Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
fertility, other
Remarks:
based on test type (migrated information)
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other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
IUCLID4 Test substance: as prescribed by 1.1 - 1.4
- Name of test material (as cited in study report): 2-methyl-2-butene
- Physical state: Clear, colourless liquid
- Volatile liquid with boiling point of between 35 and 38°C
- Analytical purity: >98%
- Lot/batch No.: A0153320
- Expiration date of the lot/batch: no data
- Stability under test conditions: stable for the duration of the study
- Storage condition of test material: In a cool, dry, well-ventilated area

Test animals

Species:
rat
Strain:
other: Crl:CD®(SD)IGS BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd., Margate, Kent, UK
- Age at study initiation: Approximately 8-9 weeks
- Weight at study initiation: 252-314 g (males), 175-240 g (females)
- Housing: 1 male:1 female during pairing; individually (females, reproductive phase)
- Diet: Pelleted UAR VRF1 certified diet ad libitum except during exposure
- Water: ad libitum
- Acclimation period: 16 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25°C
- Humidity: 40-70%
- Air changes (per hr): Each animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated.
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 28 September 2001 To: 18 April 2002 (pathology completed)

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: stainless steel and glass construction and consisted of a cuboidal body fitted with a pyramidal base and top. The internal volume of each chamber was approximately 0.75 m. At the apex of the upper pyramidal figure was the tangentially mounted air duct. Immediately below this was a perforated canister, which ensured equal distribution of the test atmosphere within the chamber.
- Method of holding animals in test chamber: Exposure cages constructed of stainless steel mesh were suspended on a framework arranged on 4 levels. Each level was able to hold four cages, with each cage capable of housing 4 rats individually.
- Source and rate of air: For all groups exposed to 2-methyl-2-butene, the vapour/air mixture produced in the vapour generators was passed into the base of the secondary dilution vessel. A further supply of clean and dry air was supplied to Groups 2 and 3 to ensure a total chamber airflow of approximately 150 L/minute. The air supply for Group 4 was provided solely by the vapour generation system. The control group was exposed using a similar system to that used for the test groups, but received compressed air only at a rate of approximately 150 L/minute.
- System of generating particulates/aerosols: The vapour generation system for each of the test groups was supplied from individual reservoirs of liquid 2-methyl-2-butene maintained at pressure. The top of each reservoir was fitted with a central, "0" - ring sealed filler cap, a system to allow pressurisation and release of the helium head pressure and a safety pressure release valve set to operate at above the study operating pressure. Except during the filling procedure, 2-methyl-2-butene in the reservoirs was maintained under a helium pressure of 10 psi.
- Temperature, humidity, pressure in air chamber: Wet and dry bulb temperatures and relative humidity were recorded at approximately 30-minute intervals throughout each exposure.
- Air flow rate: The volume flow of air to the exposure chambers was measured using calibrated flow meters and also checked approximately every 30 minutes
- Treatment of exhaust air: The chamber air extract was vented to atmosphere via an exhaust stack.

TEST ATMOSPHERE
- Brief description of analytical method used: Gas chromatography
- Chamber atmosphere was sampled in sequence from each of the four exposure chambers (Chambers 4 - 1 sampled sequentially) and from one point within each chamber. Air from each chamber was continually drawn through a transfer line, which was therefore equilibrated with the mean concentration from each chamber. When not being sampled, these transfer lines were pumped to waste.
Every six minutes, air from the transfer lines was switched to the injection loop of the gas chromatograph for automated analysis and data processing.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Proof of pregnancy: sperm in vaginal smear, referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually in solid polypropylene cage with bedding material
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The study mean analysed concentrations of 2-methyl-2-butene over the duration of the study were 584, 2026 and 7097 ppm. These levels were in good agreement with the target exposure levels.
Duration of treatment / exposure:
Over a 2-week pre-mating period, throughout mating and through to Day 19 of gestation. The females were allowed to litter and rear their offspring to Day 4 of lactation.
Frequency of treatment:
6 hours/day , daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 580, 2000 or 7000 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 1665, 5740 or 20090 mg/m3
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 584, 2026 or 7097 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
12
Control animals:
yes, concurrent no treatment

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed observations were made daily, on the days of exposure, as follows:
Pre exposure observations; Observations during exposure; Observations within ½ to 1 hour of return to home cage.
During the daily exposure, obvious signs were recorded as a group response. Due to the type of exposure system used, the ability to observe individual animals during the exposures was severely restricted.
A more detailed weekly physical examination was performed on each animal to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rat was recorded on the day that treatment commenced (Week 0), then weekly to Week 2, and then on Days 0, 7, 14 and 20 after mating, and Days 1 and 4 of lactation.
During the exposure period, bodyweights were recorded before the daily exposure.

FOOD CONSUMPTION:
- Food consumption was recorded weekly to Week 2, then between Days 0-6, 7-13, and 14-19 of gestation, and Days 1 and 4 of lactation.
Estrous cyclicity (parental animals):
Dry vaginal smears were taken from all reproductive females for 10 days before pairing in order to assess the regularity and duration of oestrous cycles.
Wet vaginal smears were taken from all females following pairing until evidence of mating was confirmed
Sperm parameters (parental animals):
Histopathological examinations of testes was sufficient to enable any changes with regard to the stage in the spermatogenic cycle and cell integrity to be evaluated.
Litter observations:
- Litter observations: All offspring were examined approximately 24 hours after birth (Day 1) and the number of offspring born (live and dead) were recorded. Live offspring were individually identified within each litter by toe tattoo and individual bodyweight, sex and any clinical observations were recorded.
- Litters were subsequently observed daily for evidence of abnormal appearance or behaviour of the offspring. Daily records were maintained of mortality and consequent changes in litter size. Wherever possible, any offspring found dead were preserved in industrial methylated spirit prior to an external examination.
- The offspring were sexed at Days 1 and 4 of lactation.
- The offspring were weighed individually on Days 1 and 4 of age.
- Sacrifice and pathology
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
- Reproductive females and their litters were killed on Day 4 of lactation. Any reproductive females failing to mate were killed approximately 14 days after the last day of pairing or day of mating.
All animals were killed by intraperitoneal injection of sodium pentobarbitone, followed by exsanguination for adults.

ORGAN WEIGHTS
- The following organs, taken from each reproductive phase female, were dissected free of adjacent fat and other contiguous tissue and the weights recorded: brain, kidneys, liver, lungs and bronchi.
- Bilateral organs were weighed together.

HISTOPATHOLOGY: Yes
- No microscopic examinations were performed on tissues from reproductive phase animals.
Postmortem examinations (offspring):
Offspring, both scheduled and unscheduled, were subjected to a macroscopic external examination then discarded.
Statistics:
All statistical analyses were carried out separately for males and females.
Data relating to food consumption was analysed on a cage basis. For all other parameters, the analyses were carried out using the individual animal as the basic experimental unit. Significant differences between control and treated groups were expressed at the 5% (p<0.05) or 1% (p<0.01) level.
Reproductive indices:
Percentage mating, conception rate; and fertility index were calculated separately for males and females.
Offspring viability indices:
Mean survival indices for each group were calculated based on individual litter values.

Results and discussion

Results: P0 (first parental animals)

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:
no effects observed
Gross pathological findings:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: estrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- There were no unscheduled deaths. Half closed eyes was seen on Day 1 of exposure in Inter dose and High dose animals. High dose animals were seen to be less responsive to outside stimuli on Days 1 and 14 of exposure.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- There was a dose-related reduction in bodyweight gain in treated reproductive females over the 2 weeks prior to pairing with significance being attained for Intermediate and High dose females. Bodyweight gain of females during gestation and lactation was unaffected by treatment.

Effect levels (P0)

Dose descriptor:
NOAEC
Effect level:
7 000 ppm
Sex:
male/female
Basis for effect level:
other: 20,090 mg/m3

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

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

There were no treatment-related effects on oestrous cycles or pre-coital interval. Mating performance and fertility were unaffected by treatment, with all but one male and female pairing resulting in a viable pregnancy. The gestation length was similar in all groups and parturition was unaffected by treatment. There were no effects on implantation counts or resultant litter size at birth and Day 4. Post implantation loss and pup survival was unaffected by treatment. Pup bodyweight was not adversely affected. There were no effects on macroscopic examination of pups at Day 4 of lactation.

Applicant's summary and conclusion

Conclusions:
It was concluded that the no effect level of 2-methyl-2-butene, for reproductive/developmental toxicity in this screening study, to rats for 4 weeks by inhalation administration, was 7000 ppm.
Executive summary:

The reproduction/developmental toxic potential of the test substance, 2-methyl-2-butene (an industrial chemical) to Crl:CD®(SD)IGS BR rats by inhalation administration was assessed. Three groups of twelve female rats were exposed over a 2-week pre-mating period, throughout mating and through to Day 19 of gestation. The females were allowed to litter and rear their offspring to Day 4 of lactation. All animals received 2-methyl-2-butene by whole body inhalation exposure at concentrations of 580, 2000 or 7000 ppm. A similarly constituted control group received air alone.

During the study, clinical condition, bodyweight, food consumption, oestrus cycles, mating performance, litter data, organ weights and macroscopic pathology were undertaken.

The study mean analysed concentrations of 2-methyl-2-butene over the duration of the study were 584, 2026 and 7097 ppm. These levels were in good agreement with the target exposure levels.

It was concluded that the no effect level of 2-methyl-2-butene, for reproductive/developmental toxicity in this screening study, to rats for 4 weeks by inhalation administration, was 7000 ppm.