Registration Dossier

Toxicological information

Repeated dose toxicity: inhalation

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Unnamed
Year:
1999
Report Date:
1999
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
Principles of method if other than guideline:
No guideline stated in publication. Publication/Study corresponds to a combined subchronic inhalation toxicity (OECD 413)/neurotoxicity study in rodents (OECD 424).
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): isobutanol, 2-methyl propanol (obtained from Union Carbide Chemicals and Plastics Company, Houston, TX, USA
- Physical state: colorless, volatile organic liquid
- Analytical purity: 99% (99.9% study report)
- Purity test date: no data
- Lot/batch No.: EH4337 (study report)
- Stability under test conditions: no data
- Storage condition of test material: no data

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc., NC, USA
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: males 285 - 365 g, females 179 - 242 g
- Fasting period before study: no data
- Housing: individually in stainless steel wire-mesh cages
- Diet (e.g. ad libitum): Purina Mills rodent Lab Chow #5002
- Water (e.g. ad libitum): yes
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 -26
- Humidity (%): 30 - 70
- Air changes (per hr): nodata
- Photoperiod (hrs dark / hrs light): 12 /12

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: no data
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 2000 L stainless steel and glass Hazelton H-2000 chambers
- Method of holding animals in test chamber: individually caged
- Source and rate of air: 500 L/minute
- Method of conditioning air: no data
- System of generating particulates/aerosols: adjustable-flow valveless metering pump (Fluid Metering Inc., Oster Bay, NY, USA) and a Laskin-typ nebulizer mounted in the supply air inlet at the top of the exposure chamber
- Temperature, humidity, pressure in air chamber: 23 to 24°C, 48 to 53%, no data
- Air flow rate: appr. 500 L/minute
- Air change rate: 15 changes/hour
- Method of particle size determination: no data
- Treatment of exhaust air: no data

TEST ATMOSPHERE
- Brief description of analytical method used: Fourier Transform infrared analyzer (FVB / Analect, Irvine, CA, USA) calibrated for isobutanol
- Samples taken from breathing zone: yes; 18 samples per exposure
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical determinations of the exposure chamber concentration yielded nominal-to-analytical ratios of means between 0.95 and 0.97
Duration of treatment / exposure:
14 weeks
Frequency of treatment:
6 hours per day, 5 days per week, with the exception of one day during the 4th, 8th, and 13th week of exposure due to neurobehavioral testing
total of exposures 70 - 73
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm (nominal)
Dose / conc.:
250 ppm (nominal)
Dose / conc.:
1 000 ppm (nominal)
Dose / conc.:
2 500 ppm (nominal)
No. of animals per sex per dose:
control and 2500 ppm groups: 20
250 ppm and 1000 ppm groups: 10
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: preliminary range finding study (5 rats/sex exposed to 0, 750, 1500, and 3000 ppm isobutanol 6 hrs/day, 5 days/week for two weeks)
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: 10 rats/sex in the control and 2500 ppm group (included in the 20 animals of these groups)
- Post-exposure recovery period in satellite groups: since no specific effects were observed, these animals were euthanized after 3 month of exposure along with the other animals in this study

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: 14th exposure week
- Dose groups that were examined: control and 2500 ppm group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination of study (week 14)
- Anaesthetic used for blood collection: Yes (carbondioxide)
- Animals fasted: Yes (overnight)
- How many animals: 5/sex and exposure group
- Parameters examined: hematocrit (HCT), hemoglobin level (HGB), total erythrocyte count (RBC), total leukocyte count, leukocyte differential, platelet count, activated partial thromboplastin time, reticulocyte count, mean corpusculart volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination of study (week 14)
- Animals fasted: Yes (overnight)
- How many animals: 5/sex and exposure group
- Parameters examined: blood urea nitrogen, creatinine, glucose, total protein, albumin, globulin, glutamic pyruvic transaminase, alkaline phosphatase, gamma glutamyl transpeptidase, glutamic oxaloacetic transaminase, creatine phosphokinase, total and direct bilirubin, cholesterol, sodium, potassium, calcium, chloride, and phosphorus.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to initiation of exposure and during week 4, 8, and 13 during exposure
- Dose groups that were examined: 15 rats/sex in the control and 2500 ppm group, 10 rats/sex for the 250 and 1000 ppm group
- Battery of functions tested: Functional Observation Battery, Motor Activity

Other:
as a second experiment a Schedule-Controled Operant Behaviour (SCOB) study was performed with an additional set of animals
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
Standardized statistical methology was used developed by the performing laboratory for routine analyses of toxicology studies.
Statistics used: Levence's Test for homogeneity of variance, Dunnett's 2-sided comparision of treatments to a control, protected Mann-Whitney Test, two factor repeated measures analysis of covariance (REPANCOVA), chi-square (one-tailed) and Cochran-Armitage Test for linear trend in proportions, Fishers' Exact Test (one-tailed).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
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:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
During exposure animals at all concentrations of isobutanol showed a slightly decreased responsiveness to external stimuli (light brush or tap on the exterior of the exposure chamber). During the first week of exposure this effect was more pronounced for a few rats at the 2500 ppm concentration. Immediately after exposure, there were no difference between control and treated animals.

No treatment related abnormal clinical signs were seen at any concentrations immediately after exposure or during the weekly, detailed clinical observations.

The conditions of one female animal at the 2500 ppm level suddenly declined after two month of exposure. It was sacrificed on day 72 of the study for human reasons since it was not eating. On day 70 this animal had paralyis in the hindlimbs and enlarged liver and spleen. After necropsy it was diagnosed to have lymphoblastic leukemia.

BODY WEIGHT AND WEIGHT GAIN
no effect

FOOD CONSUMPTION
no effect

FOOD EFFICIENCY
not examined

WATER CONSUMPTION
not examined

OPHTHALMOSCOPIC EXAMINATION
no effect

HAEMATOLOGY
slight increase in total erythrocyte count (RBC), hematocrit and hemoglobin in the 2500 ppm female group. The biological significance of these slight hematological changes is not known.

CLINICAL CHEMISTRY
no effects

URINALYSIS
not examined

NEUROBEHAVIOUR
no effects

ORGAN WEIGHTS
no effects

GROSS PATHOLOGY
no effects

HISTOPATHOLOGY: NON-NEOPLASTIC
no effects

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
-

HISTORICAL CONTROL DATA (if applicable)
no data

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEC
Effect level:
2 500 ppm (nominal)
Sex:
male/female
Basis for effect level:
other: overall effects
Dose descriptor:
NOEC
Effect level:
2 500 ppm
Sex:
male/female
Basis for effect level:
other: neurotoxicity

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Dose Dependent Effects on RBC, HGB, and HCT in Females (n=5)

Exposure Group

(ppm)

RBC

(millions/µL)

HGB

(grams/DL)

HCT (%)

0

7.68   (0.1348)

14.24   (0.3075)

41.50   (0.918)

250

8.03   (0.1536)

14.80   (0.3286)

43.28   (0.978)

1000

7.94   (0.2000)

14.92   (0.2709)

43.64   (0.644)

2500

8.42*  (0.1059)

15.52*  (0.1530)

45.30*  (0.438)

Values are expressed as mean value (SEM)

Statistically different from control (p ≤ 0.05; Dunnett’s Test)

There were two effects noted in this study. First, the responsiveness of animals of all exposed groups to external stimuli (light brush or tap on the exterior of the exposure chamber) was slightly reduced compared to controls. This effect disappeared immediately after termination of exposure. This is regarded as a non-specific acute effect due to the narcotic properties of isobutanol. It was, however, very slight and transient, and therefore it is not regarded as an adverse affect under the conditions of this study.

 

The other effect in this study was a slight change of hematological parameters in females at 2500 ppm (5 animals) where total erythrocyte count (RBC), hemoglobin (HGB), and hematocrit (HCT) was slightly but significantly increased over controls (p ≤ 0.05). The changes were however small and the biological significance is low. This effect was not seen in any of the other treatment groups.

Therefore, a level of 2500 ppm (ca. 7700 mg/m³) is considered to be the NOAEC under the conditions of this study.

Applicant's summary and conclusion

Conclusions:
No biologically and behaviorally relevant effects have been observed in this study. Therefore, the NOAEC for repeated dose toxicity and the NOEC for neurotoxicity is considered to be 2500 ppm (ca. 7700 mg/m³).
Executive summary:

In a subchronic (14 weeks) inhalation toxicity study, isobutanol (purity 99%) was administered to 20 or 10 Sprague-Dawley rats/sex/dose (controls and 2500 ppm group 20 animals, 250 and 1000 ppm group 10 animals) by dynamic whole body exposure at dose levels of 0, 250, 1000, and 2500 ppm for 6 hours per day, 5 days/week. Doses were selected on the basis of a preliminary range finding study.

 

Unspecific treatment related effects (decreased response to external stimuli on the exposure chamber) were seen for all dose levels only during exposure. This effect disappeared immediately after exposure. It is regarded as a non-specific acute effect due to the narcotic properties of isobutanol. It was very slight and transient, and therefore it is not regarded as an adverse affect under the conditions of this study.

 

In the female 2500 ppm dose group, total erythrocyte count, hemoglobin, and hematocrit were slightly elevated statistically different from controls (p ≤ 0.05). The changes were however small and the biological significance is low. This effect was not seen in any of the other groups and is estimated not to be of biological significance.

 

All other observed parameters did not show any statistically significant effects. No differences between dosed groups and controls were found for body weight, food consumption, ophthalmoscopic examination, clinical observation, clinical chemistry, neurobehavioral observations, organ weights, gross pathology, and histopathology.

 

Based on these findings, the NOAEC for repeated dose toxicity is considered to be 2500 ppm (ca 7700 mg/m) for males and females.

 

Based on neurobehavioral observation tests performed in this study, a NOEC for neurotoxicity of 2500 ppm (ca. 7700 mg/m³) for males and females can be deduced (Li/Monsanto 1999)

 

This subchronic toxicity study in the rat is acceptable and satisfies the guideline requirements for a subchronic inhalation study (OECD 413).