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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
15 Nov 2007 - 10 June 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD Guideline study conducted in compliance with GLP regulations For justification of read across please refer to section 13.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3-methylbutan-1-ol
EC Number:
204-633-5
EC Name:
3-methylbutan-1-ol
Cas Number:
123-51-3
Molecular formula:
C5H12O
IUPAC Name:
3-methylbutan-1-ol
Details on test material:
- Name of test material (as cited in study report): 3-Methylbutan-1-ol (abbreviation: IAA)
- Analytical purity: 99.8 %
- Lot/batch No.: 82934
- Supplier: Kuraray Co., Ltd.
- Stability under test conditions: Analysis of the test article, which was remaining after the end of this study and returned to the supplier, by infrared spectrophotometry revealed that it was stable during the study period

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Strain: Sprague-Dawley strain SPF rats [Crl:CD(SD)]
- Source: Atsugi Breeding Center, Charles River Laboratories Japan, Inc.
- Age at study initiation: 10 weeks of age
- Weight at study initiation: Males: 338 - 395 g; Females: 217 - 279 g
- Fasting period before study: no
- Housing: individually and subsequently in male-female pairs during the mating period
- Diet (ad libitum): NMF pelleted diet
- Water (ad libitum): tap water (Gotemba City)
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 25
- Humidity (%): 39 - 73
- Air changes (per hr): 10 to 15 times
- Photoperiod (hrs dark / hrs light): 12-hour lighting per day

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 1 w/v% CMC solution containing 1% Tween 80 in water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
A requisite amount of the test article was weighed, suspended in 1 w/v% CMC solution containing 1% Tween 80, transferred to a measuring cylinder, and the vehicle added to make the specified volume to prepare the test suspensions at specified concentrations. Preparation was done at least once in 7 days.

VEHICLE
- Preparation:
A requisite amount of CMC was weighed, dissolved in water for injection and then Tween 80 of the amount equivalent to 1 v/v% of the final volume was dissolved. The mixture was then transferred to a measuring cylinder and water for injection was added to prepare 1 w/v% CMC solution containing 1% Tween 80.

DOSE VOLUME: 10 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability:
In the stability test of the test suspensions, it was verified that the 100 mg/mL suspension (vehicle: 1 w/v% CMC solution containing 1% Tween 80) was stable at 25°C for 14 days after preparation. The 0.1 mg/mL suspension was verified to be stable at 25°C for 7 days but showed a slight decrease in the concentration after storage for 14 days. Based on these results, the frequency of preparation and storage conditions were selected.

Verification of Concentration and Homogeneity:
Each concentration of test suspensions used for males in week 1 and the final week of administration was analyzed by a GC method (detector: FID) at Gotemba Laboratory, Bozo Research Center Inc. by the method described in “Validation of the analytical method for determination of 3-methylbutan-1-ol in test suspensions by GC: Study No. C-A385”). Results showed that the concentration was 92.7 to 98.8% of the nominal concentration, c.v. value was not more than 3.0%, and both values were within the acceptable range (concentration: percentage of the nominal concentration, 100 ± 10%, c.v.: 10% or below).

Duration of treatment / exposure:
Males: 42 days (14 days before mating, 14 days during the mating period and 14 days after the end of the mating period)
Females: 41 - 53 days (14 days before mating, throughout the mating and gestation periods up to day 4 of lactation)
Satellite (recovery) groups: 42 days
Frequency of treatment:
once a day
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 30, 100, and 300 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
Main groups: 12 males and 12 females (0, 30, 100, and 300 mg/kg bw/day)
Satellite groups: 5 males and 5 females (0, and 300 mg/kg bw/day)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were set based on the results of a 14-day repeated dose oral toxicity study of IAA in Crl:CD(SD) rats at 7 weeks of age at the start of administration (Dose levels: 15, 60, 250 and 1000 mg/kg bw/day, number of animals used: 3 males and 3 females per group). In that 14-day study, administration at 1000 mg/kg bw/day was associated with sedation of activity after dosing and death of 1 female each on day 6 and day 9 of administration, but there were no toxicological abnormalities in organ weight, haematological examination or blood chemistry examination in any surviving males or females. At 250 mg/kg bw/day and below dose levels, there were no toxicological abnormalities. Based on these results, 300 mg/kg bw/day, which is approximately 1/3 of the lethal dose level in the 14-day study and at which no deaths but effects by repeated administrations were expected, was selected as the high dose level and a total of 3 dose levels including 100 and 30 mg/kg bw/day which were calculated using a common ratio of approximately 3 were selected.

- Study design:
The dose levels were set at 30, 100 and 300 mg/kg bw, and a total of 4 groups, including a control group, were provided for the study. In the main group where animals were subjected to mating, each group consisted of 12 males and 12 females. In the recovery group where animals were not subjected to mating, 5 males and 5 females were assigned each to the control group and the high dose group. For the males in the recovery group, 5 males in the main group with animal numbers 08 to 12 in the last 2 digits were transferred after the day of final administration.

Satellite groups:
For the males and females in the 0 and 300 mg/kg bw/d groups, a 14-day recovery period was provided after administration for 42 days. The females in the recovery group were not subjected to mating.
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 3 times a day (prior to dosing, immediately after and approximately 2 hours after dosing) during the administration period and once a day (in the morning) during the recovery period
- Observations: clinical signs, including abnormalities in external appearance, nutritional condition, posture, behavior and excrement

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the start of administration (all animals); once every week (males of the main and satellite groups, females of the satellite groups); once every week during the pre-mating administration period, and on the designated days (days 1, 7, 14 and 20 of gestation and day 4 of lactation) during the mating, gestation and lactation periods (females of the main groups).
- Observations: Posture, convulsion, abnormal behavior, ease of removal from cage, reactivity to handling (easiness, vocalization, etc.), fur / skin condition (staining of fur, unkempt fur, injury, skin color, etc.), eyes (exophthalmos, palpebral opening), secretions of eyes / nose, visible mucosal membrane, autonomic nervous function (lacrimation, salivation, piloerection, pupil size, abnormal respiration), arousal, gait, posture, tremor, convulsion, rearing, excretion (defecation count, urination), stereotypy (grooming, circling, etc.), abnormal behavior (self biting, retropulsion, etc.).

BODY WEIGHT: Yes
- Time schedule for examinations:
- males of the main groups: on days 1, 4, 8, 11, 15, 18, 22, 25, 29, 32, 36, 39 and 42 of administration and on the day of necropsy;
- males and females of the satellite groups: on days 1, 4, 8, 11, 15, 18, 22, 25, 29, 32, 36, 39 and 42 of administration, on days 1, 4, 8, 11 and 14 of recovery and on the day of necropsy;
- females of the main groups: on days 1, 4, 8, 11, 15 (on days 18, 22 and 25 as well for uncopulated females) of administration, on days 0, 4, 7, 11, 14, 17 and 20 of gestation, and on days 0 and 4 of lactation.

FOOD CONSUMPTION: Yes
- Time schedule for examinations:
- males of the main groups: on days 1, 4, 8, 11, 15, 32, 36, 39 and 42 of administration;
- males and females of the satellite groups: on days 1, 4, 8, 11, 15, 32, 36, 39 and 42 of administration and on days 1, 4, 8, 11 and 14 of recovery;
- females of the main groups: on days 1, 4, 8, 11 and 15 of administration, days 1, 4, 7, 11, 14, 17 and 20 of gestation and days 2 and 4 of lactation.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day following the end of the administration or on the final day of the recovery period
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes, overnight
- How many animals: 5 females and males / group
- Parameters: red blood cell count (RBC), hemoglobin (Hb), hematocrit (Ht), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), reticulocyte ratio (Reticulocyte), platelet count (Platelet), white blood cell count (WBC), differential leukocyte percentage, differential leukocyte count, prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day following the end of the administration or on the final day of the recovery period
- Animals fasted: Yes, overnight
- How many animals: 5 females and males / group
- Parameters: AlP, total cholesterol, triglyceride, phospholipids, total bilirubin, glucose, blood urea nitrogen, creatinine, sodium, potassium, chloride, calcium, inorganic phosphorus, total protein, albumin, A/G ratio, AST (GOT), ALT (GPT), LDH, γ-GTP.

URINALYSIS: Yes
- Time schedule for collection of urine:
In the final week of administration (week 6, day 37 to day 38 of administration) and in the final week of recovery (week 2, day 9 to day 10 of recovery), 5 males in each group and all males to be subjected to recovery group were individually placed in cages equipped with a urine collector, 4-hour urine was collected under fasting but with free access to water, and then 20-hour urine with free access to food and water.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters: pH, protein, ketones, glucose, occult blood, bilirubin, urobilinogen, color, sediment, urine volume (4-hour), osmolality, urine volume (20-hour), water intake (24-hour).

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations and dose groups that were examined:
5 males or 5 females per group in the final week of administration (week 6, day 39 of administration) for males in the main group, on day 4 of lactation (between day 42 and day 45 of administration) and in the final week of administration (week 6, day 39 of administration) and in the final week of recovery (week 2, day 11 of recovery) for males and females in the recovery group.
- Battery of functions tested: sensory activity / grip strength / motor activity:
1. Manipulative test: auditory response, approach response, touch response, tail pinch response, pupillary reflex, aerial righting reflex, and landing foot splay.
2. Grip strength: following the manipulative test, grip strength of the fore limb and hind limb was measured using CPU gauge MODEL-9502A (Aikoh Engineering Co., Ltd.).
3. Motor activity: following the grip strength test, motor activity was measured using a motor activity sensor for experimental animals NS-AS01 (NeuroScience Inc.). It was measured for 1 hour, at 10-minute intervals and in total of 0-60 minutes.
Sacrifice and pathology:
SACRIFICE
- Male animals: All surviving animals
- Female animals: All surviving animals

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
- Number of corpora lutea, implantation sites

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated below were prepared for microscopic examination and weighed, respectively. Organ weights were determined for the 5 males and females/group that had been subjected to hematological examination: Brain, thyroid glands (including parathyroid glands), thymus, heart, liver, spleen, kidneys, adrenals, testes, and epididymides.

The following organs were preserved for microscopic examination:
Cerebrum, cerebellum, pituitary, spinal cord (thoracic), sciatic nerve, thyroid gland, parathyroid gland, adrenal, thymus, spleen, mandibular lymph node, mesenteric lymph node, heart, lungs (including bronchus), stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver, kidney, urinary bladder, testis, epididymis, ovary, uterus, seminal vesicle, sternum (including bone marrow), femur (including bone marrow), macroscopic lesions, and the part for individual identification (ear auricle).
The organs/tissues of the 5 males and 5 females in the control group and the high dose group that were subjected to hematological/blood chemistry examinations and macroscopic lesions of all animals were subjected to microscopic examination.
Statistics:
Body weight, food consumption, water intake, data of open field observations (number of defecations, number of rearings), manipulative test (landing foot splay), grip strength and motor activity, quantitative items of urinalysis, hematological examination, blood chemistry examination and organ weight data were tested by the appropriate method, i.e. Bartlett's test (two-tailed), F-test, Dunnett’s test, a Dunnett-type mean rank test, Student's t-test, Aspin-Welch's t-test.

For the number of animals that showed normal reflex, a total number of animals that showed such item was calculated and analyzed by chi-square test with Yates’ continuity correction (levels of significance: 0.05, 0.01, two-sided). If there were cells with expected frequency of not more than 5, the data were analyzed by Fisher’s exact test (levels of significance: 0.05, 0.01, two-sided).

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
Convulsion by contact stimulus was observed in a female in the 300 mg/kg bw group on day 9 of administration. Otherwise there was no abnormality in any animal in the main group or recovery group.

BODY WEIGHT AND WEIGHT GAIN
In the 300 mg/kg bw group, body weight gain during the administration period was low in males in the main group and the difference was statistically significant 121.0 g as compared to 144.5 g in control group). The body weight was transiently low and the difference was statistically significant on day 39 of administration. During the recovery period, body weight gain of males in the 300 mg/kg bw group tended to be higher than that of the control group though it was not statistically significant. Otherwise there was no test article-related effect in males or females in the main group or recovery group.

FOOD CONSUMPTION
There were no test article-related effects in males or females in the main group or recovery group. Significantly low values were observed in males in the 300 mg/kg group on day 39 of administration and in females in the 100 mg/kg bw group on day 8 of administration; however, they were judged to be incidental since they were transient changes.

HAEMATOLOGY
- Examination at the end of the administration period:
There were no significant differences in any item between the control group and any test article group.
- Examination at the end of the recovery period:
In the 300 mg/kg bw group, a significant difference was observed in the percentage of basophils in males and females, but they were judged to be incidental since there were no significant differences in absolute count, there were no such changes at the end of the administration period, and there were no significant changes in any other white blood cell item.

CLINICAL CHEMISTRY
- Examination at the end of the administration period:
A decrease in LDH was observed in males in the 300 mg/kg bw group and the difference was statistically significant, but it was judged to be incidental since the change was not an increase, it was a mild change and there were no related changes. An increase in chloride was observed in males in the 300 mg/kg bw group and the difference was statistically significant, but it was judged to be incidental since it was a mild change and there were no related changes.
Otherwise a low value in blood urea nitrogen and a high value in inorganic phosphorus were observed and they were statistically significant in males in the 30 mg/kg bw group, but they were judged to be incidental since they were not dose-related.
- Examination at the end of the recovery period:
A decrease in phospholipid was observed in females in the 300 mg/kg bw group and the difference was statistically significant; however, it was judged to be incidental since it was a mild change which was not observed at the end of the administration period.

URINALYSIS (including measurement of water intake)
In the qualitative items, there were no abnormalities in the main group or recovery group. In the quantitative items, there were no significant differences in any item between the control group and any test article administration group.

NEUROBEHAVIOUR
- Manipulative test:
There were no abnormalities in any animal in the main group or recovery group. A significantly low value in landing foot splay was observed in females in the 300 mg/kg bw group in week 2 of recovery, but it was judged to be an incidental change since the value was higher than that recorded in week 6 of administration.
- Measurement of grip strength:
There were no abnormalities in any animal in the main group or recovery group. A significantly low value in the grip strength of hind limb was observed in females in the 30 mg/kg bw group on day 4 of lactation, but it was judged to be an incidental change since it was not dose-related.
- Measurement of motor activity:
There were no abnormalities in any animal in the main group or recovery group. A significant increase was observed in the 10-minute values of 10-20 and 20-30 minute intervals and 0-60 value in females in the 100 mg/kg bw group on day 4 of lactation; however, they were judged to be within the range of physiological variations since they were not dose-related.

ORGAN WEIGHTS
At the end of the administration period, decreases in the absolute and relative weights of the liver were observed in males in each administration group and they were statistically significant.
Otherwise significant differences from the control group were observed in the following organs; however, they were judged to be incidental since they were mild changes only in the absolute weight, and there were no test article-related changes in the relative weight or histopathological examination.
- At the end of the administration period:
Adrenal: A significantly low value in absolute weight was observed in females in the 300 mg/kg bw group.
- At the end of the recovery period:
Brain: A significantly low value in absolute weight was observed in males in the 300 mg/kg bw group.
Heart: A significantly low value in absolute weight was observed in males in the 300 mg/kg bw group.

GROSS PATHOLOGY
- At the end of the administration period:
Changes were observed in the following organs/tissues; however, they were thought to be incidental based on the incidence of their occurrences and pathological nature.
Liver: Diaphragmatic hernia nodule was observed in 1 female in the 30 mg/kg bw group.
Lung: A dark red focus was observed in 1 male in the 30 mg/kg bw group.
Stomach: A dark red focus in the glandular stomach was observed in 4 females in the control group, 4 females in the 100 mg/kg bw group and 1 female in the 300 mg/kg group.
- At the end of the recovery period:
Stomach: A dark red focus in the glandular stomach was observed in 1 female in the 300 mg/kg bw group.
Testis: Smallness was observed in 1 male in the control group

HISTOPATHOLOGY: NON-NEOPLASTIC
- At the end of the administration period:
The following changes were observed; however, they were thought to be incidental based on the incidence of their occurrences and histopathological nature.
Heart: Minimal focal cell infiltration was observed in 1 male each in the control group and the 300 mg/kg bw group.
Kidney: Minimal tubular regeneration was observed in 4 males and 1 female in the control group and 3 males and 2 females in the 300 mg/kg bw group, minimal hyaline urinary cast in 1 male in the control group and minimal interstitial mineralization in 1 female in the control group.
Liver: Mild diaphragmatic hernia nodule was observed in 1 female in the 30 mg/kg bw group, minimal microgranuloma in 5 males and 2 females in the control group, and 4 males and 2 females in the 300 mg/kg bw group.
Lung: Minimal arterial wall mineralization was observed in 1 male in the control group and 1 female in the 300 mg/kg bw group, and a minimal alveolar hemorrhage in 1 male each in the control group and 30 mg/kg bw group and 1 female in the 300 mg/kg bw group.
Stomach: Minimal or mild erosion in the glandular stomach was observed in 4 females in the control group, 4 females in the 100 mg/kg bw group and 1 female in the 300 mg/kg bw group.
Testis: Minimal seminiferous tubular atrophy was observed in 1 male in the control group.
Thyroid gland: Minimal ectopic thymic tissue was observed in 1 male in the control group.
- At the end of the recovery period:
The following changes were observed; however, they were thought to be incidental based on the incidence of their occurrences and histopathological nature.
Stomach: Mild erosion in the glandular stomach was observed in 1 female in the 300 mg/kg bw group.
Testis: Mild seminiferous tubular atrophy was observed in 1 male in the control group.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Body weight gain was decreased at 300 mg/kg bw.
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No effects up to the highest dose observed.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

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