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Toxicological information

Carcinogenicity

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Administrative data

Endpoint:
carcinogenicity: inhalation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
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:
reference to same study
Reason / purpose:
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:
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
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 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 daysENVIRONMENTAL CONDITIONS - Exposure Chambers- Temperature: 23.9-24.2°C- Humidity: 53-58% - Air changes: 15 per hr - Photoperiod: 12 hrs dark / 12 hrs lightIN-LIFE DATES: From: 11 March 1993 To: 15 March 1995

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 DESCRIPTIONIsobutene 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
Post exposure period:
none
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:0, 500, 2000, 8000 ppmBasis:other: target concentration
Remarks:
Doses / Concentrations:497±21, 1990±72,7940±313 ppmBasis: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 dayDETAILED 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: NoOPHTHALMOSCOPIC EXAMINATION: No HAEMATOLOGY: No CLINICAL CHEMISTRY: No URINALYSIS: No NEUROBEHAVIOURAL EXAMINATION: No ANALYSIS OF 2-Hydroxyisobutyric Acid (HIBA) IN URINE: YesHIBA 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 dataHISTOPATHOLOGY: 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 andglandular), 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 toassess 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 examined
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 (males: 43/49, 45/49, 46/50, 49/49; females: 44/50, 47/50, 48/50, 47/49 exposed to 0, 500, 2000 and 8000 ppm respectively); however, the severities of hyaline degeneration increased with increasing exposure concentration in males and females. In inhalation studies, hyaline degeneration is a commonly observed change in the epithelium of the nasal cavity, the incidence and severity of which may increase with increasingexposure concentration. The accumulation of these protein globules is considered a nonspecific adaptive response to prolonged inhalation of irritant material and has no adverse effect on affected animals. Hypertrophy of goblet cells lining the nasopharyngeal duct was also marginally increased with 100% incidence in males at the top dose. 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 (Table 1). The histomorphology of the carcinomas that were observed in chamber control and exposed rats was similar to the morphologic spectrum typical of spontaneously developing follicular cell carcinomas.
Relevance of carcinogenic effects / potential:
The relevance of the thyroid follicular tumours for human cancer risk is questionable as the tumours only occurred in males and there were no precursor lesions such as hyperplasia or adenoma, there was no dose-response relationship, the tumours were singular and unilateral, did not form metastases and there was no increase in liver weight indicating a secondary mechanism. In addition, the thyroid was not a target organ in repeat dose studies in rats. Although the 10% incidence of tumours was outside the historical control at the time, reported to be 0-4% (NTP 1998, Haseman et al 1998) a carcinogenicity study on propylene (NTP 1985) had a 7% incidence of thyroid follicular carcinomas, further diminishing the significance of the incidence with isobutene. Haseman JK, Hailey JR, Morris and RW (1998). Spontaneous neoplasm incidences in Fischer 344 rats and B6C3F1 mice in two-year carcinogenicity studies: a National Toxicology Program update.Toxicol Pathol, 26, 428-441NTP (1985). NTP Toxicology and Carcinogenesis Studies of Propylene (CAS No. 115-07-1) in F344/N Rats and B6C3F1 Mice (Inhalation Studies). Natl Toxicol Program Tech Rep Ser, 272, :1-146

Effect levels

open allclose all
Dose descriptor:
NOAEC
Effect level:
2 000 other: ppm (4589 mg/m3, 4.59 mg/L) nominal
Sex:
male
Basis for effect level:
other: thyroid gland: follicular cell carcinoma (1/48, 0/48, 0/48, 5/50 at 0, 500, 2000 and 8000 ppm respectively)
Remarks on result:
other: Effect type: carcinogenicity (migrated information)
Dose descriptor:
NOAEC
Effect level:
8 000 other: ppm (18359 mg/m3, 18.4 mg/L) nominal
Sex:
female
Basis for effect level:
other: no evidence of any neoplastic changes at the highest dose (8000 ppm)
Remarks on result:
other: Effect type: carcinogenicity (migrated information)

Any other information on results incl. tables

Table 1: Incidences of Follicular Cell Neoplasms and Nonneoplastic Lesions of the Thyroid Gland in Male Rats

 

Control

500 ppm

2000 ppm

8000 ppm

Number Examined Microscopically

48

48

48

50

Hyperplasia (a)

0

2 (3.0)b

0

1 (4.0)

 

Carcinoma (c)

 

 

 

 

Overall rate(d)

1/48 (2%)

0/48 (0%)

0/48 (0%)

5/50 (10%)

Adjusted rate(e)

3.0%

0 %

0 %

13.5%

Terminal rate(f)

0/7 (0%)

0/5 (0%)

0/6 (0%)

0/8 (0%)

First incidence (days)

661

 

 

618

Poly-3 test(g)

P=0.004

P=0.519N

P=0.521N

P=0.125

a  Number of animals with lesion

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

c Historical incidence for 2-year inhalation studies with chamber control groups (mean ± standard deviation): adenoma: 7/892 (0.8% ± 1.2%), range 0%-4%; carcinoma: 9/892 (1.0% ± 1.2%), range 0%-4%; adenoma or carcinoma: 16/892 (1.8% ± 1.7%), range 0%-6%

d Number of animals with neoplasm per number of animals with thyroid gland examined microscopically

e Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality

f  Observed incidence at terminal kill

g Beneath the chamber control incidence is the P value associated with the trend test. Beneath the exposed group incidence are the P values corresponding to the pairwise comparisons between the chamber controls and that exposed group. The Poly-3 test accounts for differential mortality in animals that do not reach terminal sacrifice. A lower incidence in an exposure group is indicated by N.

Applicant's summary and conclusion

Conclusions:
The NTP concluded that there was some evidence of carcinogenic activity of isobutene in male F344/N rats based on the increased incidence of follicular cell carcinoma of the thyroid gland although the relevance of this finding is questionable. There was no evidence of carcinogenic activity of isobutene in female F344/N rats exposed to 500, 2,000, or 8,000 ppm.
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. The incidences of hyaline degeneration of the olfactory epithelium and hypertrophy of goblet cells lining the nasopharyngeal duct were marginally increased in both sexes of exposed rats. The severities of hyaline degeneration increased with increasing exposure concentration in males and females but these effects are considered to be a common occurrence in long-term inhalation studies. The relevance of these finding is discussed under Section 7.5 (Repeat dose toxicity).

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. The incidences of hyaline degeneration of the olfactory epithelium were marginally increased in exposed rats; however, the severities of hyaline degeneration increased with increasing exposure concentration in males and females.

 

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.

It was concluded that there was some evidence of carcinogenic activity of isobutene in male F344/N rats based on an increased incidence of follicular cell carcinoma of the thyroid gland. There was no evidence of carcinogenic activity of isobutene in female F344/N rats exposed to 500, 2000, or 8000 ppm. The NOAEC for carcinogenicity in males was 2000 ppm (4589 mg/m3) and in females was 8000 ppm (18359 mg/m3)