Diisopropylamine

Endpoint summary

Description of key information

The signs of toxicity observed after subacute exposure to diisopropylamine were mainly related to its irritant/corrosive properties. The NOAE(C)Ls were 50 mg/kg/d by oral route, >=150 mg/kg/d by dermal route and less than 0.10 mg/l by inhalation. For an analogue substance, dimethylamine, no treatment-related effects were observed in rats exposed to 16 ppm (33.6 mg/m3) for 13 weeks.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system 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)

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

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

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

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.

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

Critical effects observed:

not specified

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, protocol similar to OECD guideline 412 1. Some tables are hardly readable due to bad copying. 2. It is not specified whether the histological examination included several nasal and laryngeal sections.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Sprague Dawley Charles River Breeding laboratories (Portage, Michigan)
- Age at study initiation: 8 weeks
- Weight at study initiation: 245g for males and 174g for females
- Fasting period before study: no
- Housing: suspended individual stainless steel wire mesh cages
- Diet (e.g. ad libitum): ad libitum Ralston Purina Rodent Chow (5002), except during the exposure period
- Water (e.g. ad libitum): ad libitum Sodium zeolite conditioned tap water (St Louis City, MO), except during the exposure period
- Acclimation period: 10 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.1 to 23.3°C (70-74°F)
- Humidity (%): 35 to 60
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: To:

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 10 m3 New York University style stainless steel chambers with a pyramidal top and bottom
- Method of holding animals in test chamber: free in its cage
- Test material was metered from a pressurized tank trough a capillary restrictor to a Laskin style nebuliser, wich vaporized the test atmosphere.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

TEST ATMOSPHERE
- Brief description of analytical method used: at least 4 samples per exposure at approx. one-hour to one and one-half hour intervals. Wilks MIRAN IA GENERAL POURPOSE GAS ANALYZER ( infrared gas analyser)
- Samples taken from breathing zone: yes

Duration of treatment / exposure:

6 hours

Frequency of treatment:

5 days/week (total 23 exposures)

Remarks:

Doses / Concentrations:
100, 620 and 2000 mg/m3
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
100, 600 and 2000 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

15 M and 15 F per group

Control animals:

yes

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: pretest, then weekly and at necropsy

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

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 data

WATER CONSUMPTION: no data
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once during last week of study
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: once at study termination
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: 10 per sex per group
- total erythrocyte count, total leukocyte count, platelet count, hematocrit, hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration,

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: once at study termination
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: 10 per sex per group
- creatinine, blood urea nitrogen, glucose, albumin, total bilirubin, electrolytes (sodium, potassium, phosphorus, calcium, and chloride), globulin (calculated), glutamic pyruvic transaminase, alkaline phosphatase, total protein, glutamic oxaloacetic transaminase, cholesterol.

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC):
- Organ weights: Testes with epididymides, liver, adrenals, brain, heart, kidneys, spleen.
- Macroscopic: tissues of the thoracic, abdominal, and cranial cavities: abdominal aorta, bone and bone marrow (femur), esophagus, nasal passages, pituitary, eyes, testes with epididymides, intestine (duodenum, colon and ileum), liver, sciatic nerve, stomach, trachea, uterus including the cervix, salivary gland (submaxillary), spinal cord (thoraco-lumbar), lymph nodes (thymic and mesenteric) , lung (two sections with two lobes and mainstem bronchi), any gross lesions determined by the pathologist to be treatment-related, adrenals, brain (longitudinal section), mammary gland, pancreas, prostate, ovaries, heart, kidneys, seminal vesicles, spleen, thymus, thyroid and parathyroid, urinary bladder, skin, skeletal muscle.
- Microscopic:
*Control: males and females all tissues
*2000 mg/m3: males and females all tissues
*100 and 600 mg/m3: males and females: eyes, lung, nasal passages, , thymus, trachea +seminal vesicles for males

Statistics:

In-life body weights, haematology and serum chemistry values: Dunnett's test (two tailed) for comparing multiple treatments with a control. Bartlett's test (one-tailed) was used also on in-life body weights. Inspections of haematology and clinical chemistry data were also performed.
Terminal body weights and absolute organ weights: Analysis of Variance and Dunnett's test.
Organ to body weights ratio: Mann Whitney with Bonferroni's Inequality Modification Procedure.
Frequencies of histological lesions: Fisher's Exact Test with Bonferronni's Inequality Modification Procedure.

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):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

effects observed, treatment-related

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

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:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY
1 male and 2 females at 2000 mg/m3; one male control was sacrificed (not test related)
During exposure: non-responsiveness (inactive state) and respiratory difficulty at 600 and 2000 mg/m3, and nasal irritation at 2000 mg/m3.
Post-exposure (mainly at 2000 mg/m3): mucous membrane irritations (red-brown perinasal encrustation, blood-like nasal discharge and periorbital encrustation), respiratory difficulties (rattling sounds, sneezing, labored breathing, gasping and rapid breathing), non-responsiveness, and spontaneous deaths.
At 600 mg/m3 a few occurrences of perinasal and periorbital encrustation and sneezing, with one case also at 100 mg/m3.

BODY WEIGHT AND WEIGHT GAIN
Decreased gain at 600 mg/m3 and decreased bod weight at 2000 mg/m3

OPHTHALMOSCOPIC EXAMINATION
corneal lesions in approximately 100, 75, and 15% of the high, mid, and low exposure level animals, respectively

HAEMATOLOGY
total erythrocyte count, hemoglobin and hematocrit were increased at 2000 and 600 mg/m3 (only females), significant except for hematocrit in females at 2000 mg/m3; mean corposcular volume was significantly decreased in females at 2000 mg/m3; significantly reduced white blood cell counts in males at all exposure levels;

CLINICAL CHEMISTRY
the following serum chemistry measurements were significantly decreased for the various female exposure groups when compared to controls: chloride (mid, high), total protein (high), albumin (high), and creatinine (mid) (considered not treatment related). The following serum chemistry measurements were significantly increased for the various exposure groups when compared to controls: alkaline phosphatase (low and high exposure males, high exposure females), SGT (high exposure females), cholesterol (mid exposure females and high exposure males) and sodium (high exposure females, not considered biologically relevant), serum cholesterol (high exposure females, not significant)

ORGAN WEIGHTS
the following serum chemistry measurements were significantly decreased for the various female exposure groups when compared to controls: chloride (mid, high), total protein (high), albumin (high), and creatinine (mid) (considered not treatment related). The following serum chemistry measurements were significantly increased for the various exposure groups when compared to controls: alkaline phosphatase (low and high exposure males, high exposure females), SGT (high exposure females), cholesterol (mid exposure females and high exposure males) and sodium (high exposure females, not considered biologically relevant), serum cholesterol (high exposure females, not significant)

GROSS PATHOLOGY
at 2000 mg/m3: enlarged adrenals (also in females at 600 mg/m3), atrophic and/or reduced spleen, seminal vesicles and thymus, gaseous distension of GI tract

HISTOPATHOLOGY: NON-NEOPLASTIC
at 2000 mg/m3: inflammation of the eye (cornea, anterior uvea, anterior chamber), corneal hyperplasia, acute necrosis of the iris; inflammation of the lung (bronchi, alveoli, peribronchi) hyperplasia/metaplasia of bronchiolar epithelium, interstitial pneumonia, granuloma/microgranuloma; inflammation of the nasal turbinates and trachea with hyperplasia/metaplasia of mucous epithelium (also seen at 100 and 600 mg/m3 for nasal turbinates); atrophy of seminal vesicular epithelium, depletion of secretory product of seminal vesicles; depletion of thymocytes;

OTHER FINDINGS
- target, analytical and nominal levels were resp. 100, 100 and 100; 600, 600 and 620, 2000, 2000 and 2000 mg/m3 - Stability: ok - Homogeneity: >96%

Dose descriptor:

LOAEC

Remarks:

eye, nasal and lung irritation

Effect level:

100 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: local effects on cornea, lung and nasal passages

Dose descriptor:

LOAEC

Remarks:

systemic toxicity

Effect level:

100 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: decreased body weight and changes in blood parameters

Critical effects observed:

not specified

Conclusions:

Since lymphocytopenia in males and lesions in the cornea and nasal passages of both sexes occurred at the lowest exposure level (100 mg/m3 in air), a "no-effect level" for DIPA could not be determined in this study.

Executive summary:

The repeated inhalation toxicity of Diisopropylamine (DIPA) was evaluated in male and female rats in a protocol similar to OECD N°412 guideline (Repeated Dose 28/14-Day inhalation Toxicity in Rodents) (Val Roloff, 1987). Sprague-Dawley rats (15/sex/concentration) were administered DIPA as a vapor at 0, 0.1, 0.6 and 2 mg/L via whole body inhalation for 6 hours per day, 5 days per week for approximately one month (maximum of 23 exposures). Rats were observed for morality twice daily and for clinical signs and body weights once a week. Ophthalmic exams were given once during the last week of the study. Hematology and clinical chemistry measurements were made. Rats were necropsied and all examined for gross pathology. Organ weights were determined for adrenals, brain, heart, kidneys, liver, spleen, and testes with epididymides. Histopathology was conducted on a full set of organs in the control and at the highest concentration, whereas eyes, lung, nasal passages, seminal vesicles, thymus and trachea were examined at all concentrations.  

Two females and one male were sacrificed at the highest concentration. In addition, one control was also sacrificed (due to an unrelated injury). During exposure, rats exhibited non-responsiveness, respiratory difficulty (middle and high-exposure rats), and nasal irritation at the highest concentration. Post-exposure observations included mucous membrane irritation (encrustation), respiratory difficulties, nonresponsiveness. At the middle concentration, a few perinasal and periorbital encrustations and episodes of sneezing occurred. One low-exposure rat had perinasal encrustation. Body weight reductions ranged from 9.6 to 40.6% at the end of the study; the differences at the high and mid-exposure levels were statistically significant from controls (p<0.05 or 0.01). Corneal lesions were seen in 13%, 75%, and 100% of rats at 0.1, 0.6 and 2.0 mg/L. At the end of the study, red blood cells, hemoglobin and hematocrit values were increased 9-11% (p<0.01) in the males at 2.0 mg/L. The same values were increased in females at 0.6 and 2.0 mg/L as well but the increase in hematocrit was not statistically significant. Mean corpuscular volume was significantly decreased (p<0.05) in the high-exposure females compared with controls while decreased white blood cell counts were seen in all exposed males (p<0.01). Chloride, total protein, and albumin and creatinine were decreased at 2.0 mg/L in females, whereas chloride and creatinine were decreased at 0.6 mg/L (p<0.05 or 0.01) compared with controls. Increases in alkaline phosphatase, SGPT, cholesterol and sodium were observed at various concentrations in males and/or females (p<0.05 or 0.01). Absolute and relative adrenal weights were increased at 2.0 mg/L. Relative spleen weights were decreased at the highest concentration, whereas increases in relative weights of brain, heart, kidneys, liver, and testes were observed (p<0.05). Decreases in several absolute weights of organs were also seen.  

At 2.0 mg/L, several statistically significant changes from controls were seen upon histopathological examination: bronchiolitis/peribronchiolitis was seen in 7 males (p < 0.01) and an additional 3 females; and lung hyperplasia/metaplasia of bronchiolar epithelium in 24 rats (p < 0.01). In the nose, hyperplasia was seen in 26, 30 and 30 rats at 0.1, 0.6 and 2.0 mg/L respectively (p < 0.01); also, necrosis and dissolution of the turbinate septum, cartilage, and bone was seen at 0.6 and 2.0 mg/L (p < 0.01); other nasal effects were of higher incidence than controls (p < 0.01). At 2.0 mg/L, the trachea exhibited inflammation (26) and hyperplasia/metaplasia (29) and mineralization (19) (p < 0.01), with 2 rats showing erosion and ulceration of the tracheal mucosa. Thymocytes were depleted in 16 rats at 2.0 mg/L (p < 0.01 in females). 

At 2.0 mg/L, additional microscopic lesions not seen in controls included cell detritus in epididymal ducts (2 males); eye problems (inflammation of the cornea (8), anterior uvea (3), and anterior chamber (2)); autolysis of the ileum (2); hepatocellular necrosis and degeneration (3); interstitial pneumonia (6), lymphoid hyperplasia (1); adrenal vacuolation (2); and additional lung effects (various numbers). Two 0.6 mg/L rats also had interstitial pneumonia. At 2.0 mg/L, the seminal vesicles of 11 rats showed secretory product depletion vs. 2 in controls (p < 0.01); 6 rats showed atrophy of the seminal vesicular epithelium vs. 2 in controls; and autolysis of the seminal vesicles occurred in one rat. The LOAEC was determined to be 0.1 mg/L based on nasal and ocular corneal lesions and moderately reduced lymphocyte counts (males only).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

100 mg/m³

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, protocol similar to OECD guideline 412 1. Some tables are hardly readable due to bad copying. 2. It is not specified whether the histological examination included several nasal and laryngeal sections.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Sprague Dawley Charles River Breeding laboratories (Portage, Michigan)
- Age at study initiation: 8 weeks
- Weight at study initiation: 245g for males and 174g for females
- Fasting period before study: no
- Housing: suspended individual stainless steel wire mesh cages
- Diet (e.g. ad libitum): ad libitum Ralston Purina Rodent Chow (5002), except during the exposure period
- Water (e.g. ad libitum): ad libitum Sodium zeolite conditioned tap water (St Louis City, MO), except during the exposure period
- Acclimation period: 10 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.1 to 23.3°C (70-74°F)
- Humidity (%): 35 to 60
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: To:

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 10 m3 New York University style stainless steel chambers with a pyramidal top and bottom
- Method of holding animals in test chamber: free in its cage
- Test material was metered from a pressurized tank trough a capillary restrictor to a Laskin style nebuliser, wich vaporized the test atmosphere.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

TEST ATMOSPHERE
- Brief description of analytical method used: at least 4 samples per exposure at approx. one-hour to one and one-half hour intervals. Wilks MIRAN IA GENERAL POURPOSE GAS ANALYZER ( infrared gas analyser)
- Samples taken from breathing zone: yes

Duration of treatment / exposure:

6 hours

Frequency of treatment:

5 days/week (total 23 exposures)

Remarks:

Doses / Concentrations:
100, 620 and 2000 mg/m3
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
100, 600 and 2000 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

15 M and 15 F per group

Control animals:

yes

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: pretest, then weekly and at necropsy

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

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 data

WATER CONSUMPTION: no data
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once during last week of study
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: once at study termination
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: 10 per sex per group
- total erythrocyte count, total leukocyte count, platelet count, hematocrit, hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration,

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: once at study termination
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes
- How many animals: 10 per sex per group
- creatinine, blood urea nitrogen, glucose, albumin, total bilirubin, electrolytes (sodium, potassium, phosphorus, calcium, and chloride), globulin (calculated), glutamic pyruvic transaminase, alkaline phosphatase, total protein, glutamic oxaloacetic transaminase, cholesterol.

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC):
- Organ weights: Testes with epididymides, liver, adrenals, brain, heart, kidneys, spleen.
- Macroscopic: tissues of the thoracic, abdominal, and cranial cavities: abdominal aorta, bone and bone marrow (femur), esophagus, nasal passages, pituitary, eyes, testes with epididymides, intestine (duodenum, colon and ileum), liver, sciatic nerve, stomach, trachea, uterus including the cervix, salivary gland (submaxillary), spinal cord (thoraco-lumbar), lymph nodes (thymic and mesenteric) , lung (two sections with two lobes and mainstem bronchi), any gross lesions determined by the pathologist to be treatment-related, adrenals, brain (longitudinal section), mammary gland, pancreas, prostate, ovaries, heart, kidneys, seminal vesicles, spleen, thymus, thyroid and parathyroid, urinary bladder, skin, skeletal muscle.
- Microscopic:
*Control: males and females all tissues
*2000 mg/m3: males and females all tissues
*100 and 600 mg/m3: males and females: eyes, lung, nasal passages, , thymus, trachea +seminal vesicles for males

Statistics:

In-life body weights, haematology and serum chemistry values: Dunnett's test (two tailed) for comparing multiple treatments with a control. Bartlett's test (one-tailed) was used also on in-life body weights. Inspections of haematology and clinical chemistry data were also performed.
Terminal body weights and absolute organ weights: Analysis of Variance and Dunnett's test.
Organ to body weights ratio: Mann Whitney with Bonferroni's Inequality Modification Procedure.
Frequencies of histological lesions: Fisher's Exact Test with Bonferronni's Inequality Modification Procedure.

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):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

effects observed, treatment-related

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

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:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY
1 male and 2 females at 2000 mg/m3; one male control was sacrificed (not test related)
During exposure: non-responsiveness (inactive state) and respiratory difficulty at 600 and 2000 mg/m3, and nasal irritation at 2000 mg/m3.
Post-exposure (mainly at 2000 mg/m3): mucous membrane irritations (red-brown perinasal encrustation, blood-like nasal discharge and periorbital encrustation), respiratory difficulties (rattling sounds, sneezing, labored breathing, gasping and rapid breathing), non-responsiveness, and spontaneous deaths.
At 600 mg/m3 a few occurrences of perinasal and periorbital encrustation and sneezing, with one case also at 100 mg/m3.

BODY WEIGHT AND WEIGHT GAIN
Decreased gain at 600 mg/m3 and decreased bod weight at 2000 mg/m3

OPHTHALMOSCOPIC EXAMINATION
corneal lesions in approximately 100, 75, and 15% of the high, mid, and low exposure level animals, respectively

HAEMATOLOGY
total erythrocyte count, hemoglobin and hematocrit were increased at 2000 and 600 mg/m3 (only females), significant except for hematocrit in females at 2000 mg/m3; mean corposcular volume was significantly decreased in females at 2000 mg/m3; significantly reduced white blood cell counts in males at all exposure levels;

CLINICAL CHEMISTRY
the following serum chemistry measurements were significantly decreased for the various female exposure groups when compared to controls: chloride (mid, high), total protein (high), albumin (high), and creatinine (mid) (considered not treatment related). The following serum chemistry measurements were significantly increased for the various exposure groups when compared to controls: alkaline phosphatase (low and high exposure males, high exposure females), SGT (high exposure females), cholesterol (mid exposure females and high exposure males) and sodium (high exposure females, not considered biologically relevant), serum cholesterol (high exposure females, not significant)

ORGAN WEIGHTS
the following serum chemistry measurements were significantly decreased for the various female exposure groups when compared to controls: chloride (mid, high), total protein (high), albumin (high), and creatinine (mid) (considered not treatment related). The following serum chemistry measurements were significantly increased for the various exposure groups when compared to controls: alkaline phosphatase (low and high exposure males, high exposure females), SGT (high exposure females), cholesterol (mid exposure females and high exposure males) and sodium (high exposure females, not considered biologically relevant), serum cholesterol (high exposure females, not significant)

GROSS PATHOLOGY
at 2000 mg/m3: enlarged adrenals (also in females at 600 mg/m3), atrophic and/or reduced spleen, seminal vesicles and thymus, gaseous distension of GI tract

HISTOPATHOLOGY: NON-NEOPLASTIC
at 2000 mg/m3: inflammation of the eye (cornea, anterior uvea, anterior chamber), corneal hyperplasia, acute necrosis of the iris; inflammation of the lung (bronchi, alveoli, peribronchi) hyperplasia/metaplasia of bronchiolar epithelium, interstitial pneumonia, granuloma/microgranuloma; inflammation of the nasal turbinates and trachea with hyperplasia/metaplasia of mucous epithelium (also seen at 100 and 600 mg/m3 for nasal turbinates); atrophy of seminal vesicular epithelium, depletion of secretory product of seminal vesicles; depletion of thymocytes;

OTHER FINDINGS
- target, analytical and nominal levels were resp. 100, 100 and 100; 600, 600 and 620, 2000, 2000 and 2000 mg/m3 - Stability: ok - Homogeneity: >96%

Dose descriptor:

LOAEC

Remarks:

eye, nasal and lung irritation

Effect level:

100 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: local effects on cornea, lung and nasal passages

Dose descriptor:

LOAEC

Remarks:

systemic toxicity

Effect level:

100 mg/m³ air (analytical)

Sex:

male/female

Basis for effect level:

other: decreased body weight and changes in blood parameters

Critical effects observed:

not specified

Conclusions:

Since lymphocytopenia in males and lesions in the cornea and nasal passages of both sexes occurred at the lowest exposure level (100 mg/m3 in air), a "no-effect level" for DIPA could not be determined in this study.

Executive summary:

The repeated inhalation toxicity of Diisopropylamine (DIPA) was evaluated in male and female rats in a protocol similar to OECD N°412 guideline (Repeated Dose 28/14-Day inhalation Toxicity in Rodents) (Val Roloff, 1987). Sprague-Dawley rats (15/sex/concentration) were administered DIPA as a vapor at 0, 0.1, 0.6 and 2 mg/L via whole body inhalation for 6 hours per day, 5 days per week for approximately one month (maximum of 23 exposures). Rats were observed for morality twice daily and for clinical signs and body weights once a week. Ophthalmic exams were given once during the last week of the study. Hematology and clinical chemistry measurements were made. Rats were necropsied and all examined for gross pathology. Organ weights were determined for adrenals, brain, heart, kidneys, liver, spleen, and testes with epididymides. Histopathology was conducted on a full set of organs in the control and at the highest concentration, whereas eyes, lung, nasal passages, seminal vesicles, thymus and trachea were examined at all concentrations.  

Two females and one male were sacrificed at the highest concentration. In addition, one control was also sacrificed (due to an unrelated injury). During exposure, rats exhibited non-responsiveness, respiratory difficulty (middle and high-exposure rats), and nasal irritation at the highest concentration. Post-exposure observations included mucous membrane irritation (encrustation), respiratory difficulties, nonresponsiveness. At the middle concentration, a few perinasal and periorbital encrustations and episodes of sneezing occurred. One low-exposure rat had perinasal encrustation. Body weight reductions ranged from 9.6 to 40.6% at the end of the study; the differences at the high and mid-exposure levels were statistically significant from controls (p<0.05 or 0.01). Corneal lesions were seen in 13%, 75%, and 100% of rats at 0.1, 0.6 and 2.0 mg/L. At the end of the study, red blood cells, hemoglobin and hematocrit values were increased 9-11% (p<0.01) in the males at 2.0 mg/L. The same values were increased in females at 0.6 and 2.0 mg/L as well but the increase in hematocrit was not statistically significant. Mean corpuscular volume was significantly decreased (p<0.05) in the high-exposure females compared with controls while decreased white blood cell counts were seen in all exposed males (p<0.01). Chloride, total protein, and albumin and creatinine were decreased at 2.0 mg/L in females, whereas chloride and creatinine were decreased at 0.6 mg/L (p<0.05 or 0.01) compared with controls. Increases in alkaline phosphatase, SGPT, cholesterol and sodium were observed at various concentrations in males and/or females (p<0.05 or 0.01). Absolute and relative adrenal weights were increased at 2.0 mg/L. Relative spleen weights were decreased at the highest concentration, whereas increases in relative weights of brain, heart, kidneys, liver, and testes were observed (p<0.05). Decreases in several absolute weights of organs were also seen.  

At 2.0 mg/L, several statistically significant changes from controls were seen upon histopathological examination: bronchiolitis/peribronchiolitis was seen in 7 males (p < 0.01) and an additional 3 females; and lung hyperplasia/metaplasia of bronchiolar epithelium in 24 rats (p < 0.01). In the nose, hyperplasia was seen in 26, 30 and 30 rats at 0.1, 0.6 and 2.0 mg/L respectively (p < 0.01); also, necrosis and dissolution of the turbinate septum, cartilage, and bone was seen at 0.6 and 2.0 mg/L (p < 0.01); other nasal effects were of higher incidence than controls (p < 0.01). At 2.0 mg/L, the trachea exhibited inflammation (26) and hyperplasia/metaplasia (29) and mineralization (19) (p < 0.01), with 2 rats showing erosion and ulceration of the tracheal mucosa. Thymocytes were depleted in 16 rats at 2.0 mg/L (p < 0.01 in females). 

At 2.0 mg/L, additional microscopic lesions not seen in controls included cell detritus in epididymal ducts (2 males); eye problems (inflammation of the cornea (8), anterior uvea (3), and anterior chamber (2)); autolysis of the ileum (2); hepatocellular necrosis and degeneration (3); interstitial pneumonia (6), lymphoid hyperplasia (1); adrenal vacuolation (2); and additional lung effects (various numbers). Two 0.6 mg/L rats also had interstitial pneumonia. At 2.0 mg/L, the seminal vesicles of 11 rats showed secretory product depletion vs. 2 in controls (p < 0.01); 6 rats showed atrophy of the seminal vesicular epithelium vs. 2 in controls; and autolysis of the seminal vesicles occurred in one rat. The LOAEC was determined to be 0.1 mg/L based on nasal and ocular corneal lesions and moderately reduced lymphocyte counts (males only).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

100 mg/m³

Study duration:

subacute

Species:

rat

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratory, Portage, MI
- Age at study initiation: Approximately 9 weeks
- Weight at study initiation: Males- 279.8 to 357.0 grams. Females- 181.0 to 211.4 grams
- Fasting period before study: no
- Housing: Individual suspended stainless steel cages, over paper bedding
- Diet: Ralston Purina RODENT CHOW No. 5002, ad libitum
- Water: ad libitum (St. Louis public water sup
- Acclimation period: 4 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 70-74
- Humidity (%): 35-60
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

open

Vehicle:

unchanged (no vehicle)

Details on exposure:

Test substance was applied daily for five days to an approximately 25 cm² shaved area of the upper back with a microliter syringe or a micropipet. The area of exposure was left unoccluded. A plastic collar was applied during the 6 hour exposure. The skin was not wiped clean of any remaining test substance after each exposure. Volme applied was based on individual animal’s body weight. Controls were treated identically, except that nothing was applied to the skin.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Gas chromatography using a flame ionization detector Test Material Stability: Analysis of samples placed on watch glass for 6 hours at 100 degrees F, or at ambient temperature (closed container, 56 days)

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

5 days/week

Remarks:

Doses / Concentrations:
0, 15, 50 and 150 mg/kg bw
Basis:
nominal per unit body weight

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale:
Diisopropylamine was applied to and left unoccluded on the shaved skin (approximately 25 cm²; approximately 10% of the total body surface area) of groups of 3 male and 3 female Sprague-Dawley rats at targeted doses of 0, 50, 150, 450, 1350 or 2000 milligrams per kilogram (mg/kg) per day, for five consecutive days. Plastic collars were used to prevent ingestion of the test material. Negative controls were handled identically to treated animals, except nothing was applied to the skin. The animals were weighed pretest, and were weighed and sacrificed on the day following the last exposure. Necropsies were not performed.
All 3 males and females at the 2000 mg/kg/day level were sacrificed after receiving three applications of the test material, due to extreme irritative effects to their skin. Body weight losses occurred in all animals at the 1350 mg/kg/day level. Clinical signs of mild to severe skin discoloration, dryness, redness, scabs and/or bleeding were seen in the majority of animals at the 450 and 1350 mg/kg/day levels.
Based on the above results, dose levels of 0, 15, 50 and 150 mg/kg/day were selected for the one month study.

- Post-exposure recovery period in satellite groups: none

Observations and examinations performed and frequency:

In-life Observations:
Checks for Mortality and Moribundity: Twice daily
Detailed Obsenations for Signs of Toxicity: Once weekly
Body Weight and Food Consumption Measurement: Once weekly

Clinical Pathology:
Frequency: Once; at termination of study
Approximate Number of Animals: 10/level/sex
Fasting: Food withheld overnight prior to blood collection
Collection Site: Posterior vena cava of anesthetized animals

Hematology Determinations:
Total erythrocyte count (RBC), total leukocyte count (WBC), platelet count (PLT), hematocrit (HCT), level of hemoglobin (HGB), and red blood cell indices [mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC)] : Whole blood treated with anticoagulant (EDTA pretreated commercial tubes) processed on a COULTER S Plus II blood cell counter using the manufacturers’ methods.
Leukocyte differential: Thin blood smears on labelled glass slides prepared$ stained with Wright’s stain, and examined microscopically
Reticulocyte count: A portion of the EDTA sample was mixed with a vital stain (methylene blue), a slide was prepared and examined microscopically

Blood Chemistry Determinations:
Albumin, total protein, blood urea nitrogen (BUN), total bilirubin, direct bilirubin, glucose, glutamic pyruvic transaminase (D-SPT/ALT), alkaline phosphatase, glutamic oxaloacetate transaminase (D-SOT/AST), gamma glutamyl transpeptidase (gamma-GT), globulin, creatinine, cholesterol (Chol), calcium, phosphate, chloride, sodium, and potassium: Serum harvested by centrifugation of samples submitted in commercial clot tubes assayed with a KDA clinical analyzer using the manufacturer’s methods. Globulin determined by subtraction of albumin value from total protein value.

Sacrifice and pathology:

- Gross Pathology
Scheduled Sacrifices:
Frequency: At study termination (approximately 4 weeks)
Number of Animals: All survivors
Organs weighed: Brain, heart, kidneys, liver, spleen, testes with epididymides
Extent of Examination: External and internal. Internal cavities opened and organs examined in situ and then removed. Hollow organs opened and examined.
Organs Retained (when present): aorta, adrenals, bone with marrow, brain, caecum, esophagus, eyes, heart, kidneys, colon, liver, lung (with mainstem bronchi), lymph node (mesenteric and e bow), mammary gland, muscle (quadriceps femoris), nasal passages, sciatic nene, pancreas, prostate, pituitary, submaxillary salivary gland, skin (treated and untreated areas), small intestine, spinal cord, spleen, stomach, testes with epididymides, thymus,
thyroid/parathyroid, trachea, uterus (corpus and cervix) , ovaries, urinary bladder
Fixative: Eyes- buffered 0.5% glutaraldehyde, 5% formalin
All other tissues- 10% neutral buffered formalin

- Histopathology
Tissues Examined: All retained tissues from all animals at control and highest dose level, target tissues at middle and low levels
Tissue Preparation: Fixed tissues washed, dehydrated, embedded in paraffin, sectioned at approximately 5 microns, and stained with hematoxylin and eosin
Examination: Light microscopy

Statistics:

The following statistical procedures were used to detect statistically significant differences between treated animals and their respective controls:
Dunnett’s Multiple Comparison Test (two-tailed)): body weights, food consumption, non categorical clinical pathology data, absolute organ weights
Mann-Whitney Test with Bonferroni Inequality Procedure: Organ weight/body weight ratios
Fisher’s Exact Test with Bonferroni Inequality Procedure: Incidence of microscopic lesions
Other statistical routines used for some data were: Bartlett’s Test to evaluate homogeneity of variances, Analysis ofVariance to determine if the sample (group) means could be considered as an estimate of a common population, and Grubb’s Test to detect outliers.

Clinical signs:

no effects observed

Dermal irritation:

effects observed, treatment-related

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

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:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no mortalities during this study.
Abnormalities of the skin at the site of application were the only clinical signs considered to be a result of testing. These consisted primarily of mild dryness and scab formation, and were most prevalent in high level females (5 of 10 animals). There was not a clear dose-response relationship for either effect, and they were probably merely the result of the repeated of a volatile agent (dryness), or the shaving of the site (scab formation).

BODY WEIGHT AND WEIGHT GAIN
Males at all treated dose levels failed to gain as much body weight as controls, although statistical significance was never achieved; all male test levels were approximately 7.5% lighter in group mean body weight than controls at the end of testing.
Females at all test levels had weight gains comparable to their respective controls.

FOOD CONSUMPTION
Food consumption for test groups of both sexes was comparable to or greater than that of controls, both on a grams/day (GM/DAY) and grams/kilogram of body weight/day (GM/KG DAY) basis throughout most of the study.

HAEMATOLOGY AND CLINICAL CHEMISTRY
All changes in hematologic and serum biochemical parameters were of small magnitude, not dose-related, were in the opposite direction of that expected from toxic injury, and/or were within established ranges of normal for rats of this strain and age. Therefore, changes in these parameters were considered to be biologically insignificant and/or unrelated to treatment.

ORGAN WEIGHTS, GROSS PATHOLOGY AND HISTOPATHOLOGY
There were no changes in relative or absolute organ weights that were considered to have been related to treatment. Decreases in absolute heart and liver weights were seen in all levels of treated males, and decreased relative heart weight was noted in high level males as well as decreased absolute brain weight in high level females. Also at the two highest treatment levels, relative testicular weights were increased. Mild splenic congestion was present in 2/10, 1/10 and 2/10 males and 4/10, 1/10 and 6/10 females from low, intermediate and high dosage groups, respectively. There were no other gross or microscopic changes considered related to treatment.

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

>= 150 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: Any obvious adverse effects were observed

Dose descriptor:

NOAEL

Remarks:

skin irritation

Effect level:

>= 150 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: Any obvious adverse effects were observed

Dose descriptor:

NOAEL

Remarks:

skin irritation

Effect level:

2.19 mg/cm² per day

Sex:

male

Basis for effect level:

other: Any obvious adverse effects were observed

Dose descriptor:

NOAEL

Remarks:

skin irritation

Effect level:

1.24 mg/cm² per day

Sex:

female

Basis for effect level:

other: Any obvious effects were observed

Critical effects observed:

not specified

Analyses of the test material stability indicated that the neat test material was stable at room temperature over a length of time exceeding that of the study. All of the test material volatilized after 30 minutes of simulated in-use conditions.

Conclusions:

dermal NOAEL in rats >= 150mg/kg (4 weeks study)

Executive summary:

The Repeated dermal toxicity of Diisopropylamine (DIPA) was evaluated in male and female rats according to a protocol similar to OECD N°410 guideline. Groups of 10 male and 10 female Charles River CD(SD)BR rats received an open dermal exposure to 15, 50, and 150 mg/kg of undiluted Diisopropylamine five times a week for 1 month. The animals were monitored forsigns of toxicity and changes in hematology and clinical chemistry parameters. Necropsy was performed on all rats. A concurrent untreated control group of animals was also monitored.

Body weight gain and feed consumption in the test groups were comparable to those of the control group. Mild skin dryness was observed at the sites of application. Because this manifestation did not appear to be dose related, the authors attributed it to the repeated applications and evaporation rather than a compound related effect. No treatment-related changes were found among the hematology and clinical chemistry parameters investigated. Decreases were found inthe absolute and relative heart weights of the male rats from the high-dose group, andincreases in the absolute and relative testes weights of the males from the mid- andhigh-dose groups. However, the authors noted that no microscopic changes were found in these organs and that mild splenic congestion was present in all groups. No dose-response relationship or concomitant changes in splenic weights and hematology parameters were seen; thus, the splenic congestion was not considered related to the treatment.

The authors concluded that there was no evidence of dermal toxicity in rats treated with doses up to 150 mg/kg/day Diisopropylamine for 1 month.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratory, Portage, MI
- Age at study initiation: Approximately 9 weeks
- Weight at study initiation: Males- 279.8 to 357.0 grams. Females- 181.0 to 211.4 grams
- Fasting period before study: no
- Housing: Individual suspended stainless steel cages, over paper bedding
- Diet: Ralston Purina RODENT CHOW No. 5002, ad libitum
- Water: ad libitum (St. Louis public water sup
- Acclimation period: 4 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 70-74
- Humidity (%): 35-60
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

open

Vehicle:

unchanged (no vehicle)

Details on exposure:

Test substance was applied daily for five days to an approximately 25 cm² shaved area of the upper back with a microliter syringe or a micropipet. The area of exposure was left unoccluded. A plastic collar was applied during the 6 hour exposure. The skin was not wiped clean of any remaining test substance after each exposure. Volme applied was based on individual animal’s body weight. Controls were treated identically, except that nothing was applied to the skin.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Gas chromatography using a flame ionization detector Test Material Stability: Analysis of samples placed on watch glass for 6 hours at 100 degrees F, or at ambient temperature (closed container, 56 days)

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

5 days/week

Remarks:

Doses / Concentrations:
0, 15, 50 and 150 mg/kg bw
Basis:
nominal per unit body weight

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale:
Diisopropylamine was applied to and left unoccluded on the shaved skin (approximately 25 cm²; approximately 10% of the total body surface area) of groups of 3 male and 3 female Sprague-Dawley rats at targeted doses of 0, 50, 150, 450, 1350 or 2000 milligrams per kilogram (mg/kg) per day, for five consecutive days. Plastic collars were used to prevent ingestion of the test material. Negative controls were handled identically to treated animals, except nothing was applied to the skin. The animals were weighed pretest, and were weighed and sacrificed on the day following the last exposure. Necropsies were not performed.
All 3 males and females at the 2000 mg/kg/day level were sacrificed after receiving three applications of the test material, due to extreme irritative effects to their skin. Body weight losses occurred in all animals at the 1350 mg/kg/day level. Clinical signs of mild to severe skin discoloration, dryness, redness, scabs and/or bleeding were seen in the majority of animals at the 450 and 1350 mg/kg/day levels.
Based on the above results, dose levels of 0, 15, 50 and 150 mg/kg/day were selected for the one month study.

- Post-exposure recovery period in satellite groups: none

Observations and examinations performed and frequency:

In-life Observations:
Checks for Mortality and Moribundity: Twice daily
Detailed Obsenations for Signs of Toxicity: Once weekly
Body Weight and Food Consumption Measurement: Once weekly

Clinical Pathology:
Frequency: Once; at termination of study
Approximate Number of Animals: 10/level/sex
Fasting: Food withheld overnight prior to blood collection
Collection Site: Posterior vena cava of anesthetized animals

Hematology Determinations:
Total erythrocyte count (RBC), total leukocyte count (WBC), platelet count (PLT), hematocrit (HCT), level of hemoglobin (HGB), and red blood cell indices [mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC)] : Whole blood treated with anticoagulant (EDTA pretreated commercial tubes) processed on a COULTER S Plus II blood cell counter using the manufacturers’ methods.
Leukocyte differential: Thin blood smears on labelled glass slides prepared$ stained with Wright’s stain, and examined microscopically
Reticulocyte count: A portion of the EDTA sample was mixed with a vital stain (methylene blue), a slide was prepared and examined microscopically

Blood Chemistry Determinations:
Albumin, total protein, blood urea nitrogen (BUN), total bilirubin, direct bilirubin, glucose, glutamic pyruvic transaminase (D-SPT/ALT), alkaline phosphatase, glutamic oxaloacetate transaminase (D-SOT/AST), gamma glutamyl transpeptidase (gamma-GT), globulin, creatinine, cholesterol (Chol), calcium, phosphate, chloride, sodium, and potassium: Serum harvested by centrifugation of samples submitted in commercial clot tubes assayed with a KDA clinical analyzer using the manufacturer’s methods. Globulin determined by subtraction of albumin value from total protein value.

Sacrifice and pathology:

- Gross Pathology
Scheduled Sacrifices:
Frequency: At study termination (approximately 4 weeks)
Number of Animals: All survivors
Organs weighed: Brain, heart, kidneys, liver, spleen, testes with epididymides
Extent of Examination: External and internal. Internal cavities opened and organs examined in situ and then removed. Hollow organs opened and examined.
Organs Retained (when present): aorta, adrenals, bone with marrow, brain, caecum, esophagus, eyes, heart, kidneys, colon, liver, lung (with mainstem bronchi), lymph node (mesenteric and e bow), mammary gland, muscle (quadriceps femoris), nasal passages, sciatic nene, pancreas, prostate, pituitary, submaxillary salivary gland, skin (treated and untreated areas), small intestine, spinal cord, spleen, stomach, testes with epididymides, thymus,
thyroid/parathyroid, trachea, uterus (corpus and cervix) , ovaries, urinary bladder
Fixative: Eyes- buffered 0.5% glutaraldehyde, 5% formalin
All other tissues- 10% neutral buffered formalin

- Histopathology
Tissues Examined: All retained tissues from all animals at control and highest dose level, target tissues at middle and low levels
Tissue Preparation: Fixed tissues washed, dehydrated, embedded in paraffin, sectioned at approximately 5 microns, and stained with hematoxylin and eosin
Examination: Light microscopy

Statistics:

The following statistical procedures were used to detect statistically significant differences between treated animals and their respective controls:
Dunnett’s Multiple Comparison Test (two-tailed)): body weights, food consumption, non categorical clinical pathology data, absolute organ weights
Mann-Whitney Test with Bonferroni Inequality Procedure: Organ weight/body weight ratios
Fisher’s Exact Test with Bonferroni Inequality Procedure: Incidence of microscopic lesions
Other statistical routines used for some data were: Bartlett’s Test to evaluate homogeneity of variances, Analysis ofVariance to determine if the sample (group) means could be considered as an estimate of a common population, and Grubb’s Test to detect outliers.

Clinical signs:

no effects observed

Dermal irritation:

effects observed, treatment-related

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

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:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no mortalities during this study.
Abnormalities of the skin at the site of application were the only clinical signs considered to be a result of testing. These consisted primarily of mild dryness and scab formation, and were most prevalent in high level females (5 of 10 animals). There was not a clear dose-response relationship for either effect, and they were probably merely the result of the repeated of a volatile agent (dryness), or the shaving of the site (scab formation).

BODY WEIGHT AND WEIGHT GAIN
Males at all treated dose levels failed to gain as much body weight as controls, although statistical significance was never achieved; all male test levels were approximately 7.5% lighter in group mean body weight than controls at the end of testing.
Females at all test levels had weight gains comparable to their respective controls.

FOOD CONSUMPTION
Food consumption for test groups of both sexes was comparable to or greater than that of controls, both on a grams/day (GM/DAY) and grams/kilogram of body weight/day (GM/KG DAY) basis throughout most of the study.

HAEMATOLOGY AND CLINICAL CHEMISTRY
All changes in hematologic and serum biochemical parameters were of small magnitude, not dose-related, were in the opposite direction of that expected from toxic injury, and/or were within established ranges of normal for rats of this strain and age. Therefore, changes in these parameters were considered to be biologically insignificant and/or unrelated to treatment.

ORGAN WEIGHTS, GROSS PATHOLOGY AND HISTOPATHOLOGY
There were no changes in relative or absolute organ weights that were considered to have been related to treatment. Decreases in absolute heart and liver weights were seen in all levels of treated males, and decreased relative heart weight was noted in high level males as well as decreased absolute brain weight in high level females. Also at the two highest treatment levels, relative testicular weights were increased. Mild splenic congestion was present in 2/10, 1/10 and 2/10 males and 4/10, 1/10 and 6/10 females from low, intermediate and high dosage groups, respectively. There were no other gross or microscopic changes considered related to treatment.

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

>= 150 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: Any obvious adverse effects were observed

Dose descriptor:

NOAEL

Remarks:

skin irritation

Effect level:

>= 150 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: Any obvious adverse effects were observed

Dose descriptor:

NOAEL

Remarks:

skin irritation

Effect level:

2.19 mg/cm² per day

Sex:

male

Basis for effect level:

other: Any obvious adverse effects were observed

Dose descriptor:

NOAEL

Remarks:

skin irritation

Effect level:

1.24 mg/cm² per day

Sex:

female

Basis for effect level:

other: Any obvious effects were observed

Critical effects observed:

not specified

Analyses of the test material stability indicated that the neat test material was stable at room temperature over a length of time exceeding that of the study. All of the test material volatilized after 30 minutes of simulated in-use conditions.

Conclusions:

dermal NOAEL in rats >= 150mg/kg (4 weeks study)

Executive summary:

The Repeated dermal toxicity of Diisopropylamine (DIPA) was evaluated in male and female rats according to a protocol similar to OECD N°410 guideline. Groups of 10 male and 10 female Charles River CD(SD)BR rats received an open dermal exposure to 15, 50, and 150 mg/kg of undiluted Diisopropylamine five times a week for 1 month. The animals were monitored forsigns of toxicity and changes in hematology and clinical chemistry parameters. Necropsy was performed on all rats. A concurrent untreated control group of animals was also monitored.

Body weight gain and feed consumption in the test groups were comparable to those of the control group. Mild skin dryness was observed at the sites of application. Because this manifestation did not appear to be dose related, the authors attributed it to the repeated applications and evaporation rather than a compound related effect. No treatment-related changes were found among the hematology and clinical chemistry parameters investigated. Decreases were found inthe absolute and relative heart weights of the male rats from the high-dose group, andincreases in the absolute and relative testes weights of the males from the mid- andhigh-dose groups. However, the authors noted that no microscopic changes were found in these organs and that mild splenic congestion was present in all groups. No dose-response relationship or concomitant changes in splenic weights and hematology parameters were seen; thus, the splenic congestion was not considered related to the treatment.

The authors concluded that there was no evidence of dermal toxicity in rats treated with doses up to 150 mg/kg/day Diisopropylamine for 1 month.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

2.32 mg/cm²

Study duration:

subacute

Species:

rat

Additional information

Repeated oral toxicity

 

The Repeated oral toxicity of diisopropylamine was evaluated in male and female rats according to OECD N°407 guideline (Repeated Dose 28-Day Oral Toxicity in Rodents) (Verschuere, 1991). DIPA was administered orally once daily to Sprague-Dawley rats for 4 weeks at doses of 0, 15, 50, and 150 mg/kg/d. Clinical examinations were realized twice daily. Animal weights and food intake were recorded twice weekly. Hematological analysis was realized on days -9and 29 and biochemistry on days -7 and 32. Urinalysis 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.

 

In the dose range-finding study, diisopropylamine was administered orally once daily to Sprague Dawley rats for 14 days at doses of 0, 10, 30, 100, 300 mg/kg/d (Verschuere, 1990). Clinical examinations were realized once daily. Animal weights and food intake were recorded twice weekly. Haematological, biochemical analysis and urinalysis were realized on day 14. All animals were necropsied on day 15 and subjected to complete macroscopic examination.

At the highest dose, all animals died between D4 and D11. A statistical difference in body weights and in food consumption was observed in the high dose group. No abnormalities were observed in haematological, biochemical, and urinalysis, but there was no surviving animal on D14 in the high dose group. Ulcerative, necrotic or subacute inflammatory multifocal lesions of stomach mucosa at 100 mg/kg animals were observed. The oral NOAEL was 30 mg/kg/d.

 

Repeated dermal toxicity

 

The Repeated dermal toxicity of diisopropylamine was evaluated in male and female rats according to a protocol similar to OECD no. 410 guideline (Naylor, 1987). Groups of 10 male and 10 femaleCD(SD)BR rats were dermally exposed to 15, 50, and 150 mg/kg of undiluted diisopropylamine five times a week for 1 month. The animals were monitored forsigns of toxicity and changes in hematology and clinical chemistry parameters. Necropsy was performed on all rats. A concurrent untreated control group of animals was also monitored.

Body weight gain and feed consumption in the test groups were comparable to those of the control group. Mild skin dryness was observed at the sites of application. Because this manifestation did not appear to be dose related, the authors attributed it to the repeated applications and evaporation rather than a compound related effect. No treatment-related changes were found among the hematology and clinical chemistry parameters investigated. Decreases were found in the absolute and relative heart weights of the male rats from the high-dose group, andincreases in the absolute and relative testes weights of the males from the mid- andhigh-dose groups. However, the authors noted that no microscopic changes were found in these organs and that mild splenic congestion was present in all groups. No dose-response relationship or concomitant changes in splenic weights and hematology parameters were seen; thus, the splenic congestion was not considered related to the treatment.

The authors concluded that there was no evidenceof dermal toxicity in rats treated with doses up to 150 mg/kg/day Diisopropylaminefor 1 month.

 

Repeated inhalation toxicity

 

The repeated inhalation toxicity of Diisopropylamine was evaluated in male and female rats in a protocol similar to OECD N°412 guideline (Repeated Dose 28/14-Day inhalation Toxicity in Rodents) (Val Roloff, 1987). Sprague-Dawley rats (15/sex/concentration) were administered DIPA as a vapor at 0, 0.1, 0.6 and 2 mg/L via whole body inhalation for 6 hours per day, 5 days per week for approximately one month (maximum of 23 exposures). Rats were observed for mortality twice daily and for clinical signs and body weights once a week. Ophthalmic exams were given once during the last week of the study. Hematology and clinical chemistry measurements were made. Rats were necropsied and all examined for gross pathology. Organ weights were determined for adrenals, brain, heart, kidneys, liver, spleen, and testes with epididymides. Histopathology was conducted on a full set of organs in the control and at the highest concentration, whereas eyes, lung, nasal passages, seminal vesicles, thymus and trachea were examined at all concentrations.

Two females and one male were sacrificed at the highest concentration. In addition, one control was also sacrificed (due to an unrelated injury). During exposure, rats exhibited non-responsiveness, respiratory difficulty (middle and high-exposure rats), and nasal irritation at the highest concentration. Post-exposure observations included mucous membrane irritation (encrustation), respiratory difficulties, non responsiveness. At the middle concentration, a few perinasal and periorbital encrustations and episodes of sneezing occurred. One low-exposure rat had perinasal encrustation. Body weight reductions ranged from 9.6 to 40.6% at the end of the study; the differences at the high and mid-exposure levels were statistically significant from controls (p<0.05 or 0.01). Corneal lesions were seen in 13%, 75%, and 100% of rats at 0.1, 0.6 and 2.0 mg/L. At the end of the study, red blood cells, hemoglobin and hematocrit values were increased 9-11% (p<0.01) in the males at 2.0 mg/L. The same values were increased in females at 0.6 and 2.0 mg/L as well but the increase in hematocrit was not statistically significant. Mean corpuscular volume was significantly decreased (p<0.05) in the high-exposure females compared with controls while decreased white blood cell counts were seen in all exposed males (p<0.01). Chloride, total protein, and albumin and creatinine were decreased at 2.0 mg/L in females, whereas chloride and creatinine were decreased at 0.6 mg/L (p<0.05 or 0.01) compared with controls. Increases in alkaline phosphatase, SGPT, cholesterol and sodium were observed at various concentrations in males and/or females (p<0.05 or 0.01). Absolute and relative adrenal weights were increased at 2.0 mg/L. Relative spleen weights were decreased at the highest concentration, whereas increases in relative weights of brain, heart, kidneys, liver, and testes were observed (p<0.05). Decreases in several absolute weights of organs were also seen.  

At 2.0 mg/L, several statistically significant changes from controls were seen upon histopathological examination: bronchiolitis/peribronchiolitis was seen in 7 males (p < 0.01) and an additional 3 females; and lung hyperplasia/metaplasia of bronchiolar epithelium in 24 rats (p < 0.01). In the nose, hyperplasia was seen in 26, 30 and 30 rats at 0.1, 0.6 and 2.0 mg/L respectively (p < 0.01); also, necrosis and dissolution of the turbinate septum, cartilage, and bone was seen at 0.6 and 2.0 mg/L (p < 0.01); other nasal effects were of higher incidence than controls (p < 0.01). At 2.0 mg/L, the trachea exhibited inflammation (26) and hyperplasia/metaplasia (29) and mineralization (19) (p < 0.01), with 2 rats showing erosion and ulceration of the tracheal mucosa. Thymocytes were depleted in 16 rats at 2.0 mg/L (p < 0.01 in females). At 2.0 mg/L, additional microscopic lesions not seen in controls included cell detritus in epididymal ducts (2 males); eye problems (inflammation of the cornea (8), anterior uvea (3), and anterior chamber (2)); autolysis of the ileum (2); hepatocellular necrosis and degeneration (3); interstitial pneumonia (6), lymphoid hyperplasia (1); adrenal vacuolation (2); and additional lung effects (various numbers). Two 0.6 mg/L rats also had interstitial pneumonia. At 2.0 mg/L, the seminal vesicles of 11 rats showed secretory product depletion vs. 2 in controls (p < 0.01); 6 rats showed atrophy of the seminal vesicular epithelium vs. 2 in controls; and autolysis of the seminal vesicles occurred in one rat.

The LOAEC was determined to be 0.1 mg/L based on nasal and ocular corneal lesions and moderately reduced lymphocyte counts (males only).

 

In a subacute inhalation study 10 male Swiss mice were exposed (whole body) to diisopropylamine at air concentrations of 62.0 (+/-11.5), 174 (+/-29.3) and 436 (+/-89.7) ppm, 6 hours/day on five days/week for 4, 9 or 14 days (Zissu, 1995). One group of five mice (control group) was exposed to filtered air with each exposed group. This study designed in order to investigate only the irritative potential on the respiratory tract.

Diisopropylamine induced histological changes in the nasal passages at concentrations of 62.0 ppm and higher. Therefore, the LOAEL (mice, 14 days) is determined to be 62.0 ppm (0.257 mg/L).

 

Valid 13-week inhalation studies in rats and mice were performed according to NTP protocols (NTP, 2011) with diethylamine, an analogue substance of diisopropylamine.

 

In a study conducted similar to OECD TG 413 (Subchronic Inhalation Toxicity: 90-Day), groups of ten Fischer 344 rats/sex were exposed to the diethylamine by whole body vapor inhalation for six hours/day, five days/week for 93 days at concentrations of 0, 8, 16, 32, 62, or 125 ppm (0, 16.8, 33.6, 67.2, 130.2, and 262.5 mg/m3) (NTP, 2011).

There were no deaths. A single occurrence of a torso lateral ulcer/abscess occurred in a 125 ppm male. Clonic seizures were seen in males (1/50, 2/50, 7/50, 9/50) and in females (2/50, 7/50, 11/50, 13/50) [doses not stated but the lowest numbers presumably were in controls – i.e., 1/50 males and 2/50 females]. There were no effects on body weights, clinical chemistry or hematology findings. The relative kidney weights of all groups of exposed females were increased and were significantly greater than those of the chamber controls (p< 0.05 or 0.01), except in the 32 ppm group (which had an n=9). The relative heart weight was increased in females at 125 ppm (p< 0.05). The relative liver weight of 125 ppm males was significantly increased (p < 0.05). Exposure-related histopathology findings in rats were limited to the nose (primarily at 62 or 125 ppm). These lesions included turbinate necrosis, suppurative inflammation, respiratory epithelial hyperplasia, squamous metaplasia of the respiratory epithelium, and olfactory epithelial atrophy.

The NOAEC for the nasal irritation and systemic toxicity was 16 ppm (33.6 mg/m3) based on histopathology findings.

 

In a study conducted similar to OECD TG 413, groups of ten B6C3F1 mice/sex were exposed to diethylamine by whole body vapor inhalation for 6 hours/day, 5 days/week for 93 days at concentrations of 0, 8, 16, 32, 62, 125 ppm (0, 16.8, 33.6, 67.2, 130.2, and 262.5 mg/m3) (NTP, 2011).

There were no deaths and no treatment-related clinical findings. Body weights and weight gain in the high concentration group were significantly reduced (p < 0.01). There were no hematology findings. The absolute weights of the liver, right kidney (the left kidney was not weighed), and thymus of 125 ppm males; heart, liver, and right kidney of 125ppm females; and thymus of 62 and 125ppm females were significantly reduced (p < 0.01 or 0.05). The relative weights of the heart, right kidney (the left kidney was not weighed), lung, and right testis of 125ppm males and the lung of 125ppm females were significantly increased (p < 0.01). There were no findings at gross necropsy. Histopathological changes were noted primarily in the nasal cavity and involved both the respiratory and olfactory epithelium of animals principally in the 62 or 125 ppm groups. These lesions included suppurative inflammation, squamous metaplasia of the respiratory epithelium, olfactory epithelial atrophy, and necrosis of the turbinates.

The NOAEC for the nasal irritation and the systemic toxicity was 16 ppm (33.6mg/m3) based on histopathology findings.

 

In a repeated exposure study, male and female Fischer 344 (F-344) rats were exposed at 25 ppm (75 mg/m3) or 250 ppm (750 mg/m3) diethylamine vapour, 6.5 hours/day, five days/week, for 24 weeks in order to assess cardiac and other organ system toxicity (Lynch et al., 1986). Scheduled sacrifices were performed following 30, 60, and 120 days of exposure.

During the first two weeks of exposure, the rats exposed at 750 mg/m3 diethylamine did not gain weight. After two weeks, however, the rate of weight gain of these rats was greater than that of controls. Nevertheless, mean body weights for both sexes of rats exposed at 750 mg/m3 diethylamine remained depressed compared to controls throughout the study. Sneezing, tearing, and reddened noses were seen in rats exposed at 750 mg/m3 diethylamine . Histopathologic examinations revealed lesions of the nasal mucosa of rats exposed at 750 mg/m3 diethylamine (rats exposed at 75 mg/m3 were not evaluated for nasal effects). These lesions of the nasal respiratory epithelium consisted of squamous metaplasia, suppurative rhinitis, and lymphoid hyperplasia. There were no pronounced treatment-related effects on organ weights, hematology, or clinical chemistry indices except for blood urea nitrogen which was evaluated in rats of both sexes exposed at 750 mg/m3 diethylamine for 24 weeks. In contrast to the high-dose animals, no treatment-related effects were observed in rats intermittently exposed at 75 mg/m3 diethylamine for up to 24 weeks. No evidence of cardiotoxicity was seen in rats exposed to either diethylamine concentration for up to 24 weeks. The results of shorter exposure (30 and 60 days, either 75 and 750 mg/m3 diethylamine, 10 animals of both sexes; 120 days 75 mg/m3 diethylamine, 50 animals of both sexes) and the pathological findings of this study were summarised in a report by Iverson (1983). Marked signs of toxicity were not seen in rats exposed to 75 mg/m3 and 750 mg/m3 diethylamine after 30 and 60 days. After 120 days of exposure to 75 mg/m3 diethylamine the incidence of slight bronchial lymphoid hyperplasia was twice as high in the exposed group than in the control group. The authors of the original study considered that this was not a reflection of diethylamine toxicity, as the lesion was also seen in control animals and there was no clear dose-response relationship across the three groups. There were no systemic treatment-related effects in rats exposed to 25 ppm (75 mg/m3) diethylamine, however nasal effects were not evaluated at this concentration.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Key study

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Key study

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Key study

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
Key study

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
Key study

Justification for classification or non-classification

According to CLP criteria, no classification is warranted for specific target organ toxicity after repeated exposure.