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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was conducted according to test guidlines and in accordance with GLP, GLP/Guidline study.
Justification for type of information:
Refer to Category Document
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Remarks:
Not specified in report
Qualifier:
according to
Guideline:
other: USEPA OPPTS 870.3650 (2000)
Deviations:
no
Remarks:
Not specified in report
Principles of method if other than guideline:
No applicable
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:

- Name of test material (as cited in study report): Diisobutyl Carbinol
- Molecular formula (if other than submission substance): C9H20O
- Molecular weight (if other than submission substance): 144.15
- Physical state: Clear, Liquid
- Analytical purity: 99.1 ± 0.06%, as the sum of two isomers
- Lot/batch No.: AA0155T3YY


 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc. (Portage, Michigan)
- Age at study initiation: Approximately eight weeks of age at initiation of treatment
- Weight at study initiation:
- Fasting period before study:
- Housing: After assignment to study, animals were housed singly in stainless steel cages, except during breeding (one male and one female) and during
the littering phases of the study. During littering, dams (and their litters) were housed in plastic cages provided with ground corn cob nesting material
from approximately GD 19 until completion of lactation.
- Diet (e.g. ad libitum): Animals were provided LabDietâ Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri) in meal form ad libitum.
- Water (e.g. ad libitum): Municipal water ad libitum.
- Acclimation period: Each animal was evaluated by a laboratory veterinarian, or a trained animal/toxicology technician under the direct supervision of a
laboratory veterinarian, to determine the general health status and acceptability for study purposes upon arrival at the laboratory (fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International - AAALAC International). The animals were housed two-three
per cage in stainless steel cages, in rooms designed to maintain adequate conditions
(temperature, humidity, and photocycle), and acclimated to the laboratory for at least one week prior to the start of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1°C (with a maximum permissible excursion of ± 3°C)
- Humidity (%): 40-70%, with the exception of one deviation of 32%
- Air changes (per hr): Room air was exchanged approximately 12-15 times/hour.
- Photoperiod (hrs dark / hrs light): A 12-hour light/dark photocycle was maintained for all animal room(s) with lightson at 6:00 a.m. and off at 6:00 p.m.

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% methylcellulose
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Oral gavage is the preferred route of exposure according to OECD Guideline 422.
- Concentration in vehicle: dose levels of 0 (control), 50, 150, or 500 mg/kg/day in vehicle.
- Amount of vehicle (if gavage): 4 ml/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity: The low- and high-dose suspensions from the first mix of the main study were analyzed to confirm homogeneous distribution of
the test material concurrent with the start of the study.
Stability: Results generated prior to the start of the study indicated that DIBC was stable in 0.5% METHOCEL at concentrations ranging from 0.25-250 mg/ml for at least 12 days. Additional stability at these concentrations was established concurrent with the start of the study.
Concentration Verification: Analysis of all dosing suspensions from the first mix of the main study were initiated prior to the start of dosing using gas chromatography with flame ionization detection (GC/FID) with external standards to determine target concentrations.
Duration of treatment / exposure:
53 days for females and 33 days for males.
Frequency of treatment:
Female rats were dosed once daily for approximately two weeks prior to breeding, continuing through breeding (two weeks), gestation (three weeks), and through postpartum day 4. Male rats were dosed daily for 14 days prior to mating and continuing throughout the mating period for a total of
33 days.
Doses / concentrations
Remarks:
Doses / Concentrations:
dose levels of 0 (control), 50, 150, or 500 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
12 male and 12 female per dose group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The high-dose level was based upon data obtained from a preliminary range-finding study and was expected to induce
some toxic effects, but not death or obvious suffering. The lower dose levels were selected to provide dose response data for any toxicity that
may have been observed among the high-dose group rats and to establish a NOEL.
- Rationale for animal assignment (if not random): Prior to test material administration, animals were stratified by body weight and then randomly
assigned to treatment groups using a computer program designed to increase the probability of uniform group mean weights and standard
deviations at the start of the study. Animals that were placed on study were uniquely identified via subcutaneously implanted transponders
(BioMedic Data Systems, Seaford, Delaware) that were correlated to unique alpha numeric identification numbers.
Positive control:
Not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily
- A cage-side examination was conducted at least twice daily. This examination was typically performed with the animals in their cages and was
designed to detect significant clinical abnormalities that were clearly visible upon a limited examination, and to monitor the general health of the
animals. The animals were not hand-held for these observations unless deemed necessary. Animals were examined for abnormalities such
as, but were not limited to: decreased/increased activity, repetitive behavior, vocalization, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor, twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in
fecal consistency, and fecal/urinary quantity. In addition, all animals were observed for morbidity, mortality, and the availability of feed and water
at least twice daily. Cage-side examinations were conducted on dams and their litters, at least twice daily. These examinations were as described as above.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations (DCO) were conducted on all rats pre-exposure and weekly throughout the study. Mated females
received DCO examinations on GD 0, 7, 14, and 20, and LD 3. The DCO was conducted at approximately the same time each examination day prior to dosing, according to an established format. The examination included cage-side, hand-held and open-field observations, which are recorded categorically or using explicitly defined scales (ranks).

BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed at least once during the pre-exposure period and on the first day of dosing. Male body
weights continued to be recorded weekly throughout the study. Females were weighed weekly during the pre-mating and mating periods. During gestation, females were weighed on GD 0, 7, 14, 17, and 20. Females that delivered litters were weighed on LD 1 and 4. Females that failed to mate
or deliver a litter were weighed at least weekly until termination. Body weight analyses were conducted for the following days: GD 0, 7, 14, and 20. Body weight gains were determined for the following intervals: GD 0-7, 7-14, 14-20, 0-20, and LD 1-4.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Feed consumed was determined weekly during the two week pre-breeding period for males and females by weighing feed crocks at the start and
end of a measurement cycle. Feed consumption was not measured for males or females due to co- housing during breeding. Following breeding,
feed consumption was not measured for males. For mated females, feed consumption was measured on GD 0, 7, 14, and 20. After parturition, feed consumption was measured on LD 1 and 4. Feed consumption was not recorded for females that failed to mate or deliver a litter. Feed
consumption was calculated using the following equation:
Feed consumption (g/day) = (initial weight of crock - final weight of crock) / (# of days in measurement cycle)

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 data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

OPHTHALMOSCOPIC EXAMINATION: Yes / No / No data
- Time schedule for examinations:
- Dose groups that were examined:

HEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were obtained from the orbital sinus following anesthesia with CO2 at the scheduled necropsy. Blood samples were not obtained from females that failed to deliver a litter.
- Anaesthetic used for blood collection: Carbon dioxide
- Animals fasted: Yes, overnight
- Sample Preparation: Blood samples were mixed with ethylenediamine-tetraacetic acid (EDTA), smears were prepared, stained with Wright-Giemsa
stain, cover-slipped and filed for possible evaluation at the discretion of the pathologist.
- Parameters: Hematologic parameters were assayed using an Advia 120 Hematology Analyzer
(Bayer Corporation, Tarrytown, New York).
- Assays include:
Hematocrit (HCT)
Hemoglobin (HGB) concentration
Red blood cell (RBC) count
Total white blood cell (WBC) count
Differential WBC count
Platelet (PLAT) count
Reticulocyte (RET) count
RBC indices:
Mean Corpuscular Hemoglobin (MCH)
Mean Corpuscular Volume (MCV)
Mean Corpuscular Hemoglobin Concentration (MCHC)

CLINICAL CHEMISTRY: Yes / No / No data
- Time schedule for collection of blood:
- Animals fasted: Yes / No / No data
- How many animals:
- Parameters checked in table [No.?] were examined.

URINALYSIS: Yes / No / No data
- Time schedule for collection of urine: Overnight (approximately 16 hours)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes / No / No data
- Parameters examined: Color, appearance, specific gravity (refractometer) and urine volume.
-Semiquantitative analyses (Multistix Reagent Strips, Bayer Corporation, Elkhardt, Indiana on the Clinitek 200+) of:
pH
Bilirubin
Glucose
Protein
Ketones
Blood
Urobilinogen
- Microscopic Exam: Urine samples were collected from each male by manual compression of the urinary bladder. The urine samples were pooled
from each group, and the microsediment were characterized microscopically.

NEUROBEHAVIOURAL EXAMINATION: Yes / No / No data
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

OTHER:
Sacrifice and pathology:
Adult Necropsy
Adult males (fasted) were submitted for necropsy after at least four weeks (actual: TD 34) of exposure. Adult females (fasted) were terminated on
LD 5, or at least 24 days after the end of the mating period for females not producing a litter. The animals were anesthetized by the inhalation of CO2and weighed. Blood was collected from the orbital sinus (all males, all females that littered), their tracheas was exposed and clamped, and the animals were euthanized by decapitation.

A complete necropsy was conducted on all animals by a veterinary pathologist or a technician qualified to recognize lesions, assisted by a team of trained individuals. The necropsy included an examination of the external tissues and all orifices. The head was removed, the cranial cavity
opened and the brain, pituitary and adjacent cervical tissues were examined. The eyes were examined in situ by application of a moistened
microscope slide to each cornea. The skin was reflected from the carcass, the thoracic and abdominal cavities were opened and the viscera
examined. All visceral tissues were dissected from the carcass, re-examined and selected tissues were incised. The nasal cavity was flushed via the
nasopharyngeal duct and the lungs were distended to an approximately normal inspiratory volume with neutral, phosphate-buffered 10% formalin using a hand-held syringe and blunt needle.

The uteri of all females were stained with an aqueous solution of 10% sodium sulfide stain (Kopf et al., 1964) for approximately two minutes and
was examined for the presence and number of implantation sites. After evaluation, uteri were gently rinsed with saline and preserved in neutral
phosphate-buffered 10% formalin.

Weights of the adrenals, brain, epididymides, heart, kidneys, liver, spleen, testes, thymus, thyroid with parathyroids (weighed after fixation) were
recorded, and organ:body weight ratios calculated.

Representative samples of tissues listed in Table 4 were collected and preserved in neutral,
phosphate-buffered 10% formalin, with the exception of the testes and epididymides that were fixed in Bouin’s fixative. Transponders were
removed and placed in jars with the tissues.

Offspring Necropsy
All pups surviving to LD 4 were euthanized by oral administration of sodium pentobarbital solution, examined for gross external alterations, and
then discarded. Any pups found dead or which were euthanized in moribund condition were examined to the extent possible and discarded.
Other examinations:
Nonel
Statistics:
Descriptive statistics only (means and standard deviations) were reported for RBC indices, and WBC differential counts. Parental body weights andparental body weight gains, litter mean body weights, feed consumption, urine volume, urine specific gravity, coagulation, clinical chemistry data
appropriate hematologic data, and organ weights (absolute and relative) were first evaluated by Bartlett's test (alpha = 0.01; Winer, 1971) for
equality of variances. Based upon the outcome of Bartlett's test, either a parametric (Steel and Torrie, 1960) or non-parametric (Hollander and
Wolfe, 1973) analysis of variance (ANOVA) was performed. If the ANOVA was significant at alpha = 0.05, a Dunnett's test (alpha = 0.05; Winer, 1971
or the Wilcoxon Rank-Sum (alpha = 0.05; Hollander and Wolfe, 1973) test with Bonferroni's correction (Miller, 1966) was performed. Feed
consumption values were excluded from analysis if the feed was spilled or scratched.

Gestation length, average time to mating, and litter size were analyzed using a nonparametric ANOVA. If the ANOVA was significant, the Wilcoxon
Rank-Sum test with Bonferroni's correction was performed. Statistical outliers (alpha = 0.02) were identified by the sequential method of Grubbs
(1969) and only excluded from analysis for documented, scientifically sound reasons. The mating, conception, fertility and gestation indices were
analyzed by the Fisher exact probability test (alpha = 0.05; Siegel, 1956) with Bonferroni's correction. Evaluation of the neonatal sex ratio on postnatal day 1 was
performed by the binomial distribution test (alpha = 0.05; Steel and Torrie, 1960).
Gender was determined for pups found dead on postnatal day 0 and these data were
included in sex ratio calculations. Survival indices, post- implantation loss, and other
incidence data among neonates were analyzed using the litter as the experimental unit by
the censored Wilcoxon test (alpha = 0.05; Hollander and Wolfe, 1973) as modified by
Haseman and Hoel

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See Table 1 Below.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
High Dose female showed significant decrease, 10.2%, on TD 1-7 of the premating phase and slight decrease the last 2 weeks of gestation (8.9%) andlactation phase (10.8%).
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Decrease in food consumption correlated with decrease in body weight in the Female 500 mg/Kg/day dose group.
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no treatment related alterations in hematologic parameters of males or females at any dose level
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Table 2. Elevated protein and cholesterol
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Table 3. Slight pH decrease.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Post mating for Males, LD-4 for Females
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See Effects Organ weights in Table 1 Below
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Table 4 below
Histopathological findings: neoplastic:
not specified
Details on results:
See Tables below

Effect levels

open allclose all
Dose descriptor:
NOEL
Remarks:
General toxicity
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOEL
Remarks:
Reproductive toxicity
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no adverse effects of DIBC on neurological or reproductive function at any dose level.
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on decreased bodyweight in the females at the top dose.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1. Final Body Weight and Organ Weight Effects

 mg/Kg/day:  0  Historical1  50  150  500
 

MALES

 Final Body Weight (g)  375.0  354.1 -447.2  385.5  401.2  383.7
 Relative Adrenals (g / 100g bw)  0.017  0.014 -0.021  0.014*  0.015  0.016
 Relative Kidneys (g / 100g bw) 0.763   0.713 -0.801  0.740  0.773  0.882
 Absolute Liver (g)  10.519  10.108 -13.420  11.469  12.278*  13.421*
 Relative Liver (g / 100b bw)  2.800  2.719 -3.298  2.972  3.062*  3.493*
 

FEMALES         

 Final Body Weight (g)  277.5 251.2 -290.4   268.0  278.3 258.8*
 Absolute Adrenals (g)  0.089  0.070 -0.094  0.084  0.079  0.132
 Relative Adrenals (g / 100g bw) 0.032   0.027 -0.037  0.031  0.029  0.051$
 Relative Kidneys (g / 100g bw)  0.686  0.696 -0.742  0.741  0.755*  0.773*
 Absolute Liver (g)  9.882  8.185 -10.433  9.714  10.315  10.860
Relative Liver (g / 100g bw)   3.557  3.155 -3.748  3.628  3.696  4.195*

*Statistically Different from Control Mean by Dunnett’s Test, Alpha = 0.05.

$Statistically Different from Control Mean by Wilcoxon’s Test, Alpha = 0.05

1Historical controls group mean range from recent OECD 422 studies.

Bold typeindicates the effects judged to be treatment related.

Table 2. Clinical Chemistry Effects

 mg/Kg/day:  0  Historical1  50  150  500
 

MALES

Total Protein (mg/dl)  6.5  6.2 -6.6  6.8  6.7  7.0*
Cholesterol (mg/dl) 38   42 -58  48$  46  69$
 

FEMALES         

 Total Protein (mg/dl)  7.1 6.5 -7.2   7.1  7.2 7.4*
Cholesterol (mg/dl)  50  42 -73  49  57  71*

*Statistically Different from Control Mean by Dunnett’s Test, Alpha = 0.05.

$Statistically Different from Control Mean by Wilcoxon’s Test, Alpha = 0.05

1Historical controls group mean range from recent OECD 422 studies.

Bold typeindicates the effects judged to be treatment related.

Table 3. Urinalysis

  0  50  150

500

 
MALES

Urine pH

7.5 (5)   7.0 (4) 6.1 (1) 6.0 (1)  
Urine pH  8.0 (2)  7.5 (6)  7.0 (8)  6.5 (3)   
Urine pH  8.5 (4)  8.0 (1)  7.5 (2)  7.0 (7)   
Urine pH  9.0 (1)  8.5 (1)  8.0 (1) 7.5 (1)

Urine pH data tabulated as tehnumber of animals (N) with the stated value

Table 4: Treatment-Related Histopathologic Effects

Sex   Male Female 
 Dosage (mg/Kg/day)  0  50  150  500  0  50  150  500
 Number of Rats 12  12  12  12  12  12  12  12
 Liver (number examined)  12  12  12  12  12  12  12  12
 Hypertrophy, hepatocyte, centrilobular,-very slight  2  0  5  9  1  1  1  10
 Nasal Tullue-Pharynx (# examined)  12  12  12  12  12  12  12  12
 Degeneration, olfactory epithelium, focal, -very slight  0  0  0  1  0  1  0
 Degeneration, olfactory epithelium, multifocal, -very slight  1  1  3  0  1  3  1
  -slight  0  0  1  0  0  0  0  0
 Degenereration, olfactory and respitory epithelium, multifocal -slight  0  0  0  1  0  0  0  1
  -moderate  0  0  0  2  0  0  0  2
 Inflamation, acute, olfactory epithelium, focal -very slight  0  0  0  1  0  0  0  0
 Inflamation, acute olfactory epithelium multifocal -very slight  0  1  1  1  0  1  0  1
 Inflammation, acute, olfactory and respiratoryepithelium, multifocal-slight  0  0  0  3  0  0  0  2
 Total aminals with nasal leisions  1  2  6  0  1  3  4

Bold type indicates the effects judged to be treatment related

Applicant's summary and conclusion

Conclusions:
Oral gavage administration of 500 mg/kg/day of DIBC resulted in decreased feed consumption and body weights in females only. The liver was the primary target organ for systemic toxicity. Treatment-related statistically significant increases in absolute liver weights were noted in males of the middle (17%) and high dose (28%) groups as well as the high dose females (10%). Corresponding increases in relative liver weights were statistically identified in these groups. The higher liver weights corresponded with very slight hypertrophy of centrilobular hepatocytes in males given 150 or 500 mg/kg/day and females given 500 mg/kg/day. Males given 500 mg/kg/day and females given 150 or 500 mg/kg/day had higher relative kidney weights that were interpreted to be treatment related. There were no corresponding clinical pathologic or histopathologic alterations for the higher kidney weights. There was no evidence of systemic toxicity in rats given 50 mg/kg/day. Additional treatment-related effects that were interpreted to be nonadverse consisted of transient salivation noted only around the time of dosing in the highdose males and females, slightly decreased urine pH in males at all dose levels, as well as increased serum total protein and cho lesterol in males or females given 500 mg/kg/day. Degeneration and/or inflammation of the olfactory and respiratory epithelium were noted in 1, 2-3, and 4-6 rats/sex in the 50, 150, and 500 mg/kg/day groups, respectively. These nasal effects were interpreted to be the result of local irritation of the test material associated with the gavage procedure. There were no adverse effects of DIBC on neurological or reproductive function at any dose level. Based on these data, the no-observed-effect level (NOEL) for general toxicity was considered to be 50 mg/kg/day. The NOEL for reproductive and neurological effects was 500 mg/kg/day, the highest dose level tested.