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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
chronic toxicity: inhalation
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, near guideline study, published in peer reviewed literature, 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
Reference Type:
publication
Title:
Unnamed
Year:
1998
Report date:
1998

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Deviations:
yes
Remarks:
limited number of parameters assessed: mortality/morbidity, clinical observations, body weights, histopathology
Principles of method if other than guideline:
NTP study
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2-methylpropene
EC Number:
204-066-3
EC Name:
2-methylpropene
Cas Number:
115-11-7
Molecular formula:
C4H8
IUPAC Name:
2-methylprop-1-ene
Details on test material:
purity of isobutene was greater than 98%

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms (Germantown, NY, USA)
- Age at study initiation: Average 6 weeks
- Weight at study initiation: mean bodyweight per group for males 146-148 g; mean bodyweight per group for females 109-110 g
- Fasting period before study: None
- Housing: Individually housed in stainless steel wire bottom cages
- Diet: NIH-07 open formula pelleted diet (Zeigler Brothers, Inc., Gardners, PA, USA), available ad libitum except during exposure and urine collection periods, changed weekly
- Water: Softened tap water (Richland municipal supply) via automatic watering system (Edstrom Industries, Waterford, WI, USA), available ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS - Exposure Chambers
- Temperature: 23.9-24.2°C
- Humidity: 53-58%
- Air changes: 15 per hr
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 11 March 1993 To: 15 March 1995

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: air
Remarks on MMAD:
MMAD / GSD: no details
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Isobutene was distributed under regulated pressure and heat lost due to isobutene vaporisation was replaced. The gas passed through a filter, then distributed to six pairs of metering valves with corresponding flow meters via 3-way solenoid valves located at the chamber end of the vapour delivery line. Isobutene vapour was diluted with conditioned air as it was injected into the chamber inlet duct. Stainless-steel chambers were used throughout the studies. The total volume of each chamber was 2.3 m3; the active mixing volume of each chamber was 1.7m3 . The inhalation exposure chamber was designed so that uniform vapour concentrations could be maintained throughout the chamber with the catch pans in place.

TEST ATMOSPHERE
- Brief description of analytical method used: Chamber concentrations of isobutene were monitored by an on-line gas chromatograph approximately every 20 minutes during exposures. Vapour concentration uniformity in the exposure chambers without animals present was measured before the study began. Chamber concentration uniformity was maintained throughout the study. The times for the exposure concentration to build up to 90% of the final exposure concentration (T90) and to decay to 10% of the exposure concentration (T10) were measured with and without animals present in the chambers. The T90 value selected was 12 minutes. No significant degradation of isobutene or enhancement of impurities was observed during the study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Isobutene was stable under the conditions used to generate and transport it to the exposure chambers and that no significant enhancement of impurities was detected in the distribution manifold or the exposure chambers over the course of a typical exposure day.
The mean concentrations achieved over the 2 year study for the 500, 2000 and 8000 ppm target concentrations were 497±21, 1990±72 and 7940±313 ppm respectively.
Duration of treatment / exposure:
2 years
Frequency of treatment:
6 h/d, 5 d/w

Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 500, 2000, 8000 ppm
Basis:
other: target concentration
Remarks:
Doses / Concentrations:
497±21, 1990±72,7940±313 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
50
Control animals:
yes, sham-exposed
Details on study design:
Dose selection rationale: Based on the lack of significant exposure-related toxicological effects in a 14 week inhalation study (exposure levels 0, 500, 1000, 2000, 4000, 8000 ppm), 8000 ppm was selected as the highest dose level in this 2 year study. A higher concentration could not be used because of the danger of explosion. The 2-year study exposure concentrations of 0, 500, 2,000, and 8,000 ppm were based on published metabolic elimination rates for Sprague- Dawley rats and B6C3F1 mice (Csanády et al., 1991). These rates indicated that 500 ppm would be within the linear range for metabolic elimination, 2000 ppm would be near the linear range, and 8000 ppm would be out of the linear range.
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice per day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Initially, every 4 weeks from week 4 through week 91, and thereafter every 2 weeks until the end of the studies

BODY WEIGHT: Yes
- Time schedule for examinations: Initially, weekly through week 12, every 4 weeks from week 15 through week 91 and every 2 weeks thereafter until the end of the studies

FOOD CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

ANALYSIS OF 2-Hydroxyisobutyric Acid (HIBA) IN URINE: Yes
HIBA is the major urinary metabolite of isobutene, and was measured in the urine of male and female rats as an indicator of isobutene exposure. Five male and five female rats from the chamber control groups and 10 male and 10 female rats from each exposed group were evaluated at 6, 12, and 18 months for determination of HIBA in urine. Rats were housed individually in metabolism cages for 16 hours after exposure while urine samples were collected over ice. Parameters evaluated included urinary excretion, creatinine, and HIBA.
Sacrifice and pathology:
GROSS PATHOLOGY: No data
HISTOPATHOLOGY: Yes. Complete histopathology was performed on all rats. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone with marrow, brain, clitoral gland, oesophagus, heart, large intestine (caecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, larynx, liver, lung, lymph nodes (mandibular, mesenteric, bronchial, mediastinal), mammary gland (with adjacent skin), nose, ovary, pancreatic islets, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, spleen, stomach (forestomach and
glandular), testes (with epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus.
Other examinations:
None. Clinical chemistry was not carried out as this was performed in the 14 week studies.
Statistics:
The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958). Statistical analyses for possible dose-related effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided.
For calculation of statistical significance, the incidences of most neoplasms and all non-neoplastic lesions are given as the numbers of animals affected at each site examined microscopically. The Poly-k test (Bailer and Portier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997) was used to
assess neoplasm and non-neoplastic lesion prevalence. Tests of significance included pairwise comparisons of each exposed group with controls and a test for an overall exposure-related trend. Continuity-corrected tests were used in the analysis of lesion incidence, and reported P values are one sided.
Body weight data, which has approximately normal distributions, was analyzed with the parametric multiple comparison procedures of Dunnett (1955) and Williams (1971, 1972).

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:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY: Survival of exposed males and females was similar to that of the chamber controls 7/50, 5/50, 6/50, and 8/50
males and 23/50, 19/50, 33/50, and 22/50 females of the control, low, mid and high dose group, respectively, were still alive after 2 years. There were no clinical findings attributed to isobutene exposure.

BODY WEIGHT AND WEIGHT GAIN: Mean body weights of exposed male and female rats were generally similar to those of the chamber controls throughout the study.

2-HYDROXYISOBUTYRIC ACID - BIOMARKER OF EXPOSURE: 2-Hydroxyisobutyric acid (HIBA), the major urinary metabolite of isobutene, was measured in the urine of male and female rats as an indicator of isobutene exposure at 6, 12, and 18 months. The amount of HIBA excreted increased with increasing exposure concentration. However, when HIBA concentration was normalized to isobutene exposure concentration, the relative amount of HIBA excreted decreased with increasing exposure concentration, implying nonlinear kinetics. Exposure to isobutene had no effect on the quantity of urine or the amount of creatinine excreted.

HISTOPATHOLOGY - NON-NEOPLASTIC: The incidences of hyaline degeneration of the olfactory epithelium were marginally increased in exposed male and female rats with 100% incidence in males at the top dose (Table 1); however, the severities of hyaline degeneration increased with increasing exposure concentration in males and females. This NTP report states that hyaline degeneration of the epithelium of the nasal cavity is a commonly observed change in inhalation studies. Hypertrophy of goblet cells lining the nasopharyngeal duct was also marginally increased with 100% incidence in males at the top dose (Table 1). Any other changes were considered not to be related to exposure to isobutene.

HISTOPATHOLOGY - NEOPLASTIC: The incidence of thyroid gland follicular cell carcinoma in male rats exposed to 8,000 ppm was increased compared to the chamber control group and exceeded the historical control range. Further details are given in Section 7.7 Carcinogenicity.

Effect levels

Dose descriptor:
NOAEC
Remarks:
Toxicity
Effect level:
2 000 other: ppm (4589 mg/m3, 4.59 mg/L) nominal.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

The incidences of non-neoplastic nasal lesions are shown below. Hyaline degeneration of the olfactory epithelium is considered a nonspecific adaptive response to prolonged inhalation of irritant material and has no adverse effect on affected animals (OECD SIDS Report for Isobutylene, 2003). The increased goblet cell hypertrophy in the was however considered to be adverse.

Table 1: Incidences of Non-neoplastic Lesions of the Nose

 

Control

500 ppm

2000 ppm

8000 ppm

Male

Number Examined Microscopically

49

49

50

49

Olfactory Epithelium, Degeneration Hyaline (a)

43 (1.3)b

45 (1.4)

46 (2.2)

49 (2.6)

Goblet Cell Hypertrophy

43

45

46

49

Female

Number Examined Microscopically

50

50

50

49

Olfactory Epithelium, Degeneration Hyaline (a)

44 (1.5)

47 (2.4)

48 (2.8)

47 (2.8)

Goblet Cell Hypertrophy

44

47

48

47

a Number of animals with lesion

b Average severity grade of lesions in affected animals: 1=minimal, 2=mild, 3=moderate, 4=marked

Applicant's summary and conclusion

Conclusions:
F344/N rats exposed by whole body inhalation to isobutene at concentrations of 0, 500, 2,000 or 8,000 ppm, (1147, 4589, 18359 mg/m3) 6 hours per day, 5 days per week, for 105 weeks showed increases in the incidences and severity of hyaline degeneration of the olfactory epithelium nasal goblet cell hypertrophy. A higher incidence was observed in males at the top dose. Male rats given 8000 ppm developed thyroid follicular cell tumours. The NOAEC for repeat dose toxicity in this study was 2000 ppm (4589 mg/m3, 4.59 mg/L).
Executive summary:

Groups of 50 male and 50 female F344/N rats were exposed to isobutene (greater than 98% pure) at concentrations of 0, 500, 2000, or 8000 ppm (1147, 4589, 18359 mg/m3) 6 hours per day, 5 days per week, for 105 weeks. Survival of exposed males and females was similar to that of the chamber controls. Mean body weights of exposed groups were generally similar to those of the chamber controls throughout the study. 

2-Hydroxyisobutyric acid (HIBA) the major urinary metabolite of isobutene, was measured in the urine of the rats as an indicator of isobutene exposure. The amount excreted increased with increasing exposure concentration but when HIBA concentration was normalized to isobutene exposure concentration, the relative amount of HIBA excreted decreased with increasing exposure concentration, implying nonlinear kinetics.

 

The incidence of thyroid gland follicular cell carcinoma in male rats exposed to 8000 ppm was increased compared to the chamber control group and exceeded the historical control range. Further discussion on these is provided in Section 7.7. The incidences and severity of hyaline degeneration of the olfactory epithelium and the incidences of nasal goblet cell hypertrophy were marginally increased in both sexes of all exposed rats (including controls). These effects are a common occurrence in long-term inhalation studies and are observed at the site of entry into the body. A higher incidence was, however, observed in males at the top dose level with 100% incidence of hypertrophy of goblet cells lining the nasopharyngeal duct. The NOAEC for repeat dose toxicity in this study was therefore 2000 ppm (4589 mg/m3, 4.59 mg/L). 

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