Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

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

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003
Reference Type:
secondary source
Title:
OECD SIDS Isobutanol
Author:
OECD
Year:
2004
Bibliographic source:
OECD SIDS, UNEP Publications

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
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

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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)

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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 / performance (P0)

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.

Effect levels (P0)

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

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

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.

Effect levels (F1)

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

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

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.

Effect levels (F2)

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

Target system / organ toxicity (F2)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

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.

Applicant's summary and conclusion