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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 16th - August 22nd, 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study: Restrictions acceptable (only 5 day/week treatment, no neurobehavioural examinations reported, only 14 animals per sex/dose)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report Date:
1986

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
(see below)
Principles of method if other than guideline:
DEVIATIONS FROM GUIDELINE
-Treatment for only 5d/week (guideline recommendation: 7d/week), no neurobehavioural examinations, not clear if fasting was performed before hematology and clinicochemical examinations.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): alpha-methyl-beta-(p-tert-butylphenyl)propionaldehyde
- Analytical purity: 97.8%
- Physical state and appearance: liquid, colourless to pale yellow
- Stabilty: 1 yr (as pure compound)
- Storage: under inert gas, closed container, room temperature (approx. 15° C)
- Batch: 109535

Test animals

Species:
rat
Strain:
other: Fuellinsdorf albino (also known as ibm:RORO (SPF))
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Institute of Biological and Medical Research, Fuellinsdorf, Switzerland
- Age at study initiation: 6-7 weeks
- Weight at study initiation: male, 134-171g; female, 104-105g
- Diet: ad libitum (NAFAG standard rat maintenance diet (ground) No.850; defined and certified for acceptable contaminant levels)
- Water: ad libitum except during urine collection period (tap water)
- Acclimation period: 12 days. Upon receipt all rats were examined for external signs of ill-health. Unhealthy animals were discarded.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: rapeseed oil (1ml/kg bw)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Each day, fresh suspensions of the test article in rape oil were prepared by the use of a magnetic stirrer. The dose volume was kept constant at 1 ml/kg body weight.


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test article from each of the dose groups was extracted from the rape oil suspension and the extracts sent to Givaudan A.G., Duebendorf, for analysis (no further details given).
Duration of treatment / exposure:
90 days
Frequency of treatment:
once daily, 5 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
0; 2; 5; 25; 50 mg/kg (Reanalysis of the test article showed a deviation from the theoretical values in the range from +9.59 % to -16.44 %)
Basis:
other: nominal
No. of animals per sex per dose:
14
Control animals:
yes, concurrent vehicle
Details on study design:
- Animal assignment: random
- Rationale for animal choice: Guideline prescribed. Furthermore, historical data of untreated animals of the strain as well as adequate references in literature was available to assist in assessment of results.
- Satellite group: Yes, 4/sex of control and additional 14/sex of 50 mg/kg bw/d dose group
- Rationale for selecting satellite groups: Test for reversibility of effects
- Length of post-exposure recovery (satellite groups): 4 weeks
The 1st treatment day was defined as day 0, week 0. The 2nd treatment day was defined as day 1, week 1. The 1st treatment week started on day 1 and terminated on day 7.

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

MORTALITY: Yes
- Check conducted continuously

BODY WEIGHT: Yes
- Time schedule for examinations: on 1st day of treatment, then every other week

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to start of treatment (control, 50 mg/kg bw/d, satellite group), week 13 (control, 50 mg/kg bw/d, satellite group)
- Dose groups that were examined: control and 50 mg/kg bw/d plus satellite group
- Comments: About 15-30 minutes prior to the examination, a mydriatic drug was instilled in each eye of the animals

FOOD CONSUMPTION : Yes
Time schedule for examinations: weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to treatment (10/sex/dose group), week 7 (all animals of satellite group), week 14 (all groups), and week 18 for the satellite group
- Anaesthetic used for blood collection: Yes, ketamine in combination with diazepam
- Animals fasted: no data
- Parameters examined: Red blood cell count, hemoglobin, mean corpuscular volume, packed cell volume, mean corpuscular hemoglobin, white blood cells, thrombocytes, reticulocytes, differential cell count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to treatment (10/sex/dose group), week 7 (all animals of satellite group), week 14 (all groups), and week 18 for the satellite group
- Animals fasted: no data
- Parameters examined: aspartate aminotransferase (AST), alanine aminotransferase (ALT), cholineesterase, alkaline phosphatase (AP), glutamate dehydrogenase (GLDH), total plasma bilirubin, plasma glucose, plasma urea, plasma creatinine, plasma cholesterol, serum sodium, serum potassium, plasma calcium, plasma phosphate, total serum protein, electrophoresis of serum proteins.

URINALYSIS: Yes
- Time schedule for collection of urine: week 6
- Dose groups that were examined: controls and 50 mg/kg bw/d and if protein > 30 mg/dl
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, during collection period, no feed or water was provided to the animals.
- Parameters examined: colour, specific gravity, pH, protein content, glucose content, occult blood, bilirubin, urobilinogen, ketone bodies, sediment compositions
Sacrifice and pathology:
SACRIFICE
- Number of animals: all survivors
- Method: exsanguination after CO2 narcosis

GROSS PATHOLOGY: Yes
- Organ weight (wet weight): brain, heart (without auricles), liver, kidneys, testes, ovaries, adrenals

HISTOPATHOLOGY: Yes
- Organs examined: All gross lesions and tumours, brain (cerebral cortex, medulla/pons, cerebellum), pituitary, thyroid and parathyroid, thymus, lung and trachea, heart, femur with bone marrow, salivary glands, liver, spleen, kidney, adrenals, pancreas, testes, uterus, oesophagus, stomach, duodenum, jejunum, ileum, caecum, colon, rectum, urinary bladder, intestinal lymph nodes, sciatic nerve, eyes and aorta.
- Groups examined: 10/sex/dose of control, 50 mg/kg bw/d. Livers were examined from all animals of both sexes, adrenals from all female rats and testes from all male rats of the study.
Statistics:
Dunnett's-test: Multiple comparisons, control versus all treated groups. Error rate = 0.1..

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS
- Male animals: Clinical signs; alopecia (1/14, 2 mg/kg bw/d; 2/14, 5 mg/kg bw/d; 1/14, 25 mg/kg bw/d; 1/14, 50 mg/kg bw/d) and incrusted eyes (1/14, 5mg/kg bw/d) and malposed tooth (2/14; control) and area with incrustation (2/14, 25 mg/kg bw/d, 1/14; 50 mg/kg bw/d) were described as not treatment related and judged to be background findings.
- Female animals: Clinical signs mostly in 50 mg/kg bw/d dose group and satellites included alopecia (2/14, 25 mg/kg bw/d; 15/28, 50 mg/kg bw/d and satellites vs 0/14 in control, considered treatment related), area with incrustation (1/14, 5 mg/kg bw/d; 5/28, 50 mg/kg bw/d and satellites vs 0/14 in control) and inflammation of skin (3/14).

MORTALITY
- 50 mg/kg bw/day: 1/14 females died in narcosis set for blood sampling (week 7). Not treatment related.

BODY WEIGHT AND WEIGHT GAIN
- no adverse effect was noted on body weight gain (see table 1 below)

FOOD CONSUMPTION AND COMPOUND INTAKE
- The feed intake was not influenced (see table 1 below)
- The results of analysis of the intake of the test article via diet showed a deviation from the theoretical values in the range from +9.59 % to -16.44 % .

HEMATOLOGY
- No treatment-related effect was apparent. All individual values of hematology parameters at weeks 0, 7, 14 and 18 were within the physiological range seen in the laboratory. At week 18, the statistical significances arose because of the small number of animals N = 4 of the control group.

CLINICAL CHEMISTRY
- Cholinesterase (male): decrease at termination by 30% and 35-60% (statistically significant) compared to control in the mid and high dose group. Effect reversible after 4 weeks recovery period reaching 78% of corresponding controls.
- Cholinesterase (female): statistically significant decrease at termination by 68% and 68-71% compared to control in the mid and high dose group, respectively. Reversible trend after 4 weeks recovery period reaching 63% of corresponding control.
- Cholestrol concentration (male): statistically significant decrease at termination: 35-39% and 60-66% of control in the high and mid dose group, respectively. Totally reversible after 4 weeks recovery period reaching 102% of corresponding control.
- Cholestrol concentration (female): statistically significant decrease at termination; 47-53% and 60-66% of control in the high and mid dose group, respectively. Totally reversible after 4 weeks recovery period reaching 114% of corresponding control.
- Aspartate aminotransferase (male): statistically significant increase (135% compared to mean of corresponding control at termination) in the high dose group. According to authors: not biologically meaningful or indicative of a treatment effect.

The lower cholesterol concentrations were categorized as treatment related but not necessarily adverse by the authors. With the exception of above mentioned parameters, all other statistically significant parameters were judged by the authors as not treatment related.

- Total proteins and electrophoresis: males dosed with 50 mg/kg bw; a trend towards increased albumin levels (absolute and relative) could be observed at week 14, which was accompanied by a slight increase of the albumin/globulin ratio. Finding was thought to reflect some adoptive changes due to treatment.
- Total proteins and electrophoresis: females dosed with 50 mg/kg; no effect was detected.

URINALYSIS
- No treatment related effect was apparent. Findings were similar and comparable to those in the respective control groups.

OPTHALMOSCOPY
- No treatment related finding as observed

ORGAN WEIGHTS
Dose dependent increase in absolute and relative weights of liver and adrenal seen at sacrifice. The effects were reversible after the 4 week recovery period (see below). The results are expressed as percentages of control values
- 25 mg/kg bw/d: male; absolute (24%) and relative (21%) increase of liver weight in comparison to controls.
- 25 mg/kg bw/d: female; absolute (57%) and relative (59%) increase of liver weight in comparison to controls. Absolute (16.4%) and relative (18%) weights of adrenals were also increased compared to controls.
- 50 mg/kg bw/d: male; absolute (45%) and relative (45%) increase of liver weight in comparison to controls.
- 50 mg/kg bw/d: female; absolute (69%) and relative (75%) increase of liver weight in comparison to controls. Absolute (30%) and relative (36%) weights of adrenals were also increased compared to controls.
- 50 mg/kg bw/d (satellite): male; after 4 weeks recovery period: absolute weight (6%) and relative (3.1 %) decrease of liver weight in comparison to controls. Absolute (5.6%) and relative (7.7%) weights of adrenals were increased compared to controls.
- 50 mg/kg bw/d (satellite): female; after 4 weeks recovery period: absolute weight (5.9%) and relative (7.7 %) increase of liver weight in comparison to controls. Absolute (6.5%) and relative (8.4 %) weights of adrenals were increased compared to controls.
GROSS PATHOLOGY
- 50 mg/kg bw/d: male; small testes (3/14 vs 1/10 control), spermatoceles (10/14 vs. 0/10 control)
- 50 mg/kg bw/d: male satellites; small testes (1/14 vs 1/4 control), spermatoceles (10/14 vs. 0/4 control)
All other findings were deemed not treatment related.

HISTOPATHOLOGY: NON-NEOPLASTIC
Testes:
The frequency of testicular atrophy was raised in males treated with 50 mg/kg. This finding persisted after the treatment-free follow -up period in control males and males treated formerly with 50 mg/kg
- 50 mg/kg bw/d: male; minimal to marked testicular atrophy (6/14 vs. 1/10 control), minimal to marked spermatoceles with inflammatory reactions (13/14 vs. 0/10 control)
- 50 mg/kg bw/d: satellites male; minimal to marked testicular atrophy (5/14 vs. 2/4 control)
Epididymides
Spermatoceles occured only in males of the 50 mg/kg bw/d dose groups. No recovery was detected after the 4 week treatment free period
- 50 mg/kg bw/d: male; minimal to marked spermatoceles with inflammatory reactions (13/14 vs. 0/10 control)
- 50 mg/kg bw/d: satellites male;minimal to marked spermatoceles with inflammatory reactions (13/14 vs. 0/4 control)

Liver:
The degree of the fat content of hepatic cells observed in all rats was higher in male and female rats treated with 50 mg/kg than in those treated with 0, 2, 5 and 25 mg/kg. After a treatment-free follow-up period of 4 weeks, there was no difference between the fat content of liver cells of control rats and that of rats treated formerly with 50 mg/kg

Adrenals:
A hypertrophy of the zona fasiculata was detected in two females treated with 50 mg/kg only. Because of the variable sections through the adrenal glands, a reliable diagnosis of this finding was not possible .

After completion of the study, a "second opinion" on the histological alterations observed in testes and epididymides of rats from this study and a tentative appraisal of possible mechanisms involved were sought from Dr Ettlin. R.A. The results of the second opinion are tabulated below (table 2)

Effect levels

open allclose all
Dose descriptor:
NOEL
Effect level:
5 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: plasma cholinesterase
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: testes with additional systemic toxicity

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Summary of average body weight change and food consumption after 13 weeks

Dose group (mg/kg bw/d)

Weight gain between begin and conclusion of study (g)

Average daily food consumption (g/day)

Male

Female

Male

Female

0

243.6

110.8

20.3

15.1

2

252.7

105.7

20.6

14.7

5

267.4

112.9

20.9

15.2

25

252

110.5

20.6

15.2

50

234.7

100.7

21.3

15.0

Satellite

237.9

110.8

21.0

16.1

For histotological findings in Testes and Epididymides (Second opinion by Dr Ettlin), see Chapter 7.8.1

Applicant's summary and conclusion