Isooctene

Toxicity to reproduction

Administrative data

Endpoint:

toxicity to reproduction

Remarks:

other: combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as published report, no restrictions, fully adequate for assessment

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

Males and females were exposed to the test substance for 10 weeks prior to pairing.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: US EPA OPPTS 870.3650 (Combined repeated dose toxicity study with the reproductive/developmental toxicity screening test)

Deviations:

yes

Remarks:

Males and females were exposed to the test substance for 10 weeks prior to pairing.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Isooctene

EC Number:

234-294-9

EC Name:

Isooctene

Cas Number:

11071-47-9

Molecular formula:

C8H16

IUPAC Name:

2-methylhept-1-ene

Details on test material:

- Name of test material (as cited in study report): isooctene
- Synonym: Di-n-butene
- Physical state: Clear, colourless liquid
- Analytical purity: 99.9%, 99.84% area (gas chromatography)
- Lot/batch No.: K402 distillate
- Expiration date of the lot/batch: Unlimited
- Stability under test conditions: Unlimited
- Storage condition of test material: room temperature; air sensitive, under N2

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: group mean range 161.7-166.6 g (males), 126.1-131.0 g (females)
- Housing: Individually in wire cages
- Diet: Milled mouse/rat laboratory diet (Provimi Kliba SA, Kaiseraugst, Basel Switzerland) ad libitum except during exposure and motor activity measurements
- Water: tap ad libitum except during exposure and motor activity measurements
- Acclimatisation period: Approximately two weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24°C
- Humidity: 30-70%
- Air changes (per hr): Not reported; air-conditioned
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 13 June 2006 To: 26 April 2007

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

other: air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: glass-steel inhalation chamber, volume of about 1.4 m³ (BASF Aktiengesellschaft).
- Air flow: Approximately 20 per hour
- Air conditions: Test group 0: A positive pressure was maintained by adjusting the airflow of the exhaust air system. This ensured that no laboratory air reached the control animals.
Test groups 1 - 3: A negative pressure was maintained by adjusting the airflow of the exhaust air system. This ensured that the laboratory was not contaminated as the result of any leakage from the inhalation chamber.
- System of generating particulates/aerosols: For each concentration the test substance was supplied to a thermostated vaporizer at a constant rate by means of the metering pump.
- Temperature, humidity, pressure in air chamber: The vapour was generated with conditioned supply air (actual range 54.6-61.1% relative humidity, temperature 20.9-22.7°C) and passed into the inhalation system.

TEST ATMOSPHERE
- Brief description of analytical method used: The nominal concentration was calculated from the study means of the test pump rates and the supply airflows used during exposure to generate the respective concentrations.
The concentrations of the inhalation atmospheres were analyzed by gas chromatography in all test groups including control.
- Total hydrocarbon analyzers were used to continuously monitor the constancy of concentrations of test substance vapours in the inhalation systems.
- No surveillance of the oxygen content in the inhalation system was performed. The air change within the inhalation systems was judged to be sufficient to prevent oxygen depletion by the breathing of the animals and the concentrations of the test substance used could not have a substantial influence on oxygen partial pressure.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 16 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 post coitum

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The vapour generation effectiveness was as expected for these high concentrations (range of 83.9-88.8%). Real time surveillance of the inhalation atmospheres with total hydrocarbon analyzers generally proved the constancy of each concentration throughout the daily exposures.

Duration of treatment / exposure:

Males were treated for approx. 13 weeks (10 weeks premating, 3 weeks mating and post mating). In females treatment was performed during premating (10 weeks), mating and gestation through day 4 after delivery (approx. 15 weeks).

Frequency of treatment:

6 hours per day / 5 days per week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 1000, 5000 and 15000 mg/m3
Basis:
other: target concentration

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 1200, 5600 and 16900 mg/m3
Basis:
nominal conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
0, 1000±100, 5000±200 and 14900±700 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

10

Control animals:

yes, sham-exposed

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The clinical conditions of the test animals were recorded at least 3 times (before, during and after exposure) on exposure days and once during the preflow period, on the day of neurofunctional test and prior to gross necropsy. During exposure only a group wise examination was possible.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were performed in all animals prior to the exposure period and thereafter in weekly intervals.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of all parental animals was determined on day -5 (start preflow period), on day 0 (start exposure period) and then in weekly intervals as well as prior to gross necropsy. Female parental animals were weighed on days 0, 7, 14 and 20 post coitum and on days 1 and 4 post partum. Parental females without litters were not weighed.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, with the following exceptions; Food consumption was not determined during the mating period, food consumption of the parental females with evidence of sperm was determined for days 0-7, 7-14 and 14-20 post coitum, food consumption of parental females, which gave birth to a litter was determined for days 1-4 post partum.

FOOD EFFICIENCY:
- Food efficiency (group means) was calculated based upon individual values for body weight and food consumption.

MATING AND REPRODUCTIVE PERFORMANCE: Yes
Male and female mating and fertility indices (%) and female gestation index (%) were calculated.

Oestrous cyclicity (parental animals):

None

Sperm parameters (parental animals):

None

Litter observations:

PUP NUMBER AND STATUS AT DELIVERY: Parameters assessed: number and sex of pups, stillbirths, live births.
PUP VIABILITY/MORTALITY: Examined once or twice/day. Numbers of dead pups on day of birth (day 0) and dying between days 1-4 of lactation determined. Viability index (%) was calculated.
SEX RATIO: Determined on day 0 and day 4
CLINICAL OBSERVATIONS: Assessed daily
PUP BODYWEIGHTS: weighed on days 1 and 4 after birth

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after the females began littering
- Maternal animals: All surviving animals when the offspring were 4 days old

GROSS NECROPSY
- Gross necropsy of all animals

HISTOPATHOLOGY / ORGAN WEIGHTS
- Selected organs were weighed (adrenals, brain, heart, kidneys, liver, lungs, ovaries, testes, spleen, thymus and uterus)
- Comprehensive range of tissues was prepared for microscopic examination (all gross lesions, brain, spinal cord (cervical, thoracic and lumbar), sciatic nerve, pituitary gland, salivary glands, thyroid/parathyroid glands, adrenal glands, prostate gland, seminal vesicles, coagulation glands, uterus, oviducts, vagina, female mammary gland, thymus, lymph nodes, spleen, trachea, lungs, heart, aorta, liver, pancreas, kidneys, oesophagus, stomach, duodenum, jejunum, ileum, caecum, colon, rectum, urinary bladder, sternum with marrow, bone marrow (femur), head (with nasal cavities), larynx, pharynx, eyes with optic nerve, femur with knee joint, skin, skeletal muscle, extraorbital lachrymal glands).

Postmortem examinations (offspring):

All surviving pups on day 4 post partum, all stillborn and any decedents were examined externally, eviscerated and organs assessed macroscopically.

Statistics:

Means and standard deviations of each test group were calculated for parameters measured. Further statistical analyses were performed as appropriate, according to the methods of Dunnett’s t-test (two-sided), Fisher’s Exact test, Wilcoxon test (one-sided), and/or Kruskal-Wallis test (two-sided).

Reproductive indices:

Male and female mating and fertility indices (%) and female gestation index (%)

Offspring viability indices:

Pup viability index (%)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
There was no mortality. Clinically, slight ataxia (from day 9 onward) and salivation (from day 8 onward) were observed at 15000 mg/m3 indicating marginal irritation and systemic toxicity of Isooctene.

BODY WEIGHT AND WEIGHT GAIN
In male animals the mean body weight and body weight change were found to be significantly decreased when compared with the controls.

FOOD CONSUMPTION
A transient decrease of food consumption was observed in male and female animals on study day 7, which did not occur again after the animals adjusted to the test substance and the exposure procedure.

FOOD EFFICIENCY
In male animals, food efficiency were found to be significantly decreased when compared with the controls.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The fertility indices for F0 males were 90, 100, 90 and 100% for the control, 1000 mg/m3, 5000 mg/m3 and 15000 mg/m3 groups respectively. The female mating index was 100% for the control, 1000 mg/m3 and 15000 mg/m3 groups and 90% for the 5000 mg/m3 group.
The gestation index was 100% in the 5000 and 15000 mg/m³ groups, 89% in the control group and 90% in the 1000 mg/m³ group. The number of liveborn and stillborn pups was comparable between all test groups. Thus, the live birth index amounted to 100% in the 5000 mg/m³ group, 99% in 15000 mg/m³ group, 98% in the control group and 96% in the 1000 mg/m³ group.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Absolute and relative liver and kidney weights of males were significantly increased in animals exposed to 5000 and 15000 mg Isooctene/m3.

HISTOPATHOLOGY (PARENTAL ANIMALS)
The treatment-related microscopic changes observed were increased hyaline cast(s) and basophilic cortical tubules in the kidneys of male animals from group 2 (5000 mg/m3) onward. In addition, in males there was suspected greater propensity for the development of large hyaline droplets related to the proximal tubules compared to controls. Mallory-Heidenhain stain of all male kidneys and immunhistochemistry of selected male kidneys with an alpha 2u globulin antibody revealed a concentration-dependent accumulation of alpha 2u globulin in cortical tubular cells in all treated groups. However, in the absence of any treatment-related morphological signs of cytotoxicity, the minimal to slight accumulation of alpha 2u globulin in the group 1000 mg/m3 males is regarded to be adaptive and non-adverse in character.
In the respiratory tract no signs of toxicity were observed.

Effect levels (P0)

Dose descriptor:

NOAEC

Remarks:

reproductive toxicity

Effect level:

15 000 other: mg/m3 (target concentration)

Sex:

male/female

Basis for effect level:

other: No effects on fertility and pre/postnatal development

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Gross pathological findings:

no effects observed

Details on results (F1)

The mean number of delivered F1 pups/dam and the rate of live- and stillborn F1 pups was not affected by the test substance. The total average of delivered F1 pups/dam was 10.7, 11.8, 11.1 and 11.3 pups/dam in the control, 1000, 5000 and 15000 mg/m³ groups.
No adverse effects on the development of offspring until postnatal day 4 were noted in the litters of all exposed groups.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under the conditions of this test, isooctene did not influence fertility or pre/postnatal development at dose levels as high as 15000 mg/m3.
The NOAEC for developmental toxicity was 15000 mg/m3 for male and female rats.

Executive summary:

Wistar rats were exposed, whole body, to 0, 1000, 5000 and 15000 mg/m³ isooctene vapour for 6 hours/day, 5 days/week. Males were exposed for approximately 13 weeks (10 weeks pre-mating, 3 weeks mating and post mating). Females were exposed during pre-mating (10 weeks), mating and gestation through day 4 after delivery (approximately 15 weeks). The parental animals were examined for their mating and reproductive performances. The pups were sexed and were weighed on the day after birth and on day 4 post partum. Their viability was recorded. All pups underwent necropsy on day 4 post partum and were examined macroscopically for external and visceral findings.

The capability to cohabitate and to generate offspring was not affected in either sex.

 

Under the conditions of this test, isooctene did not influence fertility at dose levels as high as 15000 mg/m³.