4-methylpent-1-ene

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

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 during 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 oral 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

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

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:
200 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

Details on study design:

- Dose selection rationale: The dose levels were chosen based on the results of the dose range finding study.
- Post-exposure recovery period in satellite groups: 14 days

Positive control:

No

Examinations

Observations and examinations performed and frequency:

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 and 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 nitrogen, 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

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
In treatment groups, all males were sacrificed on day 42. Females were sacrificed on nursing day 6 or pregnant day 26.
In recovery groups, all animals were sacrificed on recovery day 14.
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.

HISTOPATHOLOGY: Yes
All organs collected at necropsy were histopathologically investigated.

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.

Results and discussion

Results of examinations

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):

not examined

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

There were no unscheduled deaths considered to be attributable to test material toxicity.

Clinical Observations
All animals in treatment period showed no noteworthy findings.
Only one animals in recovery groups showed small eyeball and dark red colored eyeball throughout treatment free period.

Functional Observations
No noteworthy findings were observed.

Functional Performance Tests
Females of 1000 mg/kg on recovery group showed decreased spontaneous locomoter activity during 0-20 minutes of observation period on week 6 however, other parameters were not affected. On recovery week 2, animals in the same group showed increased distance of hindlimbs. However, other parametars were not affected.

Bodyweight
No findings were observed compared to the control group.

Food Consumption
No findings were observed compared to the control group.

Laboratory Investigations
Haematology
Males in 40 mg/kg in treatment group showed significant increase in platelets count and males in 200mg/kg showed increased APTT. No other findings were observed.

Blood Chemistry
In the treatment groups, females of 200mg/kg showed increased AST and females 1000mg/kg showed significant increased BUN. In the 1000mg/kg of recovery group, female animals showed significant increase in creatinine and chloride, decreased beta-globulin fraction.

Urinalysis
No findings were observed when compared to the control group.

Pathology
Necropsy
Malshaped right femur was observed in a male of the control group. One male showed dilated renal pelvis and another male showed splenic cyst. Control female showed enlargement spleen and adhesion between yellowish region in the spleen to adipose tissue. One female in 200mg/kg showed enlarged spleen. Not pregnant animal in 1000mg/kg showed dilated right and left uterine horns, dilated uterus cervix with white-yellow fluid and vaginal atresia. One control male in the recovery group showed small eyeball and dark-red region in fundus.

Organ weight
Male animals in 200mg/kg showed significant decrease in relative organ weight of thymus. Male of 1000mg/kg recovery group showed significant increase of absolute and relateve weight of testes.

Histopathology
KIDNEY: Slight hyaline depositions in proximal tubular epithelium were observed in three males of 40 mg/kg, seven males of 200mg/kg and six of 1000mg/kg. Slight accumulation of eosinophilic body was observed in proximal tubular epithelium in one male of 40 mg/kg, seven males of 200mg/kg and six of 1000mg/kg. These findings observed in the animals of groups of more than 200 mg/kg were significant when compared to control animals. These depositions were revealed to be positive to anti-alpha2 microglobulin immunohistochemical staining. Dilated renal pelvis was observed in one animal of 40mg/kg. In the same dose group, cyst was observed in the renal capsule of one male which had renal cystis.
TESTIS: There were no singnificant changes in all stages of spermatogenesis to control animals after treatment period. After treatment-free period, treated animals in the recovery group showed significant increases in number of sperm(round) and spermatocytes in stage I-IV pachytene stage, spermatocytes in stage VII-VIII pre-leptotene and pachytene stage, spermatocytes in stage IX-XI pachytene stage and spermatocytes in stage XII-XIV zygotene and pachytene stage.
SPLEEN: Enlarged spleen with mild congestion was observed in one female in 200mg/kg.
UTERUS: Mild infiltration of inflammatory cells in endometorium of dilated uterine horns in one female of 1000mg/kg
EYE: Moderate atrophy of lens, sever fibrosis in chroid coat, iris and ciliary body, mild hemosiderin deposition in the small eyeball in one female of control recovery group.

Effect levels

1

Dose descriptor:

NOEL

Effect level:

ca. 40 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Males in the group of 200 mg/kg showed slight increase of hyaline droplet and eosinophilic body in the proximal tubular epithelium with dose dependency and regression after recovery period.

2

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Female animals in the group of 1000mg/kg showed a significant increase in BUN on nursing day 6 and increased creatinine and Cl were evident on the end of recovery period.

Target system / organ toxicity

Critical 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. No effects were observed on reproductive performance of parents.
The NOAELs for systemic toxicity are considered to be 40 mg/kg/day for males and 200 mg/kg/day for females.