cis-4-[3-(p-tert-butylphenyl)-2-methylpropyl]-2,6-dimethylmorpholine

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19. Sep to 23. Oct 1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1980

Report date:

1980

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: The study was conducted on the basis of a 90-day study under the EPA Pesticide Program, Part II, Fed. Reg. Vol. No. 163

Version / remarks:

August 1978

GLP compliance:

no

Remarks:

predating GLP

Test material

Test material information

Constituent 1

Reference substance name:

cis-4-[3-(p-tert-butylphenyl)-2-methylpropyl]-2,6-dimethylmorpholine

EC Number:

266-719-9

EC Name:

cis-4-[3-(p-tert-butylphenyl)-2-methylpropyl]-2,6-dimethylmorpholine

Cas Number:

67564-91-4

Molecular formula:

C20H33NO

IUPAC Name:

(2R,6S)-4-[3-(4-tert-butylphenyl)-2-methylpropyl]-2,6-dimethylmorpholine

Test animals

Species:

rat

Strain:

Sprague-Dawley

Remarks:

MURA:SPRA (SPF 68 Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: MUS RATTUS, Brunnthal, FRG,
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation: 10 - 11 weeks
- Weight at study initiation: male rats: 210 +/- 15g, female rats: 180 +/- 15 g.
- Housing: singly in Makrolon wire cages (type MD III of Becker & Co., Castrop-Rauxel, FRG)
- Diet (e.g. ad libitum): HERILAN—MRH "Haltungsdiät" of H. EGGERSMANN KG, Rinteln, FRG, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 2 - 3 weeks before a 5-day preliminary flow and adaptation period was conducted

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 5
- Photoperiod (hrs dark / hrs light): 12

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: fully demineralized water and ethanol

Mass median aerodynamic diameter (MMAD):

< 1.2 µm

Remarks on MMAD:

The value given refers to the MMAD 50; the aerodynamic diameter was below 1.2 µm in 50-53 % of the particles in all treated groups.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION

- Exposure apparatus: The two-component atomizers are fitted to the upper part
of an aerodynamic exposure equipment (INA 20, head-nose exposure system) so that the aerosol is generated direct in the equipment.

- Method of holding animals in test chamber: rats used for the study were restrained in glass tubes their snouts projecting into the inhalation chamber
- Rate of air: 1500 + 300 L/h (exposure group), 1800 L/h (solvent control and air control)

- System of generating particulates/aerosols:
for technical reasons, the test substance was dissolved in analysis-grade ethanol and fully demineralized water, was supplied to two-component
atomizers (Rhema, Hofheim, FRG) by means of continuous infusion pumps (UNITA I, B. Braun, Melsungen, FRG) and atomized with compressed-air.

- Temperature, humidity, pressure in air chamber: a pressure slightly above the atmospheric pressure being set up in the equipment. Relative humidity in the equipment was adjusted to 40 - 60 % for the exposure groups by atomizing accurately metered amounts of water by means of ultrasonics or Pari nebulizers, through which a continuously measured bleed stream of the supply air was passed. Air control conditioned air (t = 21 °C, rel. humidity = 55 5 %) was used.

- Air flow rate: the flow of exhaust air was adjusted in such a way that it was 20 % less than the flow of supply air, 1440 L/h

- Method of particle size determination: The particle size analysis was carried out using a cascade impactor. Once a week a sample was taken for each group with the cascade impactor at a sampling velocity of 1.25 m/sec. The deposits in the probe and on the upper metal plate were analyzed as separate samples and evaluated together under "deposits". The glass-fiber collecting discs and the backup filter were eluted separately and analyzed by gas chromatography.

TEST ATMOSPHERE
- Brief description of analytical method used: A gas chromatographical determination of the test substance in ethanol was used. 5 mL of internal standard was added to the samples and filled with ethanol in 50-mL graduated flasks up to the calibration mark. Then 2-ml tubes were filled for the Automatic Sampler and analyzed. The volume injected was 2 µL
- Samples taken from breathing zone: yes

VEHICLE (if applicable)
- Justification for use and choice of vehicle: for technical reasons
- Composition of vehicle: 70 g analysis-grade ethanol, 2 g fully demineralized water
- Concentration of test material in vehicle: 8 g
- a solvent (aqueous ethanol) control group was included in the study in addition to a fresh-air control group.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

gas chromatographical determination

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

5 days/week - 6 hours/day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.01 mg/L air (nominal)

Remarks:

analytically verified

Doses / concentrations 2

Dose / conc.:

0.04 mg/L air (nominal)

Remarks:

analytical concentration: 0.042 mg/L

Doses / concentrations 3

Dose / conc.:

0.16 mg/L air (nominal)

Remarks:

analytical concentration: 0.165 mg/L

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The highest aerosol concentration of 0.16 mg/l was chosen taking the results of a feeding study into consideration; the amount consumed daily was used as
the basis for estimating the concentration for a 6-hour inhalation. Toxic effects were expected to occur at this concentration. The concentrations for the other
test groups Were obtained by reducing this concentration by the factor of 1/4 in each case to give the concentrations of 0.04 mg/l and 0.01 mg/l.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- The behavior and appearance of the animals was checked each day during and after exposure.

BODY WEIGHT: Yes
- Time schedule for examinations: once a week (Wednesdays) always at the same time of the day

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 7 days before administration and 28 days after the beginning of administration
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Not specified
- How many animals: all animals
- Parameters checked: hemoglobin, erythrocyte count, mean cell volume, platelet count, leukocyte count, hematocrit, mean hemoglobin content per erythrocyte, mean corpuscular hemoglobin concentration, differential blood count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see hematology
- Animals fasted: Not specified
- How many animals: all animals
- Parameters checked: total bilirubin, creatinine, urea, sodium, potassium, total protein, glucose, calcium, chloride, triglycerides, cholesterol, albumin, globulins, A/G ratio, lactate dehydrogenase, glutamic pyruvic transaminase, alkaline phosphatase, glutamic oxalacetic transaminase, plasma cholinesterase, erythrocyte cholinesterase, brain cholinesterase

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): No

LUNG BURDEN: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Organ weights: heart, liver, kidneys, brain
Organ fixation: head (including eyes, lacrymal glands, nasal cavity, nasal mucosa,
paranasal sinus, nasopharynx, oral cavity, tongue, teeth, tonsils, salivary glands, regional lymph nodes, outer, middle and inner ear, pituitary and Zymbal’s glands), heart, aorta thoracica, nasal mucosa, trachea (with larynx), lungs with mainstem bronchi (right and left), tongue, esophagus, stomach (forestomach and glandular stomach), small intestine (duodenum, jejunum and ileum), large intestine (cecum and colon), salivary glands, liver, pancreas, spleen, thymus, sternum with sternal marrow. kidneys, urinary bladder, testes, epididymides, prostate, ovaries, uterus in toto, pituitary, adrenals, thyroids (with parathyroids), brain, skeletal muscle, eye with optic nerve, skin
all changes were described macroscopically

HISTOPATHOLOGY: Yes
Microscopical examination of the above mentioned fixed organs was performed for all animals of the solvent control, air control group and animals of the highest dose group (0.16 mg/L); following organs of all animals of the dose group 0.01 and 0.04 mg/L were examined: aorta thoracica, nasal mucosa, trachea (with larynx), lungs with mainstem bronchi (right and left), tongue, esophagus, liver, kidneys, thyroids (with parathyroids), eye (with optic nerve), skin

Statistics:

Clinical examinations and pathology: The statistical evaluation was carried out using a t test generalized by WILLIAMS (1 and 2) for a simultaneous comparison of several dose groups with a control group.

Blood and plasma examinations: the individual dose groups were compared with the control group and with each of their initial values (blood sampling 0) using the t test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In test group 2 (0.01 mg/L) 3 animals had crusts on the bridge of their noses which occurred for the first time after the 14th exposure but had subsided by the day of sacrifice.
In test group 3 (0.04 mg/L) these crusts also occurred on the nose, but in 13 animals and only after 10 exposures. One female animal also had encrusted eyes with subsequent alopecia on the day of sacrifice. One male animal had crusts on the front and back extremities.
All the animals in test group 4 (0.16 mg/L) exhibited similar symptoms, but they were more pronounced. The female animals reacted more strongly than the male
ones. They had slight piloerection.

The crusts observed, are probably due to the effect of contact with the test
substance, the causes of which are, however, partially
of a technical nature. Contamination of the tubes may lead to the contact effect on the extremities. Impaction of the aerosol on the noses projecting into the inhalation apparatus also leads to contamination by the test substance. The crusts observed thus probably are a conglutination and reaction resulting from the skin-irritating test substance which is not primarily specific to inhalation.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

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:

effects observed, treatment-related

Description (incidence and severity):

A concentration-related reduction in plasma cholinesterase activity in both sexes was observed. The inhibitory effect was particularly noticeable in the female animals which in the highest test group (0.16 mg/L) exhibited only about half the enzyme activity of the control group. Whereas the statistical comparison between the lowest test group (0.01 mg/L) and the air control revealed a marked but not significant drop in activity, the evaluation in comparison with the solvent control showed a statistically significant difference in the female animals. However, it cannot be ruled out that the additional adverse effect that ethanol has on
the liver metabolism leads to a greater reduction in plasma chlolinesterase activity which is caused by metabolites of the substance.

At the end of the period of administration, there was a slight increase in alkaline phosphatase and glutamic pyruvic transaminase activity in both sexes in test
groups 3 and 4 (0.04 and 0.16 mg/L). In addition, there were signs of an increase in glutamic oxalacetic transaminase activity in the male animals of test group 4.

A reduction in the cholesterol level was detected in both sexes in test group 4 (0.16 mg/L)

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant increases in the absolute and relative liver weights which must be regarded as induced by the test substance occurred after 4-week inhalation in male and female rats in test group 3 (0.04 mg/L) and test group 4 (0.16 mg/L). Since this effect of the substance was more pronounced when statistically comparing test groups 2 — 4 with the solvent control (test group 1) than when comparing test groups 2 - 4 with the air control (test group 0), the latter method of calculation was preferred in assessing the changes in liver weight.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Focal changes to the skin around the opening of the nose which were prominent in the gross pathological findings in the rats of test groups 3 and 4 might have been induced by the test substance although most of them were only isolated findings.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Focal basal cell hyperplasia at the tip of the bifurcation or at the origin of the mainstem bronchi and the related focal basal cell hyperplasia in a mainstem bronchus of the lung specimens detected in the longitudinal sections of the trachea in test group 3 (0.04 mg/l) and test group 4 (0.16 mg/l) must be interpreted as substance-induced in the sense of an unspecific irritation response.

Histopathological findings: neoplastic:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

0.01 other: mg/l

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:

not specified