Lithium nickel potassium oxide

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 2019 to June 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Lithium nickel potassium oxide

Cas Number:

210352-95-7

Molecular formula:

Li.K.Ni.O.H2O

IUPAC Name:

Lithium nickel potassium oxide

Test material form:

solid: particulate/powder

Details on test material:

Lithium nickel potassium oxide (KDLNO)
CAS Number:
210352-95-7
Appearance/Physical state:
Black powder
Storage: Room temperature in the dark

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

This species and strain of rat is recognized as appropriate for reproduction studies. Charles River
has reproductive historical control data in this species from the same strain and source. This
animal model has been proven to be susceptible to the effects of reproductive toxicants.
The number of animals is based on the OECD Guideline for the Testing of Chemicals, Guideline
422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity
Screening Test, 29 July 2016, which recommends that evaluation of each group be initiated with
at least 10 males and 12-13 females per group. Females are evaluated for estrous cyclicity
during the pretest period and any females that fail to exhibit normal 4-5 day estrous cycles
(e.g., EDDDE), during the pretest period, are excluded from the study, therefore, the extra
females are included to yield at least 10 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.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System

Receipt
On 13 and 27 Aug 2019, Crl:CD(SD) female and male rats, respectively, were received from Charles River Laboratories, Inc., Raleigh, NC. The animals were approximately 11 weeks old and weighed between 192 and 379 g at the initiation of dosing.

Justification for Test System and Number of Animals
The Crl:CD(SD) rat is recognized as appropriate for reproduction studies. Charles River Ashland has reproductive historical control data in this species from the same strain and source. 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 the Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, 29 July 2016, which recommends that evaluation of each group be initiated with at least 10 males and 12-13 females per group. Females were evaluated for estrous cyclicity during the pretest period, and any females that fail
to exhibit normal 4-5 day estrous cycles (e.g., EDDDE) during the pretest period were excluded from the study; therefore, the extra females were included to yield at least 10 females per group. Given the possibility of nongravid animals, unexpected deaths, total litter losses, or test substance-related moribundity and/or mortality, this was an appropriate number of animals to obtain a sample size of 8 at termination.

Animal Identification
Each animal was identified using a subcutaneously implanted electronic identification chip (BMDS system). Offspring were identified by tattoo markings applied to the digits after parturition.

Environmental Acclimation
After receipt at the Testing Facility, the Crl:CD(SD) rats were acclimated prior to initiation of dosing. The testes were palpated at least once for all males during acclimation.

Selection, Assignment, and Disposition of Animals
Animals were assigned to groups by a stratified randomization scheme designed to achieve similar group mean body weights. Males and females were randomized separately. Animals at extremes of body weight range or not exhibiting normal, 4- to 5-day estrous cycles were not assigned to groups. To reduce variability among the F1 litters, 8 pups/litter of equal sex distribution, if possible, were randomly selected on PND 4. Standardization of litter size was not performed on litters with fewer than 8 pups. The remaining offspring were euthanized by an intraperitoneal injection of
sodium pentobarbital (following thyroid hormone blood collection; see Section 4.8.2.6.) and discarded. The disposition of all animals was documented in the Study Records.

Husbandry
Housing
On arrival, animals were group housed (up to 3 animals of the same sex) until cohabitation. During cohabitation, animals were paired for mating in the home cage of the male. Following the breeding period, animals were individually housed. Animals were housed in solid-bottom cages containing appropriate bedding equipped with an automatic watering valve throughout the study. Animals were separated during designated procedures/activities. Each cage was clearly labeled
with a color-coded cage card indicating study, group, animal, cage number(s), dosage level, and sex. Cages were arranged on the racks in group order.
Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). The animal facilities at Charles River Ashland are accredited by AAALAC International.

Environmental Conditions
Target temperatures of 68°F to 78°F (20°C to 26°C) with a relative target humidity of 30% to 70% were maintained. A 12-hour light/12-hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Food
PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 was provided ad libitum throughout the study, except during designated procedures. The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis are provided by the supplier and are on file at the Testing Facility. It is considered that there are no known contaminants in the feed that would interfere with the objectives of the study.

Water
Municipal tap water after treatment by reverse osmosis and ultraviolet irradiation was freely available to each animal via an automatic watering system. Water bottles were provided, if required. Periodic analysis of the water is performed, and results of these analyses are on file at the Testing Facility. It is considered that there are no known contaminants in the water that could interfere with the outcome of the study.

Animal Enrichment
Animals were socially housed for psychological/environmental enrichment and were provided with environmental enrichment as appropriate to aid in maintaining the animals’ oral health.

Veterinary Care
Veterinary care was available throughout the course of the study, and animals were examined by the veterinary staff as warranted by clinical signs or other changes. All veterinary examinations and recommended therapeutic treatments, if any, were documented in the Study Records and reviewed by the Study Director.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

methylcellulose

Details on oral exposure:

Administration of Test Materials
The test substance and vehicle were administered as a single daily oral gavage dose. Males were dosed for 14 days prior to mating, throughout mating and continuing until the day prior to euthanasia, for 28 days in total. Females were dosed for 14 days prior to mating and continuing through Lactation Day 13 for up to 54 days in total. Females with no evidence of mating were dosed through the day prior to euthanasia for at least 28 days in total. All animals were dosed at approximately the same time each day. The F1 animals were not directly exposed to the test substance at any time during the study; the offspring of the F0 parental generation were potentially exposed to the test substance in utero and while nursing.

Justification of Route and Dose Levels
The route of administration was oral (gavage) because this is a potential route of exposure to humans. Historically, this route has been used extensively for studies of this nature. The dosage levels were determined from results of the 14-Day Oral (Gavage) Toxicity Study of Lithium Nickel Potassium Oxide (KDLNO) in Rats (Sondag, 2020, 01291003 – See additional study: A 14-Day Oral (Gavage) Toxicity Study of Lithium Nickel Potassium Oxide (KDLNO) in Rats) following consultation with the Study Monitor for the Sponsor. The study showed excessive toxicity and mortality at 330 mg/kg/day and above. When the dosage was reduced to 200 mg/kg/day from either 330 or 600 mg/kg/day, clinical findings were noted but were not excessive. There were no treatment-related effects noted at 100 mg/kg/day. Therefore, 200 mg/kg/day was selected as the high dose for this study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical Method
Analyses were performed by an inductively coupled plasma method with mass spectrometric
detection using a validated analytical procedure .

Concentration Analysis
Duplicate sets of samples (0.5 mL; ) were sent to the analytical laboratory; the remaining samples were retained at the Testing Facility as backup samples. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 15% of theoretical concentration. After acceptance of the analytical results, backup samples were discarded.

Analyses of Dosing Formulations


The formulations prepared for administration during Weeks 1 and 8 of treatment were analyzed.
The analyzed dosing formulations contained 95% to 108% of the test substance, which was
within the protocol-specified range of target concentrations for solutions (85% to 115%). The
coefficients of variation of the analyzed low (Group 2) and high (Group 4) dose formulations
were 1.1% and 2.5%, respectively, which indicated that the formulations were homogeneous
(i.e. coefficient of variation ≤ 10%). A small response at the retention time of the test substance
was observed in the chromatograms of the analyzed vehicle formulation that was administered to
the control group (Group 1). It was not considered to derive from the formulation since a similar
response was obtained in the analytical blanks.

Duration of treatment / exposure:

The test substance and vehicle were administered as a single daily oral gavage dose. Males were dosed for 14 days prior to mating, throughout mating and continuing until the day prior to euthanasia, for 28 days in total. Females were dosed for 14 days prior to mating and continuing through Lactation Day 13 for up to 54 days in total. Females with no evidence of mating were dosed through the day prior to euthanasia for at least 28 days in total. All animals were dosed at approximately the same time each day. The F1 animals were not directly exposed to the test substance at any time during the study; the offspring of the F0 parental generation were potentially exposed to the test substance in utero and while nursing.

Frequency of treatment:

The test substance and vehicle were administered as a single daily oral gavage dose. Males were dosed for 14 days prior to mating, throughout mating and continuing until the day prior to euthanasia, for 28 days in total. Females were dosed for 14 days prior to mating and continuing through Lactation Day 13 for up to 54 days in total. Females with no evidence of mating were dosed through the day prior to euthanasia for at least 28 days in total. All animals were dosed at approximately the same time each day. The F1 animals were not directly exposed to the test substance at any time during the study; the offspring of the F0 parental generation were potentially exposed to the test substance in utero and while nursing.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Remarks:

KDLNO = Lithium nickel potassium oxide.
As supplied; not corrected for salt, purity and water content.

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

KDLNO = Lithium nickel potassium oxide.
As supplied; not corrected for salt, purity and water content.

Doses / concentrations 3

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Remarks:

KDLNO = Lithium nickel potassium oxide.
As supplied; not corrected for salt, purity and water content.

Doses / concentrations 4

Dose / conc.:

0 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

The test substance and vehicle were administered as a single daily oral gavage dose. Males were dosed for 14 days prior to mating, throughout mating and continuing until the day prior to euthanasia, for 28 days in total. Females were dosed for 14 days prior to mating and continuing through Lactation Day 13 for up to 54 days in total. Females with no evidence of mating were dosed through the day prior to euthanasia for at least 28 days in total. All animals were dosed at approximately the same time each day. The F1 animals were not directly exposed to the test substance at any time during the study; the offspring of the F0 parental generation were potentially exposed to the test substance in utero and while nursing.


STATISTICAL ANALYSIS
Each mean was presented with the standard deviation (S.D.) and the number of animals or cages
(N) used to calculate the mean. Where applicable, the litter was used as the experimental unit.
Statistical analyses were not conducted if the number of animals was 2 or less. 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 animals were excluded from statistical analyses.
All statistical tests for the following parameters were performed using WTDMS™. Analyses
were conducted using two-tailed tests (except as noted otherwise) for minimum significance
levels of 1% and 5%, comparing each test substance-treated group to the control group by sex.
Parental mating, fertility, copulation, and conception indices were analyzed using the Chi-square
test with Yates’ correction factor. Parental and offspring body weights and body weight
changes, parental food consumption, estrous cycle lengths, precoital intervals, gestation lengths,
former implantation sites, corpora lutea, unaccounted-for sites, live litter size on PND 0,
numbers of pups born, absolute and relative organ weights, clinical pathology values, thyroid
hormone values, anogenital distance (absolute and relative to the cube root of body weight),
number of nipples/areolae, and FOB data values were subjected to a parametric one-way
ANOVA to determine intergroup differences. If the ANOVA revealed significant (p < 0.05)
intergroup variance, Dunnett's test was used to compare the test substance-treated groups to the
control group. FOB parameters that yielded scalar or descriptive data were analyzed using
Fisher’s Exact Test. Mean litter proportions of postnatal survival and pup sexes at birth
(percentage of males per litter) were subjected to the Kruskal-Wallis nonparametric ANOVA to
determine intergroup differences. If the nonparametric ANOVA revealed significant (p < 0.05)
intergroup variance, Dunn’s test was used to compare the test substance-treated groups to the
control group.
The locomotor activity ambulatory and total counts data were statistically analyzed using SAS
Software for Windows, Release 9.4 (SAS Institute Inc., Cary, NC) or later. For the purpose of
the statistical analysis, each session consisted of the following number of levels for the factor
Time: for locomotor activity, 6 levels of 10 consecutive minutes each. For each session and each
sex, the locomotor activity data were statistically analyzed separately, using a repeated measures
analysis of variance model, with Group, Time and their interaction, as fixed factors.
The corrected Akaike’s Information Criterion was used in order to select the homoscedastic
model with the following covariance structures: Compound Symmetry, Heterogeneous
Compound Symmetry, First-Order Autoregressive, and Heterogeneous First-Order Autoregressive, for locomotor activity, and to select the heteroscedastic model that allowed for non-constant variance for one of its fixed factors.
The selection between the homoscedastic and heteroscedastic models were based on the
likelihood ratio test and the retained model was considered to conduct the inferential statistical
analysis.
The overall Group effect was assessed for all Time levels combined if the interaction was not
significant; otherwise it was assessed within each Time level. If it was found to be significant,
then the pairwise comparisons between the control group and each of the other groups were
conducted, via Dunnett’s test, for all Time levels combined in the case of no significant
interaction, and within each Time level in the case of significant interaction.
All statistical tests were conducted at the 5% significance level and all pairwise comparisons
were performed using two-sided tests.

COMPUTERIZED SYSTEMS
Critical computerized systems used in the study are listed below or presented in the appropriate
phase report. All computerized systems used in the conduct of this study have been validated
(with the exception of Microsoft Office); when a particular system has not satisfied all
requirements, appropriate administrative and procedural controls were implemented to assure the
quality and integrity of data.
As Charles River Ashland transitions between various computer systems, the study number may
appear as 01291004, 1291004, WIL-1291004, or C91004 in the data records and report.

RETENTION OF RECORDS, SAMPLES, AND SPECIMENS
All study-specific raw data, documentation, protocol, samples, specimens, and Final Reports
from this study were archived at the Testing Facility by no later than the date of Final Report
issue unless otherwise specified in the protocol. At least 1 year after issue of the Draft Report,
the Sponsor will be contacted to determine the disposition of materials associated with the study.
Electronic data generated by the Testing Facility were archived as noted above, except that the
data collected using Deviation Information Library and reporting files stored on SDMS were
archived at the Charles River Laboratories facility location in Wilmington, MA.
All records, retained samples and specimens, and reports generated from phases or segments
performed by Testing Facility-designated subcontractors were archived.

Examinations

Observations and examinations performed and frequency:

In-life Procedures, Observations, and Measurements

F0 Generation

Viability
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, once in the morning and once in the afternoon. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

Observations
The animals were removed from the cage, and a detailed clinical observation was performed once daily throughout the study. During the dosing period, these observations were performed prior to dosing. On dosing days, clinical observations were also recorded approximately 2 hours postdose. During social housing, some observations (e.g., fecal observations) may not have been attributable to an individual animal.

Body Weights
Animals were weighed individually weekly throughout the study and prior to the scheduled necropsy. 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, 4, 7, 10, and 13. A fasted weight was recorded on the day of necropsy. Terminal body weights were not collected from animals found dead or euthanized moribund.

Food Consumption
Food consumption was quantitatively measured weekly until cohabitation. 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, 4, 7, 10, and 13.


Estrous Cycles
For all females, vaginal lavages were performed daily for 14 days prior to randomization and continuing until evidence of mating was observed. The slides were microscopically examined to determine the stage of the estrous cycle. 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] for 14 consecutive days before cohabitation 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 individual mean estrous cycle length calculation. Vaginal lavages were also performed on the day of necropsy to determine the stage of the estrous cycle.

Breeding Procedures
After a minimum of 14 days of dosing, the animals were paired on a 1:1 basis within each group. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage. Vaginal lavages were performed daily during the mating period until evidence of mating was observed. If evidence of mating was not apparent after 14 days, the animals were separated, with no further opportunity for mating. Animals cohabited over a 12-hour dark cycle were considered to have been paired for 1 day.

Parturition
The day parturition was initiated was designated Lactation Day 0 (Postnatal Day [PND] 0 for pups). During the period of expected parturition, females were observed twice daily for initiation and completion of parturition and for dystocia or other difficulties. All females were allowed to deliver naturally. Beginning on the day parturition was initiated, the numbers of stillborn and live pups were recorded. Individual gestation length was calculated using the date delivery was first
observed.

Neurobehavioral Testing

FOB Assessments
FOB assessments were recorded for 5 animals/sex/group during the last week of dosing or on Lactation Day 13. The FOB used at Charles River is based on previously developed protocols (Gad, 1982; Haggerty, 1989; Irwin, 1968; Moser et al., 1988; Moser et al., 1991; O’Donoghue, 1989). Testing was performed by the same trained technicians, when possible, who did not know the animal’s group assignment and was performed at approximately the same time each day. Testing of treatment groups was balanced across the day of testing, to the extent possible.
The FOB was performed in a sound-attenuated room equipped with a white noise generator. 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 (Meyer et al, 1979)
Rotarod performance

Physiological Observations
Catalepsy
Body temperature Body weight

Motor Activity
Motor activity was assessed for 5 animals/sex/group during the last week of dosing or on Lactation Day 13. The motor activity assessment was performed in a sound-attenuated room equipped with a white-noise generator, and black enclosures were used to decrease the potential for distraction. Data were collected in 5-minute epochs over a period of 60 minutes, and the data were reported in 10-minute subintervals. Total motor activity was defined as a combination of fine motor skills (i.e., grooming; interruption of 1 photobeam) and ambulatory motor activity (e.g., interruption of 2 or more consecutive photobeams).

Clinical Pathology
Sample Collection
Animals were fasted overnight prior to blood collection. Blood samples for hematology and serum chemistry were collected from the jugular vein for males and from the retro-orbital sinus from animals anesthetized with isoflurane for females. Blood samples for coagulation parameters were collected by necropsy personnel from the inferior vena cava at the time of euthanasia from animals euthanized via carbon dioxide inhalation. K2EDTA was used for the anticoagulant on samples collected for hematology. Sodium citrate was used for samples collected for clotting determinations. Samples for serum chemistry were collected without anticoagulants.

Samples for Clinical Pathology Evaluation
Group Nos. , Sex & Time Point
1-4 Males Day 28 (males)
1-4 Females Lactation Day 14 (females)
Hematology Parameters
Differential leukocyte count -
Percent and absolute
-Neutrophil (NEU)
-Lymphocyte (LYMPH)
-Monocyte (MONO)
-Eosinophil (EOS)
-Basophil (BASO)
-Large unstained cell (LUC)
Erythrocyte count (RBC)
Hemoglobin (HGB)
Hematocrit (HCT)
Mean corpuscular hemoglobin (MCH)
Mean corpuscular volume (MCV)
Mean corpuscular hemoglobin concentration (MCHC)
Platelet count (Platelet)
Red cell distribution width (RDW)
Reticulocyte count
Percent (RETIC)
Absolute (RETIC Absolute)
Total leukocyte count (WBC)
Platelet estimatea
Red cell morphology (RBC Morphology)a

Coagulation
Blood samples were processed for plasma, and the plasma was analyzed for the relevant parameters.

Coagulation Parameters
Activated partial thromboplastin time (APTT)
Fibrinogen
Prothrombin time (PT)
Sample Quality


Serum Chemistry
Blood samples were processed for serum, and the serum was analyzed for the relevant parameters.

Serum Chemistry Parameters
Alanine aminotransferase (ALT)
Albumin
Albumin/globulin ratio (A/G Ratio) [by calculation]
Alkaline phosphatase (ALP)
Aspartate aminotransferase (AST)
Bile Acids
Calcium
Chloride
Creatinine
Gamma glutamyltransferase (GGT)
Globulin [by calculation]
Glucose
Phosphorus
Potassium
Sodium
Sorbitol dehydrogenase (SDH)
Total bilirubin (Total BILI)
Total cholesterol (Cholesterol)
Total protein
Triglycerides (Triglyceride)
Urea nitrogen
Appearance


Thyroid Hormone Analysis

Sample Collection
Blood samples for thyroid hormone analyses were collected from the jugular vein into tubes without anticoagulants.
Samples for Thyroid Hormone Evaluation
Group Nos., Sex & Time Point Thyroid Hormones
1–4 Males Day 28
1-4 Females Lactation Day 13

Sample Processing
Blood samples were maintained at room temperature and allowed to clot. Serum was isolated in
a refrigerated centrifuge and stored in a freezer set to maintain a target of -70°C.

Sample Analysis
Blood samples were analyzed for the relevant parameters.
Thyroid Hormone Parameters
Thyroxine (Total T4)
Samples to be analyzed for T4 were transferred to the Charles River Ashland Bioanalytical Chemistry Department; analyses were performed using a validated UHPLC/MS/MS assay.


F1 Litter Parameters

Viability

Litters were observed for general health/mortality and moribundity twice daily, once in the
morning and once in the afternoon. A daily record of litter size was maintained. Animals were
not removed from cage during observation, unless necessary for identification or confirmation of
possible findings.
Total litter loss was determined when the last pup in the litter was found dead or euthanized
in extremis prior to the scheduled euthanasia. Litters that were euthanized prior to scheduled
euthanasia due to reasons unrelated to test substance administration (e.g., death of the dam) were
not considered to be a total litter loss on the data tables and were not included in the pup viability
calculations.

Observations
The animals were removed from the cage, and a detailed clinical observation was performed on
PND 1, 4, 7, 10, and 13.

Sex Determination
Pups were individually sexed on PND 0, 4, and 13.

Body Weights
Pups were weighed individually on PND 1, 4, 7, 10, and 13.



Preweaning Developmental Landmarks

Anogenital Distance
The anogenital distance of all pups was measured on PND 1. Anogenital distance was defined as
the distance from the caudal margin of the anus to the caudal margin of the genital tubercle.

Assessment of Areolas/Nipple Anlagen
On PND 13, all male pups were evaluated for the presence of nipples/areolae.
The number of nipples was recorded.

Thyroid Hormone Analysis
Sample Collection
Blood samples for thyroid hormone analyses were collected via cardiac puncture from animals
anesthetized with isoflurane into tubes without anticoagulants.

Samples for Thyroid Hormone Evaluation
Group Nos.; No. of Pups & Time Point
1–4 At least 2/litter PND 4
1–4 1/sex/litter PND 13

Sample Processing
Blood samples were maintained at room temperature and allowed to clot. Serum was isolated in
a refrigerated centrifuge and stored in a freezer set to maintain a target of -70°C. The time and
date that the samples were placed in the freezer were recorded .
Sample Analysis
Blood samples were analyzed for the parameters.
Thyroid Hormone Parameters
Thyroxine (Total T4)
Samples to be analyzed for T4 were transferred to the Charles River Ashland Bioanalytical
Chemistry Department; analyses were performed using a validated UHPLC/MS/MS assay.

Terminal Procedures
F0 Generation
Unscheduled Deaths
A necropsy was conducted for animals that died on study, and specified tissues were saved.
If necessary, for humane reasons, animals were euthanized as per Testing Facility SOPs. These
animals underwent necropsy, and specified tissues were retained.
For females found dead or euthanized for humane reasons during gestation, the number and
location of viable fetuses, corpora lutea, and implantation sites were recorded. For females found
dead or euthanized during lactation, the number of corpora lutea and former implantation sites
were recorded. Recognizable fetuses were examined externally and discarded. All pups were euthanized by an intraperitoneal injection of sodium pentobarbital in the scapular region and necropsied.

Scheduled Euthanasia
All surviving animals, including females that failed to deliver or with total litter loss, were
euthanized by carbon dioxide inhalation.

Necropsy
Animals were subjected to a complete necropsy examination, which 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. The numbers of implantation
sites and former implantation sites were recorded for females that delivered or had macroscopic
evidence of implantation. 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.
Uteri of females without macroscopic evidence of implantation were opened and placed in
10% ammonium sulfide solution for detection of early implantation loss .

Organ Weights
The organs were weighed at necropsy for all scheduled euthanasia animals. Organ weights were not recorded for animals found dead or euthanized in poor condition or in extremis. Paired organs were weighed together, unless otherwise indicated. Organ to body weight ratio (using the terminal body weight) and organ to brain weight ratios were calculated.

Organs Weighed at Necropsy
Adrenal glands
Brain
Epididymidesa
Heart
Kidneys
Liver
Ovaries with oviducts
Pituitary gland
Prostate gland
Seminal vesicles (with coagulating gland and fluid)
Spleen
Testesa
Thymus gland
Thyroids with parathyroids
Paired organs were weighed separately. Care was taken to ensure separation between the left and right organs.

Tissue Collection and Preservation
Representative samples of the tissues were collected from all animals and preserved in 10% neutral buffered formalin, unless otherwise indicated.

Tissue Collection and Preservation
Adrenal glands (2)
Aorta
Bone with marrow (sternebrae)
Brain
Coagulating glands (2)
Eyes with optic nerve (2)a
Gastrointestinal tract
Esophagus
Stomach
Duodenum
Peyer’s Patches
Jejunum
Ileum
Cecum
Colon
Rectum
Heart
Kidneys (2)
Liver (sections of 2 lobes)
Lungs (including bronchi, fixed by
inflation with fixative)
Lymph node
Axillary (2)
Mandibular (2)b
Mesenteric
Ovaries and oviducts (2)
Pancreas
Peripheral nerve (sciatic)b
Pituitary gland
Prostate gland
Salivary gland (mandibular [2])b
Seminal vesicles (2)
Skeletal muscle (quadriceps)
Skin with mammary gland c
Spinal cord (cervical)
Spleen
Testes with epididymides d (2) and vas deferens
Thymus gland
Thyroids (with parathyroids, if present [2])
Trachea
Urinary bladder
Uterus e with cervix and vagina
All gross lesions
a Placed in Davidson’s solution.
b Only 1 examined.
c For females; a corresponding section of skin was taken from the same anatomic area for males.
d Placed in modified Davidson’s solution. Care was taken to ensure separation between left and right organs.
e Uterus not retained if placed in 10% ammonium sulfide solution.

Histology
Tissue trimming was performed at the Testing Facility. Tissues from 5 animals/sex in the control and high-dose groups and from all animals dying spontaneously, as well as gross lesions and target tissues (adrenal gland, stomach, duodenum, jejunum, ileum, cecum, colon, Peyer’s patches, lymph nodes, lungs, bone marrow [sternum], spleen, stomach, and thymus gland) from all groups, were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin. In addition, PAS staining was used for the testes and epididymides.

Histopathology
Pathological evaluation was performed by a board-certified veterinary pathologist. Tissues
for microscopic examination were evaluated from 5 animals/sex in the control and high-dose groups at the primary necropsy and from all animals found dead and euthanized in extremis. Gross lesions and target tissues were examined from all groups.

F1 Litters
Unscheduled Deaths
A necropsy was conducted for animals that died on study, and specified tissues were saved.
Pups euthanized due to the death of dam were euthanized as per Testing Facility SOPs. These
animals underwent necropsy, and specified tissues were retained.
Intact offspring that were found dead or euthanized for humane reasons during PND 0–4 were
necropsied using a fresh dissection technique, which included examination of the heart and major
vessels (Stuckhardt and Poppe, 1984). Findings were recorded as developmental variations or malformations, as appropriate. Representative specimens with malformations were preserved in 10% neutral-buffered formalin. A gross necropsy was performed on any pup found dead or euthanized for humane reasons after PND 4.

Scheduled Euthanasia
On PND 4, surviving animals were euthanized via an intraperitoneal injection of sodium
pentobarbital and discarded.
On PND 13, surviving animals were euthanized via an intraperitoneal injection of sodium
pentobarbital.

Necropsy
On PND 13, 1 pup/sex/litter was subjected to a complete necropsy examination, with emphasis
on developmental morphology and organs of the reproductive system. All other animals were
discarded without examination.

Tissue Collection and Preservation
Representative samples of the tissues were collected from 1 pup/sex/litter at the scheduled euthanasia and preserved in 10% neutral buffered formalin.
Tissues Collected
Thyroid (with parathyroids, if present)

Sacrifice and pathology:

See Section on "Observations and examinations performed and frequency"

Statistics:

Statistical analyses were not conducted if the number of animals was 2 or less.
Data obtained from nongravid animals were excluded from statistical analyses.
All statistical tests for the following parameters were performed using WTDMS™. Analyses
were conducted using two-tailed tests (except as noted otherwise) for minimum significance
levels of 1% and 5%, comparing each test substance-treated group to the control group by sex.
Parental mating, fertility, copulation, and conception indices were analyzed using the Chi-square
test with Yates’ correction factor. Parental and offspring body weights and body weight
changes, parental food consumption, estrous cycle lengths, precoital intervals, gestation lengths,
former implantation sites, corpora lutea, unaccounted-for sites, live litter size on PND 0,
numbers of pups born, absolute and relative organ weights, clinical pathology values, thyroid
hormone values, anogenital distance (absolute and relative to the cube root of body weight),
number of nipples/areolae, and FOB data values were subjected to a parametric one-way
ANOVA to determine intergroup differences. If the ANOVA revealed significant (p < 0.05)
intergroup variance, Dunnett's test was used to compare the test substance-treated groups to the
control group. FOB parameters that yielded scalar or descriptive data were analyzed using
Fisher’s Exact Test. Mean litter proportions of postnatal survival and pup sexes at birth
(percentage of males per litter) were subjected to the Kruskal-Wallis nonparametric ANOVA to
determine intergroup differences. If the nonparametric ANOVA revealed significant (p < 0.05)
intergroup variance, Dunn’s test was used to compare the test substance-treated groups to the
control group. The locomotor activity ambulatory and total counts data were statistically analyzed using SAS Software for Windows, Release 9.4 (SAS Institute Inc., Cary, NC) or later.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related effects on survival were observed for males at 200 mg/kg/day and for
females at all dosage levels tested.
Four males in the 200 mg/kg/day group were found dead or euthanized in extremis during Study
Days 9–20. Two, 5, and 10 females in the 30, 100, and 200 mg/kg/day groups were found dead
or euthanized in extremis near or at the end of gestation (Gestation Day 19 to Lactation Day 0),
with the exception of a single female (No. 6794) at 200 mg/kg/day that was found dead on
Gestation Day 4 following marked body weight losses. Clinical observations noted for the
majority of animals that were found dead or euthanized in extremis at 100 and 200 mg/kg/day,
including thin and/or cool body and extremities, red material around the nose, hunched posture,
partially closed eyes, decreased activity, and piloerection, were noted on the day of and/or the
day prior to death or euthanasia. For females in all dosage groups, these clinical signs were
associated with dystocia and prolonged gestation length and were considered KDLNO-related
and adverse. Only 1 of 10 gravid females in the 200 mg/kg/day group (No. 6805) initiated
parturition and delivered 2 pups on Gestation Day 21 (Lactation Day 0) and was subsequently
found dead on the same day. This female had 14 former implantations in utero. In the
100 mg/kg/day group, 4 of 10 females failed to deliver a viable litter and were found dead or
euthanized in extremis on Gestation Day 22 before parturition and 1 female (6793) was
euthanized on Lactation Day 0 due to a total litter loss, following a prolonged gestation length
(24 days) prior to initiation of parturition.
In all but 1 individual 30 mg/kg/day group female (No. 6814), gross and microscopic findings
indicated that the primary cause of morbidity was due to test substance-related effects, mainly
atrophy and necrosis in the gastrointestinal tract mucosa and/or inflammation in the lung. The
severity of the intestinal and/or lung lesions likely resulted in the dystocia event noted in several
female animals and subsequent death or euthanasia. In the 200 mg/kg/day group males, the cause
of morbidity was gastrointestinal tract toxicity with a similar pattern to what was observed in
females at all dose levels. The differences in survival between parental males and females were
likely reflective of the additional physiological demands on females during pregnancy,
parturition, and/or the longer dosing period in females. Dystocia secondary
to the test substance related lung and/or gastrointestinal lesions likely contributed to subsequent
death or euthanasia in extremis. The predominant change in both male and female groups was
necrosis of the mucosal epithelium and often atrophy of the jejunum and/or ileum. In 1 male
there was evidence for some mucosal regeneration, but generally the lesions in all animals were
acute or subacute. The lung inflammation observed in 200 mg/kg/day group females was
characterized by alveoli containing necrotic cell debris, fibrin, and mixed cell inflammatory cell
infiltrates with a multifocal to diffuse distribution. The other microscopic findings in the
lymphoid and reproductive systems of several animals were considered secondary to stress and
the poor condition of the animals.

Test substance-related clinical observations noted at the daily examinations and/or 1–2 hours
postdosing for the surviving males were limited to the 200 mg/kg/day group and included thin
body (which correlated with findings of low body weight and food consumption noted in this
group), cool body, cool and/or pale extremities, and brown and yellow material on various body
surfaces.

Mortality:

mortality observed, treatment-related

Description (incidence):

Mortality and Observations
Test substance-related effects on survival were observed for males at 200 mg/kg/day and for
females at all dosage levels tested.
Four males in the 200 mg/kg/day group were found dead or euthanized in extremis during Study
Days 9–20. Two, 5, and 10 females in the 30, 100, and 200 mg/kg/day groups were found dead
or euthanized in extremis near or at the end of gestation (Gestation Day 19 to Lactation Day 0),
with the exception of a single female (No. 6794) at 200 mg/kg/day that was found dead on
Gestation Day 4 following marked body weight losses. Clinical observations noted for the
majority of animals that were found dead or euthanized in extremis at 100 and 200 mg/kg/day,
including thin and/or cool body and extremities, red material around the nose, hunched posture,
partially closed eyes, decreased activity, and piloerection, were noted on the day of and/or the
day prior to death or euthanasia. For females in all dosage groups, these clinical signs were
associated with dystocia and prolonged gestation length and were considered KDLNO-related
and adverse. Only 1 of 10 gravid females in the 200 mg/kg/day group (No. 6805) initiated
parturition and delivered 2 pups on Gestation Day 21 (Lactation Day 0) and was subsequently
found dead on the same day. This female had 14 former implantations in utero. In the
100 mg/kg/day group, 4 of 10 females failed to deliver a viable litter and were found dead or
euthanized in extremis on Gestation Day 22 before parturition and 1 female (6793) was
euthanized on Lactation Day 0 due to a total litter loss, following a prolonged gestation length
(24 days) prior to initiation of parturition.
In all but 1 individual 30 mg/kg/day group female (No. 6814), gross and microscopic findings
indicated that the primary cause of morbidity was due to test substance-related effects, mainly
atrophy and necrosis in the gastrointestinal tract mucosa and/or inflammation in the lung. The
severity of the intestinal and/or lung lesions likely resulted in the dystocia event noted in several
female animals and subsequent death or euthanasia. In the 200 mg/kg/day group males, the cause
of morbidity was gastrointestinal tract toxicity with a similar pattern to what was observed in
females at all dose levels. The differences in survival between parental males and females were
likely reflective of the additional physiological demands on females during pregnancy,
parturition, and/or the longer dosing period in females. Dystocia secondary
to the test substance related lung and/or gastrointestinal lesions likely contributed to subsequent
death or euthanasia in extremis. The predominant change in both male and female groups was
necrosis of the mucosal epithelium and often atrophy of the jejunum and/or ileum. In 1 male
there was evidence for some mucosal regeneration, but generally the lesions in all animals were
acute or subacute. The lung inflammation observed in 200 mg/kg/day group females was
characterized by alveoli containing necrotic cell debris, fibrin, and mixed cell inflammatory cell
infiltrates with a multifocal to diffuse distribution. The other microscopic findings in the
lymphoid and reproductive systems of several animals were considered secondary to stress and
the poor condition of the animals.

Test substance-related clinical observations noted at the daily examinations and/or 1–2 hours
postdosing for the surviving males were limited to the 200 mg/kg/day group and included thin
body (which correlated with findings of low body weight and food consumption noted in this
group), cool body, cool and/or pale extremities, and brown and yellow material on various body
surfaces.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body Weights
Males
All surviving males generally gained body weight throughout the treatment period (Study
Days 0–27). Test substance-related lower mean body weight gains were noted in the
200 mg/kg/day group during Study Days 0–27 and correlated with findings of lower mean food
consumption and clinical signs of thin appearance observed in this group. Differences from the
control group were generally statistically significant. Lower (statistically significant) mean body
weight gains were noted in this group compared to the control group when the premating period
(Study Days 0–13) and the entire treatment period (Study Days 0–27) were evaluated. As a
result, the mean absolute body weight for males in the 200 mg/kg/day group was lower (5.2%)
than the control group on Study Day 27; the difference was not statistically significant.
Mean body weights and body weight gains in the 30 and 100 mg/kg/day group males were
unaffected by test substance administration throughout the study. Statistically significantly lower
mean body weight gains were noted in these groups compared to the control group during Study
Days 13–20. These differences were transient and did not affect mean absolute body weights in
these groups, and therefore were not considered test substance-related.

Females
Weekly
Females generally gained body weight throughout the premating period (Study Days 0–27). Test
substance-related lower mean body weight gains were noted in the 30, 100, and 200 mg/kg/day
group females throughout the premating period (Study Days 0–13) and generally correlated with
reduced food consumption noted in these groups; differences from the control group were
statistically significant at 30 and 200 mg/kg/day. As a result, mean absolute body weights were
5.4%, 4.2%, and 9.6% lower in the 30, 100, and 200 mg/kg/day compared to concurrent control.

Gestation
Gravid females gained body weight throughout the gestation period (Gestation Days 0–20). Test
substance-related lower mean body weight gains were noted throughout gestation in the 100 and
200 mg/kg/day groups; differences from the control group were statistically significant during
Gestation Days 17–20. As a result, mean body weight gains in these groups were lower than the
control group when the entire gestation period (Gestation Days 0–20) was evaluated; the
difference was statistically significant at 200 mg/kg/day. Mean absolute body weights in the
100 and 200 mg/kg/day groups were 7.3% and 14.4% lower than the control group on Gestation
Day 20; the difference was statistically significant at 200 mg/kg/day.
Mean body weights and body weight gains in the 30 mg/kg/day group were unaffected by test
substance administration during gestation. None of the differences from the control group were
statistically significant.

Lactation

Evaluation of female body weights at 200 mg/kg/day during the lactation period was precluded
by early euthanasia of all animals in this group.
All surviving females gained body weight from Lactation Days 1–13 of the lactation period. A
lower mean body weight was noted in the 100 mg/kg/day group (11.5%) during early lactation
(Lactation Day 1) was a continuation of the effects noted during the gestation treatment period.
However, due to mortality and moribundity, only 2 animals were available for evaluation of
body weight changes. Individual body weight changes for these females were comparable to the
control group throughout the lactation period. The individual absolute body weights in the
100 mg/kg/day group were 314 and 371 g on Lactation Day 13 compared to the control group
mean value of 351 g.
Mean body weights and body weight gains in the 30 mg/kg/day groups were unaffected by test
substance administration during lactation. Statistically significantly higher mean body weight
gains were noted in this group compared to the control group during Lactation Days 1–4 and
when the entire lactation period (Lactation Days 1–13) was evaluated.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food Consumption
Males

Test substance-related lower mean food consumption, evaluated as g/animal/day, was noted in
the 200 mg/kg/day group males throughout the premating period (Study Days 0–13); differences
from the control group were statistically significantly during Study Days 6–13. This difference
corresponded with the lower mean body weight gain noted in this group during the premating
period.
Mean food consumption in the 30 and 100 mg/kg/day group males was similar to that in the
control group throughout the premating period. No statistically significant differences were
observed.


Females
Weekly
Test substance-related lower mean food consumption, evaluated as g/animal/day, was noted in
the 200 mg/kg/day group females during the premating period (Study Days 0–13); differences
from the control group were statistically significant.
Mean food consumption in the 30 and 100 mg/kg/day group females was slightly lower than the
control group during the premating period. Differences from the control group were slight and
not statistically significant, and therefore were not considered test substance-related. These
changes corresponded with the lower mean body weight gain noted in these groups.

Gestation

Test substance-related lower mean food consumption was noted in the 200 mg/kg/day group
throughout the gestation treatment period (Gestation Days 0–20); differences were generally
statistically significant. The effects on mean food consumption in this group corresponded to the
effects on mean body weights and body weight gains in this group.
In the 100 mg/kg/day group, mean food consumption was comparable to the control group
during Gestation Days 0–17. A statistically significantly lower mean food consumption value
was noted in this group compared to the control group during Gestation Days 17–20. This
difference corresponded to the effects on mean body weight at 100 mg/kg/day during late
gestation and was considered test substance-related despite the lack of an effect on the
cumulative food consumption interval (Gestation Days 0–20).
Mean maternal food consumption in the 30 mg/kg/day group was unaffected by test substance
administration during gestation. Differences from the control group were slight and not
statistically significant.


Lactation

At 200 mg/kg/day, all females were found dead or euthanized in extremis by Lactation Day 0,
precluding the evaluation of lactation data in this group.
Mean maternal food consumption, evaluated as g/animal/day, in the 30 and 100 mg/kg/day
groups was unaffected by test substance administration during lactation. None of the differences
from the control group were statistically significant.

Haematological findings:

no effects observed

Description (incidence and severity):

Hematology

There were no test substance-related hematology findings in males at any dosage level.
Evaluation of hematology data for females at 200 mg/kg/day was precluded by mortality and
moribundity. Test substance-related minimal (not statistically significantly) decreases in white
blood cell, lymphocyte, and eosinophil counts were noted in females at 30 and/or
100 mg/kg/day. Other statistically significant difference from the control group were not
dose-related, and therefore were not considered test substance-related.

Coagulation

Evaluation of coagulation data for females at 200 mg/kg/day was precluded by mortality and
moribundity.
There were no test substance-related alterations in coagulation parameters in males and females
at 30 and 100 or in males at 200 mg/kg/day. Differences from the control group were not
statistically significant.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Serum Chemistry

Evaluation of serum chemistry findings for females at 200 mg/kg/day was precluded by
mortality and moribundity.
There were no test substance-related effects on serum chemistry in males and females at 30 and
100 mg/kg/day or in males at 200 mg/kg/day. Any statistically significant differences were not
dose-responsive, there were no similar occurrence in the opposite sex, and/or the changes were
of minimal magnitude, and therefore were considered to be the result of normal biological
variation.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Functional Observational Battery
At 200 mg/kg/day, all females were found dead or euthanized in extremis by Lactation Day 0,
precluding any collection of functional observational battery data in this group.

Home Cage Observations
Home cage parameters were unaffected by test substance administration. There were no
statistically significant differences for the test substance-treated groups when compared to the
control group during the last week of dosing (males) or on Lactation Day 13 (females).


Handling Observations
Handling parameters were unaffected by test substance administration. There were no
statistically significant differences for the test substance-treated groups when compared to the
control group during study during the last week of dosing (males) or on Lactation Day 13
(females).

Open Field Observations
Open field parameters were unaffected by test substance administration. Any statistically
significant differences were not dose-responsive, and/or the changes were of minimal magnitude,
and therefore were considered to be the result of normal biological variation.

Sensory Observations
Sensory parameters were unaffected by test substance administration. There were no statistically
significant differences for the test substance-treated groups when compared to the control group
during the last week of dosing (males) or on Lactation Day 13 (females).


Neuromuscular Observations

Neuromuscular parameters were unaffected by test substance administration. There were no
statistically significant differences for the test substance-treated groups when compared to the
control group during the last week of dosing (males) or on Lactation Day 13 (females).

Physiological Observations
Physiological parameters were unaffected by test substance administration. There were no
statistically significant differences for the test substance-treated groups when compared to the
control group during the last week of dosing (males) or on Lactation Day 13 (females).



Motor Activity

At 200 mg/kg/day, all females were found dead or euthanized in extremis by Lactation Day 0,
precluding collection of motor activity data in this group.
Motor activity patterns (total activity as well as ambulatory activity counts) in F0 animals were
unaffected by test substance administration at all concentrations when evaluated during the last
week of dosing (males) or on Lactation Day 13 (females). Values obtained from the
6 subintervals evaluated (0–10, 11–20, 21–30, 31–40, 41–50 and 51–60 minutes) and the overall
60-minute test session values were comparable to the concurrent control values and the Charles
River Ashland historical control data. Differences from the control group were slight, not
statistically significant, with the following exception. A statistically significantly lower mean
ambulatory count was noted for females in the 30 mg/kg/day group compared to the control
group during the 0–10 minute interval. This difference was not dose-responsive, and therefore
was not considered test substance-related. No remarkable shifts in the pattern of habituation
occurred in any of the test substance-treated groups when the F0 animals were evaluated during
the last week of dosing (males) or on Lactation Day 13 (females).

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Organ Weights

Test substance-related prostate, spleen, and thymus organ weight changes were observed in the 200 mg/kg/day group males.

There was minimal or moderate lung mixed cell inflammation and mild decreased cellularity in
the marginal zone of the spleen in the 200 mg/kg/day group males. These changes were similar
to those observed in females euthanized early or found dead in the 200 mg/kg/day group. The
minimal or moderate lung mixed cell inflammation was likely due to aspiration of the test
substance (an insoluble fine powder).
No other test substance-related organ weight changes were noted. There were other isolated
organ weight values that were statistically different from their respective controls. There were,
however, no patterns, trends, or correlating data to suggest these values were toxicologically
relevant. Thus, other organ weight differences observed were considered incidental and/or
related to difference of sexual maturity and unrelated to administration of KDLNO.

Gross pathological findings:

no effects observed

Description (incidence and severity):

F0 Gross Pathology

No test substance-related gross findings were noted. The gross findings observed were
considered incidental, of the nature commonly observed in this strain and age of rats, and/or
were of similar incidence in control and treated animals and, therefore, were considered
unrelated to test substance administration.
The mean numbers of unaccounted-for sites and implantation sites in the 30 and 100 mg/kg/day
groups were similar to the control group values.

F1 Gross Pathology
Unscheduled Deaths

Three (3), 5(5), and 10(2) pups (litters) in the control, 30, and 100 mg/kg/day groups,
respectively, were found dead or euthanized in extremis. No internal findings that could be

attributed to parental test substance administration were noted at the necropsies of pups that were
found dead or euthanized in extremis. Aside from the presence or absence of milk in the
stomach, internal findings included lobular dysgenesis of the lungs in Pup No. 6788-03 in the
control group. No other internal findings were noted.

Scheduled Necropsy
No internal findings were noted at the necropsy of pups euthanized on PND 13.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathology
Test substance-related lung and spleen microscopic findings were observed.

There was minimal or moderate lung mixed cell inflammation and mild decreased cellularity in
the marginal zone of the spleen in the 200 mg/kg/day group males. These changes were similar
to those observed in females euthanized early or found dead in the 200 mg/kg/day group. The
minimal or moderate lung mixed cell inflammation was likely due to aspiration of the test
substance (an insoluble fine powder). The decreased cellularity in the spleen was considered
secondary to stress and the poor condition of the animals.

Other microscopic findings observed were considered incidental, of the nature commonly
observed in this strain and age of rats, and/or were of similar incidence and severity in control
and treated animals and, therefore, were considered unrelated to administration of KDLNO.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

F1 Litter Data
In the 200 mg/kg/day group, a single female (No. 6805) was found dead and remaining pups in
her litter were euthanized in extremis on Lactation Day 0 precluding further
evaluation of litter data in this group.


PND 0 Litter Data and Postnatal Survival

Due to maternal mortality and moribundity at 100 mg/kg/day, only 4 females in this group
delivered viable litters. Lower (not statistically significant) mean number of pups born and
postnatal survival on PND 0 (relative to the number of pups born) was noted in this group
(11.3 pups/litter and 66.7% per litter, respectively) compared to the control group
(13.9 pups/litter and 98.3% per litter, respectively). In addition, a statistically significantly lower
mean live litter size on PND 0 was noted at 100 mg/kg/day (8.8 pups/litter) compared to the
control group (13.7 pups/litter). These differences were attributed to 1 female (No. 6793) that
delivered 2 pups on Gestation Day 21 (Lactation Day 0), was found dead the same day and had
15 former implantation sites, and therefore were not considered test substance-related. The mean
percentage of males at birth in the 100 mg/kg/day group was comparable to the control group.
The mean number of pups born, live litter size, percentage of males at birth and postnatal
survival in the 30 mg/kg/day group were similar to the concurrent control group

Observations
The general physical condition (defined as the occurrence and severity of clinical observations)
of all F1 pups in this study was unaffected by test substance administration. Three (3), 5(5), and
10(2) pups (litters) in the control, 30, and 100 mg/kg/day groups, respectively, were found dead.
One pup in the 30 mg/kg/day group was missing and presumed to have been cannibalized.

Offspring Body Weights
Mean male and female pup body weights and body weight changes during PND 1–13 in the
30 and 100 mg/kg/day groups were unaffected by parental administration of the test substance.
Difference from the control group were slight and not statistically significant, with the following
exception. A statistically significantly higher mean body weight gain was noted for females at
30 mg/kg/day compared to the control group during PND 4–7. This difference was transient, not
dose-responsive, and did not impact mean absolute body weight for females in this group, and
therefore was not considered test substance-related.

Anogenital Distance

The anogenital distances (absolute, relative to pup body weight, and relative to the cube root of
pup body weight) in the 30 and 100 mg/kg/day groups were similar to the control group values.
Differences from the control group were slight and not statistically significant.

Areolae/Nipple Anlagen

There were no nipples present in the F1 male pups at any dosage level when evaluated on
PND 13.

Thyroid Hormone Analysis (PND 13)

Serum total T4 was measured on samples obtained from F1 pups on PND 13. There were no test
substance-related effects on serum total T4 values in the F1 males and females at 30 and
100 mg/kg/day on PND 13. Differences from the control group were considered to be the result
of normal biological variation and were not considered to be of toxicological significance. The
serum samples were collected from F1 pups on PND 4 but not analyzed per protocol.

Gross Pathology
Unscheduled Deaths

Three (3), 5(5), and 10(2) pups (litters) in the control, 30, and 100 mg/kg/day groups,
respectively, were found dead or euthanized in extremis. No internal findings that could be

attributed to parental test substance administration were noted at the necropsies of pups that were
found dead or euthanized in extremis. Aside from the presence or absence of milk in the
stomach, internal findings included lobular dysgenesis of the lungs in Pup No. 6788-03 in the
control group. No other internal findings were noted.

Scheduled Necropsy
No internal findings were noted at the necropsy of pups euthanized on PND 13.

Details on results:

RESULTS
Analyses of Dosing Formulations
The formulations prepared for administration during Weeks 1 and 8 of treatment were analyzed.
The analyzed dosing formulations contained 95% to 108% of the test substance, which was
within the protocol-specified range of target concentrations for solutions (85% to 115%). The
coefficients of variation of the analyzed low (Group 2) and high (Group 4) dose formulations
were 1.1% and 2.5%, respectively, which indicated that the formulations were homogeneous
(i.e. coefficient of variation ≤ 10%). A small response at the retention time of the test substance
was observed in the chromatograms of the analyzed vehicle formulation that was administered to
the control group (Group 1). It was not considered to derive from the formulation since a similar
response was obtained in the analytical blanks.


F0 Generation
Mortality and Observations
Test substance-related effects on survival were observed for males at 200 mg/kg/day and for
females at all dosage levels tested.
Four males in the 200 mg/kg/day group were found dead or euthanized in extremis during Study
Days 9–20. Two, 5, and 10 females in the 30, 100, and 200 mg/kg/day groups were found dead
or euthanized in extremis near or at the end of gestation (Gestation Day 19 to Lactation Day 0),
with the exception of a single female (No. 6794) at 200 mg/kg/day that was found dead on
Gestation Day 4 following marked body weight losses. Clinical observations noted for the
majority of animals that were found dead or euthanized in extremis at 100 and 200 mg/kg/day,
including thin and/or cool body and extremities, red material around the nose, hunched posture,
partially closed eyes, decreased activity, and piloerection, were noted on the day of and/or the
day prior to death or euthanasia. For females in all dosage groups, these clinical signs were
associated with dystocia and prolonged gestation length and were considered KDLNO-related
and adverse. Only 1 of 10 gravid females in the 200 mg/kg/day group (No. 6805) initiated
parturition and delivered 2 pups on Gestation Day 21 (Lactation Day 0) and was subsequently
found dead on the same day. This female had 14 former implantations in utero. In the
100 mg/kg/day group, 4 of 10 females failed to deliver a viable litter and were found dead or
euthanized in extremis on Gestation Day 22 before parturition and 1 female (6793) was
euthanized on Lactation Day 0 due to a total litter loss, following a prolonged gestation length
(24 days) prior to initiation of parturition.
In all but 1 individual 30 mg/kg/day group female (No. 6814), gross and microscopic findings
indicated that the primary cause of morbidity was due to test substance-related effects, mainly
atrophy and necrosis in the gastrointestinal tract mucosa and/or inflammation in the lung. The
severity of the intestinal and/or lung lesions likely resulted in the dystocia event noted in several
female animals and subsequent death or euthanasia. In the 200 mg/kg/day group males, the cause
of morbidity was gastrointestinal tract toxicity with a similar pattern to what was observed in
females at all dose levels. The differences in survival between parental males and females were
likely reflective of the additional physiological demands on females during pregnancy,
parturition, and/or the longer dosing period in females. Dystocia secondary
to the test substance related lung and/or gastrointestinal lesions likely contributed to subsequent
death or euthanasia in extremis. The predominant change in both male and female groups was
necrosis of the mucosal epithelium and often atrophy of the jejunum and/or ileum. In 1 male
there was evidence for some mucosal regeneration, but generally the lesions in all animals were
acute or subacute. The lung inflammation observed in 200 mg/kg/day group females was
characterized by alveoli containing necrotic cell debris, fibrin, and mixed cell inflammatory cell
infiltrates with a multifocal to diffuse distribution. The other microscopic findings in the
lymphoid and reproductive systems of several animals were considered secondary to stress and
the poor condition of the animals.

Test substance-related clinical observations noted at the daily examinations and/or 1–2 hours
postdosing for the surviving males were limited to the 200 mg/kg/day group and included thin
body (which correlated with findings of low body weight and food consumption noted in this
group), cool body, cool and/or pale extremities, and brown and yellow material on various body
surfaces.

Body Weights
Males
All surviving males generally gained body weight throughout the treatment period (Study
Days 0–27). Test substance-related lower mean body weight gains were noted in the
200 mg/kg/day group during Study Days 0–27 and correlated with findings of lower mean food
consumption and clinical signs of thin appearance observed in this group. Differences from the
control group were generally statistically significant. Lower (statistically significant) mean body
weight gains were noted in this group compared to the control group when the premating period
(Study Days 0–13) and the entire treatment period (Study Days 0–27) were evaluated. As a
result, the mean absolute body weight for males in the 200 mg/kg/day group was lower (5.2%)
than the control group on Study Day 27; the difference was not statistically significant.
Mean body weights and body weight gains in the 30 and 100 mg/kg/day group males were
unaffected by test substance administration throughout the study. Statistically significantly lower
mean body weight gains were noted in these groups compared to the control group during Study
Days 13–20. These differences were transient and did not affect mean absolute body weights in
these groups, and therefore were not considered test substance-related.

Females
Weekly
Females generally gained body weight throughout the premating period (Study Days 0–27). Test
substance-related lower mean body weight gains were noted in the 30, 100, and 200 mg/kg/day
group females throughout the premating period (Study Days 0–13) and generally correlated with
reduced food consumption noted in these groups; differences from the control group were
statistically significant at 30 and 200 mg/kg/day. As a result, mean absolute body weights were
5.4%, 4.2%, and 9.6% lower in the 30, 100, and 200 mg/kg/day compared to concurrent control.

Gestation
Gravid females gained body weight throughout the gestation period (Gestation Days 0–20). Test
substance-related lower mean body weight gains were noted throughout gestation in the 100 and
200 mg/kg/day groups; differences from the control group were statistically significant during
Gestation Days 17–20. As a result, mean body weight gains in these groups were lower than the
control group when the entire gestation period (Gestation Days 0–20) was evaluated; the
difference was statistically significant at 200 mg/kg/day. Mean absolute body weights in the
100 and 200 mg/kg/day groups were 7.3% and 14.4% lower than the control group on Gestation
Day 20; the difference was statistically significant at 200 mg/kg/day.
Mean body weights and body weight gains in the 30 mg/kg/day group were unaffected by test
substance administration during gestation. None of the differences from the control group were
statistically significant.

Lactation

Evaluation of female body weights at 200 mg/kg/day during the lactation period was precluded
by early euthanasia of all animals in this group.
All surviving females gained body weight from Lactation Days 1–13 of the lactation period. A
lower mean body weight was noted in the 100 mg/kg/day group (11.5%) during early lactation
(Lactation Day 1) was a continuation of the effects noted during the gestation treatment period.
However, due to mortality and moribundity, only 2 animals were available for evaluation of
body weight changes. Individual body weight changes for these females were comparable to the
control group throughout the lactation period. The individual absolute body weights in the
100 mg/kg/day group were 314 and 371 g on Lactation Day 13 compared to the control group
mean value of 351 g.
Mean body weights and body weight gains in the 30 mg/kg/day groups were unaffected by test
substance administration during lactation. Statistically significantly higher mean body weight
gains were noted in this group compared to the control group during Lactation Days 1–4 and
when the entire lactation period (Lactation Days 1–13) was evaluated.

Food Consumption
Males

Test substance-related lower mean food consumption, evaluated as g/animal/day, was noted in
the 200 mg/kg/day group males throughout the premating period (Study Days 0–13); differences
from the control group were statistically significantly during Study Days 6–13. This difference
corresponded with the lower mean body weight gain noted in this group during the premating
period.
Mean food consumption in the 30 and 100 mg/kg/day group males was similar to that in the
control group throughout the premating period. No statistically significant differences were
observed.


Females
Weekly
Test substance-related lower mean food consumption, evaluated as g/animal/day, was noted in
the 200 mg/kg/day group females during the premating period (Study Days 0–13); differences
from the control group were statistically significant.
Mean food consumption in the 30 and 100 mg/kg/day group females was slightly lower than the
control group during the premating period. Differences from the control group were slight and
not statistically significant, and therefore were not considered test substance-related. These
changes corresponded with the lower mean body weight gain noted in these groups.

Gestation

Test substance-related lower mean food consumption was noted in the 200 mg/kg/day group
throughout the gestation treatment period (Gestation Days 0–20); differences were generally
statistically significant. The effects on mean food consumption in this group corresponded to the
effects on mean body weights and body weight gains in this group.
In the 100 mg/kg/day group, mean food consumption was comparable to the control group
during Gestation Days 0–17. A statistically significantly lower mean food consumption value
was noted in this group compared to the control group during Gestation Days 17–20. This
difference corresponded to the effects on mean body weight at 100 mg/kg/day during late
gestation and was considered test substance-related despite the lack of an effect on the
cumulative food consumption interval (Gestation Days 0–20).
Mean maternal food consumption in the 30 mg/kg/day group was unaffected by test substance
administration during gestation. Differences from the control group were slight and not
statistically significant.


Lactation

At 200 mg/kg/day, all females were found dead or euthanized in extremis by Lactation Day 0,
precluding the evaluation of lactation data in this group.
Mean maternal food consumption, evaluated as g/animal/day, in the 30 and 100 mg/kg/day
groups was unaffected by test substance administration during lactation. None of the differences
from the control group were statistically significant.

Reproductive Performance

F0 male and female reproductive parameters were considered.

Male Mating Index
Female Mating Index
Male Fertility Index
Female Fertility Index
Male Copulation Index
Female Conception Index
Estrous Cycle Length (days)
Pre-Coital Interval (days)

No test substance-related effects on reproductive performance were observed at any dosage
level. No statistically significant differences were noted between the control and test
substance-treated groups. One, 2, and 1 male in the control, 100, and 200 mg/kg/day groups,
respectively, did not sire a litter. One, 2, and 1 female in these same respective groups were
determined to be nongravid.

The mean numbers of days between pairing and coitus were comparable across all groups. A
statistically significantly lower mean estrous cycle length was noted in the 30 and 200 mg/kg/day
groups; however, differences from control were marginal, non-dose responsive, and values were
within the normal biological range of 4-5 days for rodents; these differences were hence
considered incidental.

Gestation Length and Parturition

Test substance-related, adverse effects on gestation length and the process of parturition were
noted in the 30, 100, and 200 mg/kg/day groups.
Nine (of 10) gravid females in the 200 mg/kg/day group failed to deliver a viable litter and were
found dead or euthanized in extremis during the time of possible parturition (Gestation
Days 19–21). The last female in this group initiated parturition and delivered 2 pups on Gestation
Day 21 (Lactation Day 0), and was subsequently found dead on the same day. In the
100 mg/kg/day group, 4 (of 8) gravid females failed to deliver a viable litter and were found dead
or euthanized in extremis on Gestation Day 22 before parturition. At 30 mg/kg/day, 2 (of 9)
gravid females were found dead or euthanized in extremis on Gestation Day 23 after failing to
deliver a litter. As a result, a mean gestation length could not be calculated for the 200
mg/kg/day group. The mean gestation lengths in the 30 and 100 mg/kg/day groups were 22.3 and
22.5 days, respectively, compared to the concurrent control group (21.8 days) and the maximum
mean value in the Charles River Ashland historical control data (22.1 days). These dystociarelated
findings indicated that test substance toxicity (injury to the gastrointestinal tract and lung)
was accentuated during pregnancy and parturition. The severity of the intestinal and/or lung
lesions likely resulted in the dystocia event and subsequent death or euthanasia. For females that
delivered viable litters, there were no significant findings noted during the process of parturition.


Functional Observational Battery
At 200 mg/kg/day, all females were found dead or euthanized in extremis by Lactation Day 0,
precluding any collection of functional observational battery data in this group.

Home Cage Observations
Home cage parameters were unaffected by test substance administration. There were no
statistically significant differences for the test substance-treated groups when compared to the
control group during the last week of dosing (males) or on Lactation Day 13 (females).


Handling Observations
Handling parameters were unaffected by test substance administration. There were no
statistically significant differences for the test substance-treated groups when compared to the
control group during study during the last week of dosing (males) or on Lactation Day 13
(females).

Open Field Observations
Open field parameters were unaffected by test substance administration. Any statistically
significant differences were not dose-responsive, and/or the changes were of minimal magnitude,
and therefore were considered to be the result of normal biological variation.

Sensory Observations
Sensory parameters were unaffected by test substance administration. There were no statistically
significant differences for the test substance-treated groups when compared to the control group
during the last week of dosing (males) or on Lactation Day 13 (females).


Neuromuscular Observations

Neuromuscular parameters were unaffected by test substance administration. There were no
statistically significant differences for the test substance-treated groups when compared to the
control group during the last week of dosing (males) or on Lactation Day 13 (females).

Physiological Observations
Physiological parameters were unaffected by test substance administration. There were no
statistically significant differences for the test substance-treated groups when compared to the
control group during the last week of dosing (males) or on Lactation Day 13 (females).



Motor Activity

At 200 mg/kg/day, all females were found dead or euthanized in extremis by Lactation Day 0,
precluding collection of motor activity data in this group.
Motor activity patterns (total activity as well as ambulatory activity counts) in F0 animals were
unaffected by test substance administration at all concentrations when evaluated during the last
week of dosing (males) or on Lactation Day 13 (females). Values obtained from the
6 subintervals evaluated (0–10, 11–20, 21–30, 31–40, 41–50 and 51–60 minutes) and the overall
60-minute test session values were comparable to the concurrent control values and the Charles
River Ashland historical control data. Differences from the control group were slight, not
statistically significant, with the following exception. A statistically significantly lower mean
ambulatory count was noted for females in the 30 mg/kg/day group compared to the control
group during the 0–10 minute interval. This difference was not dose-responsive, and therefore
was not considered test substance-related. No remarkable shifts in the pattern of habituation
occurred in any of the test substance-treated groups when the F0 animals were evaluated during
the last week of dosing (males) or on Lactation Day 13 (females).

Hematology

There were no test substance-related hematology findings in males at any dosage level.
Evaluation of hematology data for females at 200 mg/kg/day was precluded by mortality and
moribundity. Test substance-related minimal (not statistically significantly) decreases in white
blood cell, lymphocyte, and eosinophil counts were noted in females at 30 and/or
100 mg/kg/day. Other statistically significant difference from the control group were not
dose-related, and therefore were not considered test substance-related.

Coagulation

Evaluation of coagulation data for females at 200 mg/kg/day was precluded by mortality and
moribundity.
There were no test substance-related alterations in coagulation parameters in males and females
at 30 and 100 or in males at 200 mg/kg/day. Differences from the control group were not
statistically significant.

Serum Chemistry

Evaluation of serum chemistry findings for females at 200 mg/kg/day was precluded by
mortality and moribundity.
There were no test substance-related effects on serum chemistry in males and females at 30 and
100 mg/kg/day or in males at 200 mg/kg/day. Any statistically significant differences were not
dose-responsive, there were no similar occurrence in the opposite sex, and/or the changes were
of minimal magnitude, and therefore were considered to be the result of normal biological
variation.

Thyroid Hormone Analysis

Thyroid hormone (serum Total T4) was measured on samples obtained from all surviving male
animals on Study Day 28. There were no test substance-related effects on thyroid hormone
values in the F0 males at any dosage level. Differences from the control group were considered to
be the result of normal biological variation and were not considered to be of toxicological
significance. Serum samples were collected from all surviving female animals on Lactation
Day 13 but were not analyzed per protocol.

Gross Pathology

No test substance-related gross findings were noted. The gross findings observed were
considered incidental, of the nature commonly observed in this strain and age of rats, and/or
were of similar incidence in control and treated animals and, therefore, were considered
unrelated to test substance administration.
The mean numbers of unaccounted-for sites and implantation sites in the 30 and 100 mg/kg/day
groups were similar to the control group values.

Organ Weights

Test substance-related prostate, spleen, and thymus organ weight changes were observed in the 200 mg/kg/day group males.

There was minimal or moderate lung mixed cell inflammation and mild decreased cellularity in
the marginal zone of the spleen in the 200 mg/kg/day group males. These changes were similar
to those observed in females euthanized early or found dead in the 200 mg/kg/day group. The
minimal or moderate lung mixed cell inflammation was likely due to aspiration of the test
substance (an insoluble fine powder).
No other test substance-related organ weight changes were noted. There were other isolated
organ weight values that were statistically different from their respective controls. There were,
however, no patterns, trends, or correlating data to suggest these values were toxicologically
relevant. Thus, other organ weight differences observed were considered incidental and/or
related to difference of sexual maturity and unrelated to administration of KDLNO.

Histopathology
Test substance-related lung and spleen microscopic findings were observed.

There was minimal or moderate lung mixed cell inflammation and mild decreased cellularity in
the marginal zone of the spleen in the 200 mg/kg/day group males. These changes were similar
to those observed in females euthanized early or found dead in the 200 mg/kg/day group. The
minimal or moderate lung mixed cell inflammation was likely due to aspiration of the test
substance (an insoluble fine powder). The decreased cellularity in the spleen was considered
secondary to stress and the poor condition of the animals.
Other microscopic findings observed were considered incidental, of the nature commonly
observed in this strain and age of rats, and/or were of similar incidence and severity in control
and treated animals and, therefore, were considered unrelated to administration of KDLNO.

F1 Litter Data
In the 200 mg/kg/day group, a single female (No. 6805) was found dead and remaining pups in
her litter were euthanized in extremis on Lactation Day 0 precluding further
evaluation of litter data in this group.


PND 0 Litter Data and Postnatal Survival

Due to maternal mortality and moribundity at 100 mg/kg/day, only 4 females in this group
delivered viable litters. Lower (not statistically significant) mean number of pups born and
postnatal survival on PND 0 (relative to the number of pups born) was noted in this group
(11.3 pups/litter and 66.7% per litter, respectively) compared to the control group
(13.9 pups/litter and 98.3% per litter, respectively). In addition, a statistically significantly lower
mean live litter size on PND 0 was noted at 100 mg/kg/day (8.8 pups/litter) compared to the
control group (13.7 pups/litter). These differences were attributed to 1 female (No. 6793) that
delivered 2 pups on Gestation Day 21 (Lactation Day 0), was found dead the same day and had
15 former implantation sites, and therefore were not considered test substance-related. The mean
percentage of males at birth in the 100 mg/kg/day group was comparable to the control group.
The mean number of pups born, live litter size, percentage of males at birth and postnatal
survival in the 30 mg/kg/day group were similar to the concurrent control group

Observations
The general physical condition (defined as the occurrence and severity of clinical observations)
of all F1 pups in this study was unaffected by test substance administration. Three (3), 5(5), and
10(2) pups (litters) in the control, 30, and 100 mg/kg/day groups, respectively, were found dead.
One pup in the 30 mg/kg/day group was missing and presumed to have been cannibalized.

Offspring Body Weights
Mean male and female pup body weights and body weight changes during PND 1–13 in the
30 and 100 mg/kg/day groups were unaffected by parental administration of the test substance.
Difference from the control group were slight and not statistically significant, with the following
exception. A statistically significantly higher mean body weight gain was noted for females at
30 mg/kg/day compared to the control group during PND 4–7. This difference was transient, not
dose-responsive, and did not impact mean absolute body weight for females in this group, and
therefore was not considered test substance-related.

Anogenital Distance

The anogenital distances (absolute, relative to pup body weight, and relative to the cube root of
pup body weight) in the 30 and 100 mg/kg/day groups were similar to the control group values.
Differences from the control group were slight and not statistically significant.

Areolae/Nipple Anlagen

There were no nipples present in the F1 male pups at any dosage level when evaluated on
PND 13.

Thyroid Hormone Analysis (PND 13)

Serum total T4 was measured on samples obtained from F1 pups on PND 13. There were no test
substance-related effects on serum total T4 values in the F1 males and females at 30 and
100 mg/kg/day on PND 13. Differences from the control group were considered to be the result
of normal biological variation and were not considered to be of toxicological significance. The
serum samples were collected from F1 pups on PND 4 but not analyzed per protocol.

Gross Pathology
Unscheduled Deaths

Three (3), 5(5), and 10(2) pups (litters) in the control, 30, and 100 mg/kg/day groups,
respectively, were found dead or euthanized in extremis. No internal findings that could be

attributed to parental test substance administration were noted at the necropsies of pups that were
found dead or euthanized in extremis. Aside from the presence or absence of milk in the
stomach, internal findings included lobular dysgenesis of the lungs in Pup No. 6788-03 in the
control group. No other internal findings were noted.

Scheduled Necropsy
No internal findings were noted at the necropsy of pups euthanized on PND 13.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Sex:

male

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

2

Key result

Dose descriptor:

LOAEL

Effect level:

30 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Remarks on result:

not determinable

Remarks:

Under the conditions of this screening study, the no-observed-adverse-effect level (NOAEL) for F0 male systemic toxicity was determined to be 100 mg/kg/day when the test substance was administered by oral gavage to Crl:CD(SD) rats. Based on the results, a NOAEL for F0 female systemic toxicity could not be determined.

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

CONCLUSIONS
In conclusion, the test substance, lithium nickel potassium oxide (KDLNO), did not show any
adverse effects on neurobehavior, thyroid hormone or thyroid gland, reproductive performance,
or postnatal development. Mortality and moribundity caused by injury to the gastrointestinal
tract (males and females) and lungs (high dose females), adverse clinical observations, lower
body weights, and food consumption were noted for females at 30, 100, and 200 mg/kg/day and
for males at 200 mg/kg/day. Lower spleen, thymus, and prostate weights and microscopic
findings in the spleen and lung were noted for males at 200 mg/kg/day. Under the conditions of
this screening study, the no-observed-adverse-effect level (NOAEL) for F0 male systemic
toxicity was determined to be 100 mg/kg/day when the test substance was administered by oral
gavage to Crl:CD(SD) rats. Based on the results, a NOAEL for F0 female systemic toxicity could
not be determined. There were no effects on reproductive parameters for F0 males, and therefore
a dosage level of 200 mg/kg/day was considered to be the NOAEL for F0 male reproductive
toxicity. Based on the secondary dystocia and longer gestation lengths at 30, 100, and
200 mg/kg/day, a NOAEL for F0 female reproductive toxicity was not determined. The NOAEL
for F1 neonatal toxicity was 100 mg/kg/day based on the absence of any adverse effects on
postnatal development at this dosage level.

Executive summary:

SUMMARY

 

The objectives of this study were to evaluate the potential toxic effects of test substance, lithium

nickel potassium oxide (KDLNO), when administered to rats for at least 28 days and to evaluate

the potential of the test substance to affect male and female reproductive performance such as

gonadal function, mating behavior, and conception through day 13 of postnatal life.

Animals were dosed with 0, 30, 100, or 200 mg/kg/day of KDLNO in 0.25% methylcellulose

with a dose volume of 10 mL/kg via oral gavage once daily. Males were dosed for 14 days prior

to mating and continuing through 1 day prior to euthanasia for 28 days in total. Females were

dosed for 14 days prior to mating and continuing through Lactation Day 13 for up to 54 days in

total.

 

The following parameters and end points were evaluated in this study: clinical signs, body

weights, body weight gains, food consumption, estrous cycles, reproductive performance,

parturition, litter viability and survival, anogenital distance, areolae/nipple anlagen,

neurobehavior, thyroid hormones, clinical pathology, gross necropsy findings, organ weights,

and histopathologic examinations. Test substance-related effects on survival were observed for males at 200 mg/kg/day and for females at all dosage levels tested.

 

Four males in the 200 mg/kg/day group were found dead or euthanized in extremis during Study

Days 9–20. Two, 5, and 10 females in the 30, 100, and 200 mg/kg/day groups were found dead

or euthanized in extremis near or at the end of gestation (Gestation Day 19 to Lactation Day 0),

with the exception of a single female at 200 mg/kg/day that was found dead on Gestation Day 4

following marked body weight losses. Clinical observations noted for the majority of animals on

the day of and/or the day prior to death or euthanasia at 100 and 200 mg/kg/day, included thin

and/or cool body and extremities, red material around the nose, hunched posture, partially closed

eyes, decreased activity, and piloerection. For females in all dosage groups, these clinical signs

were associated with dystocia, prolonged gestation length, and were considered KDLNO-related

and adverse. Only 1 of 10 gravid females in the 200 mg/kg/day group initiated parturition and

delivered 2 pups on Gestation Day 21 (Lactation Day 0) and was subsequently found dead on the

same day. This female retained 12 dead fetuses in utero. In the 100 mg/kg/day group, 4 of

10 females failed to deliver a viable litter and were found dead or were euthanized in extremis on

Gestation Day 22 before parturition, and 1 female was euthanized on Lactation Day 0 due to a

total litter loss, following a prolonged gestation length (24 days) prior to initiation of parturition.

In all but one individual 30 mg/kg/day group female, gross and microscopic findings indicated

that the primary cause of morbidity was test substance (an insoluble fine powder) -related effects

in the gastrointestinal tract and lung. The severity of the gastrointestinal and/or lung lesions

likely resulted in the dystocia event noted in several female animals and subsequent death or

euthanasia. The effect on male survival was in the 200 mg/kg/day group only. The cause of

morbidity was gastrointestinal tract lesions with a similar pattern to what was observed in

females at all dose levels. The differences in survival between males and females was likely

reflective of the additional physiological demands on females due to pregnancy, parturition,

and/or the longer dosing regimen for the females.

 

The predominant change in both male and female groups was necrosis of the mucosal epithelium

and often atrophy of the jejunum and/or ileum. The lung inflammation observed in

200 mg/kg/day group females was characterized by alveoli containing necrotic cell debris, fibrin,

and mixed cell inflammatory cell infiltrates with a multifocal to diffuse distribution. The lung

microscopic findings in the 200 mg/kg/day group may indicate local injury secondary to reflux

, likely due to aspiration of the test substance. All other animals survived to scheduled necropsy.

 

Test substance-related lower mean body weight gains were noted for males at 200 mg/kg/day

and for females at all dosage levels tested. Correspondingly lower mean food consumption was

also noted for females at 200 mg/kg/day throughout the premating (Study Days 0–13) and for

males during the entire treatment period (Study Days 0–27). As a result, mean absolute body

weights were lower than the control group for males at 200 mg/kg/day (5.2%) on Study Day 27

and for females at 30, 100, and 200 mg/kg/day (5.4%, 4.2%, and 9.6%, respectively) on Study

Day 13. During gestation, lower mean body weight gains and lower food consumption were

noted in the 100 and 200 mg/kg/day group females compared to the control group, resulting in

mean absolute body weights that were 7.3% and 14.4% lower, respectively, on Gestation

Day 20. Evaluation of female body weights during the lactation period was precluded by early

euthanasia for all animals in the 200 mg/kg/day group. In the 100 mg/kg/day group, a lower

mean body weight (11.5%) compared to the control group was noted during early lactation

(Lactation Day 1) and was a continuation of the effects noted during the gestation treatment

period. However, due to mortality and moribundity, only 2 animals were available for further

evaluation of body weight changes. Individual body weight changes and food consumption for

these females were comparable to the control group throughout the lactation period. Mean body

weights, body weight gains, and food consumption were unaffected by test substance

administration throughout the study for males in the 30 and 100 mg/kg/day groups and during

gestation and lactation for females in the 30 mg/kg/day group.

 

There were no test substance-related effects on F0 neurobehavioral parameters (FOB and motor

activity) and reproductive performance (male and female mating and fertility, male copulation,

and female conception indices, mean estrous cycle and precoital intervals) was unaffected by test

substance administration at all dosage levels.

 

Test substance-related, adverse effects on gestation length and the process of parturition were

noted in the 30, 100, and 200 mg/kg/day groups. Nine (of 10) gravid females in the

200 mg/kg/day group failed to deliver a viable litter and were found dead or euthanized in

extremis during the time of possible parturition (Gestation Days 19–21). The last female in this

group initiated parturition and delivered 2 pups on Gestation Day 21 (Lactation Day 0), and was

subsequently found dead on the same day. In the 100 mg/kg/day group, 4 (of 8) gravid females

failed to deliver a viable litter and were found dead or euthanized in extremis on Gestation

Day 22. At 30 mg/kg/day, 2 (of 9) gravid females were found dead or euthanized in extremis on

Gestation Day 23 after failing to deliver a litter. As a result, a mean gestation length could not be

calculated for the 200 mg/kg/day group, and the mean gestation lengths in the 30 and

100 mg/kg/day groups were higher than the control group.

 

There were no test substance-related effects on hematology, coagulation, and serum chemistry

parameters in the F0 males at 30, 100, and 200 mg/kg/day and females at 30 and 100 mg/kg/day.

There were no test substance-related effects on T4 levels observed in the 30, 100, and

200 mg/kg/day group F0 males.

 

At the scheduled necropsy, there were no test substance-related macroscopic and microscopic

findings or effects on organ weight in the 30 and 100 mg/kg/day group males and females. For

the 200 mg/kg/day group males, lower mean spleen, thymus, and prostate weights, and

microscopic findings in the spleen (decreased cellularity) and lung (mixed cell inflammation)

were observed. There were no microscopic correlates for the lower mean thymus and prostate

weights.

 

The minimal or moderate lung mixed cell inflammation in the 200 mg/kg/day group observed in

males of scheduled necropsy and females found dead or euthanized in extremis may indicate

local injury secondary to reflux, likely due to aspiration of the test substance.

 

For females that delivered, the mean numbers of F0 pups born, percentage of males at birth, live

litter size on PND 0, postnatal survival, clinical condition of the pups, anogenital distance,

areola/nipple retention (males), and mean pup body weights and body weight gains in the 30 and

100 mg/kg/day groups were not affected by test substance administration to the F0 males and

females. There were no test substance-related macroscopic findings noted in F1 pups that were

found dead or at the scheduled necropsy on PND 13. There were no test substance-related effects

on serum T4 levels in the F1 pups on PND 13 at any dosage level.

 

In conclusion, the test substance, lithium nickel potassium oxide (KDLNO), did not show any

adverse effects on neurobehavior, thyroid hormone or thyroid gland, reproductive performance,

or postnatal development. Mortality and moribundity caused by injury to the gastrointestinal

tract (males and females) and lungs (high dose females), adverse clinical observations, lower

body weights, and food consumption were noted for females at 30, 100, and 200 mg/kg/day and

for males at 200 mg/kg/day. Lower spleen, thymus, and prostate weights and microscopic

findings in the spleen and lung were noted for males at 200 mg/kg/day. Under the conditions of

this screening study, the no-observed-adverse-effect level (NOAEL) for F0 male systemic

toxicity was determined to be 100 mg/kg/day when the test substance was administered by oral

gavage to Crl:CD(SD) rats. Based on the results, a NOAEL for F0 female systemic toxicity could

not be determined. There were no effects on reproductive parameters for F0 males, and therefore

a dosage level of 200 mg/kg/day was considered to be the NOAEL for F0 male reproductive

toxicity. Based on the secondary dystocia and longer gestation lengths at 30, 100, and

200 mg/kg/day, a NOAEL for F0 female reproductive toxicity was not determined. The NOAEL

for F1 neonatal toxicity was 100 mg/kg/day based on the absence of any adverse effects on

postnatal development at this dosage level.