3-methylbut-2-en-1-ol

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted September 21, 1998

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EC Commission Directive 87/302/EEC of November, 18 1987; Part B: Methods for the determination of Toxicity; Subchronic oral Toxicity Test; 90-day repeated oral dose using rodent species; Official Journal of the European Communities No. L133, pp.8-11, 1988

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3-methylbut-2-en-1-ol

EC Number:

209-141-4

EC Name:

3-methylbut-2-en-1-ol

Cas Number:

556-82-1

Molecular formula:

C5H10O

IUPAC Name:

3-methylbut-2-en-1-ol

Details on test material:

- Name of test material (as cited in study report): 3-Methylbut-2-en-1-ol (Prenol)
- Test substance No.: 00/0274-1
- Date of production: 10 Apr 2000
- Physical state: Liquid / colorless, clear
- Analytical purity: 99.1 % (method: gas chromatography)
- Homogeneity: Homogeneous
- Lot/batch No.: Ch. 00/18, Abl. Nr. 56-1706
- Stability under test conditions: Proven by reanalysis after the in life phase of the study.
- Storage condition of test material: Room temperature
- Other: The analyses were carried out at the Analytical Department of BASF Aktiengesellschaft.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Germany,
- Age at study initiation: 34 - 36 days
- Age at the start of the administration period: 41 - 43 days
- Range of weight at study initiation: males: 148.7 - 171.1 g (group mean: 159.2 g); females: 111.1 - 136.4 g (group mean: 123.5 g).
- Housing: singly
- Diet: Provimi KLIBA SA, Kaiseraugst / Switzerland, ad libitum
- Water: ad libitum
- Acclimation period: yes, 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70%
- Air changes (per hr): fully air-conditioned room
- Photoperiod (hrs dark / hrs light): 12 hours/12 hours

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was administered as a solution in drinking water. The appropriate amount of test substance was weighed, then drinking water was filled up to the desired volume and subsequently mixed using a magnetic stirrer. The test substance solutions were prepared twice a week.

VEHICLE
- Concentration in vehicle: 200, 1000 and 5000 ppm

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in drinking water over a period of 4 up to 7 days at room temperature was determined before the start of the study.
Concentration control analyses of the test substance preparations were performed in all concentrations at the start and the end of the administration period. The recovery rates were within a range of 92.1 % - 101.1 % of the target concentrations.

Duration of treatment / exposure:

90 days

Frequency of treatment:

continuously

Doses / concentrations

Remarks:

Doses / Concentrations:
5000 ppm (calculated as 243.8 mg/kg bw/d in males, 307.2 mg/kg bw/d in females) 1000 ppm (calculated as 65.4 mg/kg bw/d in males, 82.1 mg/kg bw/d in females) 200 ppm (calculated as 14.4 mg/kg bw/d in males, 21.0 mg/kg bw/d in females).
Basis:
nominal in water

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Post-exposure period: none
- Dose selection rationale: 5000 ppm as high concentration with expected toxic effects; 1000 ppm as mid concentration; 200 ppm as low concentration.

Positive control:

No data

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- 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 OBSERVATIONS: Yes
- Time schedule: daily
- 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.

BODY WEIGHT: Yes
- Time schedule for examinations: start of administration period and thereafter weekly. The difference between the body weight on the respective day of weighing and the body weight on day 0 was calculated as body weight change. Additional weights were taken on days 86, 87 and 90 of the respective animals scheduled for functional observational battery.

FOOD CONSUMPTION: weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg animal/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 AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: weekly, calculation as mean water consumption in grams per animal and day.
mean daily intake of test substance (group means) was calculated based upon individual values for body weight and water consumption.
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the start (- 4 days) and towards the end of the administration period (day 85)
- Dose groups that were examined: animals of the control and high dose group

HAEMATOLOGY: Yes, from the retroorbital venous plexus
- Time schedule for collection of blood: towards the end of the administration period (day 93 and 94).
- Anaesthetic used for blood collection: No
- Animals fasted: Yes, 16 - 20 hours
- How many animals: all animals
- The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Technicon H 1 E model; Bayer, Munich, Germany):
• leukocytes
• erythrocytes
• hemoglobin
• hematocrit
• mean corpuscular volume
• mean corpuscular hemoglobin
• mean corpuscular hemoglobin concentration
• platelets
• differential blood count
Furthermore, differential blood smears were prepared and stained according to Wright without being evaluated.
- The clotting analyses were carried out using a ball coagulometer (KC 10 A model; Amelung, Lemgo, Germany).
The following parameter was determined:
• prothrombin time (Hepato Quick's test)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: towards the end of the administration period.
- Animals fasted: Yes, 16 - 20 hours
- How many animals: all animals
- The following clinicochemical parameters were determined, using an automatic analyzer (Hitachi 917 ; Roche, Mannheim, Germany).
• alanine aminotransferase
• aspartate aminotransferase
• alkaline phosphatase
• serum-y-glutamyltransferase
• sodium
• potassium
• chloride
• inorganic phosphate
• calcium
• urea
• creatinine
• glucose
• total bilirubin
• total protein
• albumin
• globulins
• triglycerides
• cholesterol
• magnesium

URINALYSIS: Yes
- Time schedule for collection of urine: towards the end of the administration period (day 85).
- Metabolism cages used for collection of urine: Yes, for overnight collection
- Animals fasted: Yes
- The following examinations were carried out using the given methods:
With the exception of volume (graduated tubes), color (by visual evaluation), turbidity (by visual evaluation), sediment examination (microscopy) and the specific gravity (urine refractometer, the sediment was evaluated microscopically), all the other urine constituents were determined semiquantitatively using test strips (Combur-9-test M, Roche, Mannheim, Germany) and a reflection photometer (Miditron M; Roche, Mannheim, Germany).
• pH
• protein
• glucose
• ketones
• urobilinogen
• bilirubin
• blood

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: towards the end of the administration period.
- Dose groups that were examined: all animals
- Battery of functions tested: A functional observational battery (FOB) and measurement of motor activity was carried out towards the end of the administration period Day 86, 87, 90 and 91. During the measurements the animals received no food and no water.

OTHERS
Sperm analyses were performed towards the end of the administration period. Thereby for sperm analyses specimen were sampled from fasted anesthetized male animals in a randomized sequence at necropsy.
The following parameters were determined. Thereby sperm motility examinations were carried out in a randomized sequence.
• sperm motility
• sperm morphology
• sperm head count (cauda epididymis)
• sperm head count (testis)

Sacrifice and pathology:

At the end of the administration period all surviving animals were sacrificed after a fasting period (withdrawal of food) for about 16 - 20 hours. The animals were sacrificed by decapitation under CO2 anesthesia, exsanguinated and necropsied.

GROSS PATHOLOGY: Yes

ORGAN WEIGHT: Yes
- liver
- kidneys
- adrenal glands
- testes
- epididymides
- ovaries
- uterus
- spleen
- brain
- heart
- thymus
- prostate gland

HISTOPATHOLOGY: Yes
Left testis, left epididymis and both ovaries were fixed in Bouin' solution; after fixation, the organs were embedded in paraplast.
The following organs were fixed in 4% formaldehyde solution:
- all gross lesions
- salivary glands (Glandula mandibularis and Glandula sublingualis)
- esophagus
- stomach (forestomach and glandular stomach)
- duodenum, jejunum, ileum
- cecum, colon, rectum
- liver
- pancreas
- brain
- pituitary gland
- sciatic nerve
- spinal cord (cervical, thoracic and lumbar cord)
- eyes
- thyroid glands
- parathyroid glands
- trachea
- lungs
- larynx
- nose (nasal cavities)
- aorta
- heart
- bone marrow (femur)
- lymph nodes (mandibular and mesenteric)
- spleen
- thymus
- kidneys
- urinary bladder
- oviducts/uterus/vagina
- prostate gland, seminal vesicles
- female mammary gland
- skin
- skeletal muscle
- sternum with marrow
- femur with knee joint
- extraorbital lacrimal glands

Other examinations:

The scope of examinations was extended (sperm parameters, extended organ weights and histopathology) to cover also effects on reproductive organs.

Statistics:

Means and standard deviations of each test group were calculated for several parameters.
Further statistical analyses were performed:
• Dunnet test, two sided for the hypothesis of equal means (comparison of each group with the control group):
- Food consumption, water consumption, body weight, body weight change, food efficiency
• KRUSTAL-WALLIS test (two-sided) non-parametric one-way analysis (post test WILCOXON):
- Feces, rearing, grip strength length forelimbs, grip strength hindlimbs, landing foot-splay test, motor activity.
- Clinical pathology parameters, except differential blood count.
- Weight parameters
• FISHER's exact test for the hypothesis of equal proportions for pairwise comparison of each dose group with the control group:
- Urinalysis, except volume, color, turbidity and specific gravity; abnormal sperm > 4 %.
• WILCOXON-test (one sided) with Bonferoni-Holm-Adjustment for the hypothesis of equal medians for pairwise comparison of the dose groups with the control group:
- Total spermatids/g testis, total sperm/g cauda epi., % motility.

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
No animal died during the administration period.
Skin lesion was seen in one male of the mid dose group. Alopecia (both forelimbs and both body flanks) was seen in one female of the high dose group. Due to the isolated occurrence, these findings were assessed as being incidental. No other abnormal clinical signs were observed .

BODY WEIGHT AND WEIGHT GAIN
5000 ppm (243.8 mg/kg bw/d in males, 307.2 mg/kg bw/d in females):
• significantly decreased body weight on days 14 to 91 in males (12.2% below control) and on days 14 to 21, 42, 56 to 70 and 84 to 91 in females (8.8% below control).
• significant impairment of body weight change on days 7 to 91 in males (20.4% below control on day 91) and on days 14 to 28, 42 to 70 and 84 to 91 in females (19.2% below control on day 91).
• mean terminal body weight was significantly decreased in male rats (-11 .7 %) and also slightly but significantly decreased (-6 .4 %) in female rats.
These effects were assessed as being treatment-related.

1000 ppm (65.4 mg/kg bw/d in males, 82.1 mg/kg bw/d in females) and 200 ppm (14.4 mg/kg bw/d in males, 21.0 mg/kg bw/d in females):
• No changes in body weight and weight gain were observed.
FOOD CONSUMPTION
5000 ppm (243.8 mg/kg bw/d in males, 307.2 mg/kg bw/d in females):
• significantly decreased food consumption from days 7 to 28 and 42 to 91 in males (up to 18.2% below control), and females (up to 21.0% below control).

1000 ppm (65.4 mg/kg bw/d in males, 82.1 mg/kg bw/d in females):
• significantly decreased food consumption on days 77 and 91 in males (up to 9.4% below).
These effects were assessed as being treatment-related.

200 ppm (14.4 mg/kg bw/d in males, 21.0 mg/kg bw/d in females):
• No changes in food consumption were observed.
FOOD EFFICIENCY
5000 ppm (243.8 mg/kg bw/d in males, 307.2 mg/kg bw/d in females):
• significantly decreased food efficiency on days 7 to 14, 56 and 77 to 84 in males and on day 42 in females.
A relationship to treatment cannot be excluded with certainty.

1000 ppm (65.4 mg/kg bw/d in males, 82.1 mg/kg bw/d in females) and 200 ppm (14.4 mg/kg bw/d in males, 21.0 mg/kg bw/d in females):
• No changes in food efficiency were observed.

WATER CONSUMPTION AND COMPOUND INTAKE
5000 ppm (243.8 mg/kg bw/d in males, 307.2 mg/kg bw/d in females):
• decreased water consumption during the entire study in males (up to 49.9% below control) and females (up to 47.8% below control)

1000 ppm (65.4 mg/kg bw/d in males, 82.1 mg/kg bw/d in females):
• decreased water consumption on days 7 to 14, 28 to 63 and 77 to 91 in males (up to 25.1% below control) and females (up to 28.9% below control).
This was assessed as being treatment-related.

200 ppm (14.4 mg/kg bw/d in males, 21.0 mg/kg bw/d in females):
• No effects were observed in the low dose group.

The mean daily test substance intake in mg/kg body weight over the entire study period:
5000 ppm (243.8 mg/kg bw/d in males, 307.2 mg/kg bw/d in females)
1000 ppm (65.4 mg/kg bw/d in males, 82.1 mg/kg bw/d in females)
200 ppm (14.4 mg/kg bw/d in males, 21.0 mg/kg bw/d in females).
OPHTHALMOSCOPIC EXAMINATION
No substance-related effects were obtained. All findings (remainders of the pupillary membrane, corneal stipplings) were spontaneous in nature and equally distributed between treated animals and controls.

HAEMATOLOGY
There are no treatment-related changes in the hematological parameters measured.

CLINICAL CHEMISTRY
Compound-related differences in clinical chemistry parameters were not evident at any dose level in either males or females.

URINALYSIS
5000 ppm (243.8 mg/kg bw/d in males, 307.2 mg/kg bw/d in females):
• decreased urinary volume in both sexes
• increased urinary specific gravity in both sexes
These findings are assessed as being compound-related and are mainly caused by the reduced water consumption.
In the other urine parameters examined, no treatment-related changes were found.

1000 ppm (65.4 mg/kg bw/d in males, 82.1 mg/kg bw/d in females) and 200 ppm (14.4 mg/kg bw/d in males, 21.0 mg/kg bw/d in females):
• No effects were observed.

NEUROBEHAVIOUR
- Functional observational battery
• Home cage, open field observations and reflex tests: All findings were assessed as being incidental, as they occurred in single animals, only, or were equally distributed between treated groups and controls.
• Feces, Rearing, Grip strength, Landing foot-splay test: No substance-related effects were observed.
- Motor activity measurement: No substance related-findings were obtained.

ORGAN WEIGHTS
5000 ppm (243.8 mg/kg bw/d in males, 307.2 mg/kg bw/d in females)
• In male rats, the mean absolute weight of the liver was significantly decreased (-14 .4 %).
• In female rats of the high dose group, the absolute mean weight of the ovaries was slightly although significantly decreased (-13.6 %).
• The other mean absolute weight parameters did not show significant differences when compared to the control group.
• The mean relative weights of testes (+13 .7 %), epididymides (+12 .7%) and brain (+10,7 %) were significantly increased in males. This was regarded to be the consequence of the decrease of the mean terminal body weight (-11 .7%).
• In females, the mean weight of the kidneys was slightly but significantly increased (+ 10 .7 %), most likely for the same reason.

1000 ppm (65.4 mg/kg bw/d in males, 82.1 mg/kg bw/d in females).
• In male rats, the mean absolute weight of the liver was significantly decreased (-7 .9 %).

200 ppm (14.4 mg/kg bw/d in males, 21.0 mg/kg bw/d in females).
• The mean absolute weight of thymus was slightly although significantly increased in males (+16 .3 %). This was regarded incidental as there was no dose-response relationship.
• An incidental finding was the slight although significant decrease (-7 .5 %) of relative weight of the heart in males of the low dose group as there no dose-relationship was evident.
The other mean relative weight parameters did not show significant differences when compared to the control group.

All changes of mean organ weight parameters were regarded to be the consequence of the significantly decreased body weights rather than a specific treatment-related effect. Pathology (see below) did not indicate treatment-related gross lesions or microscopic findings in any of the organs
investigated.

GROSS PATHOLOGY
Only a few gross lesions were noted in the glandular stomach (erosion/ulcer, focus, hyperemia), liver (enlarged), kidneys (granular surface, pelvic dilation) and skin (lesion, sparse hair). With one exception (erosion/ulcer in 2 low dose groups) these findings occurred only once per group and they were hence interpreted to have developed spontaneously and unrelated to treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
Some grossly noted lesions of the glandular stomach (focus, ulcer/erosion), skin (sparse hair), kidneys (pelvic dilation) or liver (enlarged) lacked a microscopic correlate.
However, regardless of whether or not they had a microscopic correlate, all the gross lesions were considered to have developed spontaneously and to be unrelated to treatment.
No histological correlates were obtained for the significantly decreased absolute weights of liver in the male high dose group and in mid dose group. Further, no morphologic correlate was obtained for the significantly increased mean relative weights of the testes, epididymides and brains in male high dose group.
The increase of the relative weight of brain in male high dose group as well as the increase of the relative weights of kidneys in the female high dose group were considered to have developed spontaneously and to be unrelated to treatment.
Finally, no microscopic finding was obtained that may account for the significant decreased mean absolute weight of the ovaries.
All microscopic findings recorded were either single observations, or they were recorded at a low incidence, or they occurred in control animals only, or at comparable incidence and graded severity in control and high dose males and/or females.

OTHER FINDINGS
No effects on reproductive organs or sperm parameters were observed.

There are statistically significant intergroup differences in the results of clinical pathology testing. These deviations are marginal, incidental or inconsistent, when compared with the other sex, or lack dose-response relationship. Accordingly, these findings are considered to be of no toxicological significance.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

82.1 mg/kg bw/day (nominal)

Sex:

female

Basis for effect level:

other: decreased water consumption; corresponding to 1000 ppm.

2

Dose descriptor:

NOAEL

Effect level:

65.4 mg/kg bw/day (nominal)

Sex:

male

Basis for effect level:

other: decreased food consumption and water consumption; corresponding to 1000 ppm.

3

Dose descriptor:

LOAEL

Effect level:

307.2 mg/kg bw/day (nominal)

Sex:

female

Basis for effect level:

other: impairment of body weight; decreased food consumption, food efficiency and water consumption; urinalysis (decreased urinary volume and increased urinary specific gravity in both sexes; corresponding to 5000 ppm.

4

Dose descriptor:

LOAEL

Effect level:

243.8 mg/kg bw/day (nominal)

Sex:

male

Basis for effect level:

other: impairment of body weight; decreased food consumption, food efficiency and water consumption; urinalysis (decreased urinary volume and increased urinary specific gravity in both sexes; corresponding to 5000 ppm.

5

Dose descriptor:

NOEL

Effect level:

21 mg/kg bw/day (nominal)

Sex:

female

Basis for effect level:

other: no substance related effects; corresponding to 200 ppm.

6

Dose descriptor:

NOEL

Effect level:

14.4 mg/kg bw/day (nominal)

Sex:

male

Basis for effect level:

other: no substance related effects; corresponding to 200 ppm.

Target system / organ toxicity

Critical effects observed:

not specified