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Toxicity to reproduction

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Administrative data

Endpoint:
toxicity to reproduction
Remarks:
other: weight of evidence
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 generation
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study; conducted in general accordance with the EPA OPPTS guideline for 870.3650

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006
Reference Type:
publication
Title:
Unnamed
Year:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Tertiary-Butyl Acetate
- Source of test material: Lyondell Chemical, Deer Park, TX
- Lyondell Material: 499245 TBAc TM
- Lot/batch No.: OD50502001
- Appearance: clear, colorless liquid
- Analytical purity (when packaged): 99.68% (24 May 2005)
- Analytical purity (during testing): 99.51-99.63% (11 Oct 2005 to 20 Feb 2006)
- Stability under test conditions: stable
- Storage condition of test material: Refrigerated in sponsor supplied drums
- Purity and stability analysis under test conditions: A Certificate of Analysis provided by the sponsor states that test material purity was 99.68% (24 May 2005). Purity/stability analyses were also conducted by the Analytical Chemistry Department of WIL Research Laboratories, LLC. Test material purity was assessed using gas chromatography with flame ionization detection. Chromatograms for the test material were compared to a chromatogram of authentic tertiary butyl acetate (99.5% pure) purchased from Aldrich Chemical Co., St. Louis MO. Two test material containers were used during the study. Samples of test material were collected at the start and end of each container’s use and analyzed for purity. The mean area percent (purity) for samples collected at the start and end of use were 99.63% and 99.57%, respectively, for container #1 and 99.57% and 99.51%, respectively, for container #2. These results indicate that the test material was stable throughout the study. Samples of test material were also collected at time-zero and after 7 days of storage in the inhalation vapor generator reservoir. Comparison of purity at time-zero (99.61%) and after 7 days in the reservoir (99.61%) indicated that the test material was stable during this phase of the study.

Test animals

Species:
rat
Strain:
other: Sprague-Dawley Crl:CD®(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina
- Age at study receipt: approx. 4 weeks
- Acclimation/pretest period: 16 days
- Method of animal identification: Monel® metal eartag
- Housing (non-exposure periods): Weanling animals were housed 3 per cage (or 2 per cage, if necessary) by sex in clean, suspended, stainless steel wire-mesh cages for at least 6 days following receipt. Thereafter, all F0 animals were housed individually, except during mating, in clean, suspended, stainless steel wire-mesh cages in an environmentally controlled room. During mating, rats were paired in the home cage of the male. Following positive evidence of mating, F0 males were housed in suspended wire-mesh cages until necropsy. F0 females were housed in plastic maternity cages with nesting material, ground corncob bedding (Bed-O’Cobs®) until weaning on Lactation Day 21. Weaned F1 pups were housed together by litter until scheduled euthanasia on Postnatal Day (PND) 24 or 27. If no evidence of pregnancy, females were placed in plastic maternity cages with nesting material after the mating period.
-Housing (exposure periods): For daily exposures, rats were transferred from home cages to exposure chamber caging in the animal room, transported to the exposure room, exposed, and then returned to their home cages. Cages were sequentially rotated daily around the available rack positions within the chamber.
- Diet: PMI Nutrition International, LLC Certified Rodent Lab Diet® 5002 (meal) ad libitum except during exposures.
- Water: Reverse osmosis-treated tap water was available ad libitum.
- Method of animal distribution: Suitable animals were selected for use in a computerized randomization procedure using body weight stratification in a block design. Individual body weights at randomization were within ± 20% of the mean for each sex. All groups consisted of 10 animals/sex. Selected animals were approximately 6 weeks old at the start of exposure; body weights ranged from 182 – 219 g for F0 males and 149 – 186 g for F0 females. Body weights for the F1 animals selected for exposure beginning on PND 22 ranged from 29 -59 g for males and 21 -57 g for females.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 10 room air changes per hour, 100% fresh air except during exposure. Exposure chambers were operated under dynamic conditions at a slight negative pressure with at least 12 to 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours light/dark

IN-LIFE DATES:
- Animal receipt: 27 Sept 2005 to necropsy 20 Feb 2006

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: filtered air
Details on exposure:
Tertiary butyl acetate was generated as a vapor using a glass-bead column-type vaporization system. The liquid test material was metered to the column via a laboratory pump. Nitrogen carrier gas was metered to the bottom of the column and the column was heated to an appropriate temperature to create vaporization of the test material. The concentrated vapors were piped to the chamber inlet where the concentration was reduced by mixing with chamber ventilation air.

Exposures were conducted in four stainless steel and glass exposure chambers. Three chambers were dedicated for the test material exposure groups, while the fourth chamber was dedicated for the filtered air control group. All chambers were operated under dynamic conditions at a slight negative pressure with at least 12 to 15 air changes per hour. A HEPA filter and an activated charcoal bed were used to treat chamber air supply. Filtered-air and test material was administered as six-hour, whole-body exposures for 7 days per week. All animals were exposed at approximately the same time each day.

F0 males and females were exposed to the test atmosphere for 6 hours daily for 70 consecutive days prior to mating. Target test material concentrations were 0, 100, 400 and 1600 ppm. Mean analyzed exposure concentrations for F0 animals in this phase were 101.3, 401.3 and 1603.9 ppm. Exposure of the F0 males continued throughout mating and through the day prior to euthanasia for a total of 109-110 days of exposure. The F0 females continued to be exposed throughout mating and gestation through Gestation Day 20; after parturition, exposure of F0 females was re-initiated on Lactation Day 5 to avoid confounding effects on nursing behavior and continued through the day prior to euthanasia. The F0 females that delivered were exposed for a total of 108-119 days. Females with evidence of mating that failed to deliver were exposed through Post-mating Day 25 for a total of 97-104 days. The females with no evidence of mating were exposed through the day prior to euthanasia (Post-cohabitation Day 25) for a total of 108 days. During lactation, the dams were removed from their litters during each daily 6-hour exposure period. One F1 pup/sex/litter was selected for inhalation exposure beginning on PND 22 (following weaning) and continuing through PND 26 (total of 5 days of exposure). Mean analyzed exposure concentrations for the F1 generation were 99.8, 402.7 and 1613.1 ppm. The control group animals were exposed to clean, filtered air under conditions identical to those used for the groups exposed to the test material.

Since the study report used target rather than actual analytical concentrations in reporting results and since the target and analytical concentrations were in close agreement, all discussions and conclusions in this IUCLID document have used target concentrations.
Details on mating procedure:
The F0 animals were paired on a 1:1 basis within each exposure group after 70 days of exposure. All animals were randomly selected for pairing, avoiding sibling matings. A breeding record containing the male and female identification numbers and the start date of cohabitation was prepared. Each female was housed in the home cage of the male. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage. Each mating pair was examined daily. The day when evidence of mating was identified was termed Gestation Day 0. The animals were separated, and the female was housed in an individual plastic cage with nesting material. When evidence of mating was not apparent after 14 days, the female was placed in a plastic maternity cage with nesting material, with no further opportunity for mating.

The F0 generation was mated once to produce 1 litter (the F1 litters). Prior to the F0 pairing (Study Day 69), male body weights ranged from 392-555 g and female body weights ranged from 251-351 g. Animals were approximately 16 weeks old. All animals were randomly selected for pairing. Gestation Day 0 body weights ranged from 266-384 g. For the purpose of calculating pre-coital intervals, rats paired over a 12-hr dark cycle were considered to have been paired for 1 day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Nominal Concentrations:
A nominal exposure concentration was calculated for each daily exposure for each chamber from the total amount of tertiary butyl acetate used during the exposure and the total volume of air passed through the chamber during that day's exposure. The amount of test material used was obtained by weighing the reservoir containing the test material for each chamber prior to and after each day's exposure. The total volume of air passed through each chamber was calculated from the daily average chamber ventilation flow rate in liters per minute (LPM) and the exposure duration. The overall mean nominal concentrations (ppm) were 107, 431 and 1787 for the F0 generation and 111, 434 and 1749 for the F1 generation, respectively.

Analyzed Exposure Concentrations:
Analyzed exposure concentrations were determined at least 10 times during each 6-hour exposure using a gas chromatographic (GC) method with flame ionization detection. Samples of the exposure atmospheres were automatically collected at approximately 35-minute intervals using a sample loop and a computer-controlled multiposition valve. The overall mean analyzed concentrations (ppm) were 101.3, 401.3 and 1603.9 for the F0 generation and 99.8, 402.7 and 1613.1 for the F1 generation, respectively.

Purity Analysis:
A sample of the test material was collected from each primary test material container prior to initial use and near the end of use. The percent concentration of tertiary butyl acetate was determined by the Analytical Chemistry Department of WIL Research Laboratories using a gas chromatographic method with detection by flame ionization for each sample. Purity of tertiary butyl acetate was expressed as area percent.
Duration of treatment / exposure:
6 hrs/day
Frequency of treatment:
7 days/week
Details on study schedule:
Four groups of male and female Crl:CD(SD) rats (10/sex/group) were exposed to either clean filtered air or vapor atmospheres of the test material, tertiary butyl acetate, for 6 hours daily for 70 consecutive days prior to mating. Target test material concentrations were 0, 100, 400 and 1600 ppm. Mean measured exposure concentrations for F0 animals in this phase were 101.3, 401.3 and 1603.9 ppm. Exposure of the F0 males continued during the mating period and through the day prior to euthanasia for a total of 109-110 days of exposure. The F0 females continued to be exposed throughout mating and gestation through Gestation Day 20. After parturition, exposure of the F0 females was re-initiated on Lactation Day 5 and continued through the day prior to euthanasia. The F0 females that delivered were exposed for a total of 108-119 days. Females with evidence of mating that failed to deliver were exposed through Post-mating Day 25 for a total of 97-104 days. The females with no evidence of mating were exposed through the day prior to euthanasia (Post-cohabitation Day 25) for a total of 108 days. The F1 offspring (1/sex/litter) selected for control or test material exposure were exposed following weaning, beginning on PND 22 through PND 26 for a total of 5 days of exposure. Target test material concentrations were 0, 100, 400 and 1600 ppm. Mean measured exposure concentrations for the F1 generation were 99.8, 402.7 and 1613.1 ppm.
All F0 animals were observed twice daily for mortality and moribundity. Clinical observations, body weights and food consumption were recorded at appropriate intervals for F0 males throughout the study and for F0 females prior to mating and during gestation and lactation. Vaginal lavages were performed daily for determination of estrous cycles in F0 females beginning 3 weeks prior to pairing. All F0 females were allowed to deliver and rear their pups until weaning on Lactation Day 21. Clinical observations, body weights and sexes for F1 pups were recorded at appropriate intervals. Litters were culled to 10 pups per litter (5 per sex, when possible) on PND 4 to reduce the variability among the litters. Developmental landmarks (incisor eruption, surface righting response, hair growth, pinnal detachment and eye opening) were evaluated for each F1 pup. Offspring (1/sex/litter) from the pairing of the F0 animals were selected on PND 21 to constitute the F1 generation. F1 pups not selected for exposure were necropsied on PND 24, and exposed F1 weanlings were necropsied on PND 27 (following 5 days of exposure). F0 males were euthanized following completion of parturition and F0 females were euthanized on Lactation Day 21. Complete necropsies were conducted on all F0 animals, and selected organs were weighed for F0 males.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
100 ppm
Basis:
other: target concentration
Remarks:
Doses / Concentrations:
400 ppm
Basis:
other: target concentration
Remarks:
Doses / Concentrations:
1600 ppm
Basis:
other: target concentration
No. of animals per sex per dose:
10 animals/sex/group for F0
1 animal/sex/group for F1
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The exposure levels were based on the results of a 14-day range-finding study
- Rationale for animal assignment: At the conclusion of the acclimation period, F0 animals judged to be suitable for testing were randomly assigned to groups based on body weight. Individual body weights were within +/- 20% of the mean for each sex and there were no statistically significant differences among the groups (i.e., p-value for ANOVA not significant at p < 0.05).     

Examinations

Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS:
Animals were observed twice daily for mortality and moribundity. Clinical observations were conducted prior to exposure and 0-1 hour following the exposure on each exposure day. Detailed physical examinations were recorded weekly for all parental animals throughout the study period. 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. At approximately the midpoint of each 6-hour exposure, animals that were visible through the chamber windows were observed for appearance and behavior.

BODY WEIGHT:
Individual F0 male body weights were recorded weekly, beginning 1 week prior to the initiation of test material exposure and continuing throughout the study and prior to the scheduled necropsy. Individual F0 female body weights were recorded weekly 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, 3, 6, 9, 12, 15, 18 and 20 and on Lactation Days 1, 4, 7, 14 and 21. Body weight changes were also recorded for the entire gestation and lactation intervals (Days 0-20 and 1-21, respectively).

FOOD CONSUMPTION:
Individual F0 male and female food consumption was measured weekly, beginning 1 week prior to the initiation of test material exposure and continuing until pairing. Food intake was not recorded during the mating period. Following mating, male food consumption was measured on a weekly basis until the scheduled necropsy. Female food consumption was recorded on Gestation Days 0, 3, 6, 9, 12, 15, 18 and 20 and Lactation Days 1, 4, 7, 14 and 21. For females with no evidence of mating, food consumption continued to be recorded once weekly until euthanasia.
Oestrous cyclicity (parental animals):
Vaginal lavages were performed daily and the slides were evaluated to assess the regularity and duration of the estrous cycles of each F0 female for 21 days prior to pairing and continuing until evidence of mating was observed or until the end of the mating period. The average cycle length was calculated for complete estrous cycles.
Sperm parameters (parental animals):
Immediately upon euthanasia, the reproductive tract of each F0 male was exposed via a ventral mid-line incision. The right epididymis was excised and weighed. An incision was made in the distal region of the right cauda epididymis. The right cauda epididymis was then placed in Dulbecco's phosphate-buffered saline (maintained at approximately 37 ºC) with 10 mg/mL bovine serum albumin (BSA). After a 10-minute incubation period, a sample of sperm was loaded into a 100-μm cannula for determination of sperm motility. Because sperm motility can be affected by temperature shock, all cannulas and diluents were warmed in an incubator, and motility determinations were performed under constant temperature (approximately 37ºC) using the Hamilton-Thorne HTM-IVOS Version 12.2 computer-assisted sperm analysis (CASA) system. Analysis of a minimum of 200 motile and nonmotile spermatozoa per animal (if possible) in all groups was performed by the analyzer. The motility score (percent) for motile (showing motion only) and progressively motile (showing net forward motion) sperm was reported. Sperm morphology was evaluated by light microscopy via a modification of the wet-mount evaluation technique (Linder et al., 1992). Abnormal forms of sperm (double heads, double tails, microcephalic or megacephalic, etc.) from a differential count of 200 spermatozoa per animal, if possible, were recorded.

The left testis and epididymis from all F0 males from all test material-exposed groups were weighed, stored frozen, homogenized and evaluated for determination of homogenization-resistant spermatid count and calculation of sperm production rate (Blazak et al., 1985) using the Hamilton-Thorne CASA system. An aliquot of each sample was added to a solution containing a DNA-specific fluorescent dye (the dye stains DNA that is present in the head of the sperm). For analysis, each sample was mixed, and an aliquot was placed on a slide with a 20-μm chamber depth. Illumination from a xenon lamp within the HTM-IVOS analyzer allowed for the visualization and quantitation of the sperm. A minimum of 200 cells, if possible, or 20 fields were counted for each sample. The sperm production rate was calculated.

Sperm Indices:

-Percent Motile (or Progressively Motile) Sperm= Number of Motile (or Progressively Motile) Sperm/Total Number of Sperm Counted X 100


-Sperm Production Rate=Number of Sperm per Gram of Testis/6.1 days
6.1 days=The rate of turnover of the germinal epithelium

References:
Linder et al., 1992. Endpoints of spermatotoxicity in the rat after short duration exposures to fourteen reproductive toxicants. Reproductive Toxicology, 6:491-505.

Blazak et al., 1985. Potential indicators of reproductive toxicity: testicular sperm production and epididymal sperm number, transit time and motility in Fischer 344 rats. Fundamental and Applied Toxicology, 5:1097-1103.
Litter observations:
LITTER VIABILITY:
Each litter was examined twice daily for survival, and all deaths were recorded. All pups were individually identified by application of tattoo markings on the digits following completion of parturition on PND 0. A daily record of litter size was maintained.

LITTER REDUCTION:
To reduce variability among the litters, 10 pups per litter, 5 per sex when possible, were randomly selected on PND 4. Standardization of litter size was not performed on litters with fewer than 10 pups. All selections were performed by computerized randomization. The remaining offspring were weighed, euthanized by an intraperitoneal injection of sodium pentobarbital and discarded on PND 4.

CLINICAL OBSERVATIONS:
Litters were examined daily for survival and any adverse changes in appearance or behavior. Each pup received a detailed physical examination on PND 1, 4, 7, 14 and 21. Any abnormalities in nursing behavior were recorded. Detailed physical examinations were also recorded prior to necropsy for F1 weanlings (1/sex/litter) selected for exposure on PND 27.

BODY WEIGHTS:
Pups were individually weighed on PND 1, 4, 7, 14 and 21. Mean pup weights were presented by sex for each litter and by group. F1 weanlings (1/sex/litter) selected for exposure were also weighed daily from PND 22-27.

SEX DETERMINATION:
Pups were individually sexed on PND 0, 4 and 21
Postmortem examinations (parental animals):
MACROSCOPIC EXAMINATION:
A gross necropsy was conducted on all F0 animals euthanized in extremis by isoflurane inhalation and exsanguination, or at termination. The necropsy included examination of the external surface, all orifices, the cranial cavity, and the thoracic, abdominal and pelvic cavities, including viscera. For each F0 male euthanized at the scheduled necropsy, the right testis and epididymis were also fixed in Bouin’s solution and retained in 10% neutral-buffered formalin for possible future histopathological examination. For females that failed to deliver, a pregnancy status was determined, and specific emphasis was placed on anatomic or pathologic findings that may have interfered with pregnancy. Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss. For females that delivered or had macroscopic evidence of implantation, the numbers of former implantation sites were recorded. The number of unaccounted for sites was calculated for each female by subtracting the number of pups born from the number of former implantation sites observed.

ORGAN WEIGHTS:
The testes and epididymides (total and cauda) were weighed separately for all F0 males at the scheduled necropsy.
Postmortem examinations (offspring):
F1 LITTER:
Intact offspring dying or euthanized in extremis (by intraperitoneal injection of sodium pentobarbital) from PND 0 to 4 were necropsied using a fresh dissection technique (Stuckhardt and Poppe, 1984). Findings were recorded as either developmental variations or malformations. A detailed gross necropsy was performed on any pup dying after PND 4 and prior to weaning.

Reference:
Stuckhardt JL and Poppe SM, 1984. Fresh visceral examination of rat and rabbit fetuses used in teratogenicity testing. Teratogenesis, Carcinogenesis and Mutagenesis, 4:181-188.

EUTHANASIA OF F1 GENERATION:
F1 pups not selected for exposure were euthanized via carbon dioxide inhalation followed by exsanguination on PND 24 and examined internally for gross abnormalities. F1 weanlings selected for exposure were euthanized via carbon dioxide inhalation followed by exsanguination on PND 27 and were given a complete necropsy. This necropsy included examination of the external surface, all orifices, the cranial cavity, the external surface of the brain, the thoracic, abdominal and pelvic cavities, including viscera, and the lymph nodes, thyroid glands, mammary glands and reproductive organs. All carcasses were then discarded.
Statistics:
Analyses were conducted using two-tailed tests (except as noted otherwise) for a minimum significance level of 5%, comparing each test material-exposed group to the control group by sex. Parental mating and study indices were analyzed using the Chi-square test with Yates’ correction factor (Hollander and Wolfe, 1999). Clinical pathology data, mean body weights, body weight changes and food consumption (reproductive toxicity phase), estrous cycle lengths, pre-coital intervals, gestation lengths, implantation sites, live litter sizes, unaccounted-for sites, numbers of pups born, F1 developmental landmark data, absolute and relative organ weights, sperm production rates, epididymal and testicular sperm numbers were analyzed for heterogeneity of variance using Levene’s test (Levene, 1960) and for normality using the Shapiro-Wilk test (Royston, 1982). If the data were homogenous and normal, the data were subjected to a parametric 1-way analysis of variance (ANOVA) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test material-exposed groups to the control group. If the data were not homogeneous and normal, the Kruskal-Wallis nonparametric ANOVA test (Kruskal and Wallis, 1952) was used to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance the Dunn’s test (Dunn, 1964) was used as above.

Statistics section continued below in "Any other information on materials and methods".
Reproductive indices:
Mating, fertility, copulation and conception indices were calculated as follows:

-Male (Female) Mating Index (%) = Number of Males (Females) with evidence of Mating (or Females Confirmed pregnant)/ Total number of Males (Females) Used for mating X 100

-Male Fertility Index (%) = Number of Males Siring a Litter/Total Number of Males used for Mating X 100

-Male Copulation Index (%) =Number of Males Siring a Litter/Number of Males with Evidence of Mating (or Females confirmed pregnant) X 100

-Female Fertility Index (%) = Number of Females with Confirmed Pregnancy/ Total Number of Females used for Mating X 100

-Female Conception Index (%) =Number of Females with Confirmed Pregnancy/Number of Females with Evidence of Mating (or Females Confirmed Pregnant) X 100
Offspring viability indices:
Litter parameters were defined as follows:

-Mean Live Litter Size = Total Viable Pups on PND 0/ Number of Litters with Viable Pups on PND 0

-Postnatal Survival Between Birth and PND 0 or PND 4 (Pre-Selection) (%Per litter) =∑(Viable Pups Per Litter on PND 0 or PND 4/Number of Pups Born Per Litter)/ Number of Litter per group X 100

-Postnatal Survival for All Other Intervals (% per litter) =∑ (Viable Pups per Litter at End of Interval N/Viable Pups per Litter at Start of Interval N)/ Number of Litters per Group X 100
n=PND 0-1, 1-4 (Pre-selection), 4 (Post-selection)-7, 7-14, 14-21 or 4 (Post-selection)-21

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL OBSERVATIONS AND SURVIVAL:
One F0 male in the 400 ppm group was euthanized in extremis on Study Day 85 (following the mating period) after exhibiting reduced body weight gains and body weight losses; clinical findings consisted of red material in the cage pan and unkempt appearance during or 1 hour following exposure on the day of euthanasia. Macroscopic findings consisted of dilated pelves of the kidneys and ureters, calculi of the kidneys and bladder and a distended bladder. Based on these findings, and the lack of mortality at 1600 ppm, the moribund condition of the male in the 400 ppm group was attributed to underlying renal disease and not to test material exposure. All other F0 males and all F0 females in the control, 100, 400 and 1600 ppm groups survived to the scheduled necropsy.
There were no test material-related clinical observations at the daily examinations, at the midpoint of exposure or 1 hour following exposure. All clinical findings in the test material-exposed groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner and/or were common findings for laboratory rats of this age and strain.

BODY WEIGHTS:
A. PRE-MATING:
Statistically significant, test material-related lower mean weekly body weight gains were noted in the 1600 ppm group F0 males compared to the control group during the first 3 weeks of test material exposure. Mean weekly body weight gains in this group were similar to the control group for the remainder of the study; however, the initial reductions were of sufficient magnitude to cause slightly lower (not statistically significant) mean cumulative body weight gains compared to the control group during the premating period (Study Days 0-69) and the entire generation (Study Days 0-105). Corresponding lower mean body weights (5.1% to 9.0% lower than the control group) were noted for the 1600 ppm group males during Study Days 7-69; the differences were generally statistically significant.
No test material-related effects on mean body weights, body weight gains and cumulative body weight gains were noted in the 100 and 400 ppm group males or in the 100, 400 or 1600 ppm group females. The values in the test material-exposed groups were generally similar to the control group values for the pre-mating period (females) or the entire generation (males). None of the differences from the control group were statistically significant.

B. GESTATION:
Mean maternal body weights, body weight gains and cumulative body weight gains were unaffected by test material exposure during gestation.

C. LACTATION:
Mean body weight gain in the 1600 ppm group females was lower than the control group during Lactation Days 4-7 (4 g vs. 15 g, respectively). The difference from the control group was not statistically significant, but corresponded to the re-initiation of test material exposure and was considered test material-related, but transient and of unlikely toxicological significance as mean body weight gain in this group was similar to the control group by the next week of lactation and was slightly higher than the control group for the entire lactation period. Furthermore, mean body weights in the 1600 ppm group were similar to the control group throughout lactation; none of the differences were statistically significant.
Mean maternal body weights, body weight gains and cumulative body weight gains were unaffected by test material exposure during lactation at concentrations of 100 and 400 ppm. Differences between the control, 100 and 400 ppm groups were slight, not statistically significant and/or did not occur in an exposure-related manner.

FOOD CONSUMPTION:
A. PRE-MATING:
Slightly lower mean food consumption (2-3 g less than the control group) was observed for the 1600 ppm group males during the first 7 weeks of test material exposure, when evaluated as g/animal/day. The lower food consumption corresponded to lower body weight parameters during Study Days 0-21; however, when food consumption was normalized for body weight (i.e., g/kg/day), the values in the 1600 ppm group were slightly lower only during the first week of test material exposure. Food consumption in the 1600 ppm group was generally similar to the control group after Study Week 7. Food consumption, evaluated as g/animal/day and g/kg/day, in the 100 and 400 ppm group males and the 100, 400 and 1600 ppm group females was unaffected by test material exposure.

B. GESTATION:
Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, was unaffected by test material exposure during gestation. Statistically significantly higher food consumption was noted for the 1600 ppm group compared to the control group during Gestation Days 6-9 (g/animal/day and g/kg/day), 9-12 and 15-18 (g/kg/day); however, the differences were slight, had no effect on body weight parameters and were not considered test material-related. There were no statistically significant differences between the control, 100 and 400 ppm groups.

C. LACTATION:
Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, was unaffected by test material exposure during lactation.

ESTROUS CYCLES:
Estrous cycles were unaffected by test material exposure.

SPERMATOGENIC ENDPOINT EVALUATIONS:
No test material-related effects were observed on spermatogenesis endpoints in F0 males at any exposure concentration. Apparent reductions (not statistically significant) in mean cauda epididymal sperm concentrations were noted at 400 and 1600 ppm compared to the control group; however, the data in all groups were variable and showed a great deal of overlap, with individual animal concentrations (millions/gram) that ranged from 451.7 to 794.7 in the control group, 356.6 to 728.0 in the 400 ppm group and 290.2 to 665.4 in the 1600 ppm group. Because there were no effects on other spermatogenesis endpoints (mean testicular sperm numbers, sperm production rate, motility and progressive motility) or on sperm morphology and there were no microscopic findings noted during examination of the epididymides in the subchronic toxicity phase, the lower mean cauda epididymal sperm concentrations in the 400 and 1600 ppm groups were not considered test material-related. Rather, these apparent reductions were likely a result of the small sample size for the reproductive toxicity phase (N=9 or N=10).

REPRODUCTIVE PERFORMANCE:
No test material-related effects on F0 reproductive performance were observed at any exposure concentration. Male and female mating indices were 80.0% in the control group and 100% in the 100, 400 and 1600 ppm groups. Male and female fertility indices were 70.0%, 100.0%, 70.0% and 90.0% in the control, 100, 400 and 1600 ppm groups, respectively. Male copulation and female conception indices were 87.5%, 100.0%, 70.0% and 90.0% in the same respective groups. No statistically significant differences were noted between the control and test material-exposed groups. The mean numbers of days between pairing and coitus in the test material-exposed groups were similar to the control group value.

GESTATION LENGTH AND PARTURITION:
No test material-related effects were noted on mean gestation lengths or the process of parturition at any exposure concentration. Mean F0 gestation lengths in the test material-exposed groups were similar to the control group value. Differences were slight and were not statistically significant. The mean gestation lengths in the 100, 400 and 1600 ppm groups were 21.7, 22.3 and 22.0 days, respectively, compared to mean gestation lengths of 22.0 days in the concurrent control group and 21.9 days in the WIL comprehensive historical control data. No signs of dystocia were noted at any exposure concentration.

ORGAN WEIGHTS:
No test material-related effects on testis, epididymis or cauda epididymis weights (absolute and relative to final body weight) were observed at any exposure concentration when the test material-exposed groups were compared to the control group. Differences from the control group were slight, not statistically significant and/or did not occur in an exposure-related manner.

MACROSCOPIC EXAMINATION:
There was one F0 male in the 400 ppm group that was euthanized in extremis on Study Day 85. Macroscopic findings for this male (dilated pelves of the kidneys and ureters, calculi of the kidneys and bladder and a distended bladder) were indicative of underlying renal disease. Based on these findings, and the lack of mortality at 1600 ppm, the moribund condition of this male in the 400 ppm group was attributed to underlying pathology and not to test material exposure.
At the scheduled F0 male and female necropsies, no test material-related internal findings were observed at any exposure concentration. Macroscopic findings observed in the test material-exposed groups occurred infrequently, at similar frequencies in the control group and/or in a manner that was not exposure-related.
The mean number of unaccounted-for sites in the 1600 ppm group (2.0 per dam) was higher than the concurrent control group value (0.7 per dam), primarily due to a single female that had 8 unaccounted for sites, which is atypically high. The difference from the control group was not statistically significant; therefore, the apparent increase in the number of unaccounted-for sites in the 1600 ppm group was considered to be a function of a sampling effect, resulting from the limited number of females available for evaluation in this screening study.
No test material-related effects were observed on the number of former implantation sites at all exposure concentrations and the number of unaccounted-for sites at 100 and 400 ppm. None of the differences from the concurrent control group were statistically significant, and the values in these groups were within the range of values in the WIL historical control data for definitive inhalation studies.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEC
Effect level:
400 ppm
Sex:
male
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEC
Effect level:
1 600 ppm
Sex:
female
Basis for effect level:
other: No statistically significant test material-related adverse effects were noted for any of the parameters investigated in this screening study.
Dose descriptor:
other: NOAEC reproductive and neonatal toxicity
Effect level:
1 600 ppm
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: Generation: Parental and F1 (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

PND 0 LITTER DATA AND POSTNATAL SURVIVAL:
The mean number of pups born, live litter size, percentage of males per litter at birth and postnatal survival on PND 0 (relative to number born), PND 0-1, 1-4 (pre-selection), 4 (post-selection)-7, 7-14, 14-21, and from birth to PND 4 (pre-selection) and PND 4 (post-selection) to PND 21 were unaffected by test material exposure at 100, 400 and 1600 ppm.

CLINICAL OBSERVATIONS AND SURVIVAL:
Pups (litters) that were found dead numbered 1(1), 7(6), 2(2) and 8(3) in the control, 100, 400 and 1600 ppm groups, respectively. Five pups in the 100 ppm group and 1 pup in the 400 ppm group were missing and presumed to have been cannibalized. The general physical condition of F1 pups in this study was unaffected by parental test material exposure.

BODY WEIGHTS:
There were no test material-related effects on offspring body weights or body weight gains at any exposure level.

DEVELOPMENTAL LANDMARKS:
A. PINNAL DETACHMENT:
Pinnal detachment in the F1 male and female pups was not affected by parental test material exposure. All pups in the control, 100, 400 and 1600 ppm groups were positive for pinnal detachment by PND 4, 6, 5 and 4, respectively (males) or PND 5, 5, 4 and 5, respectively (females).

B. SURFACE RIGHTING RESPONSE:
Surface righting response in the F1 male and female pups was not affected by parental test material exposure. The mean ages of attainment were 5.2, 5.1, 5.3 and 5.2 days for F1 males, and 5.3, 5.2, 5.4 and 5.2 days for F1 females in the control, 100, 400 and 1600 ppm groups, respectively.

C. HAIR GROWTH:
Hair growth in the F1 male and female pups was not affected by parental test material exposure. The mean ages of attainment were 15.6, 15.7, 15.3 and 15.4 days for F1 males, and 15.5, 15.6, 15.2 and 15.3 days for F1 females in the control, 100, 400 and 1600 ppm groups, respectively.

D. INCISOR ERUPTION:
Incisor eruption in the F1 male and female pups was not affected by parental test material exposure. The mean ages of attainment were 10.9, 11.1, 10.8 and 11.0 days for F1 males, and 10.8, 10.9, 10.6 and 10.9 days for F1 females in the control, 100, 400 and 1600 ppm groups, respectively.

E. EYE OPENING:
Eye opening in the F1 male and female pups was not affected by parental test material exposure. The mean ages of attainment were 15.2, 15.6, 14.7 and 15.4 days for F1 males, and 15.1, 15.4, 14.7 and 15.2 days for F1 females in the control, 100, 400 and 1600 ppm groups, respectively.

NECROPSIES OF PUPS FOUND DEAD:
No internal findings that could be attributed to parental exposure to the test material were noted at the necropsies of pups that were found dead.

NECROPSIES OF WEANLINGS NOT SELECTED FOR EXPOSURE:
No internal findings that could be attributed to parental exposure with the test material were noted at the necropsy of pups euthanized on PND 24.

F1 GENERATION
CLINICAL OBSERVATIONS AND SURVIVAL:
No exposure-related clinical findings were noted at the detailed physical examinations or at the midpoint of exposure and 1-hour following exposure. The only findings in the test material-exposed groups (hypoactivity, rocking, lurching or swaying while walking and/or body cool) occurred in single females in the 100 and 1600 ppm groups 1 hour following exposure. Because these findings were noted in single females on a single occasion they were not considered test material-related.

BODY WEIGHTS:
Statistically significant reductions in mean body weight gain were noted for the 1600 ppm group F1 males and females following the initiation of exposure (PND 22-23). These reductions were considered test material-related; however, they were very slight (2 grams), transient and had no effect on mean body weights. There were no test material-related effects on mean body weights or body weight gains for F1 males and females at 100 and 400 ppm. Differences from the control group were slight and not statistically significant.

NECROPSIES OF WEANLINGS SELECTED FOR EXPOSURE:
All F1 males and females selected for direct test material exposure survived to the scheduled necropsy on PND 27. There were no internal findings noted for any animals.

Effect levels (F1)

Dose descriptor:
other: NOAEC reproductive and neonatal toxicity
Generation:
F1
Effect level:
1 600 ppm
Sex:
male/female
Basis for effect level:
other: There were no test material-related effects on survival and development of the F1 pups. Therefore, the NOAEC for reproductive and neonatal toxicity was considered to be 1600 ppm, the highest exposure level evaluated.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
There were no test material-related effects on mating performance; fertility; length of estrous cycle; sperm numbers, production rate, motility or morphology; or reproductive organs in a reproduction and developmental toxicity screening test in which adult male and female rats were exposed via inhalation to target concentrations of 0, 100, 400 or 1600 ppm tertiary butyl acetate for 6 hours/day, 7 days/week during premating, mating, gestation, and lactation for a total of 109-110 exposure days for males and 97-119 exposure days for females. There were no adverse effects on mean number of pups born, live litter size, percentage of males per litter, postnatal survival, pup body weights or body weight gains. Developmental landmarks, including pinnal detachment, attainment of surface righting response, hair growth, eye opening and incisor eruption were not affected by parental exposure to the test material and there were no gross malformations noted at pup necropsies. The NOAEC for reproductive and neonatal toxicity was considered to be 1600 ppm, the highest exposure level tested.

In a well-conducted reproductive and developmental toxicity screening study in rats, there was no clear evidence of test-substance related effects on reproduction of F0 males and females or on survival and development of F1 pups. Based on this information, tertiary butyl acetate is not a reproductive toxicant and it is not selectively toxic to the developing fetus. Tertiary butyl acetate is not classified for “Developmental or Reproductive Toxicity” according to Directive 67/548/EEC, the UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS) or the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.
Executive summary:

In a combined subchronic and reproductive toxicity screening test, groups of 10 adult Crl:CD®(SD) rats/sex/dose/test type were exposed to tertiary butyl acetate via inhalation at target concentrations of 0, 100, 400 and 1600 ppm. In the subchronic study, adult animals were exposed 6 hr/day, 7 days/wk for 13 weeks. Separate groups of adults in the reproductive toxicity screening study were similarly exposed during the premating, mating, gestation and lactation (excluding Days 0-4) phases (actual exposure concentrations were 0, 101.3, 401.3 and 1603.9 ppm for the F0 generation and 0, 99.8, 402.7 and 1613.1 ppm for the F1 generation). Selected F1 pups were directly exposed for 5 days following weaning. In the screening study, no statistically significant test material-related effects were observed in F0 females at tertiary butyl acetate concentrations up to 1600 ppm; effects in F0 males were limited to lower mean weekly body weight gains and body weights at 1600 ppm. Adverse effects in pups were limited to slight, transient reductions in mean body weight gain for F1 males and females in the 1600 ppm group exposed on PND 22-26. In the subchronic study, the most significant adverse effects were higher liver and adrenal gland weights in the 1600 ppm females and alpha-2-u-globulin nephropathy in all male groups. The NOAECs for systemic toxicity in the screening study were 400 ppm in adult males and 1600 in adult females while the NOAECs in the subchronic study were <100 ppm and 400 ppm, respectively, for males and females. The higher NOAECs in the screening study may be explained by the absence of examination of male kidney pathology and female organ weights. There were no test material-related effects on reproduction of the F0 males and females, or on survival and development of the F1 pups. Therefore, the NOAEC for reproductive and neonatal toxicity was considered to be 1600 ppm in the screening study, the highest exposure level evaluated.