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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
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:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 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 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 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
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: co-housed overnight
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually
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
Details on study schedule:
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.
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

Parental animals: Observations and examinations:
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).

BODY WEIGHT: Yes
- Time schedule for examinations:
- males of the main groups: 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: 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
Oestrous cyclicity (parental animals):
Vaginal smears were collected from all females in the main group every day (in the morning) from the day after the start of administration to the day copulation was confirmed and examined microscopically.
During the pre-mating administration period, vaginal smear pictures were classified into proestrus, estrus, metestrus and diestrus and examined for the frequency of estrus and interval between estruses (estrous cycle; 4- or 5-day interval was regarded as normal).
During the mating period, vaginal smears were examined for the presence of sperm.
Sperm parameters (parental animals):
not examined
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of liveborn pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals
- Maternal 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 haematological 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.
Postmortem examinations (offspring):
GROSS EXAMINATION OF DEAD PUPS: yes
All liveborn pups were exsanguinated under ether anesthesia after measurement of body weight on day 4 of lactation, necropsied and examined for any abnormality in organs/tissues including those in the head, thorax and abdomen. Liveborn pups were individually weighed, and the average body weight per litter was calculated by sex.
Statistics:
Body weight, food consumption, water intake, number of estruses, estrous cycle, number of days until copulation, gestation period, number of corpora lutea, number of implantation sites, number of live pups, 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 implantation index, pre-implantation loss index, post-implantation loss index, stillbirth index, partrition index, live birth index, index of external abnormalities, and viability index, litter mean was calculated first and then analyzed by the appropriate method.

For the copulation index, insemination index, fertility index, gestation index, sex ratio of liveborn pups, auditory response, approach response, touch response, tail pinch response, pupillary reflex, aerial righting reflex, number of copulated animals in each group, number of males that impregnate females, number of pregnant females, number of females that had liveborn pups, number of male pups alive, and number of female pups alive, 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).
Reproductive indices:
- Copulation index (%) = (Number of copulated animals / Number of mated animals) × 100
- Fertility index (%) = (Number of pregnant females / Number of copulated females) × 100
- Insemination index (%) = (Number of males inseminated females / Number of copulated
males) × 100
- Gestation period (days) = Number of days from day 0 of gestation to the day of delivery
- Delivery index (%) = (Number of females delivered live pups / Number of pregnant females) ×
100
- Implantation index (%) = (Number of implantation sites / Number of corpora lutea) × 100
- Pre-implantation loss (%) = [(Number of corpora lutea – Number of implantation sites) /
Number of corpora lutea] × 100
- Post-implantation loss (%) = [(Number of implantation sites – Number of liveborn pups) /
Number of implantation sites] × 100
- Stillbirth index (%) = (Number of stillborn pups / Total number of pups born) × 100
- Parturition index (%) = (Number of stillborn and liveborn pups / Number of implantation sites)
× 100
- Live birth index (%) = (Number of liveborn pups / Total number of pups born) × 100
Offspring viability indices:
- Index of external abnormalities (%) = (Number of liveborn pups with external abnormalities /
Number of liveborn pups) × 100
- Sex ratio = [Number of male pups / (Number of male pups and female pups)] × 100
- Viability index on day 4 after birth = (Number of live pups on day 4 / Number of liveborn pups
on day 0) × 100

Results and discussion

Results: P0 (first parental animals)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
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 FOOD CONSUMPTION (PARENTAL ANIMALS)
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. 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.
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.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
There were no animals showing abnormal estrous cycles, and there were no significant differences in the average length of the estrous cycle between the control group and any treatment group.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Copulation was observed in almost all couples by day 5 after the start of mating, and it was observed in all couples by day 13. All females were pregnant except 1 female in the 300 mg/kg bw group. There were no significant differences between the control group and any test article administration group in the copulation index, insemination index or fertility index.
In the delivery status, all animals had normal delivery on day 21 or 22 of gestation, and there were no significant differences between the control group and any test article administration group in the delivery index, gestation period, number of corpora lutea, number of implantation sites, implantation index, pre-implantation loss (%), post-implantation loss (%), stillbirth index, parturition index, number of liveborn pups or live birth index.
In the lactation condition, no dams had abnormalities in nesting or lactational behavior.

ORGAN WEIGHTS (PARENTAL ANIMALS)
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: adrenal, brain, heart. 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.

GROSS PATHOLOGY (PARENTAL ANIMALS)
At the end of the administration period: Some changes were observed in the following organs/tissues: liver, lung, stomach. At the end of the recovery period some changes were observed in the following organs/tissues: stomach, testis. However, they were thought to be incidental based on the incidence of their occurrences and pathological nature.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Some changes were observed; however, they were thought to be incidental based on the incidence of their occurrences and histopathological nature.
For further details, please refer to 7.5.1 Repeated dose toxicity: oral.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects on reproduction up to the highest dose.

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)
The number of pups that died during the lactation period was 18 in the control group, and 0, 2 and 1 in the 30, 100 and 300 mg/kg bw groups, respectively. The viability index on day 4 of lactation was significantly high in the 30 mg/kg bw group, but it was due to a good viability index (100%).

CLINICAL SIGNS (OFFSPRING): liveborn pups
There were no significant differences in the sex ratio or body weight at the time of birth between the control group and any treatment group, and there were no external abnormalities.

BODY WEIGHT (OFFSPRING)
There were no significant differences in the body weight at the time of birth or on day 4 of lactation between the control group and any test article group.

GROSS PATHOLOGY (OFFSPRING)
Thymic remnant in the neck was observed in 2 males in the control group, 2 males and 1 female in the 30 mg/kg bw group and 1 male in the 300 mg/kg bw group, and pelvic dilatation (bilateral) in 1 male in the 100 mg/kg bw group; however, these changes were considered to be incidental since they are known to occur spontaneously in rats and the incidence of their occurrence was small.
There were no other abnormalities in liveborn pups.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects in liveborn pups up tp the highest dose.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion