Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key 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.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
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
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:
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.
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
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

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
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.
Critical effects observed:
not specified

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

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.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
good

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
chronic toxicity: inhalation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study without detailed documentation
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 451 (Carcinogenicity Studies)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: F344/N
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic Laboratory Animals and Services (Germantown, NY)
- Age at study initiation: approximately 6 weeks old
- Housing: Individually housed
- Diet (e.g. ad libitum): NTP-2000 non-purified diet (irradiated wafers; obtained from Zeigler Brothers, Inc.) was available ad libitum except during exposure periods and changed weekly.
- Water (e.g. ad libitum): tap water was available ad libitum via an automatic watering system.
- Acclimation period: approximately 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 3°C
- Humidity (%): 55 ±15%
- Air changes (per hr): 15±2
- Photoperiod (hrs dark / hrs light): 12/12


Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
clean air
Details on inhalation exposure:
Inhalation exposures were conducted at BattelleToxicology Northwest Operations (Richland, WA). MIBK was pumped onto the heated surface of the study laboratory-designed wick generator, where it was vaporized.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The MIBK concentrations in the exposure chambers were monitored by an on-line gas chromatograph approximately every 28 min. Buildup and decay rates for chamber vapor concentrations were determined with animals present in the chambers and the time to achieve 90% of the target concentration after the beginning of vapor generation (T90) was 12 min. Evaluations of chamber uniformity and persistence and monitoring for MIBK degradation impurities were conducted periodically throughout the studies by gas chromatography. Chamber uniformity was maintained and no degradation was detected.

Duration of treatment / exposure:
2 years
Frequency of treatment:
6h/day, 5 days per week
Remarks:
Doses / Concentrations:
0, 450, 900, or 1800 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
50/sex/dose
Control animals:
yes
Details on study design:
- Rationale for animal assignment (if not random): random
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly for the first 13 weeks, monthly until the last four months of the studies, every 2 weeks thereafter, and at the end of the studies.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded initially and then weekly for the first 13 weeks, monthly until the last four months of the studies, every 2 weeks thereafter, and at the end of the studies.

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

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: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes.
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 (1972) method for testing two groups for equality and Tarone (1975) life table test to identify dose-related trends. All reported P values for the survival analyses are two sided. Average severity values were analyzed for significance with the Mann–Whitney U test. The poly-k test was used to assess neoplasm and non-neoplastic lesion prevalence. This test is a survival-adjusted quantal-response procedure that modifies the Cochran–Armitage linear trend test to take survival differences into account. Unless otherwise specified, a value of k = 3 was used in the analysis of site-specific lesions. Tests of significance included pairwise comparisons of each exposed group with controls and a test for an overall exposure related trend. Continuity-corrected poly-3 tests were used in the analysis of lesion incidence, and reported P values are one sided.

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):
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 specified
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
Survival of the 1800 ppm males was significantly less than that of the controls (0 ppm, 32/50; 450 ppm, 28/49; 900 ppm, 25/50; 1800 ppm, 19/50). Mean body weights of 900 ppm male rats were 6–8% less after week 97 and 1800 ppm males were 5–8% less after week 89, than those of the chamber control group. Mean body weights and survival (0 ppm, 35/50; 450 ppm, 34/50; 900 ppm, 26/50; 1800 ppm, 32/50) of all exposed groups of females were similar to those of the controls.

Chronic progressive nephropathy (CPN) similar to that which occurs in aged rats was observed in males and females in all groups, including controls (Tables 1 and 2). In males, significant increases in incidence were observed at 1800 ppm and in severity at all exposure concentrations. The incidence of mineralization was also significantly increased at all exposure concentrations in males; severity was generally increased in exposed groups (Table 1). In female rats, increased incidences of CPN were significant in all exposed groups. The average severity of CPN ranged from minimal to mild and was increased in exposed females at 1800 ppm. In both sexes, changes consisted of a spectrum of lesions that included varying degrees of renal tubule dilatation with and without hyaline (proteinaceous) casts, multifocal degeneration, regeneration, and hypertrophy of the tubular epithelium; thickening of the tubular and glomerular basement membranes; glomerulosclerosis; interstitial fibrosis; and variable infiltrates of mononuclear inflammatory cells within the interstitium. Minimal CPN affected less than 10% of the renal parenchyma, and consisted of focal to multifocal regenerative renal tubules surrounded by a thickened basement membrane. These regenerative tubules were small and lined by cuboidal basophilic epithelial cells. Mild CPN affected approximately 10–39% of the renal parenchyma and consisted of multifocal clusters of regenerative renal tubules, tubules that contained protein, glomeruli with thickened basement membranes, and scattered infiltrates of predominantly lymphocytes and macrophages. Moderate CPN had similar but more severe and widespread changes including glomerular atrophy and variable interstitial fibrosis. Marked CPN was diffuse and of greater severity. Mineralization was generally of minimal to mild severity and consisted of linear deposits of lamellated mineral within the lumen or epithelial cells of the collecting tubules of the renal papilla. There were exposure concentration-related increases in minimal to mild transitional epithelial hyperplasia in the renal pelvis of male rats, which were significant at 900 and 1800 ppm (Table 1). Transitional epithelial hyperplasia consisted of focal proliferation of the transitional epithelium lining the renal pelvis; the affected epithelium appeared thickened and often formed papillary projections into the urinary space. In the single section analysis (standard evaluation) of the kidney in males (Table 1), increases in renal tubule hyperplasia were significantat 450 and 1800 ppm, and the severities in these groups were elevated. There were also slight increases in renal tubule adenoma, carcinoma, and adenoma or carcinoma (combined). There were significant positive trends for adenomas and adenomas or carcinomas (combined). Although not statistically significant, the incidences of renal tubule adenoma and renal tubule adenoma or carcinoma (combined) in the 900 and 1800 ppm groups and renal tubule carcinoma in the 1800 ppm group exceeded the historical ranges for chamber controls from inhalation studies fed NTP-2000 diet. In the extended evaluation of the kidneys (Table 1), additional renal tubule hyperplasias were observed in all exposed groups such that in the combined single and step section analysis, the incidences of hyperplasia in exposed groups were significantly greater than in the controls. Additional renal tubule adenomas were observed in all groups including the controls. No additional renal tubule carcinomas were observed. In the combined single and step section analysis of renal neoplasms, there were significant positive trends for renal adenoma and adenoma or carcinoma (combined) and the incidences of these lesions were significantly increased at 1800 ppm. Hyperplasia occurred as single or multiple expanded cortical tubules composed of increased numbers of tubular epithelial cells arranged in multiple layers that partially or completely filled the tubule. Renal tubule adenomas were discrete, highly cellular, proliferative lesions that were larger than focal hyperplasias (generally greater than the combined diameter of five normal-sized renal tubules). Adenomas tended to have a more complex structure than hyperplasias and were characterized by closely packed tubules and solid nests composed of cells with large vesicular nuclei and abundant pale eosinophilic cytoplasm which sometime contained clear vacuoles. Renal tubule carcinomas were highly cellular, expansive and invasive masses composed of large basophilic to amphophilic cells that formed large multilayered tubular structures, solid nests, and sheets. Renal mesenchymal tumors occurred in two female rats in the 1800 ppm group (Table 2). Both neoplasms observed in this study were single, small to medium sized masses with poorly defined margins and were composed of sheets of mature mesenchymal (spindle) cells that infiltrated the inner cortex, medulla, and renal pelvis encircling and sequestering glomeruli, tubules, and collecting ducts. Lesions at sites other than the kidney were also observed in males. Mononuclear cell leukemia (0 ppm, 25/50; 450 ppm, 26/50; 900 ppm, 32/50; 1800 ppm, 35/50) increasedwith a significant positive trend and at 1800 ppm, the increase was significant and exceeded the historical ranges for chamber controls from inhalation studies fed NTP-2000 diet (188/399, 47±10%; range 32–66%). Adrenal medulla hyperplasia was also significantly increased at 1800 ppm (0 ppm, 13/50; 450 ppm, 18/48; 900 ppm, 18/50; 1800 ppm, 24/50). There were also exposure-related increases in benign or malignant pheochromocytoma (combined) of the adrenal gland in male rats (0 ppm, 8/50; 450 ppm, 9/48; 900 ppm, 11/50; 1800 ppm, 14/50). However, these increases were not significant and were within the historical ranges for chamber controls from inhalation studies fed NTP-2000 diet (69/398, 17±7%; range 10–28%), although the incidence in the 1800 ppm group was the upper limit of the historical range.
Dose descriptor:
NOAEC
Effect level:
450 ppm (analytical)
Sex:
male/female
Basis for effect level:
other: = 1840 mg/m3, non-neoplastic lesions observed in the kidneys at higher dose levels and the irrelevance to humans of the tumour types observed in the kidneys of male rats
Critical effects observed:
not specified
Conclusions:
A NOAEC was not reported by the study authors. Review of the study data suggests that a NOAEC of 450 ppm can be derived for neoplastic and non-neoplastic lesions, based on the non-neoplastic lesions observed in the kidneys at higher dose levels and the irrelevance to humans of the tumour types observed in the kidneys of male rats.
Executive summary:

In a whole body 2-year inhalation study in Fischer 344 rats, animals (50/sex/group) were administered the read-across substance, MIBK, at concentrations of 0 (control), 450, 900 or 1800 ppm for 6 hours per day, 5 days per week for 2 years. This GLP study was equivalent to OECD Test Guideline 451. Mortality was observed in all groups administered test article. However, survival was significantly decreased in males administered MIBK at 1800 ppm as compared to controls. Mean body weights also were decreased in males administered 900 ppm and 1800 ppm as compared to controls. The mean body weights and survival in treated females were similar to controls. The primary target of MIBK toxicity was the kidney in rats. Briefly, chronic progressive nephropathy (CPN) similar to that which occurs in aged rats also was observed in all rats (including controls). There were treatment related significant increases in both the incidence (1800 ppm) and severity in all exposed groups. Kidney lesions that typically accompany CPN also were reported in males exposed to 900 ppm and 1800 ppm MIBK. The kidney lesions observed were suggestive of α2μ-globulin nephropathy (specific to male rat), a mechanism of xenobiotic-induced renal carcinogenesis for which there is no human counterpart. A NOAEC was not identified by the authors. Review of the study data suggests that a NOAEC of 450 ppm (1840 mg/m3) can be derived for neoplastic and non-neoplastic lesions, based on the non-neoplastic lesions observed in the kidneys at higher dose levels and the irrelevance to humans of the tumour types observed in the kidneys of male rats. 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
2 650 mg/m³
Study duration:
chronic
Species:
rat
Quality of whole database:
Good

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The repeated-dose toxicity of diisobutyl carbinol (DIBC) can be assessed using the data available on DIBC itself (an OECD 422) and by making use of data on analogues (diisobutyl ketone (DIBK), methyl isobutyl carbinol (MIBC) and methyl isobutyl ketone (MIBK)). This use of analogue data is considered acceptable based on the following justifications:

1) It can be demonstrated that DIBC and DIBK share the same metabolic pathway to that of MIBK and MIBC

2) The toxicity of all of these substances is very consistent and can be attributed to their common ADME characteristics, including a common metabolic pathway and structurally related metabolites.

A detailed justification for the grouping of these substances into a category for the purposes of read across is provided in the 'read across justification' attached to section 13 of the IUCLID dossier.

Within the group of substances the following data exist:

DIBC: Repeated dose toxicity and Reproductive/Developmental screening study (OECD 422) In a range finding study 5 rats/sex were given 0, 250, 500, 750, or 1,000 mg DIBC/kg via oral gavage for 14 days. Transiently increased salivation was noted in doses greater than or equal to 500 mg/kg; increased liver and kidney weights were noted at all doses. No treatment-related gross observations were recorded at necropsy. In the full study, groups of 12 male and 12 female rats were administered 0, 50, 150, or 500 mg DIBC/kg via oral gavage. 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 beginning approximately two weeks prior to breeding and continuing through breeding (two weeks) for an exposure period of 33 days. Adecrease in bodyweight weight and feed consumption was observed in females at the 500 mg/kg level. Liver weight changes were seen in the male rats at the middle and high doses; weights were increased by 17% and 28%, respectively. The weight of the livers in the high dose females was increased by 10%. The liver effects corresponded with very slight hypertrophy of centrilobular hepatocytes in males given 150 or 500 mg/kg and females given 500 mg/kg. However there were no effects on liver enzyme markers in the blood that would indicate evidence of liver toxicity. As such it appears likely that the observed liver enlargement and associated hypertrophy were a consequence of liver enzyme induction rather than target organ toxicity. 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, however there were no corresponding clinical pathologic or histopathologic alterations for the higher kidney weights that woudl indicate evidence of toxicity. There was no evidence of systemic toxicity in rats given 50 mg/kg/day. Additional treatment-related effects that were interpreted to be non-adverse consisted of transient salivation noted only around the time of dosing in the high dose males and females, slightly decreased urine pH in males at all dose levels, as well as increased serum total protein and cholesterol in males or females given 500 mg/kg/day. Olfactory and respiratory epithelium effects were seen at the 50, 150, and 500 mg/kg groups. These effects consisted of degeneration and/or inflammation, attributed to the gavage procedure. There were no neurological effects noted. In this study the NOEL was 50 mg/kg bw/day, however the effects observed at the mid and high dose in the liver and kidneys could be considered to be adaptive in nature rather than evidence of systemic toxicity. As such, based on the decreased bodyweight in the high dose, the NOAEL for this study is considered to be 150 mg/kg bw/day. Data on supporting substances

DIBK

Oral:

In a 90-day oral gavage study in rats at a dose of 2,000 mg/kg/day for 90 days absolute and relative liver weights, relative kidney weights, absolute and relative adrenal gland weights were statistically greater than controls. No gross pathological changes were detected. Histologically, compound related changes were evident in the stomach, liver and kidneys. Gastric changes due to direct contact with the solvent included hyperkeratosis with or without pseudoepitheliomatous hyperplasia. Hepatic changes included hepatocyte hypertrophy in all animals and hepatocyte vacuolation in one animal. However, no changes in the levels of serum liver enzymes were observed in clinical chemistry assessment indicating no tissue damage in the liver. Renal changes included hyalin droplet formation in the proximal tubular epithelium, regenerating tubular epithelium and tubular dilation with casts.

Dermal:

No valid or reliable data are available.

Inhalation:

Two studies of acceptable quality (Klimisch 2) are available in rats and one in guinea pigs. The only adverse effect observed in rats was hyaline droplet nephrosis in male rats. This effect is considered to be mediated by alpha 2µ globulin accumulation as demonstrated for MIBK (methyl isobutyl ketone) and therefore not considered to be relevant for human exposure.

MIBK

Oral:

In a key study (Mulligan, 1986) groups of 30 male and 30 female Sprague-Dawley rats were administered MIBK by gavage in corn oil at daily dose levels of 0 (vehicle control), 50, 250, or 1000 mg/kg-day for 13 consecutive weeks and evaluated for exposure-related changes in body weight, food consumption, mortality, clinical signs, ophthalmological parameters, and terminal organ weights (heart, liver, spleen, brain, kidney, gonads, adrenals, thyroid, and parathyroid). The following evaluations were conducted in rats from each exposure level at interim (week 7) and final sacrifices: hematology, clinical chemistry, urinalysis, and comprehensive gross pathology. All tissue samples collected during gross necropsy in high-dose and control rats were evaluated for histopathology, and kidney samples were also histologically evaluated in mid-dose rats.

Reversible lethargy was observed in rats of both sexes receiving 1000 mg/kg-day (but not at lower dose levels) for a few hours following dosing and reportedly decreased in incidence and severity during the study. Males in the high-dose group showed a slight (9%) but significantly decreased mean body weight gain as compared to controls during the last 2 weeks of exposure, whereas female body weight gain was significantly increased during 5 of the last 6 weeks of exposure. Final bodyweights were not however different to control. Both male and female food consumption was significantly increased during the second half of the exposure period. The only potentially exposure-related hematological effects observed were slight but statistically significant increases in hemoglobin (+6%) and hematocrit (+8%) at terminal sacrifice in females administered 1000 mg/kg-day and a 15% decrease in lymphocyte count in high-dose males at terminal sacrifice. The lowest hepatic effect level that was observed in the oral exposure studies was 250 mg/kg-day for increased (+39%) serum glutamic-pyruvic transaminase (SGPT) in female rats at the terminal sacrifice. The following changes suggestive of adverse liver effects were observed at 1000 mg/kg-day at either interim and/or final sacrifice: increased SGPT (+73%, interim; +34%, terminal) in females as compared to controls, increased serum alkaline phosphatase (+84%, interim) in females, increased serum cholesterol in males (+30%, interim) and females (+59%, interim; +65%, final), increased terminal absolute (+34%, males; +39%, females) and relative (+42%, males; +38%, females) liver weights, decreased albumin/globulin ratio in males (-16%, interim), and minimally increased serum total protein in females (+9%, interim; +10%, terminal). The only renal effect occurring at 250 mg/kg-day was increased terminal absolute or relative kidney weights in males and females, ranging from 6 to 12% over controls. The following changes suggestive of adverse kidney effects were observed at 1000 mg/kg-day: increased terminal absolute and relative kidney weights (from 25 to 34% in males and from 20 to 22% in females) as compared to controls, increased blood-urea-nitrogen (BUN) in males (+37%, interim), increased serum potassium in males (+34%, terminal), decreased serum glucose in males (-27%, terminal), and a reported increase in urinary protein and ketones in males and females at terminal sacrifice (summary data were not provided). Histological examination of kidney tissues revealed an increased incidence of male rats with mild nephropathy (multifocally distributed swollen or hyperchromatic and flattened renal cortical tubular epithelial cells) at 1000 mg/kg-day (16/20) as compared to controls (4/20) but no increase in such lesions in females. Significantly increased relative adrenal weights in male (+29%) and female (+11%) rats and slightly increased relative testis weights (+9%) in males were also observed at 1000 mg/kgday. With the exception of the kidney, no exposure-related histopathologic lesions were evident in any tissue that was examined. The NOAEL was estimated to be 250 mg/kg bw/d, based on increases in relative kidney weights for male and females rats administered MIBK at doses of 250 mg/kg bw/d but without histological lesions. Effects at higher doses included kidney changes, hepatomegaly and alterations in clinical chemistry and urinalysis parameters. No treatment-related effects of any kind were observed at 50 mg/kg-day.

Inhalation:

In a whole body 2-year inhalation study in Fischer 344 rats, animals (50/sex/group) were exposed to MIBK at concentrations of 0 (control), 450 ppm, 900 ppm, or 1800 ppm for 6 hours per day, 5 days per week for 2 years (NTP, 2007; Stout et al., 2008). This GLP study was equivalent to OECD Test Guideline 451. Mortality was observed in all groups. However, survival was significantly decreased in males administered MIBK at 1800 ppm as compared to controls. Mean body weights also were decreased in males administered 900 ppm and 1800 ppm as compared to controls. The mean body weights and survival in treated females were similar to controls. The primary target of MIBK toxicity was the kidney. Briefly, chronic progressive nephropathy (CPN) similar to that which occurs in aged rats also was observed in all rats (including controls). There were treatment related significant increases in both the incidence (1800 ppm) and severity in all exposed groups. Kidney lesions that typically accompany CPN also were reported in males exposed to 900 ppm and 1800 ppm MIBK. The kidney lesions observed were suggestive of α2µ-globulin nephropathy (specific to male rat), a mechanism of xenobiotic-induced renal carcinogenesis for which there is no human counterpart. A NOAEC was not identified by the authors. Review of the study data suggests that a NOAEC of 450 ppm (1840 mg/m3) can be derived for neoplastic and non-neoplastic lesions, based on the non-neoplastic lesions observed in the kidneys at higher dose levels and the irrelevance to humans of the tumour types observed in the kidneys of male rats.

In a supporting 2-year inhalation study in B6C3F1 mice, animals (50/sex/group) were administered MIBK at concentrations of 0 (control), 450 ppm, 900 ppm, or 1800 ppm for 6 hours per day, 5 days per week for 2 years (NTP, 2007; Stout et al., 2008). This GLP study was equivalent to OECD Test Guideline 451. Survival of male and female mice was similar to controls. There were no clinical findings observed related to MIBK exposure. Mean body weights of male mice were generally similar to the controls throughout the study. Mean body weights of females exposed to 1800 ppm MIBK were 9-16% less than the controls. Eosinophilic foci in the liver were increased in all exposed groups of female mice, and the increases over the controls were significant in the 450 and 1800 ppm groups; this lesion was not significantly increased in exposed male mice. Hepatocellular adenomas and carcinomas were reported in males and females at various doses. Review of the study data suggests that a NOAEC of 450 ppm (1840 mg/m3) can be derived for neoplastic and non-neoplastic lesions, based on the reported neoplastic effects in the liver of female mice (multiple adenomas) at higher dose levels. Subsequent investigations (Geter et al., 2009) reported that MIBK-related hepatocellular findings in mice may be due to induction of cytochrome P450 enzymes following activation of the mouse constitutive androstane receptor (CAR) in a manner that is similar to Phenobarbital-like compounds. The authors of the study noted that a carcinogenic effect in mice that can be attributed to Phenobarbital-like activation of CAR is not relevant to humans.

In a supporting subchronic inhalation study in rats and mice, groups of 14 male and 14 female Fischer 344 rats and B6C3F1 mice were exposed to measured mean concentrations of 0, 50, 252, and 1002 ppm (0, 205, 1033, and 4106 mg/m3) MIBK for 6 hrs/day, 5 days/week, for 14 weeks and sacrificed following their final exposure day (Dodd and Eisler, 1983; Phillips et al., 1987). The following endpoints were evaluated: clinical signs, body weights, organ weights (kidneys, heart, liver, lungs, and testes), urinalysis, hematology, serum chemistry (including glucose and hepatic enzyme levels), complete gross pathology, targeted histopathology (nasal cavity, trachea, liver, kidneys, and lungs) in all animals and complete histopathology in control and high-exposure groups.

No effects of any kind were observed in rats or mice of either sex at 205 mg/m3. Terminal body weights were significantly increased in female rats at >=1033 mg/m3. Mouse hematology was unaffected at all exposure levels, but platelet numbers in male rats were significantly increased at 4106 mg/m3 by 13% over controls, and eosinophil number in female rats was significantly decreased at 4106 mg/m3 by 57% as compared to controls. Serum cholesterol in male rats was significantly increased at the 1033 and 4106 mg/m3 exposure levels by 23 and 35%, respectively, as compared to controls. Male rats and male mice showed a significant increase in absolute (+13%, rats; +7%, mice) and relative (+9%, rats; +11%, mice) liver weight at 4106 mg/m3; absolute, but not relative, liver weight was also slightly increased in male mice (+8%) at 1033 mg/m3. No histological lesions were observed in the liver and no changes were seen in serum liver enzymes and bilirubin in any exposure group; thus, the observed liver enlargement may have been an adaptive response to increased hepatic metabolic activity rather than a toxic effect.

Urine glucose was significantly increased in male rats at 1033 mg/m3 (+37%) and 4106 mg/m3 (+55%) and in female rats at 4106 mg/m3 (+26%). Significantly increased urine protein (+28%) was also observed in male rats at 4106 mg/m3. The only renal histological lesion observed was hyaline droplet formation in all male rats; the severity of the lesion generally increased with exposure level. The U.S. EPA has concluded that renal alpha2u-globulin hyaline droplet formation is unique to male rats and is probably not relevant to humans for the purposes of risk assessment. In conclusion, other than the male rat kidney effect, exposure of male and female rats and mice to MIBK at levels up to 1000 ppm for 14 weeks was without significant toxicological effect.

In a supporting sub-acute inhalation study, groups of six male and six female F344 rats and B6C3F1 mice were exposed for 6 hrs/day, 5 days/week, for 9 days to measured concentrations of 0, 101, 501, or 1996 ppm (0, 410, 2053, or 8178 mg/m3) MIBK (Dodd et al., 1982; Phillips et al 1987). Groups were evaluated for changes in clinical signs, body weight, organ weights (liver, lungs, kidneys, and testes), ophthalmology, gross pathology, and histopathology. The only exposure-related effects observed were periocular wetness in rats exposed to 8178 mg/m3, increased relative liver weights in male rats at 2053 and 8178 mg/m3 and in female rats and female mice at 8178 mg/m3, increased kidney weights in male rats and female mice at 8178 mg/m3, and hyaline droplet degeneration in kidneys of male rats exposed to 2053 or 8178 mg/m3, with epithelial regeneration of proximal convoluted tubules in the high-exposure group. No effects of any kind were observed in rats at 410 mg/m3 and in mice at 2053 mg/m3.

MIBC

Inhalation:

A non-GLP 6-week inhalation study was conducted in male and female Wistar rats with methyl isobutyl carbinol (MIBC) (Blair, 1982). Animals were exposed to concentrations of MIBC at 0 (control), 211, 825, or 3698 mg/m3for 6 hours per day for 5 days per week for 6 weeks. There were no adverse effects reported for the following parameters evaluated: mortality and clinical signs, hematology, gross pathology, and histopathology. The mean alkaline phosphatase value was increased in females exposed to 3698 mg/m3. Mean concentrations of ketone bodies in urine samples from all the exposed groups (except males exposed to 211 mg/m3) showed a significant increase above those of the control groups. The mean concentrations of protein were increased in all the exposed female groups and in the male group exposed to 3698 mg/m3. The mean specific gravities of both male and female groups exposed to 3698 mg/m3were increased compared to the control values as was the female group exposed to 825 mg/m3. The mean pH of urine from the male group exposed to 3698 mg/m3was reduced, and the mean glucose content of the urine of females exposed to the same concentration was increased compared to the control values. Finally, the mean urine volumes of females exposed to 211 and 3698 mg/m3were reduced compared to the control values. Although significantly increased kidney weights were noted in males exposed to 3698 mg/m3as compared to controls, there were no macroscopic or microscopic correlates associated with this finding. Although the authors did not define the NOAEC for this study, NOAEC can be considered to be the highest exposure concentration of 3698 mg/m3.

Summary

The overall data base supports the conclusion that in animal studies, liver and kidney are target organs for this set of chemicals. In the assessment of much of the data, effects on these organs have been considered adaptive given the absence of corroborating histopathological and clinical chemistry findings to indicate damage to organs. The exception is the effects on the male rat kidney. This is considered to be due to alpha 2u globulin associated nephropathy, although the quality of the evidence supporting that conclusion is limited, and currently subject to additional research. Although there are often reports of kidney enlargement in the females and in mice (to varying degrees) it is again felt that these findings are more adaptive in the absence of any other significant pathological findings. As such, in most cases, effects on the liver and kidney have been considered as non-adverse and not taken into consideration in the determination of the NOAEL.

There is no clear trend in toxicity across the group with all substances having effects on the liver and kidneys within a similar range of doses. Given the consistency in the findings and the similarity in dose response it is proposed to use the long term studies on MIBK to derive the DNELs for DIBC. An additional assessment factor of 2 is proposed to address any residual uncertainty associated with the use of a read across/weight of evidence approach for this endpoint.

With respect to the conversion of the NOAEC for MIBK into a NOAEC for DIBC, a molar conversion will be used.

With respect to the route to route extrapolation, the toxicokinetics information and physical chemical properties indicate that all category members are bioavailable via oral, inhalation and dermal routes. Comparing oral and inhalation studies (where they exist for the same substance) demonstrates a similar pattern of toxicity with the same target organs. As such it is not expected that extrapolating from an inhalation NOAEC to a dermal NOEL would result in misrepresentation of the hazards of that route of exposure. In fact, extrapolating from inhalation to dermal route is likely to represent a conservative assessment due to the slower rate of uptake via the dermal route compared to the inhalation route. i.e. the toxicokinetic differences between these routes would typically lead to a conclusion that dermal exposure would have a lower hazard potential relative to the oral and inhalation routes.

The Key study taken for the DNEL derivation will therefore be the 2 year inhalation study using MIBK.

The NOAEC from this study is 1840 mg/m3, this is 18.4mmol/m3.

18.4 mmol/m3 DIBC = 2650 mg/m3

In doing this conversion it is recognised that the converted NOAEC is greater than the highest attainable vapour concentration for DIBC (approx 1000mg/m3 based on a vapour pressure of 17 Pa). However this is not an issue for the derivation of the DNEL since the resulting DNEL would be expected to fall within the a possible vapor concentration of DIBC.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Reliability 1 repeated dose oral study (OECD 422 study design).

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Chronic toxicity/carcinogenicity study performed using an analogue (refer to endpoint summary and Section 13 of IUCLID) for justification of read across. NOAEC from study is converted based on a molar calculation to give the equivalent NOAEC of DIBC.

Justification for classification or non-classification

No classification for repeated dose toxicity is proposed. However it is acknowledged that one of the category members (MIBK) is associated with an increased tumor incidence in rats and mice (Kidneys and liver respectively). A discussion of this data and how it pertains to DIBC and other category members is included in section 7.7 of the dossier.