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

Repeated dose toxicity: dermal

Administrative data

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07. Nov - 20. Dec 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliant

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3200 (Repeated Dose Dermal Toxicity -21/28 Days)

Version / remarks:

1998

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))

Version / remarks:

1992

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1981

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesanstalt für Pflanzenbau und Pflanzenschutz Rheinland-Pfalz, 02 Jul 1997

Limit test:

no

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:

Wistar

Remarks:

CRL:WI (GLX/BRL/HAN) IGS BR

Details on species / strain selection:

Rats were selected since this rodent species is recommended in the respective test guidelines. Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation: 9 weeks
- Weight at study initiation: males: 197.5 —238.3 9 (group mean: 219.7 g); females: 143.5 —171.0 9 (group mean: 155.3 g)
- Housing: The rats were housed singly in type DK llI stainless steel wire mesh cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area about 800 cm2) in a fully air-conditioned room
- Diet (e.g. ad libitum): ad libitum, basic maintenance diet rat/mouse/ hamster, meal. supplied by Provimi Kliba SA, Kaiseraugst, Switzerland.
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Yes

DETAILS OF FOOD AND WATER QUALITY:
The food used in the study was assayed for chemical and microbiological contaminants. On the basis of the analytical findings the drinking water was found to be suitable. German Drinking Water Regulation (Trinkwasserverordnung, Bundesgesetzblatt, December 5, 1990) served as a guideline for maximum tolerable contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12

IN-LIFE DATES: From: 07. Nov 2000 (arrival) To: 20. Dec 2000

Administration / exposure

Type of coverage:

semiocclusive

Vehicle:

other: 0.05% Cremophor EL solution in doubly distilled water

Details on exposure:

TEST SITE
- Area of exposure:
- % coverage: 10 % of the body surface
- Type of wrap if used: 4 layers of porous gauze dressing ("Verbandmull Ph. Eur"., Lohmann GmbH &Co. KG) and an elastic dressing (Fixomull Stretch, Beiersdorf AG)
- Time intervals for shavings or clipplings: 5 days (males) and 6 days (females) before the first administration of the test substance, thereafter when necessary
but at least once a week.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Yes, with lukewarm water
- Time after start of exposure: 6 h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 4 mL/kg bw
- Constant volume or concentration used: yes

VEHICLE
- Concentration (if solution): 0.05 %

USE OF RESTRAINERS FOR PREVENTING INGESTION: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The mean values which were analytically analyzed were 101.3 —108.7 % of the target concentrations

Duration of treatment / exposure:

6 h

Frequency of treatment:

5/week for 4 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.2 mg/kg bw/day (nominal)

Remarks:

0.005 %

Doses / concentrations 2

Dose / conc.:

0.6 mg/kg bw/day (nominal)

Remarks:

0.015 %

Doses / concentrations 3

Dose / conc.:

2 mg/kg bw/day (nominal)

Remarks:

0.05 %

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Fasting period before blood sampling for clinical biochemistry: Before sacrifice the animals were fasted for 16 - 20 h

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) from Mondays to Fridays and once a day (in the morning) on Saturdays, Sundays and public holidays.

DETAILED CLINICAL EXAMINATIONS: Yes
conducted prior to the start of the administration period (day 0) and weekly thereafter (usually in the morning prior to test substance administration).
Parameters checked: behavior during handling, fur, skin (treated skin was not examined additionally during open field observation, as this was done daily prior to treatment), posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebralclosure, exophthalmus, feces (appearance/consistency), urine (volume/color), pupil size

DERMAL IRRITATION (if dermal study): Yes

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period in order to randomize the animals. During the administration period the body weight was determined on day 0 (start of administration) and thereafter at weekly intervals.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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: Yes

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior the start of administration and at the end of the experiment
- Dose groups that were examined: all animals at the start of administration; at the end, the animals of high dose and control group were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before necropsy
- Anesthesia: No
- Animals fasted: Yes
- How many animals: 10 animals per test group and sex
- Parameters checked: leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular, hemoglobin concentration, platelets, differential blood count, prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before necropsy
- Animals fasted: Yes
- How many animals: 10 animals per test group and sex
- Parameters checked: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-y-glutamyltransferase, serum-cholinesterase, erythrocyte-cholinesterase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
the following weights were assessed: anesthetized animals, liver, kidneys, adrenal glands, testes, epididymides, ovaries, uterus, spleen, brain, heart, thymus

HISTOPATHOLOGY: Yes
all gross lesions, salivary glands (mandibular and sublingual glands)
esophagus, stomach (glandular and non-glandular), duodenum, jejunum, ileum
cecum, colon, rectum, liver, pancreas, brain, pituitary gland, sciatic nerve, spinal cord (cervical, thoracic and lumbar cord), eyes, adrena| glands, thyroid glands, parathyroid glands, trachea, lungs, pharynx, larynx, nose, aorta, heart, bone marrow (femur), Iymph nodes (mandibular and mesenteric), spleen, thymus, kidneys, urinary bladder, testes, ovaries, oviducts, uterus/vagina, epididymides, prostate gland, seminal vesicles, fema|e mammary gland, skin (treated and untreated), skeletal muscle, sternum with marrow, femur with knee joint, extraorbita| lacrimal gland

Statistics:

Food consumption, body weight, body, weight change, food efficiency:
Parametric one-way analysis using the F-test (ANOVA) (two-sided). If the resulting
p-value was equal or less 0.05, a comparison of each group with the control group using the DUNNETT‘s test (two-sided) was performed for the hypothesis of equal means

Clinical pathology parameters, except differential blood count:
Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided).lf the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using MANN-WHITNEY U-test (two-sided) for the equal medians

Weight parameters:
Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the
resulting p-value was equal or less 0.05, a pairwise comparison of each dose group with the control group was performed using the WILCOXON test for the hypothesis of equal medians.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Incidental findings were alopecia and shearing lesions.

Dermal irritation:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption was statistically significantly increased in males of group 3 (2 mg/kg bw/d) on day 28, and statistically significantly decreased in females of group 2 (0.6 mg/kg bw/d) on day 14.

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

absolute weight changes:
In males of the high and low dose groups, the mean weight of the heart was slightly although significantly increased (+12.3% or + 9.9%, respectively), with no indication of a dose response relationship.
In female rats of the high dose group, the mean weight of the thymus was significantly increased (+ 26.3%).

relative weight changes:
In males of all treatment groups, the mean weight of the liver was significantly increased (+9.0%, + 5.5% or + 6.7% for low, mid or high dose groups, respectively), however, with no indication of a dose response relationship.
In female rats of the high dose group, the mean weight of the thymus was significantly increased (+ 22.0%).

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Gross lesions were noted in the glandular stomach (erosion/ulcer), liver (focus),
kidneys (retraction), epididymides (focus), ovaries (cyst), and skin (area with sparse hair). They were all noted at a low incidence, and they were all regarded to have developed unrelated to treatment.
No gross lesions were seen in the area of the treated skin. In the area of the nontreated skin, focal areas with sparse hair were noted in a few female animals (1/2/-/1 for dose groups 0 to 3). They were regarded to have developed spontaneously and unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Discussion of organ weight effects:

A toxicological relevance of significant liver weight effects is denied for the following reasons:
- no signicant weight change in the absolute weights
- no dose response relationship
- no such observation in female rats
- absence of a morphologic finding that may explain the weight change
- comparison with the historical control data of the same strain clearly indicates that the mean relative liver weight of the control rats (2.563 g) was the lowest in 15 other studies of the same rat strain and study duration (maximum: 4.109 g, minimum: 2.563 g, mean: 3.075 g)

The significantly increased mean absolute weight of the heart of male rats of the high dose group was also discredited as being treatment-related because
- the more reliable relative weight was not affected
- females did not show this effect
- histopathology did not indicate a finding that may have contributed to the weight
change.

The significantly increased mean absolute and relative weights of the thymus of
female rats of the high dose group were also considered incidental, as
- male rats were not so affected
- histopathology failed to show a morphologic alteration not only in the thymus but also in all other organs of the immune system.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

2 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no substance-realted toxicity was observed up to and including the highest tested dose. However, significant dermal effects can be expected for doses exceeding 0.1 % (previous study finding)

Target system / organ toxicity

Critical effects observed:

not specified