Registration Dossier

Administrative data

Description of key information

The oral NOAEL for subchronic oral toxicity was found to be approximately 1250 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
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
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
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
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
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.
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.
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.
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
Critical effects observed:
not specified

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).

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
July 2017 - Sep 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility. Analyses demonstrated the stability of the test substance preparations over a period of 10 days at room temperature.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance solutions in drinking water were prepared in intervals, which took into account the analytical results of the stability verification.
For the preparation of the administration solutions the test substance was weighed in a calibrated beaker depending on the dose group, topped up with drinking water and intensely mixed with a magnetic stirrer until it was completely dissolved.

FORM AS APPLIED IN THE TEST (if different from that of starting material): mixed with drinking water
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The test guideline requires the rat to be used as the animal species. This rat strain was selected since extensive historical control data are available for Wistar rats.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany GmbH
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 11-12 weeks (male animals); 10 weeks (female animals)
- Weight at study initiation: males: 390 g (mean); females: 215 g (mean)
- Housing: up to 5 animals per sex and cage during pretreatment; individually during the study period
- Diet: ad libitum; ground Kliba maintenance diet mouse-rat “GLP” (supplied by Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: ad libitum
- Acclimation period: 21 days

DETAILS OF FOOD AND WATER QUALITY: The drinking water was regularly assayed for chemical contaminants as well as for the presence of (pathogenic) microorganisms. 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. With regard to the analytical findings of chemical and microbiological contaminants and the duration of application, the diet was found to be suitable.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: 27 June 2017 To: 17 Aug 2017 (males); 14 Sept 2017 (females)
Route of administration:
oral: drinking water
Vehicle:
water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
At the beginning (during pre-mating), twice during gestation and once during lactation of the study each 1 sample were taken from the low, mid and high concentrationfor a concentration control analysis. The samples of the gestation were analyzed only if any imprecision occurs during the analysis of the samples from the beginning and lactation of the study.

The samples collected from the beginning of the administration period and during the lactation period were analyzed via capillary gas chromatography with internal standard quantification. The analytical investigations of the test substance preparations were carried out in compliance with the Principles of Good Laboratory Practice.

The test substance concentrations in the drinking water were found to be in the range of 90-110 % of the nominal concentration.
Duration of treatment / exposure:
males: 29 days; females: 58/64 days
The duration of treatment covered a 2-weeks premating period and mating in both sexes (mating pairs were from the same test group), 2 days postmating in males as well as the entire gestation and approximately 3 weeks of lactation period in females up to the day of scheduled sacrifice of the animals.
Frequency of treatment:
continuously via drinking water
Dose / conc.:
12 500 ppm
Remarks:
corresponds to a mean daily intake of approx. 785 mg/kg bw/d in males and 1273 mg/kg bw/d in females;
During lactation the concentration was reduced to 50% (6250 ppm) in females.
Dose / conc.:
3 750 ppm
Remarks:
corresponds to a mean daily intake of approx. 229 mg/kg bw/d in males and 359 mg/kg bw/d in females;
During lactation the concentration was reduced to 50% (1875 ppm) in females.
Dose / conc.:
1 250 ppm
Remarks:
corresponds to a mean daily intake of approx. 71 mg/kg bw/d in males and 116 mg/kg bw/d in females;
During lactation the concentration was reduced to 50% (625 ppm) in females.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: A test study was performed in male and female Wistar rats. The test substances (2-Methylbutanol, 3-Methylbutanol, 1-Hexanol and 2-Hexanol) were orally (gavage) applied at dose levels of 0 mg/kg bw/day (corn oil; test group 0), 1000 mg/kg bw/day (2-Methylbutanol; test group 1), 1000 mg/kg bw/day (3-Methylbutanol; test group 2), 500 mg/kg bw/day (1-Hexanol; test group 3) and 500 mg/kg bw/day (2-Hexanol; test group 4) over a period of 14 days to 3 female Wistar rats per test group. After treatment with 3-Methylbutanol (1000 mg/kg bw/d) one animal was found dead on day 5. No mortality occurred in the other test groups. In all treatment groups treatment-related changes of the body weights were observed.


Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes (for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity; parturition and lactation behavior of the dams)
- Time schedule: at least once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to the first administration (day 0) and at weekly intervals during the administration period
- Examined parameters (outside of the cage in a standard arena (50 × 37.5 × 25 cm)): abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/ consistency), assessment of the urine discharged during the examination, pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: twice a week at the same time of the day (in the morning) until sacrifice, with the following exceptions:
• During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 4, 7, 10, 14, 17 and 20.
• Females with litter were weighed on the day after parturition (PND 1), 4, 7, 10 and 13.
• Body weight was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: twice a week with the following exceptions:
• Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental animals).
• Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0 - 4, 4 - 7, 7 - 10, 10 - 14, 14 - 17 and 17 - 20.
• Food consumption of F0 females which gave birth to a litter was determined on PND 1 - 4, 4 - 7, 7 - 10 and 10 - 13.
•Food consumption was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.

WATER CONSUMPTION: Yes
- Time schedule for examinations: twice a week with the following exceptions:
• During pregnancy, water consumption of the females with evidence of sperm was determined for GD 0 - 1, 3 - 4, 6 - 7, 9 - 10, 13 - 14, 16 - 17 and 19 - 20.
• During lactation, water consumption of the females, which gave birth to a litter was determined for PND 1 - 3, 3 - 4, 6 - 7, 9 - 10 and 12 - 13.
•Water consumption was not determined for females without positive evidence of sperm during mating and gestation and for females without litter during lactation.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination (males); at PND 14 (females)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (about 16 to 20 hours)
- How many animals: the first 5 surviving parental males and the first 5 females with litters (in order of delivery) per group
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination (males); at PND 14 (females)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (about 16 to 20 hours)
- How many animals: the first 5 surviving parental males and the first 5 females with litters (in order of delivery) per group
- Parameters checked in table 2 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes; functional observational battery and motor activity assessment (see "OTHER")

OTHER:
Functional observational battery (FOB):
- A functional observational battery was performed in the first five parental male animals per test group and the first five surviving females with litter (in order of delivery) of all test groups at the end of the administration period starting at about 10.00 h on study day 28 (males) and 55 (females).
- Examined parameters:
• Home cage observations: posture, tremors, convulsions, abnormal movements, gait, other findings
• Open field observations: behavior on removal from the cage, fur, skin, salivation, nasal discharge, lacrimation, eyes/ pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/ stereotypes, gait, activity/ arousal level, feces excreted within 2 minutes (appearance/ consistency), urine excreted within 2 minutes (amount/ color), rearing within 2 minutes, other findings
• Sensory motor tests/ reflexes: reaction to an object being moved towards the face (approach response), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (auditory startle response), coordination of movements (righting response), behavior during handling, vocalization, pain perception (tail pinch), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test, other findings

Motor activity measurement:
- measured from 14:00 h onwards on the same day as the FOB was performed

Estrous cycle determinations:
- In all parental females in the premating phase, estrous cycle normality was evaluated by preparing vaginal smears during a minimum of 2 weeks prior to premating. The evaluation continued throughout the pairing period until the female showed evidence of copulation. Additionally, on the day of scheduled sacrifice, the estrous cycle stage was also determined in all female F0 rats.

Male reproduction data:
- The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs.

Female reproduction and delivery data
- The pairing partners, the number of mating days until vaginal sperm were detected and gestational status were recorded for F0 females.

Thyroid hormones (males only)
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (about 16 to 20 hours)
- How many animals: all surviving males at termination
- Parameters checked in table 3 were examined.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 4, Organ weights)

HISTOPATHOLOGY: Yes (see table 4)
Statistics:
see table 5
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
Water consumption of F0 parental female rats of test group 1 (1250 ppm) was comparable to the concurrent control values throughout the entire study. During gestation, but not premating and lactation, an overall slightly higher water consumption was noted for the the mid- (3750 ppm) and high-dose F0 females (12500 ppm, statistically significant during GD 6 - 7 and 16 - 17). This phenomenon may be related to an increased thirst of the females due to the taste/texture of high-concentrated test item solutions. As there were no corroborative signs of toxicity, no adversity is assumed for this finding.
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In males of test groups 1, 2 and 3 (1250, 3750 and 12500 ppm) platelet counts were significantly higher compared to controls, which was due to low values in the controls. Additionally, in females of test group 3 (12500 ppm) hemoglobin values were significantly increased. All mentioned values were within historical control ranges (males, platelets 594-776 Giga/L; females, hemoglobin 8.4-9.2 mmol/L). Therefore, these changes were regarded as incidental and not treatment-related. In males of test group 1 (1250 ppm), mean corpuscular hemoglobin (MCH) content and relative neutrophil counts were significantly lower whereas relative lymphocyte counts were significantly higher compared to controls. However, the values were not dose-dependently changed and therefore the changes regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In males of test group 1 (1250 ppm) alanine aminotransferase (ALT) activities were significantly increased, but the values were not dose-dependently changed. Therefore, this alteration was regarded as incidental and not treatment-related.
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):
In males of test group 3, the mean absolute brain weight was slightly (-4%), but significantly (p<= 0.05) decreased. Because the mean relative weight was not significantly changed and the mean absolute brain weight (2.066 g) was in the range of historical control data (2.066 g – 2.158 g), a treatment-related effect was considered unlikely.
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Estrous cycle data, generated during the last 2 weeks prior to mating for the F1 litter, revealed regular cycles in the females of all test groups 0 - 3. The mean estrous cycle duration was similar: 4.0 / 3.9 / 3.9 and 4.0 days in test groups 0 - 3, respectively.
Dose descriptor:
NOAEL
Effect level:
12 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to the highest dose tested
Remarks on result:
other: = about 875 mg/kg bw/d for males and 1273 mg/kg bw/d for females
Critical effects observed:
no
Conclusions:
Under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats the no observed adverse effect level (NOAEL) for general systemic toxicity, fertility and reproductive performance and for developmental toxicity in the offspring was 12500 ppm for male (about 785 mg/kg bw/d) and female (about 1273mg/kg bw/d) Wistar rats, the highest concentration tested.
Executive summary:

In an OECD 422 study, the test item was administered daily as addition to the drinking water in different concentrations to groups of 10 male and 10 female Wistar rats to screen for potential systemic, reproductive and developmental toxicity. After a two-week premating period, these parental animals were paired and the females were allowed to give birth and bring up the offspring until sacrifice on PND 4 or PND 13. In males the overall mean dose of the test substance throughout the study was approx. 71 mg/kg bw/d in the 1250 ppm group, approx. 229 mg/kg bw/d in the 3750 ppm group and approx. 785 mg/kg bw/d in the 12500 ppm group; in females it was approx. 116 mg/kg mg/kg bw/d in the 1250 ppm group, approx. 359 mg/kg bw/d in the 3750 ppm group and approx. 1273 mg/kg bw/d in the 12500 ppm group.

Analyses confirmed the overall accuracy of the prepared concentrations in the drinking water. The stability of these preparations was also demonstrated over a period of 10 days under ambient conditions.

In the clinical examinations of the F0 parental animals no clinical symptoms were caused by the test compound up to the high-concentration of 12500 ppm. In the in-depth investigations including the detailed clinical observation, the functional observational battery and the measurement of motor activity no treatment-related differences to control were observed at any concentration. Clinical chemistry, haematology, water consumption, food consumption and body weights / body weight gain did not show important test substance-related changes.

Concerning clinical pathology (including thyroid hormone measurement) no treatment-related, adverse effects were observed up to a concentration of the compound of 12500 ppm in the drinking water.

Regarding pathology, there were no treatment-related organ weight changes, gross lesions, and histopathological findings in male and female Wistar rats. Regarding fertility and reproductive performance, as well as pre-postnatal development no signs of toxicity were observed in male or female parental animals or their offspring of all test groups (1250, 3750, and 12500 ppm) during the entire study. Most F0 parental animals across all test groups proved to be fertile and those individuals failing to generate offspring did not show any specific gross or histopathological findings. Mating behavior, conception, implantation and parturition were not influenced. Neither determination of anogenital distance/index not the count of nipple/areola anlagen revealed any treatment-related changes up to and including a concentration of the test item of 12500 ppm in the drinking water.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Several study results on the test substance itself as well as read-across data were considered reliable and suitable for assessment in a weight of evidence approach.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Oral route:

The toxicity after repeated dosing of 3-methylbutan-1-ol was evaluated. For this purpose, it was tested in two combined repeated-dose / reproductive developmental toxicity study similar to OECD TG 422 and in compliance with GLP regulations (Kuraray Co. Ltd. 2008 and BASF 2018). For the older study, the substance was administered to groups of 12 male and 12 female Sprague-Dawley strain SPF rats at dose levels of 0 (control group), 30, 100 or 300 mg/kg bw for a total of 42 days to males (for 14 days before mating throughout the mating period up to the day before necropsy) and for a total of 41 to 53 days to females (for 14 days before mating throughout the mating and gestation periods up to day 4 of lactation) to examine its repeated dose toxicity and reproductive and developmental toxicity. For 5 males and 5 females in the 0 and 300 mg/kg bw groups, a 14-day recovery period was provided after administration for 42 days to examine reversibility of the toxic changes. The females in the recovery group were treated additionally to the main groups and were not subjected to mating. No deaths occurred in any group, and there were no test article-related effects in clinical observation, detailed clinical observation, manipulative test, measurement of grip strength, measurement of motor activity, food consumption, urinalysis (including water intake), haematological examination, blood chemistry examination, organ weight, histopathological findings or gross pathological examination. In the measurement of body weight, a low value in body weight gain (from day 1 to day 42) during the administration period was observed in males of the 300 mg/kg bw group. During the 2-week recovery period, the body weight gain of males in the recovery group of the 300 mg/kg bw group was higher than that of the control group. Based on the results described above, it was judged that the no adverse effect levels for repeated dose toxicity of 3-methylbutan-1-ol were 100 mg/kg bw /day in males and 300 mg/kg bw /day in females. As the changes were slight, not accompanied by any further alterations, and these effects were not observed in two 90 day studies at higher doses, these values were not chosen as key parameter for the assessment of repeated dose toxicity.

To resolve remaining uncertainities about the test substances' toxicity up to limit doses, i

n the more recently performed OECD 422 study, the test item was administered daily as addition to the drinking water in different concentrations to groups of 10 male and 10 female Wistar rats to screen for potential systemic, reproductive and developmental toxicity. As a revised version of the OECD testing guideline as followed, in this study more paramters are investigated and pups are observed for a longer period of time. In addition, dosages are higher and reach the limit concentrations prescribed in the guideline.

After a two-week premating period, these parental animals were paired and the females were allowed to give birth and bring up the offspring until sacrifice on PND 4 or PND 13. In males the overall mean dose of the test substance throughout the study was approx. 71 mg/kg body weight/day (mg/kg bw/d) in the 1250 ppm group, approx. 229 mg/kg bw/d in the 3750 ppm group and approx. 785 mg/kg bw/d in the 12500 ppm group; in females it was approx. 116 mg/kg body weight/day (mg/kg bw/d) in the 1250 ppm group, approx. 359 mg/kg bw/d in the 3750 ppm group and approx. 1273 mg/kg bw/d in the 12500 ppm group.

Analyses confirmed the overall accuracy of the prepared concentrations in the drinking water. The stability of these preparations was also demonstrated over a period of 10 days under ambient conditions.

In the clinical examinations of the F0 parental animals no clinical symptoms were caused by the test compound up to the high-concentration of 12500 ppm. In the in-depth investigations including the detailed clinical observation, the functional observational battery and the measurement of motor activity no treatment-related differences to control were observed at any concentration. Clinical chemistry, haematology, water consumption, food consumption and body weights / body weight gain did not show important test substance-related changes.

Concerning clinical pathology (including thyroid hormone measurement) no treatment-related, adverse effects were observed up to a concentration of the compound of 12500 ppm in the drinking water.

Regarding pathology, there were no treatment-related organ weight changes, gross lesions, and histopathological findings in male and female Wistar rats. Regarding fertility and reproductive performance, as well as pre-postnatal development no signs of toxicity were observed in male or female parental animals or their offspring of all test groups (1250, 3750, and 12500 ppm) during the entire study. Most F0 parental animals across all test groups proved to be fertile and those individuals failing to generate offspring did not show any specific gross or histopathological findings. Mating behavior, conception, implantation and parturition were not influenced. Neither determination of anogenital distance/index not the count of nipple/areola anlagen revealed any treatment-related changes up to and including a concentration of the test item of 12500 ppm in the drinking water.

Furthermore, the read across substance reaction mass of 2-methylbutan-1-ol and pentan-1-ol was tested in an GLP-compliant OECD 422 study (BASF 2018). It

was administered daily as addition to the drinking water in different concentrations to groups of 10 male and 10 female Wistar rats to screen for potential systemic, reproductive and developmental toxicity. After a two-week premating period, these parental animals were paired and the females were allowed to give birth and bring up the offspring until sacrifice on PND 4 or PND 13. In males the overall mean dose of the test substance throughout the study was approx. 77 mg/kg body weight/day (mg/kg bw/d) in the 1250 ppm group, approx. 254 mg/kg bw/d in the 3750 ppm group and approx. 842 mg/kg bw/d in the 12500 ppm group; in females it was approx. 117 mg/kg body weight/day (mg/kg bw/d) in the 1250 ppm group, approx. 372 mg/kg bw/d in the 3750 ppm group and approx. 1239 mg/kg bw/d in the 12500 ppm group.

Analyses confirmed the overall accuracy of the prepared concentrations in the drinking water. The stability of these preparations was also demonstrated over a period of 10 days under ambient conditions.

In the clinical examinations of the F0 parental animals no clinical symptoms were caused by the test compound up to the high-concentration of 12500 ppm. In the in-depth investigations including the detailed clinical observation, the functional observational battery and the measurement of motor activity no treatment-related differences to control were observed at any concentration. Water consumption, food consumption and body weights / body weight gain did not show important test substance-related changes. A small decrease in food consumption of high-dose males as well as temporary increase of water consumption in high-dose females were not accompanied by any body weight changes or other clinical findings and thus not noteworthy enough to be considered adverse.

Concerning clinical pathology (including thyroid hormone measurement) no treatment-related, adverse effects were observed up to a concentration of the compound of 12500 ppm in the drinking water.

Regarding pathology, there were no treatment-related organ weight changes, gross lesions, and histopathological findings in male and female Wistar rats. Regarding fertility and reproductive performance, as well as pre-postnatal development no signs of toxicity were observed in male or female parental animals or their offspring of all test groups (1250, 3750, and 12500 ppm) during the entire study. Most F0 parental animals across all test groups proved to be fertile and those individuals failing to generate offspring did not show any specific gross or histopathological findings. There were no test article-related effects on estrous cycle, number of days until copulation, copulation index, insemination index, or fertility index. There were no test article-related effects on delivery index, length of gestation period, number of corpora lutea, number of implantation sites, implantation index, index of pre-implantation loss, index of post-implantation loss, index of stillborn pups, parturition index, number of liveborn pups, live birth index or sex ratio, and there were no abnormalities in the lactation condition during the lactation period. In live born pups, there were no test article-related changes in body weight, external observation, gross pathological findings or viability index on day 4 of lactation. Neither determination of anogenital distance/index not the count of nipple/areola anlagen revealed any treatment-related changes up to and including a concentration of the test item of 12500 ppm in the drinking water. This results in a calculated NOAEL of 1000 mg/kg bw for the assessment of adverse effects on reproduction at the screening level.

Repeated dose toxicity of 3-methylbutan-1-ol was further investigated in a 90 day drinking water study performed according to OECD guideline 408 and in compliance with GLP regulations (BG-Chemie 1990). Ten Wistar rats per sex and dose received nominal concentrations of 1000, 4000 and 16000 ppm 3-methylbutan-1-ol corresponding to approx. 80, 340, 1250 mg/kg bw /day in the drinking water for 3 months. For comparison, one group of untreated animals (10 males, 10 females) was used as a control. Feed consumption, water consumption and body weight were determined once a week. The animals' state of health was checked each day, and once a week they were also subjected to an additional exact clinical examination. Prior to the beginning and towards the end of the study, ophthalmological examinations were carried out in the animals of the control and 16000 ppm group. One clinicochemical and one haematological examination were carried out toward the end of the study. At the end of the 3-month administration period all animals were sacrificed by decapitation and were assessed by gross pathology. Subsequently, a histopathological examination was carried out. There were no clinical signs in the sense of a toxic effect notable during the study period. Although an increased water consumption of males and females in the 1000 and 4000 ppm groups was observed, it was assessed as being no toxic effect since there was no clear dose response relationship apparent and it varied at different times in degrees of intensity. The administration of 3-methylbutan-1-ol caused an increase in the red blood cell count and a decrease in the mean corpuscular volume and the mean corpuscular haemoglobin content of the male rats at 16000 ppm (about 1250 mg/kg bw/d). There was also a slight increase in red blood cell number in the males after treatment with 4000 ppm. 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 and 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). Under the experimental conditions of the present study, the NOAEL of 3-methylbutan-1-ol was thus found to be 16000 ppm (about 1250 mg/kg bw /d) for the male and female rats.

Additionally, a publication is available where a NOAEL was derived after repeated oral exposure for 119 days in rats (Carpanini et al.1973). Groups of 15 male and 15 female rats were given daily doses of 0 (control), 150, 500 or 1000 mg 3-methylbutan-1-ol/kg body weight for 17 weeks. There were no effects associated with treatment in the results of the haematological examinations, serum analyses, urinary cell counts, renal concentration tests or organ weights. A slightly reduced rate of body weight gain at the highest dose level in males was shown to be due to a reduced food intake. Two rats given 1000 mg/kg bw/day died, but histopathological examination showed that these deaths were due to dosing into the lungs and not to any toxic effects of isoamyl alcohol. The no adverse effec level in this study was 1000 mg/kg bw/day.

Additionally, the read-across substance pentan-1 -ol was tested according to a protocol largely equivalent to OECD guideline 408 (Butterworth et al. 1978). 15 ASH/CSE rats per sex and dose received volumes of 5 mL/kg bw/day pentan-1-ol at doses of 50, 150 and 1000 mg/kg bw/day in corn oil via gavage for 13 weeks. During the study, the animals were weighed initially, at days 1, 2 and 6 and then at intervals of not more than 1 week up to day 91 of the study. Also, food and water consumptions were measured over the 24-hr period preceding the day of weighing. At the end of the study, blood and urine were collected and haematological examination and serum analyses as well as urinalysis were performed. At autopsy all the tissues were examined for gross abnormalities and selected organs were examined by histopathology. As result no abnormalities in appearance or behaviour were observed during the study and no significant differences between the treated and control rats in body weight or in food and water consumption were found. Although some isolated changes were observed in haematology pattern, like a reduction in haemoglobin concentration in the male rats after 13 weeks, no consistent pattern with respect to dose-response, sex or time relationships was found. As this was also the case for isolated changes in organ weights, and no other effects on renal function, organ weights or histopathology could be detected, the highest dose of 1000 mg/kg bw was established to be the NOAEL of pentan-1-ol.

As a conclusion, the oral NOAEL for repeated dose toxicity was determined to be at least 1000 mg/kg bw/day for all read-across category members. A detailed read-across justification is attached in IUCLID chapter 13. 

 

Inhalation:

There are no valid data available concerning repeated dose toxicity by the inhalation route.

 

Dermal route:

There are no data available concerning repeated dose toxicity by the dermal route.

 

Other routes:

In a publication (Stoner 1973) it was described how the maximum tolerable dose (MTD) of the read-across substance pentan-1-ol in 5 female mice was determined after 6 ip injections over a 2-week period. The post exposure period was 1-2 months. Doses of 50 and 250 mg/kg bw (nominal) were applied. As a result, the MTD was found to be 6000 mg/kg bw in the female mice.

Justification for classification or non-classification

The available data are considered reliable and suitable for classification purposes under Regulation (EC) No 1272/2008 (CLP).

As a result, the substance is not classified for repeated dose toxicity under Regulation (EC) No 1272/2008.