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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted in compliance with GLP regulations For justification of read across please refer to sction 13.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991
Reference Type:
publication
Title:
Subchronic toxicity studies of 3-Methyl-1-butanol and 2-Methyl-1 -propanol in rats
Author:
Schilling K et al.
Year:
1997
Bibliographic source:
Human & Experimental Toxicology ( 1997) 16, 722-726

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
test substance in drinking water
GLP compliance:
yes
Remarks:
Department of Toxicology
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 3-methyl-1-butanol
- Test substance No. 88/56
- Lab. J. No.: H 21670
- Physical state: liquid/colourless
- Analytical purity: >98%
- Stability under test conditions: The stability was ensured for the study period under the specified storage conditions by reanalysis (see report of Nov 25 1988)
- Storage condition of test material: at room temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Dr. K. Thomae GmbH, D-7950 Biberach/Riss, FRG
- Age at study initiation: 42 d
- Identification: ear tattoo
- Weight at study initiation: mean 173 g (males), 150 g (females)
- Housing: singly in wire cages (type D III of Becker & Co, Castrop-Rauxel, FRG; floor area about 900 cm2))
- Diet (e.g. ad libitum): KLIBA maintenance diet rat/mouse/ hamster, 343 meal, Klingentalmuehle AG, CH-4303 Kaiseraugst, Switzerland; during the exposure-free observation period
- Water (e.g. ad libitum): tap water
- Acclimation period: at least 7 days

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

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on oral exposure:
DRINKING WATER PREPARATION
- Rate of preparation (frequency): the drinking water solutions were prepared twice a week
- Mixing appropriate amounts with: the test substance was weighed for each particular test group and the specific quantity of drinking water (also weighed) added. To obtain a homogeneous solution of the test substance in the drinking water the mixture was then stirred for about 30 minutes using a magnetic stirrer.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Capillary gas chromatography using the area percentage method (under consideration of the water content).
Duration of treatment / exposure:
3 months
Frequency of treatment:
continuous exposure via drinking water
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1000, 4000 and 16000 ppm
Basis:
nominal in water
Remarks:
Doses / Concentrations:
80, 340, 1250 mg/kg bw
Basis:
nominal in water
Remarks:
Doses / Concentrations:
appr. 73, 295 and 1068 mg/kg bw
Basis:
other: calculated, actually ingested in male animals
Remarks:
Doses / Concentrations:
appr. 90, 385 and 1431 mg/kg bw
Basis:
other: calculated, actually ingested in female animals
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: On the basis of the data of a prior study with doses of 0 and 20000 resp. 16000 ppm to guarantee a procedure parallel to the study with a similar substance, the doses have been selected using a factor of 4 for the subchronic study with administration of the test substance in the drinking water.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: a check was made twice (mondays to fridays) and once a day (saturdays, sundays and puplic holidays) for general observations. Furthermore, the animals were subjected once a week to an additional exact clinical examination.

BODY WEIGHT: Yes
- Time schedule for examinations: the body weights of the animals were determined once a week during the study and in each case on the same day of week (Tuesday). The animals were additionally weighed prior to the start of the study for randomization.

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: once a week during the administration period for a period of 4 days (Friday - Tuesday).
The mean daily intake of test substance (in mg) per kg body weight was calculated at the intervals at which water consumption was determined according to the following formula: (WTR CONS * D) / body weight on day x
WTR CONS = mean daily water consumption (in g) within 4 days of the study (from day x-4 to day x); D = dose in ppm
The values listed in the tables are group means, determined from the intake of test substance by the individual animals.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before the start of the study and toward the end of the study
- Dose groups that were examined: the eyes of the animals in the test group 0 (control) and in the test group 3 (16000 ppm) were examined for any changes to the refracting media using a HEINE FOCALUX hand-held slit lamp.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 87 days
- Animals fasted: No
- Parameters examined: leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets. The differential blood count and the reticulocytes were counted visually. The data were transferred into the computer. Clotting analyses were carried out by determining the thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Parameters examined: enzymes (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-γ-glutamyltransferase); blood chemistry (sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

The exsanguinated animals were necropsied and assessed by gross pathology. The anesthezited animals, liver, kidneys, adrenal glands and testes were weighed.
Subsequently, the following organs and tissues were fixed in 4% formaldehyde solution:
brain, thyroid/parathyroid glands, trachea, heart, salivary glands (gl. mandibularis, gl. sublingualis), spleen, adrenal glands, testes / ovaries, prostate and seminal vesicle, esophagus, duodenum, jejunum, ileum, urinary bladder, female mammary gland, sciatic nerve, eyes, spinal cord (cervical, thoracic, lumbar), pituitary gland, thymus, lungs, aorta, liver, kidneys, pancreas, uterus, skin, stomach, cecum, colon, rectum, mesenteric lymph node, skeletal muscle, femur (with joint and marrow), sternum with marrow, extraorbital lacrimal glands, all gross lesions.

Fixation was followed by histotechnical processing carried out by EPS-UK (Hereford, England) and examination by light microscopy and assessment of findings. In the control and high dose groups all organs and tissues were examined; at the low and medium dose level only lungs, liver, kidneys and all gross lesions were assessed.
Other examinations:
A check was made for dead or moribund animals twice a day (mondays to fridays) or once a day (saturdays, sundays, and on public holidays).
Statistics:
The statistical evaluation of the data was carried out on the computer systems of the testing laboratory. Means and standard deviation were calculated for the variables feed consumption, drinking water consumption, body weight and test substance intake for the animals in each test group. The statistical significance of the clinical data (body weight) was determined using an analysis of variance (ANOVA) with subsequent DUNNETT's test.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
No signs attributable to the administration of the test substance were observed throughout the study period.

BODY WEIGHT AND WEIGHT GAIN
The body weight gain of all dosed animals of both sexes was, within the biological range, analogous to the controls.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
In the males of test groups 1 (1000 ppm) and 2 (4000 ppm) there were signs of an increased drinking water consumption as of about the fifth week of the study, which at different times varied in degrees of intensity and showed no dose dependency.
The female animals of the dose groups showed a deviated drinking water consumption during the administration period, which was in the last 3 weeks of the study, in dose groups 1 (1000 ppm) and 2 (4000 ppm) not clear dose-dependent increased.
- The amount of test substance intake (in mg) consumed each day by the animals per kilogram body weight was calculated at the times at which the drinking water consumption was also determined.

OPHTHALMOSCOPIC EXAMINATION
The ophthalmological examinations carried out before the beginning of administration and at the end of the study using a hand-held slit lamp revealed no substance-induced impairment of the refracting media; except one animal showed at the end of the study in the right eye reddish areas in the lower temporal quadrant. This effect was assessed as being not substance induced, i.e. spontaneous in nature.

HAEMATOLOGY AND CLINICAL CHEMISTRY
All the parameters for which a substance-induced change is even merely suspected are assessed below:
- Red blood cells: at the end of the 3-month administration period, there was in the blood of the males of test group 3 (16000 ppm) an increase in the erythrocyte values, a decrease in the mean corpuscular volume and a decrease in the mean corpuscular hemoglobin content. The authors of the study assumed that these changes might have been attributable to the test substance administration and could be an indication of a marginal hemotoxic potential of the test substance. However, these effects were generally mild in extent and occurred in only one sex. They were all in the range of biological variation, e.g. the count of red blood cells between 7.76 – 8.94 E+12/L. Since the slightly increased erythrocyte count of the males in test group 2 (4000 ppm) was also in the range of historically observed biological variations, it is questionable if these effects were related to the test substance. Moreover, the deviations in haematology parameters from those of the controls did not correlate with other biochemical, haematological or histopathology results, and are thus not characteristic for a specific toxic effect.
- Other examinations: the other examinations exhibited no changes which are causally related to the substance administration.
Deviations from the control group figures were found in isolated cases but are not regarded as being related to the test substance administered.
ORGAN WEIGHTS
No statistically significant organ (liver, kidneys, adrenal glands and testes) weight changes when compared with the control group were observed.

GROSS PATHOLOGY
Macroscopy did not reveal any substance-induced changes.

HISTOPATHOLOGY: NON-NEOPLASTIC
In the histological examination, ectopia of thymus tissue in the region of the thyroid glands was found in 5 males of test group 3. One male control animal and two female control animals had this finding, too. The ectopic thymus tissue is congenital and, hence, not attributable to the administration of the test substance. Also all other changes diagnosed by light microscopy are assessed as being not substance-related.

OTHER FINDINGS
No animal died intercurrently during the entire study.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: overall effects: no toxic effects found
Dose descriptor:
NOAEL
Effect level:
1 250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: overall effects: no toxic effects found haematology: effects, although within historical control data range

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Haematological data (male rats) and prothrombin time (female rats) after administration of 3-methylbutan-1-ol:

 

Dose

 

WBC

RBC

HGB

HCT

MCV

MCH

MCHC

PLT

Prothrombin time HQT

[mg/kg bw/d]

 

[E+09/L]

[E+12/L]

[mmol/L]

[L/L]

[E-15L]

[E-15mol]

[mmol/L]

[E+09/L]

[seconds]

0

M

7.29

7.76

9.52

0.378

48.63

1.23

25.18

995

32.4

 

SD

0.92

0.20

0.34

0.017

1.81

0.04

0.32

88

2.2

80

M

8.65*

8.10

9.77

0.391

48.23

1.21

24.98

1051

33.2

 

SD

0.68

0.37

0.39

0.019

1.48

0.05

0.63

75

3.0

340

M

7.73

8.18*

9.81

0.392

47.88

1.20

25.02

1069

35.3*

 

SD

0.92

0.34

0.42

0.018

1.23

0.02

0.41

81

2.2

1250

M

7.84

8.41**

9.79

0.392

46.55*

1.16**

24.98

1038

35.6*

 

SD

1.28

0.38

0.33

0.016

1.60

0.03

0.36

86

1.8

* p<0.05; ** p<0.01

M: mean; SD: standard deviation

WBC: white blood cells; RBC: red blood cells

HGB: haemoglobin; HCT: haematocrit

MCV: mean corpuscular volume; MCH: mean corpuscular haemoglobin; MCHC: mean corpuscular haemoglobin concentration

PLT: platelets; HQT: Hepato Quick’s test

 

 

The administration of 3-Methylbutanol-1 to male and female Wistar rats via their drinking water at dose levels of 1000, 4000 and 16000 ppm corresponding to approx. 80, 340, and 1250 mg/kg bw/day led to no clinical signs in the sense of a toxic effect.

The increased water consumption of males and females in the dose groups 1 (1000 ppm) and 2 (4000 ppm) is possibly substance-related, but it shows no clear dose response relationship and varied at different times in degrees of intensity. With respect to this, it is assessed as being no toxic effect.

In addition, a statistically significant increase in red blood cells, a decrease in the mean corpuscular volume and mean corpuscular haemoglobin in male animals at 16000 ppm was observed. These effects were generally mild in extent and occurred in only one sex. They were all in the range of biological variation, e.g. the count of red blood cells between 7.76 – 8.94 E+12/L. Since the slightly increased erythrocyte count of the males in test group 2 (4000 ppm) was also in the range of historically observed biological variations, it is questionable if these effects were related to the test substance. Moreover, the deviations in haematology parameters from those of the controls did not correlate with other biochemical, haematological or histopathology results, and are thus not characteristic for a specific toxic effect.

To conclude, it can be stated that from the clinical point of view a dose level which causes clear signs of toxicity in male and female rats is above 16000 ppm (= 1250 mg/kg bw/day).

Applicant's summary and conclusion