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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
not applicable
GLP compliance:
Limit test:

Test material

Constituent 1
Reference substance name:
Constituent 2
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
- Name of test material (as cited in study report): isobutylene
- Physical state: colourless gas
- Analytical purity: 99.9%
- Lot/batch No.: 295469
- Stability under test conditions: not specified
- Storage condition of test material: Cylinders kept tightly closed, away from heat, sparks or open flame, in a ventilated area.

Test animals

other: Alpk:APfSD (Wistar-derived)
Details on test animals or test system and environmental conditions:
- Source: Rodent Breeding Unit (RBU), Alderley Park, Macclesfield, Cheshire, UK
- Age at study initiation: 10-12 weeks
- Weight at study initiation: 215-293g
- Housing: individually housed in metal cages with solid bottoms
- Diet: CT1 diet (supplied by Special Diets Services Limited, Stepfield, Witham, Essex, UK ad libitum except during exposure
- Water: Mains water ad libitum except during exposure
- Acclimation period: Animals were acclimatised to the holding chambers for 4 days before the start of exposure. However, as the animals were time-mated, the day of arrival was the first day of the study.

- Temperature: 22±3°C
- Humidity: 30-70%
- Air changes (per hr): At least 12 changes/hr
- Photoperiod: 12 hrs dark / 12 hrs light)

IN-LIFE DATES: From: 28 February 2001 To: 6 June 2002

Administration / exposure

Route of administration:
inhalation: vapour
other: air
Details on exposure:
- Exposure apparatus: The animals were housed in circular whole body exposure chambers. The chambers consisted of a perspex circular section subdivided into 10 equal segments by wire mesh partitions. The chamber was covered by an aluminium lid and stood on an aluminium base plate. The internal volume of each chamber was approximately 150 litres.
- System of generating atmosphere: Test atmospheres were generated by metering appropriate amounts of test substance through a copper coil heated to 40°C using a Techne TE-WA water bath. The resultant vapour was passed through a distribution plenum to which flowmeters were attached. The vapour was passed through the flowmeters to a copper transfer line to the top of each exposure chamber for groups 2, 3 and 4. Clean dry air was used to dilute to the required concentration for each group.
- Temperature/humidity in air chamber: Temperatures were within the range of 22±3°C and the relative humidity within 15-70%.
- Air flow rate: Flow rates through the chamber gave at least 12 air changes per hour. The air flow rates were monitored continuously using variable area flowmeters and were recorded at approximately 30 minute intervals during the study. The optimum air flow rates through the chamber necessary to conduct the study were determined during trial generations.

- Brief description of analytical method used: Grab samples were taken from each exposure chamber using a 5 mL gas-tight syringe equipped with integral on-off valve. Samples were directly introduced into a gas chromatograph equipped with flame ionisation detection using an automated gas-sampling valve. Absolute atmosphere concentrations of isobutylene (in units of ppm v/v) were directly calculated after suitable system calibration and reprocessing.
- Samples taken from breathing zone: yes, approximately hourly, during each exposure period.

Clean, dry air (dried and filtered using equipment supplied by ATLAS-COPCO, Sweden), was supplied to the control group.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
The mean analysed atmosphere concentrations were found to be 505 ± 18.2, 1963 ± 56.4 and 8014 ± 183 ppm for target concentrations of 500, 2000 and 8000 ppm respectively.
No isobutylene was detected in the control or room atmospheres.
The limit of detection was calculated to be approximately 4 ppm (v/v) isobutylene in the atmosphere concentration.
Details on mating procedure:
At the Rodent Breeding Unit, female rats were paired overnight with males of the same strain. The following morning, vaginal smears from the females were examined for the presence of spermatozoa. The day on which sperm were detected in a vaginal smear was designated as day 1 of gestation.
Duration of treatment / exposure:
6 hours per day
Frequency of treatment:
On days 5 to 21 (inclusive) of gestation
Duration of test:
The day of mating was designated day 1 and the rats were terminated on day 22 of gestation
Doses / concentrationsopen allclose all
Dose / conc.:
250 ppm
Dose / conc.:
1 000 ppm
Dose / conc.:
8 000 ppm
No. of animals per sex per dose:
Control animals:
yes, concurrent no treatment
Details on study design:
The exposure concentrations were selected by the Sponsor on the basis of the results obtained from an inhalation toxicity study in which groups of 10 male and 10 female rats were exposed to 250, 1000 or 8000 ppm isobutylene for 6 hrs/day, 5 days/wk for 13 consecutive wks. Although no toxicologically significant effects were observed at 8000 ppm, higher exposure concentrations were not possible because of the risk of explosion. Isobutylene forms explosive mixtures with air.
On day 1 of gestation, females were delivered to CTL. The females were supplied over 4 consecutive days in each of 2 weeks.


Maternal examinations:
- Time schedule: Detailed observations were recorded for each animal at least once a day throughout the study (days 1 - 22 of gestation). On exposure days, clinical observations were recorded prior to exposure. The rats were observed during and following exposure.

- Time schedule for examinations: The bodyweight of each rat was recorded on days 1, 5, 6, 7, 10, 13, 16, 19 and 22 of gestation. Bodyweights were recorded prior to daily exposure.

- The amount of food consumed by each rat was measured by giving a weighed quantity of food on days 1, 5, 7, 10, 13, 16 and 19 and calculating the amount consumed from the residue on days 5, 7, 10, 13, 16, 19 and 22 respectively. Food was not available to the rats during each 6 hour period of exposure.

- Killed on gestation day 22 of gestation
- Organs examined: All rats were given an examination post mortem which involved an external examination and an examination of the thoracic and abdominal viscera and uterine contents. All macroscopic findings were recorded. Pregnancy status was determined.

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Position of implantations: Yes
- Number of live foetuses: Yes
- Number of intra-uterine deaths, early and late
Fetal examinations:
- External examinations: Yes: [all per litter] including examination of the oral cavity
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter] including assessment of ossification of the manus and pes
- Head examinations: Yes: [all per litter]
All analyses were carried out in SAS (1996). For Fisher’s Exact Tests the proportion in each treated group was compared to the control group proportion. The percentage of male foetuses was analysed by analysis of variance following double arcsine transformations of Freeman and Tukey (1950). All statistical tests were two sided.
Historical control data:
not cited

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
no effects

Effect levels (maternal animals)

Key result
Dose descriptor:
Effect level:
8 000 other: ppm (18359 mg/m3, 18.4 mg/L) nominal
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Fetal body weight changes:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
no effects

Effect levels (fetuses)

Key result
Dose descriptor:
Effect level:
8 000 other: ppm (18359 mg/m3, 18.4 mg/L) nominal.
Basis for effect level:
other: Developmental Toxicity

Fetal abnormalities

not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

There was no effect of isobutylene on the number, growth or survival of the foetuses in utero. 

There was no effect of isobutylene on foetal development. Although cleft sternebrae were observed only in foetuses in the isobutylene groups, the incidence of foetuses affected was small and not dose-related and there were no minor changes in the appearance or ossification of the sternebrae to indicate that this area of the skeleton was adversely affected by isobutylene. Also, there was no evidence for an adverse effect of isobutylene on other ossification centres of the skeleton. Isolated differences from control were considered to be incidental. Thus isobutylene at exposure concentrations of up to 8000 ppm, is considered not to have any adverse effect on foetal development.

Applicant's summary and conclusion

Isobutylene at exposure concentrations of 500, 2000 and 8000 ppm had no effect on female rats during gestation. There was no effect of isobutylene on the number, growth or survival of the foetuses in utero and no effect on foetal development.
Executive summary:

Pregnant rats were exposed to isobutylene (2-methylpropene) at exposure concentrations of 500, 2000 and 8000 ppm (1147, 4589, 18359 mg/m3), from gestation day 5-21. Isobutylene had no effect on the females during gestation. There was no effect of isobutylene on the number, growth or survival of the foetuses in utero and no effect on foetal development. The NOECs for maternal and developmental toxicity were 8000 ppm (18359 mg/m3), the highest concentration tested.