Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
54074-94-1
Cas Number:
54074-94-1
IUPAC Name:
54074-94-1
Constituent 2
Reference substance name:
isopropyl-3-hydroxybutyrate
IUPAC Name:
isopropyl-3-hydroxybutyrate
Test material form:
gas under pressure: refrigerated liquefied gas
Details on test material:
The test item, Isopropyl-3-Hydroxybutyrate (IPHB), was received from the Sponsor, on
30 July 2012, as follows:

IDENTIFICATION: IPHB; Lot no. 30705-45-df; Exp. date: 17 July 2014; [WIL ID no. 120125]
PHYSICAL DESCRIPTION: Colorless, clear liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
Sexually mature male and virgin female Sprague Dawley [Crl:CD(SD)] rats were used as the test system on this study. The animal model, the Crl:CD(SD) rat, is recognized as appropriate for reproductive toxicity studies. WIL Research has reproductive historical control data in the Crl:CD(SD) rat. This animal model has been proven to be susceptible to the effects of reproductive toxicants. The number of animals selected for this study was based on the OECD Guideline for Testing of Chemicals: Guideline 422, Combined Repeated Dose Toxicity Study with Reproduction/Developmental Toxicity Screeing Test, 22 March 1996, which recommends evaluation of at least 8 pregnant females per group. Given the possibility of nongravid animals, unexpected deaths, total litter losses, or treatment-related moribundity and/or mortality, this is an appropriate number of animals to obtain a sample size of 8 at termination.

Crl:CD(SD) rats (53 males and 106 females) were received in good health from Charles River Laboratories, Inc., Raleigh, NC on 24 July 2012. The animals were approximately 58 days old upon receipt. Each animal was examined by a qualified biologist on the day of receipt. The day following receipt, all animals were weighed and clinical observations were recorded. Each rat was uniquely identified by a Monel® metal ear tag displaying the animal number. The animals were housed for an acclimation period of 14 days prior to the first day of treatment. During the acclimation period, the animals were observed twice daily for mortality and general changes in appearance and behavior.

HOUSING
Following receipt and until pairing, all F0 animals were housed individually in clean, stainless steel wire-mesh cages suspended above cage-board. The cage-board was changed at least 3 times per week. The toxicity phase females were not paired and remained in clean, stainless steel wire-mesh cages until euthanasia. The males and reproductive phase females were paired for mating in the home cage of the male. Following positive evidence of mating, the males were housed in suspended wire-mesh cages until the scheduled necropsy, and the females were transferred to plastic maternity cages with nesting material, ground corncob bedding (Bed-O'Cobs®; The Andersons, Cob Products Division, Maumee, OH). The nesting material is periodically analyzed by the manufacturer for contaminants. No contaminants were present in the bedding at concentrations sufficient to interfere with the outcome of the study. The results of these analyses are maintained at WIL Research. The dams and their litters were housed in these cages until euthanasia on lactation day 4. Females with no evidence of mating or that failed to deliver were housed in plastic maternity cages until post-cohabitation or post-mating day 25. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). The animal facilities at WIL Research are accredited by AAALAC International. Enrichment devices (Nylabones®) were provided to all animals as appropriate throughout the study, except during the mating period. Testes were palpated at least once.

DIET, DRINKING WATER, AND MAINTENANCE
The basal diet used in this study, PMI Nutrition International, LLC Certified Rodent LabDiet® 5002, was a certified feed with appropriate analyses performed by the manufacturer and provided to WIL Research. Feed lots used during the study were documented in the study records. Feeders were changed and sanitized once per week. Municipal water supplying the facility was sampled for contaminants according to WIL Research’s SOPs. The results of the diet and water analyses are maintained at WIL Research. No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study. Reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system, and the basal diet
were provided ad libitum throughout the acclimation period and during the study, with the following exception. All males and toxicity phase females (including those not scheduled for clinical pathology assessments) were fasted overnight prior to the scheduled clinical pathology blood collections.

ENVIRONMENTAL CONDITIONS
All rats were housed throughout the acclimation period and during the study in an environmentally controlled room. The room temperature and humidity controls were set to maintain environmental conditions of 71°F ± 5°F (22°C ± 3°C) and 50% ± 20%, respectively. Room temperature and relative humidity data were monitored continuously and were scheduled for automatic collection on an hourly basis. These data are summarized in Appendix D. Actual mean daily temperature ranged from 71.0°F to 73.0°F (21.7°C to 22.8°C) and mean daily relative humidity ranged from 40.8% to 74.6% during the study (see Deviations from the Protocol). Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. The light status (on or off) was recorded once every 15 minutes. Air handling units were set to provide a minimum of 10 fresh air changes per hour.

ASSIGNMENT OF ANIMALS TO TREATMENT GROUPS
At the conclusion of the acclimation period, all available males and females were weighed and examined in detail for physical abnormalities. At the discretion of the Study Director, each animal judged to be in good health and meeting acceptable body weight requirements was selected for use in the computerized randomization procedure based on body weight stratification in a block design. At that time, the individual body weights and corresponding animal identification numbers were entered into WTDMS™. A printout containing the animal numbers, corresponding body weights, and individual group assignments was generated. The animals then were arranged into groups according to the printout. Animals not assigned to study were transferred to the WIL Research stock colony or euthanized by carbon dioxide inhalation and discarded. The experimental design for this study consisted of 1 control and 3 test item-treated groups composed of 12 male rats/group and 24 females rats/group; the 24 females/group were separated into 2 phases (toxicity phase females and reproductive phase females). At the initiation of dose administration (study day 0), the selected animals were approximately 10 weeks old. Body weights on study day 0 ranged from 311 g to 417 g for the males, 221 g to 273 g for the toxicity phase females, and 212 g to 273 g for the reproductive phase females. The males and reproductive phase females were approximately 12 weeks old when paired on study day 14; reproductive phase female body weights ranged from 228 g to 299 g on gestation day 0.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
The vehicle and test item formulations were administered orally by gavage, via an appropriately sized flexible, Teflon®-shafted, stainless steel ball-tipped dosing cannula (Natume, Japan) once daily. The males were dosed from study day 0 through study day 27 or 28 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 28-29 doses. The toxicity phase females were dosed from study day 0 through study day 27 or 28 for a total of 28-29 doses. The reproductive phase females were dosed during study day 0 through the day prior to euthanasia (14 days prior to pairing through lactation day 3) for a total of 40-52 doses. Females assigned to the reproductive phase but with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (post-mating or post-cohabitation day 25) for a total of 39-52 doses. The dosage volume for all groups was 10 mL/kg. Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose. All animals were dosed at approximately the same time each day.

The following table presents the study group assignment:

Group Treatment Dosage level Dosage volume Males Females
Number (mg/kg/day) (mL/kg) toxicity phase reproductive phase
1 Vehicle 0 10 12 12 12
2 IPHB 250 10 12 12 12
3 IPHB 500 10 12 12 12
4 IPHB 1000 10 12 12 12

Dosage levels were selected based on the results of a previous 14-day tolerability study (Charlap, 2012, WIL-387027) and were provided by the Sponsor Representative after consultation with the WIL Study Director. In that study, dosages up to the highest level tested (1000 mg/kg/day) were well tolerated. The selected route of administration for this study was oral (gavage) because this is the potential route of exposure for humans. Historically, this route has been used extensively for studies of this nature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyzed dosing formulations were within WIL Research’s SOP range for solutions
(90% to 110%). The test item was not detected in the vehicle formulation that was
administered to the control group (Group 1).
Results of the analyses of dosing formulations are summarized below.

Mean concentration, mg/mL (% of target)
date of preparation group 2 (25 mg/mL) group 3 (50 mg/mL) group 4 (100 mg/mL)
06 August 2012 24.3 (97.1) 49.4 (98.7) 105 (105)
28 August 2012 26.1 (105) 53.5 (107) 103 (103)
20 September 2012 23.5 (94.1) 48.1 (96.1) 97.6 (97.6)
Duration of treatment / exposure:
males: 28 days
females: up to 52 days
Frequency of treatment:
The males were dosed from study day 0 through study day 27 or 28 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 28-29 doses. The toxicity phase females were dosed from study day 0 through study day 27 or 28 for a total of 28-29 doses. The reproductive phase females were dosed during study day 0 through the day prior to euthanasia (14 days prior to pairing through lactation day 3) for a total of 40-52 doses. Females assigned to the reproductive phase but with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (post-mating or post-cohabitation day 25) for a total of 39-52 doses.
Doses / concentrationsopen allclose all
Dose / conc.:
250 mg/kg bw (total dose)
Dose / conc.:
500 mg/kg bw (total dose)
Dose / conc.:
1 000 mg/kg bw (total dose)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
PARAMETERS EVALUATED

SAMPLE COLLECTION FOR BETA-HYDROXYBUTYRATE EVALUATION
Blood samples (approximately 0.5 mL each) for β-Hydroxybutyrate evaluation were collected from each male or toxicity phase female on study days (pre-dose), 1, 2, 3, 4, 7, 14, and 28 at approximately 1 hour prior to dose administration. Blood was collected via the jugular vein using the hand-held restraint method into tubes without anti-coagulant. Serum was isolated in a refrigerated centrifuge, transferred to uniquely labeled polypropylene tubes, and stored frozen at approximately -70°C for future analysis.

CLINICAL OBSERVATIONS AND SURVIVAL (ALL PHASES)
All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Detailed physical examinations were conducted weekly throughout the study and prior to euthanasia. Each male and female was also observed for signs of toxicity approximately 1 hour following dose administration. The absence or presence of findings was recorded for all animals. In addition, the presence of findings at the time of dose administration was recorded for individual animals. 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 WEIGHTS (ALL PHASES)
Individual male and toxicity phase female body weights were recorded weekly (beginning 1 week prior to test item administration) throughout the study, on the days FOB and locomotor activity evaluations were performed, and prior to the scheduled euthanasia. Individual reproductive phase female body weights were recorded weekly (beginning 1 week prior to test item administration) until evidence of copulation was observed. Mean weekly body weights and body weight changes are presented for each interval. In addition, cumulative mean body weight changes are presented for the pre-mating period (males and reproductive phase females) and for the entire generation (males and toxicity phase females). Once evidence of mating was observed, female body weights were recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 0 (when possible), 1, and 4. Mean gestation body weights and corresponding mean body weight changes are presented for these intervals and for the overall gestation interval (days 0-20); mean lactation body weight changes are presented for lactation days 1-4. Weekly body weights for females for which there was no evidence of mating are presented in the individual report tables until necropsy. When body weights could not be determined for an animal during a given interval (as
females enter gestation, weighing error, etc.), group mean values were calculated for that interval using the available data.

FOOD CONSUMPTION (ALL PHASES)
Individual food consumption was recorded on the corresponding weekly body weight days during the entire treatment period for the toxicity phase females. Individual food consumption was recorded on the corresponding weekly body weight days until pairing for males and reproductive phase females. Food intake was not recorded during the mating period for males and reproductive phase females. Once evidence of mating was observed, female food consumption was recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 1 and 4. Following mating, food consumption for females with no evidence of mating was measured on a weekly basis until the scheduled euthanasia and is presented in the individual report tables until necropsy. Food consumption was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals. Calculation of the comprehensive intervals excludes all erroneous values such as total food spillage. When food consumption could not be determined for an animal during a given interval (due to a weighing error, food spillage, etc.), group mean values were calculated for that interval using the available data.

REPRODUCTIVE PERFORMANCE (REPRODUCTIVE PHASE FEMALES)

BREEDING PROCEDURES: The males and reproductive phase females were paired on a 1:1 basis within each treatment group following 14 days of treatment with the test item. A breeding record containing the male and female identification numbers and the start date of cohabitation was maintained. 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 following a vaginal lavage and verified by a second biologist. Each mating pair was examined daily. The day when evidence of mating was identified was termed gestation day 0. If evidence of copulation was not detected after 14 days of pairing, any females that had not shown evidence of mating were placed in plastic maternity cages. For the purpose of calculating pre-coital intervals, rats paired over a 12-hour dark cycle
were considered to have been paired for 1 day.

PARTURITION: All reproductive phase females were allowed to deliver naturally and rear their young to PND 4. During the period of expected parturition, the females were observed twice daily for initiation and completion of parturition and for signs of dystocia. On the day parturition was initiated (PND 0), pups were sexed and examined for gross malformations, and the numbers of stillborn and live pups were recorded. Individual gestation length was calculated using the date delivery started.

BEHAVIORAL EVALUATIONS (MALES AND TOXICITY PHASE FEMALES)

FOB ASSESSMENTS: FOB assessments were recorded for 6 males/group and 6 toxicity phase females/group following approximately 28 days of dose administration. The FOB used at WIL Research is based on previously developed protocols (Gad, 1982, Haggerty, 1989, Irwin, 1968, Moser et al., 1988, Moser et al., 1991, and O’Donoghue, 1989). FOB Testing was performed by the same biologists, to the extent possible, without knowledge of the animal’s group assignment. The FOB was performed in a sound-attenuated room equipped with a white noise generator set to operate at 70 ± 10 dB. All animals were observed for the following parameters as described below:

HOME CAGE OBSERVATIONS
(Posture, Convulsions/Tremors, Feces consistency, Biting, Palpebral (eyelid) closure)
HANDLING OBSERVATIONS
(Ease of removal from cage, Lacrimation/Chromodacryorrhea, Piloerection, Palpebral closure, Eye prominence, Red/Crusty deposits, Ease of handling animal in hand, Salivation, Fur appearance, Respiratory rate/character, Mucous membranes/Eye/Skin color, Muscle tone)
OPEN FIELD OBSERVATIONS
(Mobility, Rearing, Convulsions/Tremors, Grooming, Bizarre/Stereotypic behavior, Time to first step (seconds), Gait, Arousal, Urination/Defecation, Gait score, Backing)
SENSORY OBSERVATIONS
(Approach response, Startle response, Pupil response, Forelimb extension, Air righting reflex, Touch response, Tail pinch response, Eyeblink response, Hindlimb extension, Olfactory orientation)
NEUROMUSCULAR OBSERVATIONS
(Hindlimb extensor strength, Hindlimb foot splay, Grip strength-hind and forelimb, Rotarod performance)
Forelimb and hindlimb grip strength were measured using a device similar to the one described by Meyer et al. (1979). The animal was allowed to grip a T-shaped grip bar with its forepaws and was pulled back gently along a platform until its grip was broken. As the backward locomotion continues, the animal’s hindpaws reach a T-shaped rearlimb grip bar, which it is allowed to grasp and then forced to release by continued pulling. Mark-10 series EG digital force gauges (Mark-10 Corporation, Copiague, NY) were used to record the maximum strain required to break forelimb and hindlimb grip. The average of 3 valid measurements was taken as the animal’s score for each grip strength measure.
PHYSIOLOGICAL OBSERVATIONS
(Catalepsy, Body temperature, Body weight)

LOCOMOTOR ACTIVITY: Locomotor activity counts were recorded for the same 6 males/group and 6 toxicity phase females/group that were tested in the FOB following approximately 28 days of dose administration. Locomotor activity, recorded after the completion of the FOB, was measured automatically using a personal computer-controlled system that utilizes a series of infrared photobeams surrounding a clear plastic, rectangular cage to quantify each animal’s motor activity. Four-sided black plastic enclosures were used to surround the clear plastic boxes to decrease the potential for distraction from extraneous environmental stimuli or stimuli from biologists or adjacent animals. The black enclosures rested on top of the photobeam frame and did not interfere with the path of the beams. The testing of treatment groups was done according to replicate sequence. Each animal was tested separately. Data were collected in 5-minute epochs and the test session duration was 60 minutes. These data were compiled as six, 10-minute subintervals for tabulation. Data for ambulatory and total motor activity were tabulated. Total motor activity was defined as a combination of fine motor skills (i.e., grooming, interruption of 1 photobeam) and ambulatory motor activity (interruption of 2 or more consecutive photobeams).

CLINICAL PATHOLOGY (MALES AND TOXICITY PHASE FEMALES)

Blood samples for clinical pathology evaluations (hematology and serum chemistry) were collected from 6 males/group and 6 toxicity phase females/group. The same animals used for FOB and motor activity assessments were used for clinical pathology evaluations. The animals were fasted overnight prior to blood collection. Blood for serum chemistry and hematology was collected from the retro-orbital sinus following isoflurane anesthesia. Blood for coagulation parameters was collected from the vena cava at the time of necropsy. Blood was collected into tubes containing EDTA (hematology), sodium citrate (clotting determinations) or no anticoagulant (serum chemistry).

Sacrifice and pathology:
ANATOMIC PATHOLOGY

MACROSCOPIC EXAMINATION (ALL PHASES): All adults were euthanized by carbon dioxide inhalation. Males and toxicity phase females were euthanized following a minimum of 28 days of dose administration (completion of the mating period for males). Reproductive phase females that delivered were euthanized on lactation day 4; the numbers of former implantation sites and corpora lutea were recorded. Females that failed to deliver were euthanized on post-mating day 25 (females with evidence of mating) or post-cohabitation day 25 (females with no evidence of mating). Uteri from reproductive phase females with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss (Salewski, 1964). Necropsy included examination of the external surface, the external surface of the brain, all orifices,and the cranial, thoracic, abdominal, and pelvic cavities, including viscera.

ORGAN WEIGHTS (ALL PHASES)

The following organs were weighed from all males and toxicity phase females at the scheduled necropsies:
Adrenal glands Ovaries with oviducts
Brain Spleen
Epididymidesa Testesa
Heart Thymus gland
Kidneys Thyroids with parathyroidsb
Liver
a = These paired organs were weighed separately.
b = Fixed in 10% neutral-buffered formalin prior to weighing.
Except as noted, paired organs were weighed together. Absolute weights, organ to final body weight ratios, and organ to brain weights ratios were reported. For reproductive phase females, ovary, uterus, and brain (for females that failed to deliver) weights were recorded at the lactation day 4 necropsy. Absolute weights and organ to final body weight ratios were reported.

MICROSCOPIC EXAMINATIONS (MALES AND TOXICITY PHASE FEMALES)

After fixation, protocol-specified tissues were trimmed according to WIL Research’s SOPs and the protocol. Trimmed tissues were processed into paraffin blocks, sectioned at 4 to 8 microns, mounted on glass microscope slides, and stained with hematoxylin and eosin. Testes were stained with PAS and hematoxylin. Microscopic examination was performed on all tissues listed in Section 5.8.1. for all males and toxicity phase females in the control and 1000 mg/kg/day groups at the scheduled necropsies and all gross lesions irrespective of groups. Missing tissues were identified as not found at necropsy, lost at necropsy, lost during processing, not in plane of section, or other reasons as appropriate. Tissues may appear on the report tables as not examined due to the tissue not being in the plane of section, not present at trimming, etc.

F1 LITTER PARAMETERS (REPRODUCTIVE PHASE)

LITTER VIABILITY AND DEATHS: Each litter was examined 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. A daily record of litter size was maintained. Intact offspring that were found dead were necropsied using a fresh dissection technique, which included examination of the heart and major vessels (Stuckhardt and Poppe, 1984). Tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as deemed necessary by the gross findings. The carcass of each pup was then discarded.

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 and 4. Any abnormalities in nursing behavior were recorded.

BODY WEIGHTS: Pups were individually weighed on PND 1 and 4. Mean pup weights were presented by sex for each litter and by dose group. When body weights could not be determined for a pup during a given interval (due to an unscheduled death, weighing error, etc.), group mean values were calculated for that interval using the available data. The time periods a given pup was not weighed were designated as “NA” on the individual report tables.

SEX DETERMINATION: Pups were individually sexed on PND 0 and 4.
Statistics:
Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data tables. Data obtained from nongravid females were excluded from statistical analyses following the mating period. Where applicable, the litter was used as the experimental unit. All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test item-treated group to the control group by sex. Parental mating, fertility, conception, and copulation indices were analyzed using the Chi-square test with Yates’ correction factor (Hollander and Wolfe, 1999). Parental body weights (weekly, gestation, and lactation), body weight changes, and food consumption, offspring body weights and body weight changes, gestation length, numbers of corpora lutea and implantation sites, number of pups born, live litter size on PND 0, unaccounted-for sites, absolute and relative organ weights, clinical pathology values, pre-coital intervals, and continuous FOB data values were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test item-treated groups to the control group. Histopathological findings and FOB parameters that yield scalar or descriptive data in the test item-treated groups were compared to the control group using Fisher’s Exact test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Details on results:
All animals survived to the scheduled necropsies. No test item-related clinical observations were noted in the test item-treated groups at the daily examinations or approximately 1 hour following dose administration during the study. A test item-related, lower mean body weight gain and corresponding reduction in mean food consumption were noted for the 1000 mg/kg/day group males during the first week of test item administration and resulted in lower mean body weight gains when the pre-mating (study days 0-13) and entire treatment (study days 0-27) periods were evaluated. However, as the effects on mean body weight gains were not of sufficient magnitude to affect mean body weights for the 1000 mg/kg/day group males, the differences were not considered to be adverse. Mean body weights, body weight gains, and food consumption for males in the 250 and 500 mg/kg/day groups and for toxicity phase and reproductive phase females in the 250, 500, and 1000 mg/kg/day groups were unaffected by test item administration. No test item-related effects were noted during the FOB assessments or locomotor activity evaluations at any dosage level for males and toxicity phase females. There were no test item-related gross necropsy observations, final body weight alterations, or organ weight differences in the males, reproductive phase females, and repeat-dose toxicity phase females. There were no test item-related clinical pathology (hematology, coagulation, serum chemistry) alterations or histologic changes in the males and repeat-dose toxicity phase females.

Male and reproductive phase female mating and fertility, male copulation, and reproductive phase female conception indices, mean number of days between pairing and coitus, gestation length, and the process of parturition were unaffected by test item administration at all dosage levels.
Mean numbers of corpora lutea, unaccounted-for sites, and implantation sites, mean number of pups born, live litter size, the percentage of males at birth, and postnatal survival in the 250, 500, and 1000 mg/kg/day groups were similar to the control group values. There were no adverse test item-related effects on mean pup body weights and body weight gains and no remarkable clinical findings or macroscopic findings for F1 pups at all dosage levels.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
food consumption and compound intake
gross pathology
haematology
histopathology: non-neoplastic
organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the conditions of this screening study, no test item-related effects on reproductive performance, gestation length, parturition, reproductive organs, or neurobehavioral parameters were noted at any dosage level. Based on these results, a dosage level of 1000 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of Isopropyl-3-Hydroxybutyrate when administered orally by gavage to Crl:CD(SD) rats. Test item-related lower mean body weight gains and reduced mean food consumption were observed for the 1000 mg/kg/day group males during the first week of treatment, but were considered to be transient and not adverse as absolute mean body weights were not affected. There were no test item-related clinical findings noted for males or females at any dosage level and no effects on mean body weights, body weight gains, and food consumption for males at 250 or 500 mg/kg/day or females at 250, 500, or 1000 mg/kg/day. In addition, there were no test item-related effects on clinical pathology, organ weights, and macroscopic and histopathological findings at any dosage level. Hence, the NOAEL for systemic toxicity was considered to be 1000 mg/kg/day. In the absence of effects on the general physical condition of the F1 pups, the NOAEL for neonatal toxicity was 1000 mg/kg/day.
Executive summary:

This study was designed to investigate the potential toxic effects of n-butyl-3 -hydroxybutyrate, using data from a related ether alcohol, isopropyl-3 -hydroxybutyrate (IPHB), when administered to rats for 28 days and to evaluate the potential of the test item to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition, and early postnatal development.

This study included 4 groups of Crl:CD(SD) rats consisting of 12 males, 12 toxicity phase females, and 12 reproductive phase females per group. IPHB, in the vehicle (deionized water) was administered orally by gavage once daily at dosage levels of 250, 500, and 1000 mg/kg/day. A concurrent control group received the vehicle on a comparable regimen. The dosage volume for all groups was 10 mL/kg. Males and females were approximately 10 weeks of age at the beginning of test item administration. Males received 14 daily doses prior to mating and were dosed throughout the mating period through the day prior to euthanasia for a total of 28 -29 doses. Toxicity phase females were dosed daily from study day 0 through the day prior to euthanasia for a total of 28-29 doses. Reproductive phase females received 14 daily doses prior to pairing and were dosed through lactation day 3 for a total of 40 -52 doses; females with no evidence of mating/that failed to deliver were dosed through the day prior to euthanasia (post-mating or post-cohabitation day 25) for a total of 39-52 doses. All animals were observed twice daily for mortality and moribundity. Clinical

observations, body weights, and food consumption were recorded at appropriate intervals. Blood samples were collected at appropriate intervals throughout the study from males and toxicity phase females for future serum beta-Hydroxybutyrate analysis.

All F0 reproductive phase females were allowed to deliver and rear their pups until lactation day 4. F1 clinical observations and body weights were recorded on PND 1 and 4. Pups were necropsied on PND 4. FOB and locomotor activity data were recorded for 6 males/group and 6 toxicity phase females following approximately 28 days of dose administration. Clinical pathology evaluations (hematology and serum chemistry) were performed on 6 males/group and 6 toxicity phase females/group at necropsy. F0 males

and toxicity phase females were euthanized following a minimum of 28 days of dose administration (following completion of the mating period for males). F0 reproductive phase females were euthanized on lactation day 4. Complete necropsies were conducted

on all F0 animals and selected organs were weighed. Selected tissues were examined microscopically from all males and toxicity phase females in the control and high-dose groups.

All animals survived to the scheduled necropsies. No test item-related clinical observations were noted in the test item-treated groups at the daily examinations or approximately 1 hour following dose administration during the study. A test item-related, lower mean body weight gain and corresponding reduction in mean food consumption were noted for the 1000 mg/kg/day group males during the first week of test item administration and resulted in lower mean body weight gains when the pre-mating (study days 0-13) and entire treatment (study days 0-27) periods were evaluated. However, as the effects on mean body weight gains were not of sufficient

magnitude to affect mean body weights for the 1000 mg/kg/day group males, the differences were not considered to be adverse. Mean body weights, body weight gains, and food consumption for males in the 250 and 500 mg/kg/day groups and for toxicity

phase and reproductive phase females in the 250, 500, and 1000 mg/kg/day groups were unaffected by test item administration.

No test item-related effects were noted during the FOB assessments or locomotor activity evaluations at any dosage level for males and toxicity phase females. There were no test item-related gross necropsy observations, final body weight alterations, or organ weight differences in the males, reproductive phase females, and repeat-dose toxicity phase females. There were no test item-related clinical pathology (hematology, coagulation, serum chemistry) alterations or histologic changes in the males and repeat-dose toxicity phase females. Male and reproductive phase female mating and fertility, male copulation, and reproductive phase female conception indices, mean number of days between pairing and coitus, gestation length, and the process of parturition were unaffected by test item

administration at all dosage levels. Mean numbers of corpora lutea, unaccounted-for sites, and implantation sites, mean number of pups born, live litter size, the percentage of males at birth, and postnatal survival in the 250, 500, and 1000 mg/kg/day groups were similar to the control group values. There were no adverse test item-related effects on mean pup body weights and body weight gains and no remarkable clinical findings or macroscopic findings for F1 pups at all dosage levels.

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