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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Remarks:
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other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment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:
- 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), 178-252 g (females)
- Housing: Maximum of 4 rats/sex/cage (toxicity phase)
- Diet: Pelleted UAR VRF1 certified diet ad libitum except during exposure and overnight prior to blood sampling.
- 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:
whole body
Vehicle:
other: air
Details on inhalation 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.
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:
4 weeks
Frequency of treatment:
6 hours/day, 7 days per week
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

Observations and examinations performed and frequency:
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 throughout the treatment period, and before necropsy.
During the exposure period, bodyweights were recorded before the daily exposure.

FOOD CONSUMPTION:
- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for each week throughout the treatment period. From these records the mean weekly consumption per animal (g/rat/week) was calculated for each cage.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During Week 5 of treatment (before exposure), blood samples were obtained from all toxicity phase animals.
- Anaesthetic used for blood collection: Yes. Animals were held under light general anaesthesia induced by isoflurane and blood samples (0.5 mL) were withdrawn from the retro-orbital sinus.
- Animals fasted: Yes
- How many animals: 12 per sex/dose
- The following parameters were examined: haematocrit, haemoglobin, red blood cell count, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, mean cell volume, total white cell count, differential white cell count, large unstained cells, platelet count. A blood film (Romanowsky stained) was examined by light microscopy for abnormal morphology and unusual cell types, including normoblasts.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the same time and using the same toxicity phase animals as for peripheral haematology, further blood samples (nominally 0.7 mL) were collected into lithium heparin as anticoagulant.
- Animals fasted: Yes
- How many animals: 12 per sex/dose
- The following parameters were examined: alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, bile acids, urea, creatinine, glucose, total cholesterol, triglycerides, sodium, potassium, chloride, calcium, inorganic phosphorus, total protein, albumin, A/G ratio (calculated from total protein concentration and analysed albumin concentration).

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Before commencement of treatment and during Weeks 1, 2, 4 and 5 of treatment, a functional observational battery FOB) was performed.
- Dose groups that were examined: All males and toxicity females.
- Battery of functions tested: sensory activity / grip strength / motor activity.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- All animals were killed by intraperitoneal injection of sodium pentobarbitone, followed by exsanguination for adults.

ORGAN WEIGHTS
- The following organs were dissected free of adjacent fat and other contiguous tissue and the weights recorded: adrenals, lungs and bronchi, brain, ovaries, epididymides, pituitary, heart, spleen, kidneys, testes, liver, thymus.
- Bilateral organs were weighed together.

HISTOPATHOLOGY: Yes
- A wide range of tissues were examined for all toxicity phase animals of Groups 1 (Control) and 4 (7000 ppm) sacrificed on completion of the scheduled treatment period, and for all animals killed during the study.
Tissues reported at macroscopic examination as being grossly abnormal were examined for all animals.
The following tissues, which were considered to exhibit a reaction to treatment at the high dosage, were examined for all animals; spleen, nasal passages, liver, heart, kidneys.

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.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
There was one unscheduled death unrelated to treatment (group 2 (Low dose) female). Half closed eyes were seen on Day 1 of exposure in Intermediate 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 WEIGHT GAIN
Bodyweight gain in the high dose animals was transiently lower than control (statistically significant in females). Bodyweight gain of females during gestation and lactation was unaffected by treatment.

HAEMATOLOGY
Group mean prothrombin time was statistically significantly longer for intermediate dose females and high dose animals, and activated partial thromboplastin time was longer for high dose males.

CLINICAL CHEMISTRY
Group mean cholesterol levels were statistically significantly higher in high dose toxicity phase females following 4 weeks of treatment.

ORGAN WEIGHTS
Bodyweight adjusted group mean liver weights were slightly yet statistically significantly higher for high dose toxicity phase females following 4 weeks of treatment.

HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment-related findings were seen in several tissues of animals exposed to 2-methyl-2-butene, as follows: in the liver, centrilobular hepatocyte hypertrophy in high dose females; in the spleen, decreased extramedullary haemopoiesis in high dose males and females; in the nasal passages, increased goblet cell hyperplasia in the respiratory epithelium in high dose males; in the heart, a slightly greater severity of myocardial inflammatory lesions in high and intermediate dose males; in the kidneys, marginal increases in incidence and/or degree of cortical and medullary tubular basophilia in high and intermediate dose males.

Effect levels

Dose descriptor:
NOAEC
Effect level:
580 ppm
Sex:
male/female
Basis for effect level:
other: 1665 mg/m3 (slight, general systemic effects)

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Functional observational battery and motor activity: There were no treatment-related changes.

Applicant's summary and conclusion

Conclusions:
The no effect level of 2-methyl-2-butene for general systemic toxicity to rats for 4 weeks by inhalation administration was 580 ppm.
Executive summary:

The general systemic 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 male and twelve female rats were exposed for 6 hours per day for a period of 4 weeks. 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, detailed functional observational battery, motor activity, bodyweight, food consumption, haematology, blood chemistry, organ weight and macroscopic and microscopic pathology investigations 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 general systemic toxicity to rats for 4 weeks by inhalation administration was 580 ppm.