Lithium nickel potassium oxide

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2020 to 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (28-Day (Subacute) Inhalation Toxicity Study

Principles of method if other than guideline:

This study was performed in accordance with the United States Code of Federal Regulations,
Title 40, Parts 160 and 792: Good Laboratory Practice Standards, as accepted by Regulatory
Authorities throughout the European Union (OECD Principles of Good Laboratory Practice),
Japan (MAFF and METI), and other countries that are signatories to the OECD Mutual
Acceptance of Data Agreement, with the following exceptions:
- A Certificate of Analysis for the test substance was provided by the Sponsor, but the
characterization analyses were not conducted in accordance with GLP standards; and
-The blood plasma sample analysis, and supporting control plasma sample collection for
exploratory bioanalytical method development at BASF SE were coordinated by the
Sponsor as a supplemental bioanalytical phase of this study and were not performed or
reported in accordance with GLP.

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:

Sprague-Dawley

Remarks:

Crl:CD(SD) rats

Details on species / strain selection:

Justification for Test System and Number of Animals:


At this time, studies in laboratory animals provide the best available basis for extrapolation to
humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.

The Sprague Dawley rat was chosen as the animal model for this study as it is an accepted rodent
species for nonclinical toxicity testing by regulatory agencies. The total number of animals used in this study was considered to be the minimum required to properly characterize the effects of the test substance. This study was designed such that it did not require an unnecessary number of animals to accomplish its objectives.


Animal Identification:
Each animal was identified using a subcutaneously implanted identification chip.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal Identification:
Each animal was identified using a subcutaneously implanted identification chip.

Environmental Acclimation:
Each animal was inspected by qualified personnel upon receipt. Animals judged to be in good
health were placed in acclimation for at least 7 days. To screen animals for poor tolerance to
restraint and to limit potential effects on respiration of the novel environment/conditions of
restraint, the animals were acclimated to restraint in nose-only exposure tubes 4 times
(1 acclimation/day) prior to their first day of exposure by increasing the restraint time over the
pretreatment period (first day – 1 hour, second day – 2 hours, third day – 4 hours, fourth day – 6
hours; times were approximate). Following the restraint period, each animal was observed for
clinical signs of injury or stress.

Selection, Assignment, and Disposition of Animals:
Main and Recovery Study animals were assigned to groups by a stratified randomization scheme
designed to achieve similar group mean body weights. Males and females were randomized
separately. An animal with ophthalmic findings was not assigned to a group. Individual body
weights at randomization were within ± 20% of the mean for each sex.
The disposition of all animals was documented in the study records.


Housing: Group housed 2–3 animals of the same sex and same exposure group.
Caging: Polycarbonate, solid-bottom cages containing appropriate bedding
material (Bed-O-Cobs® or other suitable material).

Cage Identification: Color-coded cage card indicating study, group, animal number(s), and
sex.
Housing set-up was as specified in the Guide for the Care and Use of Laboratory Animals
(National Research Council, 2011). Animals were separated during designated
procedures/activities. Cages were arranged on the racks in group order. Where possible, control
group animals were housed on a separate rack from the test substance-treated animals.




Environmental Conditions:
The targeted conditions for the animal room environment were as follows:
Temperature: 66°F to 77°F (19°C to 25°C)
Humidity: 30% to 70%
Light Cycle: 12 hours light and 12 hours dark.

Food:
Diet: PMI Nutrition International, LLC Certified Rodent LabDiet 5002.
Type: Meal.
Frequency: Ad libitum, except during the exposure period, acclimation to nose-only
restraint tubes, and during periods of fasting. For those animals
scheduled for fasting prior to necropsy, food was offered for at least
2 hours after each animal’s final exposure and then removed for the
overnight fasting period.
Analysis: Results of analysis for nutritional components and environmental
contaminants were provided by the supplier and are on file at the Testing
Facility. It was considered that there were no known contaminants in the
feed that would interfere with the objectives of the study.

Water:
Type: Municipal tap water, treated by reverse osmosis and ultraviolet
irradiation.
Frequency/Ration: Freely available to each animal via an automatic watering system, except
during the exposure period and acclimation to nose-only restraint tubes.
Water bottles were provided, if required.
Analysis: Periodic analysis of the water was performed, and results of these
analyses are on file at the Testing Facility. It was considered that there were no known contaminants in the water that could interfere with the
outcome of the study.

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:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

> 2 - < 3 µm

Geometric standard deviation (GSD):

2.63

Remarks on MMAD:

The test material was milled to achieve MMAD in the respirable range.

During the course of generation trials prior to initiation of animal exposures, an iterative process
was performed to attempt to obtain the protocol-specified target for particle size (≤ 2.0 microns
for the MMAD) at the desired aerosol concentrations. Setups attempted included the use of
different airflows, various transitional tubing, settling box, and cyclone. The atmosphere
generation system provided a consistent, fairly monodisperse aerosol. However,
the mean MMAD was between 2.0 and 3.0 microns across all groups. The differences from the
protocol specified range did not affect the ability of the aerosol to be respirable.

Details on inhalation exposure:

Administration of Test Substances:

Animals were exposed via nose-only exposure units for 6 hours/day
on a 5-day per week basis for 4 weeks (minimum of 20 exposures for each animal).
The first exposure was designated as Day 1.



Justification of Route and Dose Levels:

The inhalation route of exposure was selected because this is considered by the United States
Environmental Protection Agency (EPA) to be a potential route of human exposure. The results
of this study will be used to inform higher-tier testing required by the EPA.
In a previous acute inhalation study, KDLNO was administered to 5 rats/sex via nose-only
inhalation for 4 hours. Mortality was observed at all exposure levels (1 female at 0.532 mg/L, 2
males and 2 females at 2.037 mg/L, and 4 females at 5.081 mg/L). Clinical signs at all exposure
levels included accelerated respiration, intermittent and/or labored respiration sounds, and
piloerection. All animals had lower body weights the day following exposure.
In consultation with the Study Monitor for the Sponsor, the maximum target exposure
concentration for Phase 1 of the range-finding study was 250 mg/m3. Additional exposure levels
of 100 and 10 mg/m3 were used to elicit a dose response for any effects observed. In Phase 1,
KDLNO was administered to 5 rats/sex via nose-only inhalation for 6 hours. One female in the
250 mg/m3 group died during exposure on Day 1. Additionally, 7 animals in the 100 mg/m3
group (4 males and 3 females) and 4 animals in the 250 mg/m3 group (3 males and 1 female)
died on Day 3. Also, 2 females in the 10 mg/m3 group died on Day 4 prior to scheduled terminal
euthanasia. Clinical signs observed in all test substance-treated groups included labored
breathing, abnormal breathing sounds, and shallow breathing. In addition, some animals within all test substance-treated groups were observed with observations of cold to touch and decreased
activity.

In consultation with the Study Monitor for the Sponsor, the maximum target exposure
concentration for Phase 2 of the range-finding study was selected to be 2 mg/m3. An additional
exposure concentration of 0.5 mg/m3 was used to assist in determination of a dose response for
any effects observed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Inhalation Exposure and Characterization Methods:

Control (filtered air) and test substance atmospheres were administered as 6-hour, nose-only
inhalation exposures on a 5-day per week basis for 4 weeks (minimum of 20 exposures for each
animal). To accommodate protocol-specified activities, exposures were staggered as necessary.
Prior to each exposure, the animals selected for exposure were placed into nose-only exposure
restraint tubes and transported to the exposure room. Following exposure, the animals were
returned to their home cages. Food and water were withheld during the animal exposure periods.
For exposure of the filtered air control group, supply air was delivered to maintain an airflow
similar to that in the test substance-exposed groups.

For exposure of the test substance-treated groups, a dry powder aerosol exposure was generated
using a fluidized bed aerosol generator.
The exposure atmosphere concentrations were characterized by actual aerosol concentration
measurements using standard gravimetric techniques.





Sampling Methods:
Exposure concentrations were determined using standard gravimetric methods. Samples were
collected on pre-weighed, 25-mm glass-fiber filters (Type A/E, PALL Corporation; Ann Arbor,
MI) held in a closed-face filter holder. Sample flow was controlled using a needle valve attached
to the in-house vacuum system and was measured using a mini-BUCK calibrator (A.P Buck Inc.;
Orlando, FL). Following each sample collection, the filters were reweighed and the mass
concentration (mg/m3) was calculated by dividing the gravimetrically determined mass of
aerosol by sample volume. Sample volume was calculated by multiplying the sample flow rate
by the length of the sampling period. Exposure concentrations were determined three times per
week for CNOS 1 and 2, two times per exposure for CNOS 3, three times per exposure for
CNOS 4, and five times per exposure for CNOS 5. For a minimum of 2 exposure days/week,
following standard gravimetric collection, the full set of filter samples (filtered air control plus
test substance treated groups, as applicable) was placed in separate tubes and stored at a target
temperature of 18-24°C for possible future analysis.




Aerosol Particle Size Measurement:
Aerosol particle size measurements were conducted using a 7-stage stainless-steel Cascade
impactor (Model No. 02-007, 02-100-2L, or 02-100-2L-A, IN-TOX Products; Moriarty, NM)
positioned in the animal-breathing zone of the test substance CNOS. Pre-weighed coated 22-mm
stainless steel collection substrates were used for stages 1-7. A pre-weighed, 25-mm glass fiber
filter (Type A/E) was used as the final collection substrate. Following sample collection, the
substrates and filter were re-weighed and the particle size was calculated based on the impactor
stage cut-offs. Particle size was expressed as mass median aerodynamic diameter (MMAD) and
geometric standard deviation (GSD). Aerosol particle size measurements were conducted at least
weekly for the test substance exposure systems during the exposure period and were collected for
approximately 1800, 720, 360, and 360 minutes for CNOS 2, 3, 4 and 5, respectively. Due to a
low collection weight for CNOS 2 during week 3, this impactor was run for an additional
1800 minutes, which is a total of 3960 minutes. The sample flowrate was measured using a
mini-Buck Calibrator and was approximately 1.907-2.074 LPM.

Duration of treatment / exposure:

Exposure Regimen:
Filtered air (control) and the test substance, Lithium Nickel Potassium Oxide, was administered
as 6-hour, nose-only inhalation exposures to rats on a 5-day per week basis for 4-weeks
(minimum of 20 exposures).


Inhalation Exposure System Description:
Filtered air control and test substance exposures were conducted using 11.0-L conventional nose only exposure systems (CNOS). Air supplied to the nose-only systems was provided from the Inhalation Department breathing
quality, in-house compressed air source and a HEPA- and charcoal-filtered, temperature- and
humidity-controlled supply air source. All nose-only system exhaust passed through a Solberg
canister filter (Solberg Manufacturing, Inc.; Itasca, IL) prior to entering the facility exhaust
system, which consists of redundant exhaust blowers preceded by activated-charcoal and
HEPA-filtration units.

Frequency of treatment:

Exposure Regimen:
Filtered air (control) and the test substance, Lithium Nickel Potassium Oxide, was administered
as 6-hour, nose-only inhalation exposures to rats on a 5-day per week basis for 4-weeks
(minimum of 20 exposures).


Inhalation Exposure System Description:
Filtered air control and test substance exposures were conducted using 11.0-L conventional nose only exposure systems (CNOS). Air supplied to the nose-only systems was provided from the Inhalation Department breathing
quality, in-house compressed air source and a HEPA- and charcoal-filtered, temperature- and
humidity-controlled supply air source. All nose-only system exhaust passed through a Solberg
canister filter (Solberg Manufacturing, Inc.; Itasca, IL) prior to entering the facility exhaust
system, which consists of redundant exhaust blowers preceded by activated-charcoal and
HEPA-filtration units.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/m³ air

Doses / concentrations 2

Dose / conc.:

0.1 mg/m³ air

Doses / concentrations 3

Dose / conc.:

0.25 mg/m³ air

Doses / concentrations 4

Dose / conc.:

0.5 mg/m³ air

Doses / concentrations 5

Dose / conc.:

1 mg/m³ air

No. of animals per sex per dose:

Experimental Design

Treatment Group 1: Control - Filtered Air: 10males 5females: 5 recovery males 5recovery females
Treatment Group 2: 0.10mg/m3 KDLNO: 10males 5females: 5 recovery males 5recovery females
Treatment Group 3: 0.25mg/m3 KDLNO: 10males 5females: 5 recovery males 5recovery females
Treatment Group 4: 0.50mg/m3 KDLNO: 10males 5females: 5 recovery males 5recovery females
Treatment Group 5: 1.00mg/m3 KDLNO: 10males 5females: 5 recovery males 5recovery females

Control animals:

yes, concurrent vehicle

Details on study design:

The objective of this study was to evaluate the potential toxicity of the test substance, lithium
nickel potassium oxide (KDLNO) when administered by nose-only inhalation to Sprague
Dawley rats for 6 hours per day, 5 days per week, 4 weeks (minimum of 20 exposures for each
animal), as well as to evaluate the recovery, persistence or progression of any effects following a
2-week recovery period. The results of this study may be used to select exposure concentrations
for a subsequent subchronic (90-day) repeat-exposure inhalation toxicity study (OECD Guideline
No. 413). Four target aerosol exposure concentrations were selected based on the results of a
preliminary 9-day repeat-exposure inhalation toxicity study.

Experimental Design

Treatment Group 1: Control - Filtered Air: 10males 5females: 5 recovery males 5recovery females
Treatment Group 2: 0.10mg/m3 KDLNO: 10males 5females: 5 recovery males 5recovery females
Treatment Group 3: 0.25mg/m3 KDLNO: 10males 5females: 5 recovery males 5recovery females
Treatment Group 4: 0.50mg/m3 KDLNO: 10males 5females: 5 recovery males 5recovery females
Treatment Group 5: 1.00mg/m3 KDLNO: 10males 5females: 5 recovery males 5recovery females

Examinations

Observations and examinations performed and frequency:

Parameter: Cageside Observations

Population(s): All Main and Recovery Study animals

Frequency (minimum required): Prior to exposure. 0.5–2 hours post exposure. At least once daily on nonexposure/recovery days.
Cageside observations were not required to be conducted on days that the detailed clinical observations were performed during the nonexposure and/or recovery
period.

Comments: Animals were observed within their cage unless necessary for identification or confirmation of possible findings. The absence or presence of findings was recorded for individual animals. Findings noted outside the above-specified observation periods were also recorded. Only the presence of unscheduled observations was recorded; the absence of findings was thus not recorded.


Parameter: Detailed Clinical Observations

Population(s): All Main and Recovery Study animals

Frequency (minimum required): Within 4 days of receipt. One week prior to randomization (± 2 days). On the day of randomization.
On Day 1 (prior to exposure). Weekly (± 2 days) during the study period. On the day of the scheduled
necropsies.
Comments:Animals were removed from the cage. The absence or presence of findings was recorded for individual animals.

Parameter: Individual Body Weights

Population(s): All Main and Recovery Study animals

Frequency (minimum required): Within 4 days of receipt. One week prior to randomization (± 2 days). On the day of randomization.
On Day 1 (prior to exposure). Twice weekly during Weeks 1 and 2 of the study period and weekly (± 2 days) during the remainder of the study period. On the day prior to the first day of scheduled necropsies.
On the day of the scheduled necropsy.

Comments: Fasted weight collected on the day of necropsy.



Parameter: Food Consumption

Population(s): All Main and Recovery Study animals

Frequency (minimum required): Recorded once weekly (± 2 days) throughout the study period, beginning on Day 1

Comments: Quantitatively measured.

Parameter: Mortality

Population(s): All animals

Frequency (minimum required):
At least twice daily (morning and afternoon), beginning upon arrival through termination/release.

Comments: Animals were observed within their cage unless necessary for identification or confirmation of possible findings.

Sacrifice and pathology:

Necropsy:

Main and Recovery Study animals were subjected to a complete necropsy examination, which
included evaluation of the carcass; all external surfaces and orifices; the cranial cavity and
external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated
organs and tissues.

Necropsy procedures were performed by qualified personnel with appropriate training and
experience in animal anatomy and gross pathology. A veterinary pathologist, or other suitably
qualified person, was available.

Other examinations:

Ophthalmic Examinations:
Frequency: Once during the pretreatment period (to include unused alternate animals).
Near the end of the exposure period (Day 25 ± 2 days).
Near the end of the recovery period, if necessary, (Day 41 ± 2 days) if findings are noted at the end of the exposure period.
Population(s): All Main Study and Recovery animals.
Procedure: Ophthalmic examinations will be conducted by a board-certified veterinary ophthalmologist using an indirect ophthalmoscope and slit lamp biomicroscope. Prior to examination, animals will be treated with a mydriatic agent.

Statistics:

Constructed Variables:
Data collected during the pretreatment period were presented on individual data tables but not
summarized or statistically analyzed. All statistical analyses were performed within the
respective study phase, unless otherwise noted. Numerical data collected on scheduled occasions
were summarized and statistically analyzed as indicated below according to sex and occasion.
Values may also be expressed as a percentage of pretreatment period or control values, or fold
change of control values, when deemed appropriate. Calculated values on Provantis tables may
not be reproducible from the individual values presented because all calculations were conducted
using non-rounded values.
Body Weight Gains: Calculated between each scheduled interval.
Food Consumption: Calculated between each scheduled interval.
Organ Weight Relative to Body Weight: Calculated against the terminal body weight for the scheduled
intervals.
Organ Weight Relative to Brain Weight: Calculated against the brain weight for the scheduled intervals.


Descriptive Statistical Analyses:
Means, standard deviations, ratio, percentages, numbers, and/or incidences were reported as
appropriate by dataset.

Inferential Statistical Methods:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were
conducted using two-sided tests and are reported at the 1% and 5% levels, unless otherwise
noted.
The pairwise comparisons of interest are listed below:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Group 5 vs. Group 1

Parametric/Non-parametric (Provantis):
Levene’s test was used to assess the homogeneity of group variances.
The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not
significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis
test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or
Dunn’s test, respectively.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

Clinical Observations:
There were no test substance-related clinical observations. All clinical observations in the test
substance-treated groups were noted with similar incidence in the control group, were limited to
single animals, were not noted in a dose-related manner, and/or were common findings for
laboratory rats of this age and strain.

Mortality:

no mortality observed

Description (incidence):

Mortality:
All animals survived until scheduled necropsy.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body Weights and Body Weight Gains:
Body weights were unaffected by test substance administration.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food Consumption:
Food consumption was unaffected by test substance administration.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmic Examinations:
No ophthalmic lesions indicative of toxicity were observed in any of the test substance-treated
groups. All findings observed were typical in prevalence and appearance for laboratory rats of
this age and strain.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematology:
Test substance-related changes in hematology values were limited to minimally increased
neutrophil counts in males and females at ≥ 0.50 mg/m3 at Day 29/30.
Following a 2-week recovery period, increased neutrophils persisted in the 0.50 and 1.00 mg/m3
group males but were of lesser magnitude compared with values at Day 29. In all female test
substance-exposed groups, at least one animal per dose group was noted with minimally to
mildly increased neutrophil count at the end of the recovery period when compared with the
range of the concurrent filtered air control group.
The increased neutrophils at Day 29/30 and the end of the recovery period were consistent with
inflammation and correlated with the BAL cytology findings.

The remaining differences in hematology parameters, regardless of statistical significance, were
consistent with biological variation and were considered unrelated to test substance exposure.

Coagulation:
No test substance-related coagulation changes were noted at any exposure concentration levels.
All differences in coagulation parameters, none of which attained statistical significance, were
consistent with biological variation and were considered unrelated to test substance exposure.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical Chemistry:
There were no test substance-related changes in clinical chemistry values at any exposure
concentration levels.
One male exposed at 1.0 mg/m3 (Animal No. 5009) was noted with increased gamma glutamyl
transferase activity, urea nitrogen and creatinine concentration at Day 29. Although the specific
cause of these changes was unclear, they were considered unrelated to KDLNO exposure due to
the single incidence.
All other differences in clinical chemistry values, none of which attained statistical significance,
were consistent with biological variation and were considered unrelated to test substance
exposure.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinalysis
There were no test substance-related changes in urinalysis values at any exposure concentration
levels.
All differences in urinalysis values, regardless of statistical significance, were consistent with
biological variation and were considered unrelated to test substance exposure.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Organ Weights

Terminal Euthanasia Animals (Day 29):

Test substance-related higher mean lung weights (absolute and relative to terminal body and
brain weights) were observed in the 0.25, 0.50, and 1.00 mg/m3 group males and females at the
terminal euthanasia. Higher mean lung weights were dose-responsive, statistically significant,
and correlated microscopically with increased alveolar macrophages, mixed cell inflammation,
cellular debris, and BALT lymphoid hyperplasia in the 0.25, 0.50, and 1.00 mg/m3 group males
and females.
No other test substance-related organ weight changes were identified. Of note, lower mean
thymus weights (absolute and relative to terminal body and brain weights) were observed in the
KDLNO-exposed males (0.25, 0.50, and 1.00 mg/m3 groups) but were considered incidental as
values lacked statistical significance, were non-dose-responsive, and there were no histologic
correlates.
Remaining organ weight changes not discussed exhibited 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 KDLNO exposure.

Recovery Euthanasia Animals (Day 43):
Test substance-related organ weight changes noted at the terminal euthanasia were observed at
the end of the recovery period (Day 43).

Test substance-related higher mean lung weights (absolute and relative to terminal body and
brain weights) were observed in the 0.10, 0.25, 0.50, and 1.00 mg/m3 groups at the recovery
euthanasia. Higher mean lung weights were dose-responsive, statistically significant (except the
0.10 mg/m3 groups), and correlated microscopically with increased alveolar macrophages, mixed
cell inflammation, cellular debris, and/or BALT lymphoid hyperplasia in the 0.10, 0.25, 0.50,
and 1.00 mg/m3 group males and females.

No other test substance-related organ weight changes were noted. Remaining differences
exhibited 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 KDLNO exposure.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross Pathology

Terminal Euthanasia Animals (Day 29):

Test substance-related enlargement of the tracheobronchial lymph nodes was observed in the
0.50 and 1.00 mg/m3 group males at the terminal euthanasia and correlated microscopically with
minimal lymphoid hyperplasia in these dose groups.
Remaining gross findings observed were considered incidental, of the nature commonly
observed in this strain and age of rat, and/or were of similar incidence in control and treated
animals and, therefore, were considered unrelated to KDLNO exposure.

Recovery Euthanasia Animals (Day 43):
Test substance-related gross pathology findings noted in the tracheobronchial lymph nodes at the
terminal euthanasia were observed at the end of the recovery period (Day 43). Additionally, test substance-related gross observations were also observed in the lungs at the recovery euthanasia.

Test substance-related enlargement of the tracheobronchial lymph nodes was observed in a
single 1.00 mg/m3 group female (Animal No. 5509) at the recovery euthanasia, and correlated
microscopically with minimal lymphoid hyperplasia.
Test substance-related gross findings observed in the lungs at the recovery euthanasia consisted
of failure to collapse and pale focus (multifocal). Failure to collapse was observed in a single
1.00 mg/m3 group male (Animal No. 5012) and correlated microscopically with moderately
increased alveolar macrophages and cellular debris. Pale focus was observed grossly in a single
0.50 mg/m3 group male (Animal No. 4013) and also correlated microscopically with moderately
increased alveolar macrophages and cellular debris.
Other gross findings observed were considered incidental, of the nature commonly observed in
this strain and age of rat, and/or were of similar incidence in control and treated animals and,
therefore, were considered unrelated to KDLNO exposure.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathology:

Terminal Euthanasia Animals (Day 29):
Test substance-related microscopic changes in the lungs consisted of increased alveolar
macrophages, mixed cell inflammation, cellular debris, and bronchial-associated lymphoid tissue
(BALT) lymphoid hyperplasia. Minimal to moderate, increased alveolar macrophages were
observed in the 0.10, 0.25, 0.50, and 1.00 mg/m3 group males and females, and were
characterized as accumulations of vacuolated/foamy macrophages within alveolar spaces that
occasionally contained intracytoplasmic cellular debris. Minimal to marked, mixed cell
inflammation was observed in the 0.25, 0.50, and 1.00 mg/m3 group males and females and was
characterized by increased numbers of neutrophils and mononuclear cells centered on alveolar
septae with associated thickening of the septae, occasional type II pneumocyte hyperplasia, and
rare hemoglobin crystals. Minimal to marked, cellular debris was present in the 0.25, 0.50, and
1.00 mg/m3 group males and females and was characterized as accumulations of amorphous
basophilic, often lacey material within the alveolar spaces and occasionally within macrophages.
Finally, minimal BALT lymphoid hyperplasia was observed in the 0.25, 0.50, and 1.0 mg/m3
group males and females, and was characterized by increased aggregates of lymphocytes located
adjacent to bronchi/bronchioles and/or vessels.

Test-substance related microscopic changes in the tracheobronchial lymph nodes consisted of
minimal lymphoid hyperplasia in the 0.25, 0.50, and 1.00 mg/m3 group males and females.
Lymphoid hyperplasia was characterized as enlargement of the tracheobronchial lymph node
secondary to increased lymphocyte accumulations.
There were no other test-substance related microscopic changes. Testicular degeneration/atrophy
within the seminiferous tubules was observed in two 1.00 mg/m3 group males (Animal
Nos. 5006 and 5009) at the terminal euthanasia, but following evaluation of the testes from all
animals this change was considered to be incidental given the lack of a dose response, lack of
effect in the 1.00 mg/m3 group males at the recovery euthanasia, unilateral distribution in Animal
No. 5006, and frequent observation of this change in toxicology studies.
Remaining microscopic findings observed were considered incidental, of the nature commonly
observed in this strain and age of rat, and/or were of similar incidence and severity in control and
treated animals and, therefore, were considered unrelated to KDLNO exposure.

Recovery Euthanasia Animals (Day 43):
Test substance-related microscopic changes in the lungs consisted of increased alveolar
macrophages, mixed cell inflammation, cellular debris, and BALT hyperplasia and resembled
microscopic findings at the terminal euthanasia. Minimal to marked, increased alveolar
macrophages were observed in the 0.10, 0.25, 0.50, and 1.00 mg/m3 group males and females.
Minimal to mild, mixed cell inflammation was observed in the 0.10 (males only), 0.25, 0.50, and
1.00 mg/m3 group males and females. Minimal to moderate, cellular debris was observed in the
0.10 (females only), 0.25, 0.50, and 1.00 mg/m3 group males and females. Finally, minimal
BALT lymphoid hyperplasia was observed in the 0.50 and 1.00 mg/m3 group males.
Test substance-related changes in the tracheobronchial lymph node were similar to findings at
the terminal euthanasia and consisted of minimal lymphoid hyperplasia in the 0.50 and
1.00 mg/m3 group females.
There were no other test substance-related microscopic changes observed at the recovery
euthanasia. Of note, marked degeneration/atrophy within the seminiferous tubules of the testes
was observed in a single 0.25 mg/m3 group male (Animal No. 3015; with correlating gross
observation of small testes) but was considered incidental given the lack of effect in the higher
dose groups and frequent observation of this finding in toxicology studies.
Remaining microscopic findings observed were considered incidental, of the nature commonly
observed in this strain and age of rat, and/or were of similar incidence and severity in control and
treated animals and, therefore, were considered unrelated to KDLNO exposure.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Bronchoalveolar Lavage Fluid Evaluation:
After excluding cell counts for two control group females (No. 1504 and 1505) due to BALF
smears with insufficient cellularity, moderately to markedly increased percentages of neutrophils
were observed at Day 29/30 in males at ≥ 0.25 mg/m3 and females at ≥ 0.10 mg/m3 when
compared with control group means. Additionally, there was an increased incidence of
vacuolated macrophages which were frequently associated with a large amount of cellular debris
in males at ≥ 0.25 mg/m3 and females at ≥ 0.10 mg/m3.
At Day 29/30, there was markedly increased LDH activity and markedly increased TPROT
concentration in males and females at ≥ 0.25 mg/m3. The changes in LDH and TPROT were
consistent with acute inflammation.
At the end of the recovery period, increased numbers and percentages of neutrophils, and
increased LDH and TPROT persisted in females at all exposure levels indicating a lack of
recovery. There was also increased incidence and severity of vacuolated macrophages and
cellular debris at the end of the recovery period in females at all exposure concentrations when
compared with the concurrent filtered air control group. The cellular debris was suggestive of
necrosis.

Effect levels

Key result

Dose descriptor:

LOAEC

Effect level:

ca. 0.1 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

other: Bronchoalveolar Lavage Fluid Evaluation:

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

0.1 mg/m³ air

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

presumably yes

Applicant's summary and conclusion

Conclusions:

In conclusion, administration of lithium nickel potassium oxide (KDLNO) by nose-only
inhalation to Crl:CD(SD) rats at exposure concentrations of 0.10, 0.25, 0.50, and 1.0 mg/m3 for
6 hours/day on a 5-day per week basis for 4 weeks (minimum of 20 exposures for each animal)
resulted in higher lung weights and increased alveolar macrophages, mixed cell inflammation,
and/or cellular debris in the 0.10 mg/m3 group at the primary and/or recovery necropsies. Based
on these findings, the adaptive response of increased macrophages to inhaled particle was
exceeded and therefore was considered adverse. Based on these results, a lowest-observed adverse - effect-concentration (LOAEC) was considered to be 0.10 mg/m3.

Executive summary:

The objective of this study was to evaluate the potential toxicity of the test substance, lithium
nickel potassium oxide (KDLNO) when administered by nose-only inhalation to Sprague Dawley
rats for 6 hours per day, 5 days per week, for 4 weeks (minimum of 20 exposures for each animal),
as well as to evaluate the recovery, persistence or progression of any effects following a 2-week
recovery period. The results of this study may be used to select exposure concentrations for a
subsequent subchronic (90-day) repeat-exposure inhalation toxicity study (OECD Guideline No.
413). Four target aerosol exposure concentrations were selected based on the results of a
preliminary 9-day repeat-exposure inhalation toxicity study.

The study design was as follows:

Treatment Group 1: Control - Filtered Air: 10males 5females: 5 recovery males 5recovery females
Treatment Group 2: 0.10mg/m3 KDLNO: 10males 5females: 5 recovery males 5recovery females
Treatment Group 3: 0.25mg/m3 KDLNO: 10males 5females: 5 recovery males 5recovery females
Treatment Group 4: 0.50mg/m3 KDLNO: 10males 5females: 5 recovery males 5recovery females
Treatment Group 5: 1.00mg/m3 KDLNO: 10males 5females: 5 recovery males 5recovery females

 

Achieved mean actual exposure concentrations in the 0.10, 0.25, 0.50, and 1.00 mg/m3 groups

were 0.087, 0.29, 0.50, and 1.3 mg/m3, respectively.

 

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

weights, body weight gains, food consumption, ophthalmology, clinical pathology parameters

(hematology, coagulation, clinical chemistry, and urinalysis), bronchoalveolar lavage fluid

parameters (chemistry and cytology), lung and plasma bioanalysis, gross necropsy findings,

organ weights, and histopathologic examinations.

 

All animals survived to the scheduled necropsy. There were no test substance-related clinical

observations or effects on body weight, food consumption, coagulation, clinical chemistry, or

urinalysis. There were no test substance-related ophthalmic findings. Test substance-related effects on hematology parameters included increased neutrophil counts in males and females at ≥ 0.50 mg/m3 at Day 29/30. At the end of the recovery period, the increase in circulating neutrophils persisted in males at ≥ 0.50 mg/m3.

 

Test substance-related effects on bronchoalveolar lavage fluid parameters included increased

percentages of neutrophils in males at ≥ 0.25 mg/m3 and females at ≥ 0.10 mg/m3, increased

incidence of vacuolated macrophages in males at ≥ 0.25 mg/m3 and females at ≥ 0.10 mg/m3,

and increased lactate dehydrogenase (LDH) activity and increased total protein (TPROT)

concentration in males and females at ≥ 0.25 mg/m3 at Day 29/30. At the end of the recovery

period, increased numbers and percentages of neutrophils, and increased LDH and TPROT

persisted in females at all exposure levels indicating a lack of recovery. There was also increased

incidence and severity of vacuolated macrophages and cellular debris at the end of the recovery

period in females at all exposure concentrations when compared with the concurrent filtered air

control group.

 

Test substance-related macroscopic findings included enlarged tracheobronchial lymph nodes in

the 0.50 and 1.00 mg/m3 group males at the terminal necropsy (Day 29). These changes persisted

at the end of the recovery period (Day 43).

 

Test substance-related effects on organ weights included higher mean lung weights (absolute and

relative to terminal body and brain weights) in the 0.25, 0.50, and 1.00 mg/m3 group males and

females at the terminal necropsy. These changes persisted at the end of the recovery period.

Test substance-related microscopic changes included increased alveolar macrophages in the 0.10,

0.25, 0.50, and 1.00 mg/m3 group males and females, and mixed cell inflammation, cellular

debris, and bronchial-associated lymphoid tissue (BALT) lymphoid hyperplasia in the 0.25, 0.50,

and 1.00 mg/m3 group males and females, at the terminal necropsy. At the end of the recovery

period, increased alveolar macrophages persisted in the 0.10, 0.25, 0.50, and 1.00 mg/m3 group

males and females, mixed cell inflammation was observed in the 0.10 (males only), 0.25, 0.50,

and 1.00 mg/m3 group males and females, cellular debris was observed in the 0.10 (females

only), 0.25, 0.50, and 1.00 mg/m3 group males and females, and BALT lymphoid hyperplasia

was observed in the 0.50 and 1.00 mg/m3 group males.

 

In conclusion, administration of lithium nickel potassium oxide (KDLNO) by nose-only

inhalation to Crl:CD(SD) rats at exposure concentrations of 0.10, 0.25, 0.50, and 1.0 mg/m3 for

6 hours/day on a 5-day per week basis for 4 weeks (minimum of 20 exposures for each animal)

resulted in higher lung weights and increased alveolar macrophages, mixed cell inflammation,

and/or cellular debris in the 0.10 mg/m3 group at the primary and/or recovery necropsies. Based

on these findings, the adaptive response of increased macrophages to inhaled particle was

exceeded and therefore was considered adverse. Based on these results, a lowest-observed adverse-

effect-concentration (LOAEC) was considered to be 0.10 mg/m3.