4-methylpent-1-ene

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Start date: Aug 4th 2004. Test complete: Dec 27 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Conducted under OECD guidelines and OECD good laboratory practices

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2005

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-methylpent-1-ene

EC Number:

211-720-1

EC Name:

4-methylpent-1-ene

Cas Number:

691-37-2

Molecular formula:

C6H12

IUPAC Name:

4-methylpent-1-ene

Details on test material:

Description : Clear liquid
Lot number : 3B24A

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Japan) Limited, Atsugi, Kanagawa
- Age at study initiation: approximately ten weeks old
- Fasting period before study: No
- Housing: The animals were housed one animal during treatment, one male and one female durig mating period, one maternal animal during pregnant, one maternal animal and its litter in each metal bracket cage with metallic mesh floor. Female animals from the day of mating to after 4 days of nursing were housed with bedding for experimental animal(Charles River).
- Diet (e.g. ad libitum): A pelleted diet (gamma-ray irradiated CRF-1 by Oriental Yeast Co., Ltd. was used
- Water (e.g. ad libitum): Community tap water at Sapporo-city was supplied from auto-supply system attached to the cage. The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study
- Acclimation period: The animals were acclimatized for 14 days during which time their health status was assessed


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3ºC
- Humidity (%): 50 ± 20%
- Air changes (per hr): from ten to fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light and twelve hours darkness

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

VEHICLE
- Concentration in vehicle: 8 mg/ml for low dose group, 40 mg/ml for middle dose group, 200 mg/ml for high dose group.
- Amount of vehicle (if gavage):5ml/kg.
- Lot/batch no. (if required): 3B24A
- Purity: 98.36%

MAXIMUM DOSE VOLUME APPLIED: 5 ml/kg

DOSAGE PREPARATION (if unusual): Not applicable

Details on mating procedure:

Non-recovery animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days from day 14. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug or sperma in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperma within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of concentration of preparations were conducted at start, during and end of the study.

Duration of treatment / exposure:

Male: 42 days
Female: From 14 days before mating to 5 days after delivery

Frequency of treatment:

once a day

Details on study schedule:

Dose Groups 1 to 4 (non-recovery)
i) Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii) All females had a daily vaginal smear prepared during the pre-mating period.
iii) Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioural toxicity.
iv) One day prior to pairing (Day 14), blood samples were taken from five males and five females, randomly selected from each dose group and analyzed for haematological and blood chemical assessment.
v) On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
vi) Following positive evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
vii) At the end of treatment (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli and urinalysis was performed.
viii) Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Evaluation of each litter size, litter weight, mean offspring weight by sex, clinical observations and surface righting were also performed during this period.
ix) At Day 4 post partum, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
x) Additional blood samples were taken from five males from each dose group for haematological and blood chemical assessments on Day 42. After that the male dose groups were killed and examined macroscopically.
xi) Additional blood samples were taken from five randomly selected females from each dose group for haematological and blood chemical assessment on Day 4 post partum, (or in the case of the high dose females, samples were taken prior to or at termination) At Day 5 post partum, all surviving non-recovery females and surviving offspring were killed and examined macroscopically.
Dose groups 5 and 6 (recovery)
i) Groups of five male and five female rats were dosed according to dose group continuously up to the point of sacrifice of non-recovery males at which time treatment was discontinued.
ii) The males and females were maintained without treatment for a further fourteen days.
iii) Urinalysis was performed for all males during the final week of recovery.
iv) Blood samples were taken for haematological and blood chemical assessment on Day 56.
v) After fourteen days of recovery, males and females were killed and examined macroscopically.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 mg/kg/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
40 mg/kg/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
20 mg/kg/day
Basis:
actual ingested

Doses / concentrations 4

Remarks:

Doses / Concentrations:
1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

Control-12 males and 12 females 0 mg/kg/day
Low- 12 males and 12 females 40 mg/kg/day
Middle-12 males and 12 females 200 mg/kg/day
High-12 males and 12 females 1000 mg/kg/day

Recovery Group
Control-5 males and 5 females 0 mg/kg/day
High-5 males and 5 females 1000 mg/kg/day

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes

All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before and after dosing in the morning, and once in the afternoon, during the treatment period. During treatmen-free period, recovery animals were observed twice daily. All observations were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
Prior to the start of treatment and at weekly intervals during the treatment period, all animals were observed for signs of behavioural toxicity. Functional performance tests such as posture, eyelid closing, respiration, twitches, abnormal behaviour, body tone, alertness, pilo-erection,exophthalmos,pupil diameter, visible mucosa, lacrimation, salivation and body temperature in home cage, tremor, gait, arousal response, urination, stereotyped behaviour, abnormal behaviour and respiration in the arena were also performed on all animals prior to dosing and on day 7, 14, 21, 28, 35 and 42 in treatment groups and once weekly during treatment free period in the recovery period.

Behavioural Assessments
Detailed individual clinical observations were performed for each animal using a purpose-built arena. The following parameters were observed:
Gait
Tremors
Twitches
Convulsions
Bizarre/Abnormal/Stereotypic behaviour
Salivation
Pilo-erection
Exophthalmia
Lacrimation
Hyper/Hypothermia
Skin colour
Respiration
Palpebral closure
Urination
Defecation
Transfer arousal
Tail elevation

Functional Performance Tests
Motor Activity: Five animals per each groups were randomly measured responses to visual, tactile, auditory and pain stimuli, and proprioceptive and righting reflex response on day 36, recovery day 11, nursing day 4.
Forelimb/Hindlimb Grip Strength: A CPU gage meter (by Aiko Engeneering) was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal.
Distance of hindlimbs was also measured. Animal was fallen down from 30 cm in the air after black ink was put on their hindlimbs. Distance of hindlims were calculated on the footprints on the floor.
Spontaneous locomoter activity was measured by automated system, CompACT AMS for one hour.

BODY WEIGHT: Yes
- Time schedule for examinations:before dosing day 1, 3, 5, 7, 10, 14, 21, 28, 35 and 42 , recovery day 7 and 14. Treatment Day 1, 3, 5, 7, 10, 14 before dosing and gestation day 1, 3, 5, 7, 10, 14 adn 20, nursing day 0, 1, 4 before dosing and at the day of sacrifice for females in main study.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food consumption were measured on the same schedule of body weight measurement.

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

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: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the termination
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes (overnight)
- How many animals:
- Parameters: RBC, hematocrit, hemoglobin, MCV, MCH, MCHC, reticulocyte, platelet, WBC, differential count of WBC, prothronbin time, activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: Yes (overnight)
- How many animals: five animals from each groups
- Parameters: AST, ALT, ALP, gamma-GTP, glucose, choline esterase, total cholesterol, phospholipid, triglyceride, total bilirubin, urea ntrogen, creatinine, Na, K, Cl, Ca, inorganic phosphorus, total protein, protein fraction, albumin, A/G ratio.

URINALYSIS: Yes
- Time schedule for collection of urine: day 40-41 and recovery day 12-13, nursing day 4-5
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters: pH, protein, glucose, ketone body, urobilinogen, bilirubin, occult blood, color, urinary sediments, urine volume, specific gravity, water consumption

Oestrous cyclicity (parental animals):

A vaginal smear was prepared and the stages of oestrous cycle was recorded.

Sperm parameters (parental animals):

Not examined

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
[number of corpora lutea, number of implantation sites, number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, other: Clinical condition of offspring from birth to Day 4 post partum, Inviduals litter and offspring weights on Day 1 and 4 post partum

GROSS EXAMINATION OF DEAD PUPS:
Yes, full external and internal examination.

Postmortem examinations (parental animals):

SACRIFICE

- Male animals: Males were killed by ether anesthesia followed by exsanguination on Day43.
- Maternal animals: Any females that failed to achieve pregnancy were killed on Day 52. Any females that failed to deliver until pregnant day 25 after mating were killed on pregnant day 26. Any females produce a litter were killed on nursing day 6.

GROSS NECROPSY
- Adult non-recovery males were killed by ether anesthesia followed by exsanguination on Day 43. Adult non-recovery females were killed by ether anesthesia followed by exsanguination on Day 5 post partum. Surviving offspring were terminated under the inhalation of carbone dioxide . Any females that failed to achieve pregnancy or produce a litter were killed on or after Day 26 post coitum.
For all females the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded.
Recovery animals were killed by ether anesthesia followed by exanguination fourteen days after the termination of treatment (Day 57).
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
Following organs were investigated:
Brain(cerebrum, cerebellum), spine, pituitary, thymus, thyroid with parathyroid, adrenal, spleen, heart, esophagus, stomach, liver, pancreas, duodenum, ileum, jejunum, cecum, colon, rectum, trachea, bronchus and lung, kidney, bladder, testis, epididymis, prostate, ovary, uterus, eyeball with harderian gland, skin, sternum, femur, mesenteric lymph node, mandibular lymph node, skeletal muscle, sciatic nerve.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 5 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, other: Results of the surface righting assessments on Day 1 post partum, Clinical condition of offspring from birth to Day 4 post partum, Individual litter and offspring weights on Day 1 and 4 post partum.

Statistics:

Where variances were shown to be homogenous by Bartlett's test, one way analysis of variance (ANOVA) were conducted. Where Bartlett's test showed unequal variances the data was analyzed using non-parametric methods: Kruskal-Wallis. Where ANOVA showed statistical significance Dunnet's test were conducted to compare with control. Where Kruskal-Wallis showed significance Mann-Whitney U test was conducted.
Incidence of abnormal estrous cycle, mating rate, fertility rate, delivery rate, nursing rate on nursing day4 were analyzed by multiple chi-squared test. Once the significance was shown, these data were compared to control animals using normal chi-squared test. In the case that normal chi squared test could not be appropriate, Fishcer's exact test could be used. Five percent in probability values was considered to be significant.

Reproductive indices:

Mating performance and Fertlity
The following parameters were calculated from the individual data during the mating period of the parental generation.

Pre coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.
Copulation index = (number of animales mated/number of animales paired) x 100
Fertility Indices = (number of fertilized females/number of mated pairs) x 100

For each group the following were calculated:

Gestation Index (%) = (number of live offspring/number of oregnant females) x 100
Mating Index (%) = (Number of animals mated/Number of animals paired) x 100
Pregnancy Index(%) = (Number of pregnant females/Number of animals mated) x 100
Implantation Index (%) = (number of implantation sites/number of corpus luteum) x 100

Offspring viability indices:

Live Birth Index(%) = (Number of offspring alive on Day 1/ Number of offspring born) x 100
Viability Index (%) = (Number of offspring alive on Day 4/ Number of offspring alive on Day 1) x 100
Nursing index (%) = (number of nusring femals on Day 4/number of females deliver live offspring) x 100
Sex ration (%) = (number of male offspring/number of male and female offspring) x 100
Viability index (%) = (number of live offspring on Day4/number of live offspring) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

There were no affected parameters in reproductive performance by test item treatment.
One female in the group of 200 mg/kg and control group showed continued anestrous.
One pair in the control group did not achieve mating.
One female treated with 200mg/kg and three females with 1000mg/kg did not become pregnant.

Effect levels (P0)

Dose descriptor:

NOEL

Effect level:

ca. 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test item related findings were observed in both sexes.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

in 1000mg/kg

Gross pathological findings:

no effects observed

Details on results (F1)

Body weight of offspring in the group of 1000mg/kg showed a tendency of lower than those of other groups and showed statistically significant on Day 1.

Autolysed dead fetuses were observed in the group of control (one male, one female), 40mg/kg (one male), 200mg/kg( one female).

Investigation of live offspring on day 4 showed ventricular enlargement and malshaped thoracic cavity in one each of 40mg/kg group, yellow region in left lobe of the liver of one male of 1000 mg/kg group.

Except above findings, no any other findings were observed.

Effect levels (F1)

Dose descriptor:

NOEL

Generation:

F1

Effect level:

ca. 200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Tendency of decreased body weight were observed in the offspring in 1000 mg/kg gourp.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Increase of urea nitrogen was observed at the end of the administration period, and increases of creatinine and chloride were observed at the end of the recovery period in females given 1000 mg/kg/day. Hyaline droplets and eosinophilic bodies in proximal tubular epithelium were observed in males given 200 mg/kg or more. However no effects were observed on reproductive performance of parents or on developmental performance in pups.
The NOAELs for systemic toxicity for parents are considered to be 40 mg/kg/day for males, 200 mg/kg/day for females, but for reproductive and developmental toxicity are considered to be 1000 mg/kg/day for parents and pups.