Registration Dossier

Administrative data

Description of key information

For isobutyric acid, no data on repeated dose toxicity could be located.
To compensate for this lack of data, information resulting from isobutanol and isobutyl isobutyrate as supporting substances will be used as substitute. Data are available for oral and inhalation repeated dose toxicity.
Repeated dose toxicity: oral (gavage)
Supporting substance isobutanol: In a valid subchronic (90 d) toxicity studies, a NOAEL of 316 mg/kg bw/day has been observed (EPA/Res. Triangle Inst. 1987).
The NOAEL for isobutyric acid (conversion using the respective molecular weight) is ca. 375 mg/kg bw/day
Supporting substance isobutyl isobutyrate: In a valid subchronic (90 d) toxicity studies, a NOAEL of 1000 mg/kg bw/day has been observed (Drake 1978).
The NOAEL for isobutyric acid (conversion using the respective molecular weight) is ca. 610 mg/kg bw/day
Repeated dose toxicity: inhalation
Supporting substance isobutanol: In a valid subchronic (90 d) toxicity study, the NOAEL was derived to be 7700 mg/m³ (Li/Monsanto 1999).
The NOAEC for isobutyric acid (conversion using the respective molecular weight) is ca. 9150 mg/m³.
The findings of all three studies do not indicate a specific target organ.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
As working hypothesis for the read-across approch from iso-butanol to iso-butyric acid information on toxicokinetics for isobutanol showing a readily absorbtion from the gastrointestinal as well as from the respiratory tract. Isobutanol is metabolized in the liver and it undergoes rapid oxidation (Hedlund 1969, Saito 1975, Ehrig 1988, Sinclair 1990). Isobutyraldehyde and subsequently isobutyric acid is formed (Saito 1975, Poet and Corley (OPP/ACC) 2004).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemicals Isobutanol was investiagate as a pure compound without significant impurities. The target substance iso-butyric acid is as well regarded and evaluated as pure compound and mono-constituent substance under REACh Legislation. No significant impurities need to be assessed according to analytical data.

3. ANALOGUE APPROACH JUSTIFICATION
To verify the rapid metabolism of isobutanol and isobutyl isobutyrate to intermediate isobutyric acid, information on toxicokinetics and metabolism of these two substances are presented below. The information is taken from the endpoint summary of the OXEA IUCLID 5 dataset of isobutanol (2009) and from the OECD SIDS/SIAR of isobutyl isobutyrate (last update 2005-09-08). Detailed Information on references can be found in these sources.

Isobutanol is readily absorbed from the gastrointestinal as well as from the respiratory tract. Maximum blood levels are reached within 15 minutes (rat, respiration; Poet and Corley (OPP/ACC) 2004), 45 to 60 minutes (human, rabbit, oral application; Bilzer 1990, Saito 1975) or 90 minutes (rat, oral application; Gaillard 1965). Elimination from blood is considerably fast as well. In humans, an elimination half-life of 1.45 h was determined (Bilzer 1990). In rabbits, isobutanol could not be detected any more 6 hours after application (Saito 1975).

Isobutanol is metabolized in the liver and it undergoes rapid oxidation (Hedlund 1969, Saito 1975, Ehrig 1988, Sinclair 1990). Isobutyraldehyde and subsequently isobutyric acid are formed (Saito 1975, Poet and Corley (OPP/ACC) 2004) by oxidation with alcohol dehydrogenase and aldehyde dehydrogenase respectively (Hedlund 1969, Saito 1975, Rüdell 1983), Beta-oxidation and decarboxylation of isobutyric acid results in propionyl-coenzyme A (Saito 1975) which can enter the tricarbxylic acid cycle. Accordingly, propionaldehyde, propionic acid, and succinic acid were identified as additional metabolites (Rüdell 1983).

Only minor amounts of applied isobutanol doses are excreted unchanged (rabbit 0.54 %, Saito 1975; rat 0.27%, Gaillard 1965). Small amounts have been found as glucuronides in the urine of rabbits (4.4 %, Kamil 1953).

In expired air, no metabolites (aldehyde, ketone) could be detected (Kamil 1953).

In conclusion, isobutanol is rapidly metabolized to isobutyraldehyde and further on to isobutyric acid with higher levels of isobutyric acid detectable in blood.

The repeated dose toxicity of isobutanol has been evaluated in two valid studies of high reliability which are used as key studies (oral administration: EPA/Res. Triangle Inst. 1987 and inhalation exposure: Li/Monsanto 1999).
In a valid subchronic toxicity study (90 days) similar to OECD TG 408, isobutanol (purity not stated) was administered to 30 rats/sex/dose by gavage at dose levels of 0, 100, 316, and 1000 mg/kg bw/day on the basis of a preliminary range finding study.

Treatment related effects were seen only at a dose level of 1000 mg/kg bw/day, but not at dose levels of 100 or 316 mg/kg bw/day. At the 1000 mg/kg bw/day dose level, hypoactivity was observed in all rats and body weight gain in the males was 18% below the control average during week 1. Food consumption averages were below those of the controls for both males and females during week 1 and 2. In addition, serum potassium averages (both sexes) were 11-15% below control averages at the interim evaluation (day 29) only and this may have been related to treatment.

No difference between dosed groups and controls were found for ophthalmoscopic examination, hematology, urinalysis, organ weights, gross pathology and histopathology.
Results do not indicate a specific target organ.


Reason / purpose:
read-across source
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories Inc., Portage, MI, USA
- Age at study initiation: 22 - 23 days
- Weight at study initiation: 45-55 g
- Fasting period before study: no
- Housing: individually in wire-bottom cages
- Diet (e.g. ad libitum): Purina Certified Rodent Laboratory Chow No. 5002 (pellet)
- Water (e.g. ad libitum): filtered municipal water
- Acclimation period: 7days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1
- Humidity (%): 43 ± 5
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 /12
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: dissolution in water

DIET PREPARATION
- Rate of preparation of dosing solution (frequency): weekly

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: as required by dose
- Amount of vehicle (if gavage): 10 ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
GC analysis with a Varian 2100 GC with flame ionization detector connected to a Spectra Physics Model 4100 recording integrator.
Analysis of the test material was performed at week 1, 3, 8, 9, and 13. Measured concentrations were found to in acceptable accordance with nominal values.
Duration of treatment / exposure:
92 days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
100, 316, 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
30 in total (10 for interim sacrifice)
In addition, one group of 10 animals per sex (group V) for baseline observation (sacrifice prior to initiation of dosing)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: preceding rage finding toxicity study (TLR study #032-001)
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: no satellite groups
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: No data
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly

FOOD EFFICIENCY: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during pretreatment period and at week 13
- Dose groups that were examined: all rats were examined

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to initiation of dosing, at week 4 or 5, and at week 13 or 14
- Anaesthetic used for blood collection: Yes, CO2
- Animals fasted: No data
- How many animals: prior to dosing 10 animals per sex ( group V), at week 4 or 5 (interim sacrifice) and at week 13 or 14 (final sacrifice) 10 animals per sex from each group
- Parameters examined: hemoglobin (HGB), hematocrit (PCV), erythrocyte count (RBC), mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), total and differential leucocyte counts (WBC), estimated platelet count (PLT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to initiation of dosing, at week 4 or 5, and at week 13 or 14
- Anaesthetic used for blood collection: Yes, CO2
- Animals fasted: No data
- How many animals: prior to dosing 10 animals per sex ( group V), at week 4 or 5 (interim sacrifice) and at week 13 or 14 (final sacrifice) 10 animals per sex from each group
- Parameters examined: alkaline phosphatase (Alk phos), blood urea nitrogen (BUN), glutamate pyruvate transaminase (SGPT), glutamate oxaloacetate transaminase (SGOT), glucose (Gluc), total protein (TP), albumin (Alb), A/G ratio (calculated), globulin (calculated), total bilirubin (Tot. bili.), sodium (Na), potassium (K), chloride (Cl), calcium (Ca), creatinine, inorganic phosphate (phos.), total carbon dioxide (TCO2), total serum cholesterol (Chol)

URINALYSIS: Yes
- Time schedule for collection of urine: prior to initiation of dosing, at week 4 or 5, and at week 13 or 14
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters examined. pH, specific gravity, glucose, protein, ketones, bilirubin, urobilinogen, microscopy of sediment.

NEUROBEHAVIOURAL EXAMINATION: No special examinations
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
body weight, food consumption, clinicopathologic, and organ weight data were tested for homogeneity of variance by Bartlett's method (Steel and Torrie, 1980).
If the data were found to be homogeneous, differences between control and treatment means were tested for statistical significance by the method of Dunnett (Dunnett, 1964).
If the data were found not to be homogeneous, the method of Gill (modified Dunnett's) was employed (Gill, 1977)
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):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
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
Treatment related clinical signs were restricted to the high-dose group and included hypoactivity, ataxia, and salivation. Hypoactivity decreased in the course of the experiment and was only sporadically observed after week 4.
The mortality rate was 1/60, 1/60, 2/60 and 11/60 for the control, 100, 316, and 1000 mg/kg/day dose group, respectively. In combination with the results of the gross necropsy and histologic findings, the increased number of death in the high-dose group are not related to a systemic toxicity of orally administered isobutyl alcohol.

BODY WEIGHT AND WEIGHT GAIN
During week 1 the males of the 1000 mg/kg/day dose group gained 10% less body weight than the controls.

FOOD CONSUMPTION
For the first two weeks of the study, food consumption averages of the 1000 mg/kg day dose group males and females were significantly lower than controls ( p ≤ 0.01 - males, p ≤ 0.05 - females week 1 and p ≤ 0.01 - males/females week 2).

FOOD EFFICIENCY
no examined

WATER CONSUMPTION
not examined

OPHTHALMOSCOPIC EXAMINATION
no effects

HAEMATOLOGY
no effects

CLINICAL CHEMISTRY
There was no definite treatment related effect on clinical pathologic parameters. However, the possibility of treatment-related effects was suggested by differences between control and treatment group mean serum potassium, albumin, and total protein concentrations. Group mean serum potassium concentration for males and females in the 1000 mg/kg/day group was 11-15% lower than the control group means at the interim evaluation, but it was similar for treated and control groups at the final clinical pathologic evaluation. Slightly lower than control group means for femals of the 316 mg/kg/day dose group (7%, p ≤ 0.05)and for females of the 1000 mg/kg/day dose group (9% p ≤ 0.01) appeare to be a result of higher than expected values in the control group rather than abnormally low values in the treated groups.

URINALYSIS
no effects

NEUROBEHAVIOUR
not examined

ORGAN WEIGHTS
no effects

GROSS PATHOLOGY
no effects

HISTOPATHOLOGY: NON-NEOPLASTIC
no effects

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
not examined

HISTORICAL CONTROL DATA (if applicable)
Key result
Dose descriptor:
NOEL
Remarks:
calculated from RAC substance via mole weights ratio
Effect level:
375 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: overall effects clinical signs; body weight; food consumption; clinical chemistry
Critical effects observed:
not specified

Four rats were determined to have died from perforation of the esophagus (incurred during dosing). The case of death of another ten rats was acute tracheitis (or bronchial hemmorhage), which was apparently due to the regurgitation and subsequent aspiration of the isobutyl alcohol and/or its vapors into the trachea (and probably the nasal cavity). The acute lesions of the trachea (inflammation and/or necrosis) seen histologically support this theory.

Executive summary:

In a subchronic toxicity study (90 days) isobutanol (purity not stated) was administered to 30 rats/sex/dose by gavage at dose levels of 0, 100, 316, and 1000 mg/kg bw/day on the basis of a priliminary range finding study.

 

Treatment related effects were seen only at a dose level of 1000 mg/kg bw/day, but not at dose levels of 100 or 316 mg/kg bw/day. At the 1000 mg/kg bw/day dose level, hypoactivity was observed in all rats and body weight gain in the males was 18% below the control average during week 1. Food consumption averages were below those of the controls for both males and females during week 1 and 2. In addition, serum potassium averages (both sexes) were 11-15% below control averages at the interim evaluation (day 29) only and this may have been related to treatment.

No difference between dosed groups and controls were found for ophthalmoscopic examination, haematology, urinalysis, organ weights, gross pathology and histopathology.

The NOEL is 316 mg/kg bw/day for males and females for isobutanol (EPA/Res. Triangle Inst. 1987). Within the read-across approach it was transfered via mole weight ratio into a NOEL for iso-butyric acid accounting to 375 mg/kg bw/day

This subchronic toxicity study in the rat is acceptable and satisfies the guideline requirements for a subchroic oral study (OECD 408).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
375 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
As working hypothesis for the read-across approch from iso-butanol to iso-butyric acid information on toxicokinetics for isobutanol showing a readily absorbtion from the gastrointestinal as well as from the respiratory tract. Isobutanol is metabolized in the liver and it undergoes rapid oxidation (Hedlund 1969, Saito 1975, Ehrig 1988, Sinclair 1990). Isobutyraldehyde and subsequently isobutyric acid is formed (Saito 1975, Poet and Corley (OPP/ACC) 2004).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source chemicals Isobutanol was investiagate as a pure compound without significant impurities. The target substance iso-butyric acid is as well regarded and evaluated as pure compound and mono-constituent substance under REACh Legislation. No significant impurities need to be assessed according to analytical data.

3. ANALOGUE APPROACH JUSTIFICATION
In a valid subchronic (14 weeks) inhalation toxicity study according to OECD TG 413, isobutanol (purity 99%) was administered to 20 or 10 Sprague-Dawley rats/sex/dose. Controls and 2500 ppm group consisted of 20 animals, 250 and 1000 ppm group were 10 animals in size. Test substance administration was 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 based on 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 effect 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 it 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.

Results do not indicate a specific target organ.

Isobutanol is readily absorbed from the gastrointestinal as well as from the respiratory tract. Maximum blood levels are reached within 15 minutes (rat, respiration; Poet and Corley (OPP/ACC) 2004), 45 to 60 minutes (human, rabbit, oral application; Bilzer 1990, Saito 1975) or 90 minutes (rat, oral application; Gaillard 1965). Elimination from blood is considerably fast as well. In humans, an elimination half-life of 1.45 h was determined (Bilzer 1990). In rabbits, isobutanol could not be detected any more 6 hours after application (Saito 1975).

Isobutanol is metabolized in the liver and it undergoes rapid oxidation (Hedlund 1969, Saito 1975, Ehrig 1988, Sinclair 1990). Isobutyraldehyde and subsequently isobutyric acid are formed (Saito 1975, Poet and Corley (OPP/ACC) 2004) by oxidation with alcohol dehydrogenase and aldehyde dehydrogenase respectively (Hedlund 1969, Saito 1975, Rüdell 1983), Beta-oxidation and decarboxylation of isobutyric acid results in propionyl-coenzyme A (Saito 1975) which can enter the tricarbxylic acid cycle. Accordingly, propionaldehyde, propionic acid, and succinic acid were identified as additional metabolites (Rüdell 1983).

Only minor amounts of applied isobutanol doses are excreted unchanged (rabbit 0.54 %, Saito 1975; rat 0.27%, Gaillard 1965). Small amounts have been found as glucuronides in the urine of rabbits (4.4 %, Kamil 1953).

In expired air, no metabolites (aldehyde, ketone) could be detected (Kamil 1953).

In conclusion, isobutanol is rapidly metabolized to isobutyraldehyde and further on to isobutyric acid with higher levels of isobutyric acid detectable in blood.
Reason / purpose:
read-across source
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
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
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
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).
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
Key result
Dose descriptor:
NOAEC
Remarks:
concluded from RAC substance via mole weight
Effect level:
9 150 mg/m³ air (nominal)
Sex:
male/female
Basis for effect level:
other: overall effects
Dose descriptor:
NOEC
Remarks:
concluded from RAC substance via mole weight
Effect level:
9 150 mg/m³ air (nominal)
Sex:
male/female
Basis for effect level:
other: neurotoxicity
Critical effects observed:
not specified

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.

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 isobutanol repeated dose toxicity is considered to be 2500 ppm (ca 7700 mg/m) for males and females. Via mole weight ration this results in a NOAEC for isobutyric acid for repeated dose toxicityis considered to be 2500 ppm (ca 9150 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).

Via mole weight ration this results in a NOEC for isobutyric acid for neurotoxicity is considered to be2500 ppm (ca 9150 mg/m) for males and females.

 

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

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
9 150 mg/m³

Additional information

For isobutyric acid, no study concerning repeated dose toxicity could be identified. As substitute, data for isobutanol and isobutyl isobutyrate will be used based on following reasons (for more detailed information on toxicokinetics/metabolism and justification of supporting substances see Section 7.1, endpoint summary).

 

After administration, isobutyl isobutyrate will be rapidly metabolized in vivo in blood and various tissues by ubiquitous esterases. Half-life of this transformation is short resulting in complete cleavage of the ester. Isobutanol and isobutyric acid are formed, the latter being identical with the test substance. Isobutanol is rapidly metabolized in vivo to isobutyraldehyde and subsequently to isobutyric acid by alcohol and aldehyde dehydrogenases respectively. Thus, in the course of metabolic transformation of isobutyl isobutyrate and isobutanol, isobutyric acid is generated rapidly and predominant as intermediary metabolite. Thus, it is justified to use isobutanol as well as isobutyl isobutyrate as supporting substance in the evaluation of the systemic toxic effects of isobutyric acid.

 

Effect levels derived for the supporting substances isobutanol and isobutyl isobutyrate will be transformed to effects levels for isobutyric acid using the respective molecular weights. These values are used to evaluate the toxic potential of isobutyric acid.

 

The repeated dose toxicity of isobutanol has been evaluated in two valid studies of high reliability which are used as key studies (oral administration: EPA/Res. Triangle Inst. 1987 and inhalation exposure: Li/Monsanto 1999). The study with isobutyl isobutyrate (oral exposure: Drake 1978) is used as supporting study.

 

Oral administration (gavage)

 

Supporting substance: isobutanol

In a valid subchronic toxicity study (90 days) similar to OECD TG 408, isobutanol (purity not stated) was administered to 30 rats/sex/dose by gavage at dose levels of 0, 100, 316, and 1000 mg/kg bw/day on the basis of a preliminary range finding study.

 

Treatment related effects were seen only at a dose level of 1000 mg/kg bw/day, but not at dose levels of 100 or 316 mg/kg bw/day. At the 1000 mg/kg bw/day dose level, hypoactivity was observed in all rats and body weight gain in the males was 18% below the control average during week 1. Food consumption averages were below those of the controls for both males and females during week 1 and 2. In addition, serum potassium averages (both sexes) were 11-15% below control averages at the interim evaluation (day 29) only and this may have been related to treatment.

 

No difference between dosed groups and controls were found for ophthalmoscopic examination, hematology, urinalysis, organ weights, gross pathology and histopathology.

Results do not indicate a specific target organ.

The NOEL of isobutanol is 316 mg/kg bw/day for males and females (EPA/Res. Triangle Inst. 1987)

 

Deduction of the NOEL (oral) for isobutyric acid

The NOEL of isobutyric acid will be calculated on basis of the NOEL of isobutanol (EPA/Res. Triangle Inst. 1987) using the respective molecular weights (88.11 and 74.12).

 

The deduced oral NOEL for isobutyric acid is 375 mg/kg bw/day.

 

Supporting substance: isobutyl isobutyrate

In a valid subchronic toxicity study (18 weeks) similar to OECD TG 408, isobutyl isobutyrate (purity >= 98%) was administered to 15 male and female Wistar rats per group by gavage at dose levels of 0, 10, 100, and 1000 mg/kg bw/day. Observation parameters were slightly reduced compared to OECD TG 408 (no behavioral/neurological observations, limited blood parameters, no clinical chemistry).

 

No treatment related effects were observed even at the highest dose level in male and female rats.

 

Results do not indicate a specific target organ.

 

The NOAEL is 1000 mg/kg bw/day for males and females based on overall effects (Drake 1978)

 

Deduction of the NOAEL (oral) for isobutyric acid

The NOAEL of isobutyric acid will be calculated on basis of the NOAEL of isobutyl isobutyrate (Drake 1978) using the respective molecular weights (88.11 and 144.21).

 

The deduced oral NOAEL for isobutyric acid is 610 mg/kg bw/day.

 

Inhalation exposure

 

Supporting substance: isobutanol

In a valid subchronic (14 weeks) inhalation toxicity study according to OECD TG 413, isobutanol (purity 99%) was administered to 20 or 10 Sprague-Dawley rats/sex/dose. Controls and 2500 ppm group consisted of 20 animals, 250 and 1000 ppm group were 10 animals in size. Test substance administration was 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 based on 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 effect 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 it 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.

 

Results do not indicate a specific target organ.

 

Based on these findings, the NOAEC for repeated dose toxicity is considered to be 2500 ppm (ca. 7700 mg/m³) 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).

 

Deduction of the NOAEC (inhalation) for isobutyric acid

 

The NOAEC of isobutyric acid will be calculated on basis of the NOAEC of isobutanol (Li/Monsanto 1999) using the mass concentration/m³ and the respective molecular weights (88.11 and 74.12).

 

The deduced NOAEC for isobutyric acid is 9150 mg/m³ (2500 ppm).

Justification for classification or non-classification

It is concluded that no classification for repeated dose toxicity is necessary because adverse effects after repeated exposure were only observed at doses above the guidance values for classification.