 1 male died accidentally and 4 females died on days 7, 23, 34 and 36.

Treatment induced digestive lesions at 150 mg/kg/d : erosive or ulcerative purulent laryngotracheitis, gastritis with epithelial hyperplasia (from 50 mg/kg/d upwards), gastric erosion or ulceration, and haemorrhagia or necrotic areas in the mucosa. These lesions, which indicate a local irritative effect, can be related to other modifications observed during the study : ptyalism and respiratory disorders at 150 mg/kg/d,. weight loss in males at 150 mg/kg/d, neutrophilia from 50 mg/kg/d upwards, decrease in hemoglobin levels in females and slight thrombocytosis in males at 150 mg/kg/d. The other modifications imputed to treatment were : at 150 mg/kg/d, somnolence, piloerection and increase in chloride and sodium plasma concentrations in both sexes; decrease in sodium and chloride urinary, excretion in males only; slight decrease in albumin levels and increase in cholesterol levels in both sexes; increase in triglyceride and potassium levels in males; slight increase in liver weight in females, and slight decrease in thymus weight and histological lesions indicating health deterioration in both sexes (in addition to the previously described digestive lesions); at 50 mg/kg/d, increase in chloride and sodium plasma concentrations (also seen at 150 mg/kg/d). No observable effects were noted at 15 mg/kg/d. At higher doses, the main target organs belonged to the digestive tract (larynx, stomach, small intestine). Excluding the local gastro-intestinal irritation and the directly related effects, the NOAEL can be estimated to be 50 mg/kg/d.