Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

inhalation, rat, 2-gen: NOAEL P/F1/F2 >= ca. 7.5 mg/L (=2500 ppm; GLP, EPA guideline OPPTS 870.3800; WIL Res. Lab. Inc., 2003)

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): Isobutanol
- Physical state: liquid
- Analytical purity: 99.9 %
- Stability under test conditions: the test substance was proven stable under the storage conditions
- Storage condition of test material: room temperature
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Inc.
- Age at study initiation: (P) ca. 7 wks; (F1) ca. 4 wks
- Weight at study initiation: (P) Males: 236-350 g; Females: 159-213 g; (F1) Males: post natal day 32: means: 84-97 g; Females: post natal day 32: means: 78-88 g
- Housing: individually
- Diet: ad libitum (no food during exposure)
- Water: ad libitum (no water during exposure)
- Acclimation period: 21 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3 °C
- Humidity (%): 30-70 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Exposures were conducted in four 2.0 m3 stainless steel and glass whole-body inhalation chambers. One chamber was dedicated for each group for the duration of the study. Chamber supply air was provided from a HEPA- and charcoal-filtered, temperature- and humidity-controlled source. Treatment of exhaust air consisted of charcoal- and HEPA filtration.

The generation system was operated as follows: Vapors of the test article were generated using a heated bead column. The test article was introduced to the top of the column, while nitrogen entering the bottom of the column served as the carrier gas. For Groups 2 and 3 the column was 2.4 cm in diameter (ID), 40 cm long filled with 2, 3 and 4 mm beads. For Group 4, the column was 5 cm in diameter (ID) and 68 cm long filled with 3, 6, 8 and 12-mm beads. The columns were wrapped with heating tapes (Omega Engineering). Temperatures were set to approximately 100°C for chambers 2 and 3, and approximately 170-185ºC for chamber 4 using an Omega CN370 temperature controller. The chamber 4 temperature was significantly higher due to the need to maintain an internal temperature of at least 70ºC to ensure vaporization of the test article. The test article was vaporized as it dripped from 1/16-inch Teflon tubing onto the glass beads contained within each heated column.
The test article was metered from an amber glass reservoir to the column using an FMI pump (Fluid Metering, Inc., Oyster Bay, New York). Calibrated FMI pumps included 2 model no. QG-6 pumps with a 1/4-inch piston for chamber 2, and a 3/8-inch piston for chamber 3 and a model no. QG-20 pump with a 1/4-inch piston for chamber 4.
Vaporization nitrogen was delivered from the facility nitrogen generation system and was controlled using calibrated flowmeters (Gilmont Instruments, Barrington, Illinois). The vaporization nitrogen carried the isobutanol vapor through Teflon delivery lines (3/8-inch O.D. tubing for chambers 2 and 3, 1-inch O.D. tubing for chamber 4) to the chamber inlet where the concentration was reduced to the desired level with chamber ventilation air. The animal exposure was initiated by switching the FMI pumps and the compressed nitrogen on simultaneously.

TEST ATMOSPHERE
- Brief description of analytical method used: Exposure concentrations within each chamber were measured 9 to 10 times (approximately every 35 minutes) during each daily exposure period by a validated gas chromatographic method. At least one standard was analyzed each day prior to exposure to confirm gas chromatographic calibration. Chamber temperature, relative humidity, ventilation rate, and negative pressure within the chambers were monitored continuously and were recorded approximately every 35 minutes. Oxygen content within the chamber was measured during the pre-study method development phase. Nominal chamber concentrations were determined daily. Total air volume was calculated by multiplying mean chamber ventilation rate (in liters per minute) by the exposure duration (in minutes). Test atmosphere homogeneity was demonstrated during pre-study method development. There were no detectable aerosols at any evaluation interval.
- Samples taken from breathing zone: yes
Details on mating procedure:
- M/F ratio per cage: 1/1
- Proof of pregnancy: [vaginal plug or sperm in vaginal smear] referred to as [day 0] of pregnancy
- After 14 days of unsuccessful pairing the female was placed into a plastic maternity cage with nesting material (also no evidence of mating was apparent)
- After successful mating each pregnant female was caged: in a plastic maternity cage with nesting material
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Exposure concentrations within each chamber were measured 9 to 10 times (approximately every 35 minutes) during each daily exposure period by a validated gas chromatographic method.
Duration of treatment / exposure:
Premating exposure period (P, males): at least 10 weeks
Premating exposure period (P, females): at least 10 weeks
7 days/week prior to mating, during mating and gestation; treatment was suspended during lactation days 0-4 and re-initiated on lactation day 5
Frequency of treatment:
daily for 6 hours per day

Details on study schedule:
- F1 parental animals not mated until 11-12 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 28 days of age.
- Age at mating of the mated animals in the study: 15-16 weeks
Dose / conc.:
500 ppm (analytical)
Dose / conc.:
1 000 ppm (analytical)
Dose / conc.:
2 500 ppm (analytical)
No. of animals per sex per dose:
30
Control animals:
yes
Details on study design:
- Dose selection rationale: Exposure levels were selected based upon a reduction in response to external stimuli (during exposure) observed in a previous 90-day neurotoxicity study and a non-statistically significant reduction in body weights and body weight changes during postnatal days 4-7 and 7-14 in pups from dams exposed to 2500 ppm isobutanol in a probe study.
Positive control:
no data
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS/DETAILED CLINICAL OBSERVATIONS
Detailed physical examinations were recorded weekly for all parental animals throughout the study period. All animals were observed twice daily for moribundity and mortality; in addition, the animals were observed for appearance, behavior and pharmacotoxic signs within one hour after completion of exposure. Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labor) or other difficulties.

BODY WEIGHT
Time schedule for examinations: Individual F0 and F1 male body weights were recorded on study days 0, 1, 4 and 7, weekly thereafter throughout the study, and prior to the scheduled necropsy. Individual F0 and F1 female body weights were recorded on study days 0, 1, 4 and 7 and weekly thereafter until evidence of copulation was observed. Mean weekly body weights and body weight changes are presented for each interval. Once evidence of mating was observed, female body weights were recorded on gestation days 0, 4, 7, 11, 14 and 20 and on lactation days 1, 4, 7, 14 and 21; for the F0 females, body weights were also recorded on lactation day 28. Body weight changes are presented for each of these intervals. After weaning (lactation day 28), weekly body weights were recorded for these F0 females until the scheduled necropsy.

FOOD CONSUMPTION
Individual F0 and F1 male and female food consumption was measured on study days 0, 1, 4 and 7 and weekly thereafter until pairing. Food intake was not recorded during the mating period. Male food consumption was measured after mating on a weekly basis until the scheduled necropsy. Female food consumption was recorded on gestation days 0, 4, 7, 11, 14 and 20 and lactation days 1, 4, 7, 14 and 21; for the F0 females, food consumption was also recorded on lactation day 28. For the F0 generation, the last scheduled interval for weekly recording of food consumption was study day 126. Since final body weights and clinical observations were recorded on the scheduled necropsy days (after study day 126), food consumption was manually recorded at that time. These data are not presented in the report tables, but will be maintained in the raw data. Food consumption was calculated and reported as g/animal/day and g/kg/day for the corresponding body weight change intervals. Food efficiency (body weight gained as a percentage of food consumed) was also calculated and reported for these intervals.

Oestrous cyclicity (parental animals):
Vaginal smears were prepared daily to determine the stage of estrous for each female, beginning 21 days prior to pairing and continuing until evidence of mating was observed. For females with no evidence of mating, smearing was continued until termination of the mating period. The average cycle length was calculated for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P] beginning 21 days prior to initiation of the mating period and until the detection of evidence of mating). Estrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. The cycle during which evidence of mating was observed for a given animal was not included in the mean individual estrous cycle length calculation. Vaginal smears were also performed on the day of necropsy to determine the stage of estrus.
Sperm parameters (parental animals):
sperm motility, morphology and numbers, mean testicular and epididymal sperm numbers, sperm production rate, motility, progressive motility and the percentage of morphologically normal sperm
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.


PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving adults were euthanized following the selection of the F1 generation and completion of a detailed clinical observation.
- Maternal animals: All surviving adults were euthanized following the selection of the F1 generation and completion of a detailed clinical observation.

GROSS NECROPSY
The necropsy included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal and pelvic cavities including viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
organs collected: Adrenals (2); Aorta; Bone with marrow (sternebrae); Brain (forebrain, midbrain, hindbrain); Coagulating gland; Eyes with optic nerve (2); Gastrointestinal tract (Esophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum); Heart; Kidneys (2); Liver (sections of two lobes); Lungs (including bronchi, fixed by inflation with fixative); Lymph node (mesenteric); Ovaries and oviducts (2); Pancreas; Peripheral nerve (sciatic); Pituitary; Prostate; Salivary gland [submandibular (2)]; Seminal vesicles (2); Skeletal muscle (rectus femoris); Skin with mammary gland; Spinal cord (cervical); Spleen; Testes with epididymidesa (the right testis and epididymis were fixed in Bouin's solution) and vas deferens; Thymus; Thyroids [with parathyroids, if present (2)]; Trachea; Urinary bladder; Uterus with cervix and vagina; All gross lesions
Organ weights: Adrenals; Brain; Epididymides (total and caudal; these paired organs were weighed seperately); Kidneys; Liver; Ovaries; Pituitary; Prostate; Seminal vesicles with coagulating glands (with accessory fluids), Spleen, Testes (these paired organs were weighed seperately), Thymus gland; Uterus with oviducts and cervix
Organs examined: Adrenal glands: cortex and medulla; Brain; Cervix; Epididymis (right): caput, corpus and cauda; Kidneys; Liver; Ovaries; Pituitary, Prostate; Seminal vesivles with coagulatin glands (with accessory fluids); Spleen; Testis (right); Thymus, Uterus (with oviducts); Vagina; All gross (internal) lesions
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [28 (F1) and 21 (F2)] days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
Prior to weaning, 30 F1 pups/sex/group were randomly selected for the F1 parental generation and for evaluation of developmental landmarks. Additional F1 pups were retained as potential replacement animals, and were euthanized by CO2 inhalation and necropsied on PND 34, 36 or 37. In addition, one F1 and one F2 pup/sex/litter (when available) were selected from the F1 and F2 weanlings for complete necropsy on PND 28 and PND 21, respectively; brain, spleen and thymus gland weights were recorded. All remaining non-selected F1 and F2 weanlings were euthanized by CO2 inhalation and necropsied on PND 28 and PND 21, respectively, with emphasis on developmental and reproductive system morphology. All gross lesions from F1 and F2 weanlings were preserved in 10% neutral-buffered formalin for possible future histopathologic examination; all other tissues were discarded.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
- All gross lesions from F1 and F2 weanlings were preserved in 10% neutral-buffered formalin for possible future histopathologic examination; all other tissues were discarded.
Statistics:
detailed statistical analyses were performed (see other information on materials and methods)
Reproductive indices:
Mating and fertility indices were calculated:
Male (Female) Mating Index (%) = [No. of Males (Females) with Evidence of Mating (or Confirmed Pregnancy) / Total No. of Males (Females) Used for Mating] x 100
Female Fertility Index (%) = [No. of Females with Confirmed Pregnancy / Total No. of Females Used for Mating] x 100
Male Fertility Index (%) = [No. of Males Siring a Litter / Total No. of Males Used for Mating] x 100
Offspring viability indices:
survival indices were calculated (not further specified)
Clinical signs:
no effects observed
Description (incidence and severity):
PARENTAL ANIMALS
There were no test article-related clinical observations at the weekly detailed physical examinations or one hour following exposure. The response to novel stimulus was similar in all groups, including the control group.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
PARENTAL ANIMALS
After 91 days of exposure to the test article, female no. 74805 in the 500 ppm group had pale eyes and ears and appeared to be having difficulty during parturition (gestation day 23) and was euthanized in extremis on lactation day 0. This female delivered 12 pups and had four pups retained in utero. On the day of euthanasia, this female had pale ears and eyes and red discharge from the vagina. Microscopically, the cause of death for this female was determined to be severe acute renal tubular necrosis and moderate acute hepatic necrosis. Therefore, this death was not attributed to the test article; no evidence of dystocia was observed in females in the 1000 or 2500 ppm groups. All other animals survived to the scheduled necropsy.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
PARENTAL ANIMALS
Mean F0 body weights, body weight gains and cumulative body weight gains in the 500,
1000 and 2500 ppm groups were unaffected by exposure to the test article during the premating period and one week following weaning (females) and throughout the study (males). The only statistically significant (p<0.05) difference from the control group was
a decreased mean body weight gain in the 1000 ppm group males during study days 84-91. A similar reduction was not observed in the 2500 ppm group; therefore, this transient decrease was not attributed to the test article. No test article-related effects on maternal body weights or body weight gains were observed in the test article-exposed groups during the F0 gestation period. Differences from the control group were slight and were not statistically significant.
No test article-related effects on F0 lactation body weights or body weight gains were observed in the test article-exposed groups. The only statistically significant (p<0.01) differences from the control group were a mean body weight loss of 33 grams in the 500 ppm group compared to a loss of 53 grams in the control group during lactation days 21-28, resulting in a statistically significant (p<0.05 or p<0.01) increase in mean body weight on lactation day 28 and in mean body weight gain during the entire lactation period (days 1-28). Since the control group lost more weight than the 500 ppm group, this difference was not considered to be an adverse change.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
PARENTAL ANIMALS
Food consumption, evaluated as g/animal/day and g/kg/day, and food efficiency in the 500, 1000 and 2500 ppm groups were unaffected by exposure to the test article during the pre-mating period and one week following weaning (females) and throughout the study (males). Occasional statistically significant (p<0.05 or p<0.01) reductions in food consumption (primarily g/kg/day) were observed in the 2500 ppm group males compared to the control group values during study days 14-21 through 112-119. Food consumption (g/animal/day) in these males was generally not affected during these intervals. Mean body weights and food efficiency in the 2500 ppm group males and females were similar to or greater than the control values. Therefore, these sporadic reductions were not attributed to exposure to the test article. The only other statistically significant differences (p<0.05 or p<0.01) from the control group were a decrease in food consumption (g/animal/day) in the 2500 ppm group males during study days 28-35, an increase in food consumption (g/kg/day) during day 0-1 in the 1000 ppm group males and a decrease in food efficiency in the 1000 ppm group males during study days 1-4. Similar differences were not observed in the 2500 ppm group males during this interval; therefore, no relationship to exposure was evident.
F0 maternal food consumption and food efficiency during gestation were unaffected by test article exposure in the 500, 1000 and 2500 ppm groups. The only statistically significant difference from the control group was a slight reduction in food consumption (g/kg/day) in the 2500 ppm group when the entire gestation period (days 0-20) was evaluated. Food efficiency and gestation body weight gain in this group was unaffected during this interval. Therefore, the reduction was not attributed to test article exposure.
F0 maternal food consumption and food efficiency in the test article-exposed groups were similar to the control group values throughout lactation. The only statistically significant (p<0.01) differences were a smaller decrement in food efficiency in the 500 ppm group during lactation days 21-28 than the control group and an increase in food efficiency in this group during the entire lactation period (days 1-28). Because similar effects were not observed at higher exposure levels, these differences were not considered test article related.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
PARENTAL ANIMALS
No test article-related effects on organ weights were observed in the 500, 1000 and 2500 ppm group males or females. The only statistically significant (p<0.05 or p<0.01) differences from the control group were increases in mean absolute prostate weights in the 1000 ppm group males and mean relative prostate weights in the 500 and 1000 ppm group males and a decrease in mean absolute pituitary weight in the 500 ppm group females. Because similar effects were not observed in the 2500 ppm group males and females, these changes were not attributed to the test article.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
PARENTAL ANIMALS
Female no. 74805 in the 500 ppm group was euthanized on lactation day 0 due to difficulties during parturition. This female delivered 12 pups and had four fetuses with no apparent malformations retained in utero. This animal had dark red contents in the ileum and jejunum and a pale pituitary gland. Similar findings were not observed in females at the scheduled necropsy in the higher exposure groups. Therefore, these findings were not attributed to the test article.
At the scheduled necropsy, no exposure-related trends were observed in the macroscopic findings noted in the test article-exposed groups. Findings were observed similarly in control group animals, were noted infrequently and/or did not occur in an exposure-related manner. The mean number of implantation sites and the mean number of unaccounted sites in the F0 test article-exposed females were similar to the control group values; no statistically significant differences were observed.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
PARENTAL ANIMALS
Female no. 74805 was euthanized in extremis during parturition. Microscopic findings for this female included severe acute renal tubular necrosis, moderate acute hepatic necrosis, lymphoid depletion of the spleen and lymphoid necrosis of the thymus. The cause of the moribund condition of this female was renal and liver necrosis with multiple organ failure.
At the scheduled necropsy, no test article-related microscopic findings were observed including for animals that failed to breed or produce a litter. The only statistically significant (p<0.05) differences from the control group values were an increase in the incidence of hydronephrosis in the 500 ppm group females and a decrease in the incidence of dilatation of the uterine lumen in the 2500 ppm group females. A slightly increased incidence (not statistically significant) of basophilic tubules was observed in the kidneys of the 2500 ppm group males; however, the severity of this lesion was minimal, and a similar increase was not observed in the 2500 ppm group females. Therefore, this common, spontaneous alteration was not considered to be test article-related. Other microscopic findings were typical of spontaneous conditions in young rats.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
PARENTAL ANIMALS
GESTATION LENGTH AND PARTURITION:
Mean F0 gestation lengths in the test article-exposed groups were unaffected by exposure to the test article. Differences from the control group value were slight and were not statistically significant. The mean gestation lengths in the 500, 1000 and 2500 ppm groups were 21.9, 21.9 and 21.8 days, respectively, compared to a mean gestation length of 22.0 days in the control group. Female no. 74805 in the 500 ppm group was euthanized in extremis due to apparent difficulty during parturition. The cause of moribundity for this animal was determined to be renal and liver necrosis with multiple organ failure. Because dystocia was not observed in females in the 1000 and 2500 ppm groups, the difficulty during delivery was not attributed to the test article.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
PARENTAL ANIMALS
The mean lengths of estrous cycles in the test groups were similar to that in the control group. None of the differences were statistically significant.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
PARENTAL ANIMALS
No test article-related effects were observed on F0 spermatogenesis endpoints (mean testicular and epididymal sperm numbers, sperm production rate, motility, progressive motility and morphology) in males at any exposure level. Differences from the control group were slight and were not statistically significant. A slight reduction in the percentage of morphologically normal sperm in the 2500 ppm group (95.9%) compared to the control group value (97.9%) was attributed to a single male (no. 74628) with an abnormally low percentage of morphologically normal sperm (0.5%). The values for the remaining males in this group were comparable to those in the control group. Therefore, the reduction in morphologically normal sperm in the single male in the 2500 ppm group was attributed to biological variation.
Reproductive performance:
no effects observed
Description (incidence and severity):
PARENTAL ANIMALS
No test article-related effects on reproductive performance were observed. Male and female mating indices were 86.7%, 100.0%, 96.7% and 100.0% in the control, 500, 1000, and 2500 ppm groups, respectively. Male and female fertility indices were 73.3%, 96.7%, 93.3% and 93.3% in the same respective groups. The only statistically significant (p<0.05) differences from the control group were increases in the male and female fertility indices in the 500 ppm group. It should be noted that the fertility index for the control group (73.3%) was reduced compared to the mean value in the WIL historical control data (90.4%). However, the values in the isobutanol-exposed groups exceeded the mean value in the WIL historical control data. Males that did not sire a litter numbered 8, 1, 2 and 2 in the control, 500, 1000 and 2500 ppm groups, respectively. Females that had evidence of mating but did not deliver numbered 4, 1, 2 and 3 in the same respective groups.
The mean numbers of days between pairing and coitus in the test article-exposed groups were similar to the control group value. The mean lengths of estrous cycles in these groups were similar to that in the control group. None of the differences were statistically significant.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 7.5 mg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: original value: 2500 ppm; no effects observed
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
The numbers of pups (litters) available for clinical signs were 299 (22), 390 (29), 390 (27) and 388 (27) in the control, 500, 1000 and 2500 ppm groups, respectively. The numbers of pups found dead during the lactation period were 13, 22, 21 and 30 in the control, 500, 1000 and 2500 ppm groups, respectively. In the same respective exposure groups, 3, 19, 9 and 11 pups were missing and presumed cannibalized. The incidence of dead/missing pups was slightly higher in the 2500 ppm group, although this was not considered exposure-related. In general, larger-sized litters tend to have increased postnatal deaths due to competition with littermates for access to maternal resources (nursing, thermoregulation, grooming, etc.). The dams in the 2500 ppm group had approximately one additional pup/litter compared to the control group. The pups from female no. 74805 (500 ppm group) were euthanized on PND 0 due to the euthanasia of the dam. The general physical condition of the F1 pups during lactation was generally similar in all groups, including the control group.
All F1 adults survived to the scheduled necropsy. There were no test article-related clinical observations at the detailed physical examinations or one hour following exposure. The response to novel stimulus in the test article-exposed groups was similar to that in the control group.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The mean numbers of F1 pups born, live litter sizes and percentages of males per litter were similar among all groups; no statistically significant differences were noted. Postnatal survival on PND 0 (relative to the number born) and during PND 0-1, 1-4, 4-7, 7-14, 14-21, 21-28, birth to PND 4 and PND 4-28 were unaffected by test article exposure. Statistically significant (p<0.05 or p<0.01) decreases in postnatal survival were observed in the test article-exposed groups relative to the control group values. These reductions occurred in the 500 ppm group during PND 0-1, 4-7 and 4-28 and in the 1000 ppm group during PND 0-1. Reductions (not statistically significant) were observed in the 2500 ppm group during PND 0-1, birth to PND 4 and PND 4-28. Because no exposure concentration-dependent response was observed, these decreases in postnatal survival were not considered test article-related.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No exposure-related effects on mean pup body weights or body weight gains were observed during the pre-weaning period (PND 1-28). Numerous statistically significant (p<0.05 or p<0.01) decreases in mean pup body weights and/or pup body weight gains were observed in the 500 and 1000 ppm group F1 males and females, primarily during PND 4-28. Mean pup body weights in the test article-exposed groups were similar to the control group value on PND 1. Mean pup body weights in the 2500 ppm group were also reduced (not statistically significant) between PND 14 and 28 (6.4%-8.1% for the males and 4.9%-7.7% or the females). Mean pup body weight gains in the 2500 ppm group were similar to those in the control group during PND 1-4, 4-7 and 7-14. Mean pup body weight gains in this group were slightly reduced during PND 14-21 and 21-28 (statistically significant at p<0.05 during PND 14-21, males only). These reductions in mean body weights and mean body weight gains followed neither a concentration-dependent nor a time-course response. The earliest, most severe reductions were observed in the 500 ppm group and began on PND 4, which was prior to the re-initiation of maternal exposure and continued throughout the pre-weaning periods.
The onset of reductions in mean body weight in the 1000 ppm group occurred, at a lesser extent, on PND 14; statistical significance in this group was not observed after PND 21. There were no reductions in mean body weight observed during the pre-weaning period in the 2500 ppm group; only slight reductions (statistically significant only in males at one time point) in mean body weight gains were observed during the last half of the pre-weaning period. Because of the lack of an exposure-related effect, none of the decreases were attributed to the test article. No exposure-related effects on mean pup body weights or body weight gains were observed during the post-weaning period (PND 28-32 and 32-35). Statistically significant (p<0.05 or p<0.01) decreases in mean pup body weights and/or pup body weight gains were observed in the 500 and 1000 ppm group F1 males and females during this period. The reductions did not occur in a concentration-dependent manner; therefore, the decreases were not attributed to the test article. Mean pup body weights and body weight gains in the 2500 ppm group were similar to those in the control group. No statistically significant differences were noted.
No test article-related trends in F1 mean body weights, body weight gains or cumulative body weight gains were observed in the test article-exposed groups. Numerous statistically significant (p<0.05 or p<0.01) differences (primarily decreases) in mean body weights and body weight gains were observed in males and females in the test article-exposed groups. The majority of the differences were observed in the 500 and 1000 ppm group males. Mean body weights in these males were 10.3% and 9.7%, respectively, lower than the control group value at the end of the pre-mating period (study day 204). These decrements appeared to be a continuation of the decreases in mean body weight gain observed in the 500 and 1000 ppm groups during the pre-weaning and early post-weaning periods. The effects were most pronounced at the low-exposure level, less pronounced at the mid-exposure level and not expressed at the high-exposure level. Because the changes were not concentration-dependent, they were not attributed to exposure to the test article.
No test article-related differences in mean maternal body weights or body weight gains were observed in the F1 females during gestation. Differences from the control group values were slight, and none were statistically significant. No test article-related differences in mean maternal body weights or body weight gains were observed in the F1 females during lactation. Differences from the control group values were slight, and none were statistically significant.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No test article-related trends in F1 food consumption (g/animal/day and g/kg/day) or food efficiency were observed in the test article-exposed groups. Occasional statistically significant (p<0.05 or p<0.01) differences from the control group were noted, but no test article-related trends were apparent. Statistically significant (p<0.05 or p<0.01) decreases in food consumption evaluated as g/animal/day were observed in the 500 and 1000 ppm group males. Because the changes observed in the test article-exposed groups were not concentration-dependent, there were no exposure-related changes in food efficiency and food consumption in the males, evaluated as g/kg/day, was generally similar to that in the control group, the decreases were not attributed to test article exposure. These reductions in food consumption (g/animal/day) were attributed to the smaller size of the males in these groups. In the 2500 ppm group males and females, food consumption (g/animal/day and/or g/kg/day) was occasionally reduced (statistically significant at p<0.05 or p<0.01) compared to the control group values. Specifically, food consumption (g/kg/day) in the 2500 ppm group females was generally reduced throughout the pre-mating period and also during the post-mating period (study days 260-266). The differences from the control group were often statistically significant (p<0.05 or p<0.01).
However, food efficiency in the 2500 ppm group males and females was similar to that in the control group and no trends were apparent that would be consistent with a test article-related effect.
F1 maternal food consumption and food efficiency during gestation in the test article exposure groups was unaffected by test article exposure. The only statistically significant (p<0.05 or p<0.01) differences from the control group were reductions in food consumption (evaluated as g/kg/day) in the 2500 ppm group during gestation days 0-4 and 11-14. Because similar decreases were not observed in the g/animal/day values, they were not attributed to exposure.
F1 maternal food consumption and food efficiency in the test article exposure groups were unaffected by test article exposure during lactation. The only statistically significant (p<0.05 or p<0.01) differences from the control group were reductions in food consumption (evaluated as g/animal/day and/or g/kg/day) in the 500, 1000 and 2500 ppm groups during lactation days 14-21. Because of the absence of a concentration-dependent response, these decreases were not attributed to test article exposure.
Sexual maturation:
no effects observed
Description (incidence and severity):
There were no test article-related differences in the mean day of acquisition of balanopreputial separation or mean body weight on the day of acquisition. The only statistically significant difference (p<0.05) from the control group was a reduced mean body weight on the day of balanopreputial separation in the 500 ppm group. However, because similar decreases were not observed in the 1000 and 2500 ppm groups, the decrease was not attributed to the test article. There were no test article-related differences in the mean day of acquisition of vaginal patency or mean body weight on the day of acquisition. The only statistically significant difference (p<0.05) from the control group was a delay in the day of acquisition of vaginal patency in the 500 ppm group. Similar effects were not observed in the 1000 and 2500 ppm groups; therefore, the delay was not attributed to the test article. Reproductive performance was unaffected by test article exposure at concentrations of 500, 1000 and 2500 ppm.
F1 male and female fertility indices were 73.3%, 83.3%, 76.7% and 80.0% in the control, 500, 1000 and 2500 ppm groups, respectively. Male and female mating indices in the same respective groups were 96.7%, 93.3%, 90.0% and 83.3%. None of the differences from the control group were statistically significant. Males that did not sire a litter numbered 8, 5, 7 and 6 in the control, 500, 1000 and 2500 ppm groups, respectively. Females that had evidence of mating but did not deliver numbered 7, 3, 4 and 1 in the same respective groups.
The mean numbers of days between pairing and coitus in the test article-exposed groups were similar to the control group value. The mean lengths of estrous cycles in these groups were similar to that in the control group. None of the differences were statistically significant. No test article-related effects on F1 spermatogenic endpoints (mean testicular and epididymal sperm numbers, sperm production rate, sperm motility, progressive motility and the percentage of morphologically normal sperm) were observed at any exposure level. Differences from the control group were slight and were not statistically significant.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No test article-related changes in mean organ weights (absolute or relative to final body weight) were observed in F1 pups in the 500, 1000 or 2500 ppm groups. The only statistically significant (p<0.05) differences from the control group were decreases in the mean absolute thymus weight in the 500 ppm group males and the mean absolute brain weight in the 500 ppm group females. Mean body weights on PND 28 was reduced in the 500 ppm group males and females. In addition, similar decreases were not observed in the 1000 or 2500 ppm groups nor in the relative organ weights in the 500 ppm group animals; therefore, these reductions were not attributed to the test article.
No test article-related changes in mean organ weights (absolute or relative to final body weight) of the F1 adults were observed. The only statistically significant (p<0.05 or p<0.1) differences from the control group values were reductions in mean absolute liver weight in the 500 ppm group males and mean absolute pituitary weight in the 500 and 1000 ppm group females, and increases in the mean relative (to final body weight) thymus and brain weights in the 500 ppm group males. Mean final body weights in these groups were reduced compared to the control group values, resulting in reduced absolute weights for the liver and pituitary. Because the brain is unaffected by reductions in body weight, increased mean relative brain weights would be expected. Similar changes were not observed in the 2500 ppm group males and females. Therefore, these changes were not attributed to the test article.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test article-related internal findings noted at the necropsies of pups that were found dead. Pup no. 74782-14 in the 500 ppm group had dark red discoloration of the brain. Pup no. 74782-08 in the same group had lungs that were not fully collapsed (PND 29). In the 2500 ppm group, pup no. 74803-14 had a mechanical injury (fractured right parietal bone with associated hemorrhage). Aside from the presence or absence of milk in the stomach, no other internal findings were noted in pups that died.
No test article-related internal findings were observed in the weanlings necropsied on PND 28, 34, 36 or 37, or in the pups euthanized due to the death of the dam. One pup in the 500 ppm group had a raised, white area on the median lobe of the liver. Two pups in the 1000 ppm group had a dilated renal pelvis (unilateral). In the 2500 ppm group, one pup had a cystic oviduct. One pup in the same group had a mechanical injury (the frontal and parietal bones were fractured). No other internal findings were observed.
No internal findings were observed at the necropsy of pups selected for organ weights.
No test article-related internal findings were observed at the scheduled necropsy of F1 adult males and females. Findings observed were noted similarly in the control group, did not occur in an exposure concentration-related manner and/or were common to laboratory rats. The mean numbers of implantation sites and the numbers of unaccounted sites in the test article-exposed groups were similar to the control group values.
Histopathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test article-related histopathologic alterations observed in F1 animals at exposure levels of 500, 1000 and 2500 ppm, including for animals that failed to breed or produce a litter. Findings in the test article-exposed groups occurred similarly in the control group and were considered spontaneous or incidental in nature. The only statistically significant (p<0.05) difference from the control group was a decrease in the number of males in the 2500 ppm group with subacute inflammation in the kidneys. A decrease in the number of animals with microscopic lesions is not considered adverse. Therefore, these differences were not considered test article-related. No test article-related effects on primordial follicle counts or corpora lutea counts were observed in the 2500 ppm group females.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
GESTATION LENGTH AND PARTURITION
F1 maternal food consumption and food efficiency in the test article exposure groups were unaffected by test article exposure during lactation. The only statistically significant (p<0.05 or p<0.01) differences from the control group were reductions in food consumption (evaluated as g/animal/day and/or g/kg/day) in the 500, 1000 and 2500 ppm groups during lactation days 14-21. Because of the absence of a concentration-dependent response, these decreases were not attributed to test article exposure.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 7.5 mg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: original value: 2500 ppm; no effects observed
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The numbers of pups (litters) litters available for clinical signs were 286 (22), 348 (25), 316 (23) and 334 (24) for the control, 500, 1000 and 2500 ppm groups, respectively. The numbers of pups found dead over the entire lactation period numbered 6, 11, 10 and 10 in the control, 500, 1000 and 2500 ppm groups, respectively. In the same respective exposure groups, 1, 4, 4 and 1 pups were missing and presumed cannibalized. The general condition of the pups in the test article exposure groups was similar to that in the control group; findings were noted infrequently and/or did not occur in an exposure concentration-related manner.
Mortality / viability:
no mortality observed
Description (incidence and severity):
Postnatal survival, the mean number of pups born, the percentage of males at birth and live litter size on PND 0 in the 500, 1000 and 2500 ppm groups were unaffected by exposure to the test article. Differences from the control group were slight and were not statistically significant.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No test article-related effects were observed on mean F2 pup body weights or body weight gains. The F2 control group litters were slightly smaller by approximately one pup than the 500 (PND 1 only), 1000 and 2500 ppm group litters (PND 1 and 4); however, the statistical analyses on mean body weight and body weight gains were performed using the litter size as the covariant. The F2 control group pups generally weighed more than the F1 control group pups in this study and the pups in the WIL historical control data for inhalation studies.
The slightly increased litter sizes (0.8-1.1 pups/dam) in the 1000 and 2500 ppm groups correlated with concomitant reductions in F2 mean pup body weights (7% and 3%, respectively, in the males; 7% and 4%, respectively, in the females) on PND 1. Mean pup F2 body weights in the 500 ppm group males and females were 7% and 9%, respectively, lower than the concurrent control group values on PND 1. The differences in the 500 and 1000 ppm groups were statistically significant (p<0.05) compared to the control group values. However, the F2 mean male and female pup weights in the concurrent control group on PND 1 were %-6% greater than the mean male and female pup weights in the inhalation studies in the WIL historical control data and 6% greater than the concurrent control group pups in the F1 generation in this study. Mean body weight gain in the 1000 and 2500 ppm group males and females were similar to the control group values during PND 1-4. Mean body weight gains in the 500 ppm group males and females were decreased (statistically significant at p<0.05) compared to the control group values during PND 1-4. On PND 4, mean male pup body weights were 12%, 8% and 1% lower than the concurrent control group value in the 500, 1000 and 2500 ppm groups, respectively; mean female pup weights were 12%, 7% and 2% lower in the same respective groups.
The differences in both genders were statistically significant (p<0.01) only in the 500 ppm group. However, the concurrent F2 control group male and female mean body weights were 8% greater than those in the WIL historical control data for inhalation studies and 11% and 9% greater than the mean F1 male and female pup weights, respectively, on PND 4. Due to the lack of effects in the 1000 and 2500 ppm groups, the apparent influence of litter size on mean pup weights and the difference of the concurrent control group values compared to the historical control data for inhalation studies, the differences in mean body weights and body weight gains in the F2 pups during PND 1-4 were not attributed to test article exposure.
Following standardization of litters (to eight pups/dam) on PND 4, mean male and female pup weights in the 500 ppm group were 17% and 15%, respectively, lower than the concurrent control group values (statistically significant at p<0.01) on PND 7. Mean male and female pup weights in the 1000 ppm group were 9% lower than the concurrent control group values; the difference was statistically significant (p<0.05) only for the females. Mean pup body weights in the 2500 ppm group were unaffected by F1 maternal exposure on PND 7. Mean body weights gains in the 500 and 1000 ppm groups were reduced during PND 4-7. The differences from the control group were statistically significant (p<0.01) only for the 500 ppm group males and females. Mean body weight gains in the 2500 ppm group males and females was slightly (not statistically significant) less than those in the concurrent control group, but consistent with the calculated gain in the WIL historical control data for inhalation studies. The mean body weight gains in the 1000 and 2500 ppm groups were comparable to those in the F1 control group. Due to the lack of effect at the highest exposure concentration and the difference of the concurrent control group values from the historical control values, these differences in F2 postnatal body weights and body weight gains were not considered exposure-related.
On PND 14, mean male and female pup body weights in the 500 ppm group were 11% and 10%, respectively, lower than the concurrent control group values; the differences were statistically significant (p<0.05 or p<0.01). The mean body weights for the 1000 and 2500 ppm group males and females were similar to the historical control values for inhalation studies, but were lower (5%-8%) than the concurrent control group values (not statistically significant). In addition, the PND 14 mean body weights for the 500, 1000 and 2500 ppm group males and females were comparable to or greater than the values for the F1 control group pups. Mean body weight gains in the 500, 1000 and 2500 ppm group males and females were slightly reduced (not statistically significant) compared to the concurrent control group values during PND 7-14. The concurrent control group values during this interval were 3% higher than the calculated mean gain in the WIL historical control data for inhalation studies. In addition, mean F2 body weight gains in all groups during PND 7-14 were equal to or higher than the gain in the F1 control group pups at this same interval. Due to the lack of effect in the 1000 and 2500 ppm groups and the difference of the concurrent F2 control group values compared to the F1 control group values, the differences in mean body weights on PND 14 was not considered exposure-related.
On PND 21, mean male and female pups body weights in the 500, 1000 and 2500 ppm groups were reduced (statistically significant at p<0.05 or p<0.01) compared to the concurrent control group values. However, the F2 control group values on this day were 14% higher than the F1 pups on PND 21 and 5% higher than the mean body weight on PND 21 in the WIL historical control data for inhalation studies. The 2500 ppm group values were higher than the F1 control group values, while the 500 and 1000 ppm group values were lower than the F1 control group and the inhalation historical control group values. Mean body weight gains in the 500, 1000 and 2500 ppm group males and females were also reduced (statistically significant at p<0.01) compared to the concurrent control group value. Mean body weight gains in these groups were also reduced compared to the F1 control group value and the calculated value in the WIL historical control data. However, the most severe reductions occurred in the 1000 ppm group males and females and the least severe reductions were observed in the 2500 ppm group males and females. Therefore, these reductions were not considered exposure-related due to the lack of an exposure-response relationship.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No test article-related effects on mean F2 pup organ weights (absolute and relative to final body weight) were observed. Occasional statistically significant (p<0.05 or p<0.01) differences from the control group were noted in the 500, 1000 and 2500 ppm groups, but no exposure-related trends were evident.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test article-related internal findings were observed in F2 pups that were found dead.
One pup in the control group had a mechanical injury consisting of fractured pubis, ischium and ilium bones and all sacral vertebrae.
One pup in the 1000 ppm group had dark red discoloration of all lobes of the lungs. Aside from the presence or absence of milk in the stomach, no other internal findings were observed.
No test article-related internal findings were observed in the F2 weanlings not selected for organ weights. Two pups each in the 500 and 1000 ppm groups had a dilated renal pelvis (unilateral). No other internal findings were observed.
No test article-related internal findings were observed in the F2 weanlings selected for organ weights. One pup in the 2500 ppm group had a dilated renal pelvis (unilateral). No other internal findings were observed.
Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
>= 7.5 mg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: original value: 2500 ppm; no effects observed
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

F0 and F1 parental survival were unaffected by test article exposure in all exposure groups. No test article-related effects were observed on F0 and F1 reproductive performance (estrous cycles, mating and fertility indices, the mean number of days between pairing and coitus and the mean length of gestation), body weights, food consumption and food efficiency in the 500, 1000 and 2500 ppm group males or females. Spermatogenic endpoints (mean testicular and epididymal sperm numbers, sperm production rate, motility, progressive motility and the percentage of morphologically normal sperm) in the F0 and F1 test article-exposed groups were unaffected by test article exposure. There were no test article-related macroscopic findings or changes in mean organ weights in the F0 or F1 males and females in the 500, 1000 and 2500 ppm groups. Microscopic evaluation of the F0 and F1 males and females revealed no test article-related histopathologic lesions, including for animals that failed to breed or produce a litter. No test article-related effects on primordial follicle counts and corpora lutea counts were observed in the F1 2500 ppm group females. F1 and F2 pup survival and the general physical condition of the pups were unaffected by exposure to the test article. No test article-related effects on mean pup body weights were observed in the 500, 1000 or 2500 ppm group F1 or F2 pups. No test article-related macroscopic findings were observed in F1 or F2 pups that were found dead or euthanized at the scheduled necropsy. There were no test article-related changes in mean organ weights for the F1 or F2 pups.

The mean day of acquisition of balanopreputial separation or vaginal patency and mean body weights on the day of acquisition in the F1 test article-exposed pups were not affected by the test article.

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
7 500 mg/m³
Study duration:
subchronic
Species:
rat
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A GLP conform two-generation reproductive toxicity study has been conducted by inhalation with isobutanol according to EPA OPPTS 870.3800 (ACC 2003). Groups of 30 male and 30 female Sprague-Dawley rats were exposed by inhalation (6 hours/day, seven days/week) to 0, 500, 1000, or 2500 ppm (ca. 1.5, 3.0, or 7.5 mg/L) of the test substance for two generations. Daily treatments were continuous with the exception of the period between gestation day 21 to postnatal day 4 (removal of the dams from the pups during this period typically causes pup mortality). Exposure to 500 to 2500 ppm (ca. 1.5 to 7.5 mg/L) of the test substance did not cause any parental systemic, reproductive, or neonatal toxicity in both the F1 and F2 generation when administered for two generations via whole-body exposure. The NOAEL was considered to be at least 7.5 mg/L (2500 ppm).

Effects on testes of rats after subchronic inhalative exposure should be studied in a study with focus on neurotoxicity of isobutanol (CMA 1996). In the process of shipping the testis to the lab for histological processing, the testes were placed in plastic bags with fixative. Unfortunately, the plastic bags were compressed in the shipping container and flattened the tissue, distorting the three-dimensional architecture of the testes, which is essential for determining the stage of the testes on cross-section. The pathologist tried to conduct the testicular staging exercise with the flattened testes anyway. No substance related effects were observed. The lack of histological findings in the other testes, the lack of dose-dependent effects on spermatid head counts, and the lack of interpretable changes in stage frequency indicate that isobutanol did not affect testicular function in these animals.

Effects on developmental toxicity

Description of key information
inhalation, rat, gestation day 6-15: NOAEL maternal, teratogenicity and fetotoxicity >= 10 mg/L (GLP; OECD 414; BG Chemie 1990). 
inhalation, rabbit, gestation day 7-19: NOAEL maternal = 2.5 mg/L due to slight impairment of body weight gain; NOAEL teratogenicity and fetotoxicity = 10 mg/L (GLP; OECD 414; BG Chemie 1990).
Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): 2-Methylpropan-1-ol
- Physical state: liquid
- Analytical purity: 99.8 %
- Storage condition of test material: room temperature
- Storage condition of test material: The stability was ensured for the period of the study under the specified storage conditions
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Dr. K. Thomae GmbH
- Age at study initiation: ca. 10-11 weeks
- Weight at study initiation: mean: 216 g
- Housing: individually
- Diet: ad libitum (no food during exposure)
- Water: ad libitum (no water during exposure)
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 30-70 %
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Concentrations of 0.5, 2 .5 and 10 mg/liter were achieved by supplying the test substances via continuously operating pumps to evaporators maintained at 50-70 °C by a water circulation thermostat. The vapors were diluted with clean air. This vapor-air mixture was distributed to a horizontal-flow whole-body exposure chamber at a flow rate corresponding to about 15 air exchanges per hour (inhalation chamber glass/steel construction with volumes of approximately 1100 l; manufactured by BASF AG, Ludwigshafen, Germany).
Supply and exhaust air flows were adjusted by flow meters, in order to achieve a minimal negative pressure in the inhalation chambers. Pressure and temperature (21-24 °C) were measured continuously. The relative humidity (49-64 %) was checked with a hygrometer (Humicap, Vaisala, Finland).


TEST ATMOSPHERE
- Brief description of analytical method used: Samples of the inhalation atmospheres were analyzed hourly by gas chromatography (Hewlett-Packard gas chromatograph Model 5840 A with an automatic sampler Model 767 1A, FID ; column, 2 m x 2 mm with 15% Ucon LB 550 x on Chromosorb W/HP; 80/100 mesh; oven temperature, 90 °C).
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the inhalation atmospheres were analyzed hourly by gas chromatography (Hewlett-Packard gas chromatograph Model 5840 A with an automatic sampler Model 767 1A, FID; column, 2 m x 2 mm with 15% Ucon LB 550 x on Chromosorb W/HP; 80/100 mesh; oven temperature, 90 °C).
Details on mating procedure:
- Impregnation procedure: [cohoused]
- If cohoused:
- M/F ratio per cage: 1/4
- Length of cohabitation: 4 pm to 7.30 am of the following day (15.5 hours)
- Verification of same strain and source of both sexes: [yes]
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 of pregnancy
Duration of treatment / exposure:
day 6-15 of gestation
Frequency of treatment:
6 hours/day, daily
Duration of test:
until day 20 of gestation
Dose / conc.:
0.5 mg/L air (nominal)
Dose / conc.:
2.5 mg/L air (nominal)
Dose / conc.:
10 mg/L air (nominal)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In the range-finding study 10 pregnant rats per group were exposed to 0.5, 2.5, or 5.0 mg/liter for 6 hr each day, from Day 6 to Day 15 of gestation. No maternally toxic effects (clinical signs, body weight, various hematological, and clinicochemical parameters, gross pathological findings at necropsy) could be observed up to 5 mg/liter. This is the highest concentration (limit concentration) recommended by guidelines (EPA/TSCA, 1985). For the following full-scale studies 10 mg/liter was used as the highest concentration.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations: The behaviour and state of health of the test animals were checked at least 3 times on exposure days and, as a rule, once during the preflow period and the post-exposure observation period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily


BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of the animals was checked on day 0 (= day of detection of sperm) and on days 3, 6, 9, 12, 15, 18 and 20 p.c. As a rule, the animals were weighed at the same time of the day.


FOOD CONSUMPTION: No data

WATER CONSUMPTION: No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: On day 20 p.c. the dams were sacrificed in randomized order by cervical dislocation and the fetuses removed by cesarean section. After the dams had been sacrificed, they were necropsied and assessed by gross pathology.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Number of live and dead fetuses; calculation of conception rate and pre- and postimplantation losses
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: No data
Statistics:
The Dunnett test (Dunnett, 1955, 1964) was used to statistically compare body weight, body weight changes, corrected body weight gain, intact uterine weight, fetal and placental weights, the number of corpora lutea, implants, resorptions, live fetuses, and pre- or postimplantation losses. The Fisher's exact test (Dixon, 198 1) was used for evaluating the conception rate, maternal mortality, and all fetal findings.
Indices:
no data
Historical control data:
historical control data was available
Details on maternal toxic effects:
Details on maternal toxic effects:
The body weights of all test groups compared to the control were not statistically significantly influenced. The body weight change of all test groups compared to the control was not statistically significantly influenced. The results of the corrected body weight gain (body weight on day 20 p.c. minus body weight on day 6 p.c. minus weight of the uterus before it was opened) do not show any differences of statistical significance or biological relevance between the groups. There were no substance-induced clinical signs or findings in all test groups at any time of the study period (preflow, exposure, post-exposure observation). No deaths were recorded throughout the study period.
There were no remarkable observations at necropsy in any of the dams, which could be attributed to the test substance exposure. Only hydrometra was recorded for two animals of the control, one animal of test group 2 (2.5 mg/l) and for one animal of test group 3 (10 mg/l); all these animals did not become pregnant. Edema of lungs, which has to be related to the sacrifice of the animals, was noted for one control and one animal of the 2.5 mg/l group. There were no substantial differences concerning the uterus weights between the controls and the substance treated groups. All values lie within the range of biological relevance.
The conception rate varied between 76 and 92%.
Concerning all groups, there were no substance-related and/or statistically significant differences in conception rate, in the mean number of corpora lutea and implantation sites as well as in the value calculated for the preimplantation loss, the number of resorptions and viable fetuses.
The statistically significant dereases in postimplantation loss, total and late resorptions in the high dose females are without any biological relevance with regard to adverse effects, but considered incidental findings. All differences evident are considered to be incidental and within the normal range of deviations for animals of this strain and age.
Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/L air (analytical)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Abnormalities:
no effects observed
Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
The sex distribution of the fetuses in test groups 1 - 3 (0 .5 - 10 mg/l) was comparable with the control fetuses. The differences observed in comparison to the control are without any biological relevance. The mean placental weights in test groups 1 - 3 (0.5, 2.5 and 10 mg/l) were not influenced by the test substance exposure to the dams. The differences observed in comparison to the control are without any biological relevance. The mean fetal weights were not influenced by the test substance exposure. All values are within the range of biological variation.
The external examination of the fetuses revealed two kinds of malformations. Anasarca was recorded for two high dose fetuses, one of them showing in addition cleft palate. Both malformations are also present at a low incidence in the historical control data.
There were no external varations.
Several so-called unclassified observations (placentae fused or necrobiotic) were recorded in 4 control and in 2 fetuses of test group 3 (10 mg/l).
The examination of the organs of the fetuses revealed only one kind of malformation (dilatation of both heart ventricles) in one fetus of the high dose group. Variations were detected in each group including the control. The very common finding (dilated renal pelvis) in the rat strain used in this study and the other kind of variation (hydroureter) occurred without a clear dose-response relationship. The differences observed in comparison to the control group including the statistically significantly decreased number of soft tissue variations in the low and in the intermediate dose are without any biological relevance.
No so-called unclassified observations (like blood coagulum around the bladder) were recorded.
Various malformations of the sternebrae and/or the vertebral column were seen in 5 fetuses of the control group, in 9 fetuses of the 0.5, in 2 fetuses of the 2.5 and in 1 fetus of the 10 mg/l group. The variations exhibited were related to the ribs (shortened 13th or accessory 14th ribs or rudimentary cervical ribs) and the sternum (sternebra(e) of irregular shape, bipartite or accessory sternebra) and were found in all groups. They occurred without any dose-response relationship. In all groups, signs of retardations (incomplete or missing ossification of hyoid, skull bones, vertebral bodies, sternebra(e), and/or metacarpal and metatarsal bones) were found. The number of fetuses with incompletely or not ossified sternebrae is statistically significantly decreased in test groups 1 (0 .5 mg/l) and 3 (10 mg/l). Furthermore, the number of fetuses with only one ossification center in the sternebra(e) is statistically significantly increased in the intermediate dose group (2.5 mg/l) and the overall number of high dose skeletal retardations is lowest in comparison to the other groups.
All differences between the groups, however, are within the range of biological variation and/or occur without a clear dose-response relationship.
Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/L air (analytical)
Based on:
test mat.
Basis for effect level:
other: fetotoxicity
Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/L air (analytical)
Based on:
test mat.
Basis for effect level:
other: teratogenicity
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

The only statistically significant differences between the groups are the lower number of low and intermediate dose fetuses with soft tissue variations and the lower number of high dose fetuses with skeletal retardations which are, of course, without any biological relevance. The external malformations (i.e. cleft palate and/or anasarca) noted for two high dose fetuses (out of 250) and the rare soft tissue finding (i.e. globular shaped heart) observed in one high dose fetus (out of 122) are finally assessed as being of incidental nature and not related to the test substance exposure, because they occurred with very low incidences only and there were no indications of an increased malformation rate in the high dose fetuses if all classified findings are summarized. Moreover, the spontaneous occurrence of both external malformations is documented in the historical control data. All other observations, which are summarized in the relevant tables occurred in all test groups including the control to about the same amount, without any clear, dose-related differences between the groups and/or are present in the same magnitude in the historical control data.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Specific details on test material used for the study:
- Name of test material (as cited in study report): 2-Methylpropan-1-ol
- Physical state: liquid
- Analytical purity: 99.8 %
- Storage condition of test material: room temperature
- Storage condition of test material: The stability was ensured for the period of the study under the specified storage conditions
Species:
rabbit
Strain:
Himalayan
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Dr. K. Thomae GmbH
- Age at study initiation: 24-29 weeks
- Weight at study initiation: 2.5-2.7 kg
- Housing: individually
- Diet: ad libitum (no food during exposure)
- Water: ad libitum (no water during exposure)
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 30-70 %
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Concentrations of 0.5, 2.5 and 10 mg/liter were achieved by supplying the test substances via continuously operating pumps to evaporators maintained at 50-70 °C by a water circulation thermostat. The vapors were diluted with clean air. This vapor-air mixture was distributed to a horizontal-flow whole-body exposure chamber at a flow rate corresponding to about 15 air exchanges per hour (inhalation chamber glass/steel construction with volumes of approximately 1100 l; manufactured by BASF AG, Ludwigshafen, Germany).
Supply and exhaust air flows were adjusted by flow meters, in order to achieve a minimal negative pressure in the inhalation chambers. Pressure and temperature (21-24 °C) were measured continuously. The relative humidity (49-64 %) was checked with a hygrometer (Humicap, Vaisala, Finland).


TEST ATMOSPHERE
- Brief description of analytical method used: Samples of the inhalation atmospheres were analyzed hourly by gas chromatography (Hewlett-Packard gas chromatograph Model 5840 A with an automatic sampler Model 767 1A, FID ; column, 2 m x 2 mm with 15% Ucon LB 550 x on Chromosorb W/HP; 80/100 mesh ; oven temperature, 90 °C).
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the inhalation atmospheres were analyzed hourly by gas chromatography (Hewlett-Packard gas chromatograph Model 5840 A with an automatic sampler Model 767 1A, FID; column, 2 m x 2 mm with 15% Ucon LB 550 x on Chromosorb W/HP; 80/100 mesh; oven temperature, 90 °C).
Details on mating procedure:
- Impregnation procedure: [artificial insemination]
- Day of insemination referred to as [day 0] of pregnancy
Duration of treatment / exposure:
days 7 - 19 following artificial insemination (= Day 0)
Frequency of treatment:
6 hours/day, daily
Duration of test:
until day 29 of gestation
Dose / conc.:
0.5 mg/L air (nominal)
Dose / conc.:
2.5 mg/L air (nominal)
Dose / conc.:
10 mg/L air (nominal)
No. of animals per sex per dose:
15
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In the range-finding study 5 pregnant rabbits per group were exposed to 0.5, 2.5, or 5.0 mg/liter for 6 hr each day, from Day 7 to Day 19 (rabbits) of gestation. No maternally toxic effects (clinical signs, body weight, various hematological, and clinicochemical parameters, gross pathological findings at necropsy) could be observed up to 5 mg/liter. This is the highest concentration (limit concentration) recommended by guidelines (EPA/TSCA, 1985). For the following full-scale studies 10 mg/liter was used as the highest concentration.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations: The behavior and state of health of the test animals were checked at least 3 times on exposure days and, as a rule, once during the preflow period and the post-exposure observation period.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily


BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of the animals was checked on day 0 (= day of insemination) and on days 3, 7, 10, 13, 16, 19, 21, 24, 27 and 29 p.i . As a rule, the animals were weighed at the same time of the day.


FOOD CONSUMPTION: No data

WATER CONSUMPTION: No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: On day 29 p.i., the dams were sacrificed in randomized order by intravenous injection of pentobarbital and the fetuses removed by cesarean section. After the dams had been sacrificed, they were necropsied and assessed by gross pathology.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Number of live and dead fetuses; calculation of conception rate and pre- and postimplantation losses
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: No data
Statistics:
The Dunnett test (Dunnett, 1955, 1964) was used to statistically compare body weight, body weight changes, corrected body weight gain, intact uterine weight, fetal and placental weights, the number of corpora lutea, implants, resorptions, live fetuses, and pre- or postimplantation losses. The Fisher's exact test (Dixon, 198 1) was used for evaluating the conception rate, maternal mortality, and all fetal findings.
Indices:
no data
Historical control data:
historical control data was available
Details on maternal toxic effects:
Details on maternal toxic effects:
The body weights of all test groups compared to the control were not statistically significantly influenced. The body weight change of all test groups compared to the control was not statistically significantly influenced. But on the first days of the exposure period the animals of test group 3 showed a slight retardation in body weight gain. Over the total exposure period (days 7 - 19 p.i.) a slight retardation in body weight gain, which was not statistically significant, was observed. This finding can be seen as a marginal indication of a maternally toxic effect, especially at the start of exposure. This is confirmed by the observation, that the body weight increase in the post-exposure observation period is higher compared with the other groups (recovery process).
The results of the corrected body weight gain (body weight on day 29 p.i. minus body weight an day 7 p.i . minus weight of the uterus before it was opened) do not show clearly dose-related differences between the groups.
There were no abnormal clinical signs or findings in the control animals at any time of the study (preflow, exposure, post-exposure observation).
The low dose animals showed no abnormal clinical signs or findings during preflow and exposure. One animal had an abortion on day 20 p.i. and was therefore sacrificed on day 21 p.i. The other animals of the test group were without abnormal clinical signs or findings during post-exposure observation.
The mid dose animals showed no abnormal clinical signs or findings during preflow. The bedding of one animal was reddish on days 18 and 19 p.i. The other animals showed no abnormal clinical signs or findings during exposure. One animal was found dead in cage on day 24 p.i. (abortion).
The other animals of the test group showed no abnormal clinical signs or findings during post-exposure observation.
The high dose animals showed no abnormal clinical signs or findings during preflow. One animal had aqueous discharge from eyes on days 11 - 27 p.i. The other animals of the test group were without abnormal clinical signs and findings during exposure and postexposure observation.
All of the findings which were recorded for the does with scheduled sacrifice are spontaneous ones (e.g. blind ending or absence of uterine (horn(s)) or are causally related to the sacrifice of these animals (e.g. lung with edema and/or petechiae). The other necropsy findings, which were noted for the dams which died intercurrently or had to be sacrificed after abortion were also of incidental nature.
The uterus weights of the animals of the substance treated groups do not show any significant differences in comparison to the controls.
The conception rate varied between 93 and 100%.
As mentioned before, 1 doe of the low dose group (0 .5 mg/l) was sacrificed after abortion and one doe of the mid dose group (2.5 mg/l) died intercurrently after abortion in the post-treatment period. No further substance-related and/or statistically significant differences in the conception rate, in the mean number of corpora lutea, total implantations, resorptions and live fetuses as well as in the values calculated for the pre- and postimplantation loss could be noted. The differences evident were considered to be incidental and within the normal range of deviations for animals of this strain and age.
Key result
Dose descriptor:
NOAEL
Effect level:
2.5 mg/L air (analytical)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Abnormalities:
no effects observed
Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
The sex distribution in test groups (0.5 mg/l, 2.5 mg/l and 10 mg/l) was comparable with the control group. The observable differences are without any biological relevance.
The mean placental weights in the test groups (0.5, 2.5 and 10 mg/l) were not influenced by the administration of the test substance to the does. The differences observed in comparison to the control are without any dose-response relationship and without any biological relevance. The slightly increased mean placental weights in the mid dose group (2.5 mg/l) are due to the incidentally lower number of fetuses/doe in this group and not related to exposure.
The mean fetal weights in test groups (0.5, 2.5 and 10 mg/l) were not influenced by the exposure of the test substance.
The external examination of the fetuses revealed no malformations or variations in any group.
Only one unclassified observation (placentae necrobiotic) was recorded for two fetuses of mid dose group (2 .5 mg/l).
The examination of the organs of the fetuses revealed no malformations in any group.
Variations were detected in each group including the control. The very common finding (separated origin of carotids) in the rabbit strain used in this study occurred without any dose-response relationship. Traces of the interventricular foramen/septum membranaceum were recorded for several fetuses of the control group and the low and high dose test groups (0, 0.5 and 10.0 mg/l), but were most frequently seen in the highest dose group, the differences in relation to the control group being statistically significant; however, if the relevant values are compared with the corresponding historical control values from other prenatal toxicity studies in Himalayan rabbits, which were carried out in BASF's Department of Toxicology between 1988 and 1990, the number/percentage of high dose fetuses/litters with traces of interventricular foramen/septum membranaceum is fully in the range of biological variation. Therefore, the statistically significantly increased number of high dose fetuses showing this variation of the heart is assessed as being of incidental nature.
Moreover, hypoplasia of gallbladder and dilated renal pelvis were observed in a very low incidence in the different groups without a dose-response relationship and/or are to be found in the same kind of magnitude in the historical control data.
Furthermore, several so-called unclassified observations (focal liver necrosis, blood coagulum around bladder) were seen in all groups, excluding the high dose group, with no relation to the dose.
Various malformations of the sternebrae and/or the vertebral column (lumbar vertebra absent) were seen in 2 fetuses of the control group, in 1 fetus of the 0.5 mg/l and in 1 fetus of the 10 mg/l group. They appeared without any dose-response relationship. The variations exhibited were related to the ribs (accessory 13th rib(s) or flying rib(s)), the sternum (sternebra(e) of irregular shape or fused) and the skull (epectal bone between nasal and frontal bones or splitting of skull bones) and were found in all groups without a clear dose-response relationship.
In all groups, including the controls, signs of retardations (incomplete or missing ossification of sternebra(e), vertebral column, the skull and/or the distal extremities) were found. They appeared without any dose-response relationship. The diminished number of high dose fetuses with incomplete ossification of sternebra(e) and overall fetal skeletal retardations is without any biological relevance.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 10 mg/L air (analytical)
Based on:
test mat.
Basis for effect level:
other: teratogenicity
Key result
Dose descriptor:
NOAEL
Effect level:
>= 10 mg/L air (analytical)
Based on:
test mat.
Basis for effect level:
other: fetotoxicity
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

There is only a statistically significant increase in the high dose group (10 mg/l) concerning overall fetal variations and a statistically significant decrease in this test group concerning skeletal retardations, while the number of fetal malformations shows no differences of biological relevance between the groups.

The increased number of high dose fetuses showing variations is mainly caused by the higher occurrence of one variation of the heart. This is assessed as being without biological significance, because of the similar incidence of this finding in the historical control data. The same is true for the incidentally diminished number of 10 mg/l fetuses, for which skeletal retardations were recorded.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
10 000 mg/m³
Species:
rat
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In two GLP conform developmental toxicity studies according to OECD 414, groups of pregnant female rats (25/group) or rabbits (15/group) were exposed via inhalation to 0, 0.5, 2.5 or 10 mg/L test substance for 6 hours/day during gestation (rats - days 6-15; rabbits - days 7-19; BG Chemie 1990a, BG Chemie 1990b). Rabbit dams exposed to 10 mg/L had slight decreases in body weight gain during gestation while exposures in rat dams had no treatment-related effects. No evidence of teratogenicity or fetotoxicity was reported in either the rats or the rabbits fetuses. In the rat study, the NOAEL for maternal, teratogenicity and fetotoxicity was 10 mg/L. In the rabbit study, the NOAEL for maternal toxicity was 2.5 mg/L and the NOAEL for developmental toxicity and fetotoxicity was 10 mg/L.


Justification for selection of Effect on developmental toxicity: via inhalation route:
No effects were observe in rats and rabbits. The same NOAEC was obtained for both species (corresponding to the highest dose tested).

Toxicity to reproduction: other studies

Additional information

There is no indication given for isobutanol for a relevant reprotoxic potential.

Justification for classification or non-classification

Due to the lack of toxicity on fertility and development in definite studies with isobutanol, there is no need for classification according to reproductive toxicity according to 1272/2008/EC (CLP) requirements.