Lithium nickel potassium oxide

Endpoint summary

Description of key information

Acute Oral Toxicity;

Under the conditions of this study the median lethal dose of KDLNO after oral administration was found to be greater than 500 mg/kg bw and less than 2000 mg/kg bw in rats.

 

Acute Inhalation Toxicity:

Under the current study conditions, the LC50 value for female Wistar rats was 2.048 mg/L (calculated based on analytical concentration) after a 4-hour inhalation exposure to the dust aerosol of KDLNO. The LC50 value for male Wistar rats is considered to be > 2.037 mg/L (based on analytical concentration) after a 4-hour inhalation exposure to the dust aerosol of KDLNO.

 

Acute Dermal Toxicity:

The acute dermal median lethal dose (LD50) was determined to be LD50, dermal, rat > 2000 mg/kg bw.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study completed March 20, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Japan MAFF Testing Guideline of 12 Nousan No. 8147, November 24, 2000 as this in line with OECD 423.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

no

GLP compliance:

yes

Test type:

acute toxic class method

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Rats were selected since this rodent species is recommended in the respective test guidelines. Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.

Sex:

female

Details on test animals or test system and environmental conditions:

Nulliparous and non-pregnant female animals were used for the test as suggested by the OECD guideline when there is no indication that male animals are likely to be more sensitive to the acute effects of the test item. They were young adult animals (female animals approx. 10 weeks and of comparable weight (± 20% of the mean weight)). There was an acclimatization period of at least 5 days before the beginning of the experimental phase; during the acclimatization period, the animals were accustomed to the environmental conditions of the study and to the diet. Rats were identified by individual identification by cage cards and tail marking.

The animals were housed in fully air-conditioned rooms. Central air-conditioning guaranteed a range of 22°C ± 3°C for temperature and of 30 – 70% for relative humidity. There were no deviations from these ranges, which influenced the results of the study.

Air changes per hour
Approx. 10
Day / night rhythm:
12 h / 12 h (6.00 a.m. – 6.00 p.m. / 6.00 p.m. – 6.00 a.m.)
Type of cage:
Makrolon cage, type III
Number of animals per cage:
Single housing
Feeding:
VRF1(P); SDS Special Diets Services, 67122 Altrip, Germany, ad libitum
Drinking water:
Tap water ad libitum
Bedding:
H 15005-29; Ssniff, Spezialdiäten GmbH (Experimental Animal Diets Inc., 59494 Soest, Germany)
Enrichment:
Wooden gnawing blocks (Type NGM E-022); ABEDD® LAB & VET Service GmbH, Hasnerstraße 84/6; 1160 Wien – Austria

The feed used in the study was assayed for chemical and microbial contaminants by the manufacturer in quarterly intervals. Results are archived by Bioassay.

The drinking water was regularly assayed for contaminants by the municipal authorities of Heidelberg. The German Drinking Water Regulation of Dec. 5, 1990 served as the guideline for maximum tolerable contaminants.

The bedding and enrichment were regularly assayed for contaminants (chlorinated hydrocarbons and heavy metals).

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

Chosen due to good homogeneity of the test material in corn oil

Details on oral exposure:

The test item formulation was prepared shortly before application by stirring with a high speed homogenizer (Ultra-Turrax) and a magnetic stirrer. Additionally, the homogeneity of the test item preparation during application was ensured by stirring with a magnetic stirrer. The stability of the test item in the vehicle was determined indirectly by concentration control analysis. For this purpose, the samples taken were stored at room temperature over the maximum duration of the administration period, subsequently deep-frozen and sent for analysis.

The homogeneity of the test item preparation was determined indirectly by concentration control analysis.

Route of administration:
Single oral administration by gavage.
Fasting period:
Feed was withdrawn from the animals at least 16 hours before administration, but water was available ad libitum.
Time of day of administration:
In the morning
Observation period:
14 days
Body weight determination:
Individual body weights shortly before administration (day 0), weekly thereafter and on the last day of observation and on the day of death starting with study day 1.

Doses:

SELECTION OF DOSES/CONCENTRATIONS
A starting dose of 2000 mg/kg bw was chosen in the first step with 3 female animals.
As all animals died, a dose of 500 mg/kg bw was administered to 3 female rats in the second step.
Because no mortality occurred, a further dose of 500 mg/kg bw was administered to another group of 3 female animals in the third step.

No. of animals per sex per dose:

3 females per dose

Control animals:

no

Details on study design:

The objective of this study was to assess the acute oral median lethal dose following a single oral administration to female Wistar rats followed by a 14 day observation period.
The study was performed in rats, because this species has been shown to be suitable for this type of study and is recommended by the test guidelines.

Statistics:

In the single 2000 mg/kg bw test group all animals died on day 3 or 5 after administration.
No mortality occurred in both 500 mg/kg bw test groups.

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 500 - < 2 000 mg/kg bw

Based on:

test mat.

Mortality:

In the single 2000 mg/kg bw test group all animals died on day 3 or 5 after administration.
No mortality occurred in both 500 mg/kg bw test groups.

Clinical signs:

other: Clinical signs in the single 2000 mg/kg test group revealed in all animals an impaired general state and piloerection from hour 2 or 4 until hour 5 and again from day 2 until day 3 after administration. Black discolored faeces was seen on day 1 in all ani

Gross pathology:

The following macroscopic pathologic findings were observed in the animals that died (single 2000 mg/kg bw test group, 3 females):
o Stomach filled with blackish liquid, discoloration of the stomach contents
o Dark discolored small and large intestine
There were no macroscopic pathological findings in any animal sacrificed at the end of the observation period (6 females of both 500 mg/kg bw. groups).

Other findings:

The following test substance-related clinical observations were recorded, clinical signs occurred within the first 5 days after administration:
2000 mg/kg (single test group):
 Mortality in all animals
 Impaired general state in all animals
 Dyspnea in all animals
 Piloerection in all animals
 Black discolored faeces in all animals
 Reduced defecation in all animals
 Exsiccosis in all animals
 Macroscopic pathological findings in the animals that died:
o Filled stomach with blackish liquid, discoloration of the stomach contents
o Dark discolored small and large intestine

500 mg/kg (both test groups):
 No clinical signs were observed

The body weights of the surviving animals in both 500 mg/kg bw test groups increased within the normal range throughout the study period.
There were no macroscopic pathological findings in any animal sacrificed at the end of observation period (6 females of both 500 mg/kg bw test groups).
The acute oral LD50 was calculated to be
LD50, oral, rat > 500 < 2000 mg/kg bw

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

Under the conditions of this study the median lethal dose of KDLNO after oral administration was found to be greater than 500 mg/kg bw and less than 2000 mg/kg bw in rats.

Executive summary:

In an acute oral toxicity study performed according to the Acute Toxic Class Method, doses of 2000 and 500 mg/kg bw of the test item KDLNO (preparations in corn oil Ph.Eur.) were administered by gavage to three test groups of three fasted Wistar rats each (2000 mg/kg bw in 3 females, 500 mg/kg bw in 6 females).

 

Under the conditions of this study the median lethal dose of KDLNO after oral administration was found to be greater than 500 mg/kg bw and less than 2000 mg/kg bw in rats. The acute oral LD50 was calculated to be LD50, oral, rat > 500 < 2000 mg/kg bw

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

> 500 - < 2 000 mg/kg bw

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 2017 to April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1300 (Acute inhalation toxicity)

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

GLP compliance:

yes

Test type:

traditional method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST SYSTEM
Test species / strain: Rat / Wistar / Crl:WI(Han)
The female animals were nulliparous and non-pregnant.
Reasons for selection of the
test species:
Rats were selected since this rodent species is recommended in the respective test guidelines. Wistar rats were selected since there is extensive experience available in the laboratory
with this strain of rats.
The Wistar strain was also selected because extensive historical control data are available for this strain.
Age at day 0: Young adult animals (male animals approx. 8 weeks, female animals approx. 10 weeks)
Sex: Male / female
Supplier: Charles River Laboratories,
Research Models and Services, Germany GmbH,
Sandhofer Weg 7,
97633 Sulzfeld
Arrival in the testing facility: Acclimatization for at least 5 days before exposure.
Identification: Individual identification by cage cards and tail markings.
Body weight at day 0: Animals of comparable weight (± 20% of the mean weight,
actual weights)

Randomization: The animals were randomly selected from a pool of animals.

HOUSING AND DIET
Room temperature /
relative humidity:
The animals were housed in air-conditioned rooms. Central airconditioning
guaranteed a range of 20 – 24°C for temperature
and of 45 – 65 % for relative humidity. There were no deviations from these ranges, which influenced the results of the study.
There were 15 air changes per hour.
Day / night rhythm: 12 h / 12 h (6.00 a.m. – 6.00 p.m. / 6.00 p.m. – 6.00 a.m.)
The room was completely disinfected using a disinfector ("AUTEX", fully automatic, formalin ammonia- based terminal disinfector) before the start of the study. The floor and the walls
were cleaned once a week with water containing an appropriate disinfectant.

Type of cage: During the acclimatization period before exposure:
Typ 2000P: ca. 2065 cm2 (polysulfone cages) supplied by TECNIPLAST, Germany (caged in groups)
After exposure:
Typ III: (polycarbonate cages) (floor area about 800 cm2)
(for single housing)
Enrichment: Wooden gnawing blocks (Lignocel Block Large) J. Rettenmaier
& Söhne GmbH Co KG, Rosenberg, Germany and Play Tunnel,
large (Art. 14153); PLEXX b.v., Elst, Netherlands
Number of animals per cage: Single housing or up to 5 animals (caged in groups)
Feeding: Kliba laboratory diet, mouse/rat maintenance “GLP”, 12 mm
pellets, Provimi Kliba SA, Kaiseraugst, Basel Switzerland, ad
libitum
Drinking water: Tap water ad libitum
Bedding: Dust-free wooden bedding was used in this study (the present supplier is documented in the raw data).

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Mass median aerodynamic diameter (MMAD):

> 2.5 - < 3 µm

Details on inhalation exposure:

EXPOSURE
Nose-only inhalation system INA 10 (glass-steel construction, BASF SE, volume V ≈ 34 L): the
animals were restrained in glass tubes and their snouts projected into the inhalation system.
The homogenous distribution of test substance atmosphere/s in this inhalation system has
been verified with model aerosols.
Conditioned air:
The central air conditioning system provides cold air of about 15°C. This cold air passes
through an activated charcoal filter, is adjusted to room temperature of 20 to 24°C and passes
through a second particle filter (H13 (HEPA) Camfil Farr, Germany). The generated
conditioned air was used to generate inhalation atmospheres.
Compressed air:
Compressed air was produced by an oil-free compressor (HT 6, Josef Mehrer GmbH & Co KG,
Germany). For this purpose, air is filtered by an inlet air strainer and introduced into the
compressor. After passing through a second ultra-filter (SMF 5/3, 108 mm, Donalson), the
compressed air (15 bar) is stored in a storage of 1500 or 5000 L. The compressed air is
conducted to the laboratories via pipes, where the pressure is reduced to 6 bar. In the
laboratory, the compressed air can be taken as required.
Exhaust air:
The exhaust air was filtered and conducted into the exhaust air of the building.
The exposure system was located inside an exhaust cabin in an air-conditioned laboratory.
During each exposure, the following scheduled parameters were recorded four times at about
1-hour intervals:
Supply air flows: (compressed air plus conditioned air from a central air-conditioning system)
Test group 1: 0.8 m³/h (compressed air)
0.7 m³/h (conditioned air)
The flows were adjusted and continuously measured with a flowmeter (Yokogawa).
Supply air flows: (compressed air from a central air-conditioning system)
Test group 2 and 3: 1.0 m³/h
The flows were adjusted and continuously measured with a flowmeter (Yokogawa).
Exhaust air flows: Test group 1: 1.3 m³/h
Test group 2 and 3: 0.8 m³/h
The flows were adjusted and continuously measured with a flowmeter (Yokogawa).

The lower amounts of exhaust air, which were adjusted by means of a separate exhaust air
system, achieved positive pressures inside the exposure systems. This ensured that the
mixtures of test substance and air were not diluted with laboratory air in the breathing zones of
the animals.
Air changes of about 44 (test group 1) and 29 (test groups 2 and 3) times per hour can be
calculated by dividing the supply air flows by the volume of the inhalation systems.
The animals were exposed to the inhalation atmosphere for 4 hours plus equilibration time of
the inhalation systems (t99 about 6 min (test group 1) and 10 min (test groups 2 and 3)).
No surveillance of the oxygen content in the inhalation system was performed. The air change
was judged to be sufficient to prevent oxygen depletion by the breathing of the animals, and
the concentration of the test substance used could not have a substantial influence on oxygen
partial pressure.
Temperatures: The temperatures in the inhalation system were measured with a digital thermometer (Testo).
Relative humidities: The relative humidities in the inhalation system were measured with a dielectric probe (Testo).

Duration of exposure:

4 h

Remarks on duration:

The animals were exposed to the inhalation atmosphere for 4 hours plus equilibration time of
the inhalation systems (t99 about 6 min (test group 1) and 10 min (test groups 2 and 3)).

Concentrations:

The test was run with actual measured concentrations of 0.532 mg/L, 2.037 mg/L and
5.081 mg/L (analytical concentration).

No. of animals per sex per dose:

Five male and five female Wistar rats were used for each test group.

Details on study design:

TEST GROUPS
Five male and five female Wistar rats were used for each test group.

GENERATION OF THE INHALATION ATMOSPHERE
Test-substance preparation: The test substance was dosed unchanged.
Equipment: - Balance (Mettler)
- Flat-Tray Feeder (ZD 5 FB-C-1M-50, Three-Tec GmbH, Seon, Switzerland)
- Conducting tube made of stainless steel
- Pneumatic vibrator NCT 1 (NetterVibration, Mainz-Kastel, Germany)

Generation technique: For each concentration, appropriate amount of test substance
was dosed by the above mentioned flat-tray feeder. Applying compressed air, dust aerosols atmospheres were generated inside the inhalation system. The concentrations were adjusted by setting the pace of the double concave screw of the flat-tray feeder on a control panel.

EXPOSURE
Nose-only inhalation system INA 10 (glass-steel construction, BASF SE, volume V ≈ 34 L): the
animals were restrained in glass tubes and their snouts projected into the inhalation system.
The homogenous distribution of test substance atmosphere/s in this inhalation system has
been verified with model aerosols.
Conditioned air:
The central air conditioning system provides cold air of about 15°C. This cold air passes
through an activated charcoal filter, is adjusted to room temperature of 20 to 24°C and passes
through a second particle filter (H13 (HEPA) Camfil Farr, Germany). The generated
conditioned air was used to generate inhalation atmospheres.
Compressed air:
Compressed air was produced by an oil-free compressor (HT 6, Josef Mehrer GmbH & Co KG,
Germany). For this purpose, air is filtered by an inlet air strainer and introduced into the
compressor. After passing through a second ultra-filter (SMF 5/3, 108 mm, Donalson), the
compressed air (15 bar) is stored in a storage of 1500 or 5000 L. The compressed air is
conducted to the laboratories via pipes, where the pressure is reduced to 6 bar. In the
laboratory, the compressed air can be taken as required.
Exhaust air:
The exhaust air was filtered and conducted into the exhaust air of the building.
The exposure system was located inside an exhaust cabin in an air-conditioned laboratory.
During each exposure, the following scheduled parameters were recorded four times at about
1-hour intervals:
Supply air flows: (compressed air plus conditioned air from a central air-conditioning system)
Test group 1: 0.8 m³/h (compressed air)
0.7 m³/h (conditioned air)
The flows were adjusted and continuously measured with a flowmeter (Yokogawa).
Supply air flows: (compressed air from a central air-conditioning system)
Test group 2 and 3: 1.0 m³/h
The flows were adjusted and continuously measured with a flowmeter (Yokogawa).
Exhaust air flows: Test group 1: 1.3 m³/h
Test group 2 and 3: 0.8 m³/h
The flows were adjusted and continuously measured with a flowmeter (Yokogawa).

The lower amounts of exhaust air, which were adjusted by means of a separate exhaust air
system, achieved positive pressures inside the exposure systems. This ensured that the
mixtures of test substance and air were not diluted with laboratory air in the breathing zones of
the animals.
Air changes of about 44 (test group 1) and 29 (test groups 2 and 3) times per hour can be
calculated by dividing the supply air flows by the volume of the inhalation systems.
The animals were exposed to the inhalation atmosphere for 4 hours plus equilibration time of
the inhalation systems (t99 about 6 min (test group 1) and 10 min (test groups 2 and 3)).
No surveillance of the oxygen content in the inhalation system was performed. The air change
was judged to be sufficient to prevent oxygen depletion by the breathing of the animals, and
the concentration of the test substance used could not have a substantial influence on oxygen
partial pressure.
Temperatures: The temperatures in the inhalation system were measured with a digital thermometer (Testo).
Relative humidities: The relative humidities in the inhalation system were measured with a dielectric probe (Testo).

Statistics:

STATISTICAL EVALUATION
LC50 values, 4-hour exposure
LC50 (female rats)1): 2.048 mg/L
While mortality of female animals increased in the concentration-related manner, no males
died at the highest tested concentration of about 5 mg/L, but two of five males died at the mid
concentration of about 2 mg/L. None of the males died at the lowest concentration of about
0.5 mg/L. This patter is considered as biological variability, demonstrated a flat dose-response
relationship in the males. The overall data of males were consistent to those of the females.
Due to the missing dose-response relation in the lethality rate of male Wistar rats, meaningful
statistical evaluation is not possible. LC50 of male animals is considered to be > 2.037 mg/L.
_____________
1) Probit analysis for female animals.
No statistical evaluation for males and both sexes combined.

Key result

Sex:

female

Dose descriptor:

LC50

Effect level:

2.048 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Key result

Sex:

male

Dose descriptor:

LC50

Effect level:

> 2.037 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Mortality:

Test group 1 (0.532 mg/L):
One of five female animal were sacrificed in a moribund state on study day 6. No lethality was
observed in males.
Test group 2 (2.037 mg/L):
Two of the five male animals and two of the five females died or were sacrificed in a moribund
state after exposure on study days 4 or on study day 7 during the post-exposure observation
period.
Test group 3 (5.081 mg/L):
Four of the five female animals and none of the five males died or were sacrificed in a moribund
state on study days 5 or 8 during the post-exposure observation period.

Clinical signs:

bodyweight loss

Body weight:

Test group 1 (0.532 mg/L)
The mean body weights of the male animals decreased on the first post-exposure observation
days but increased thereafter. The mean body weights of the surviving female animals
decreased on the first post-exposure observation week but increased thereafter.
Test group 2 (2.037 mg/L)
The mean body weights of the surviving male animals decreased on the first post-exposure
observation days but increased thereafter. The mean body weights of the surviving female
animals decreased on the first post-exposure observation week but increased thereafter.
Test group 3 (5.081 mg/L)
The mean body weights of the male animals decreased on the first post-exposure observation
days but increased thereafter. The mean body weights of the surviving female animals
decreased on the first post-exposure observation week but increased thereafter.

Other findings:

Clinical signs of toxicity in animals exposed to 0.532 mg/L comprised accelerated and
intermittent respiration, labored respiration, respiration sounds, no feces, poor general state,
piloerection and substance-contaminated fur. Findings were observed from hour 3 of exposure
until the end of the post-exposure observation period of 14 days. The mean body weights of
the male animals decreased on the first post-exposure observation days but increased
thereafter. The mean body weights of the surviving female animals decreased on the first post exposure observation week but increased thereafter. During necropsy of the dead female
animal, dark-red discoloration and partly surface sunken was seen in all lobes of the lung. At
termination of the study, in two female animals few dark-red foci and partly sunken surface of
the lung was noted. The remaining male and female animals did not show any gross
pathological abnormality.

Clinical signs of toxicity in animals exposed to 2.037mg/L included accelerated and intermittent
respiration, abdominal respiration, respiration sounds, semiclosed eyelid, red discharge and
red encrusted nose, poor general state, piloerection, substance like encrusted nose and
substance-contaminated and discolored (substance like) fur. Findings were observed from
hour 3 of exposure until the end of the post-exposure observation period of 14 days. The mean
body weights of the surviving male animals decreased on the first post-exposure observation
days but increased thereafter. The mean body weights of the surviving female animals
decreased on the first post-exposure observation week but increased thereafter. During
necropsy of the dead animals (two males and two females), only one female showed dark-red
discoloration of the lung. The remaining dead animals did not show any gross pathological
abnormality. At the termination of the post exposure observation period gross
pathology revealed partly swelling in lungs of all males, or dark-red foci and sunken surface
of the lung in all females.

Clinical signs of toxicity in animals exposed to 5.081 mg/L comprised accelerated and
intermittent respiration, respiration labored, abdominal respiration, red discharge of the nose,
poor general state, hypothermia, hunched posture, piloerection, diarrhea, black discolored feces, substance-contaminated and discolored (substance like) fur. Findings were observed
from hour 2 of exposure until the end of the post-exposure observation period of 14 days. The
mean body weights of the male animals decreased on the first post-exposure observation days
but increased thereafter. The mean body weights of the surviving female animals decreased
on the first post-exposure observation week but increased thereafter. During necropsy of the
four dead female animals, only one of them showed macroscopic changes characterized by
dark-red discoloration and partly sunken surface in all lobes of the lung, and additional black
foci in the stomach. The other dead females did not show any gross pathological abnormality.
Remaining male and female animals sacrificed at the end of the post-exposure observation
period showed no gross pathological abnormalities.

Histopathological examination of the lungs was conducted in animals Nos. 172 and 178
(sacrificed on 14 days after exposure to 2.037 mg/L). Histopathology revealed an interstitial
inflammation accompanied by alveolar fibrosis, interstitial and alveolar edema and additional
histiocytosis (with black particles) in the lungs of both animals.

Interpretation of results:

GHS criteria not met

Executive summary:

SUMMARY

To determine the acute inhalation toxicity (single 4-hour exposure, nose only) of KDLNO as a dust aerosol, a study was performed in male and female Wistar rats according to OECDGuideline method 403, as well as EC.

The test was run with actual measured concentrations of 0.532 mg/L, 2.037 mg/L and 5.081 mg/L (analytical concentration).

Cascade impactor measurements resulted in particle size distributions with mass median aerodynamic diameters (MMADs) between 2.5 and 3.0 μm, which are well within the respirable range.

One of the five females and no males were sacrificed in a moribund state at 0.532 mg/L. Two of five males and two of five females died or were sacrificed in a moribund state at 2.037 mg/L. At 5.081 mg/L no males and four of five female animals died or were sacrificed in a moribund state. Mortality was observed from study day 4 up to 8 days after the end of the exposure.

Clinical signs of toxicity in animals exposed to 0.532 mg/L comprised accelerated and intermittent respiration, labored respiration, respiration sounds, no feces, poor general state, piloerection and substance-contaminated fur. Findings were observed from hour 3 of exposure until the end of the post-exposure observation period of 14 days. The mean body weights of the male animals decreased on the first post-exposure observation days but increased thereafter. The mean body weights of the surviving female animals decreased on the first postexposure
observation week but increased thereafter. During necropsy of the dead female
animal, dark-red discoloration and partly surface sunken was seen in all lobes of the lung. At termination of the study, in two female animals few dark-red foci and partly sunken surface of the lung was noted. The remaining male and female animals did not show any gross pathological abnormality.

Clinical signs of toxicity in animals exposed to 2.037mg/L included accelerated and intermittent respiration, abdominal respiration, respiration sounds, semiclosed eyelid, red discharge and red encrusted nose, poor general state, piloerection, substance like encrusted nose and substance-contaminated and discolored (substance like) fur. Findings were observed from hour 3 of exposure until the end of the post-exposure observation period of 14 days. The mean body weights of the surviving male animals decreased on the first post-exposure observation days but increased thereafter. The mean body weights of the surviving female animals
decreased on the first post-exposure observation week but increased thereafter. During necropsy of the dead animals (two males and two females), only one female showed dark-red discoloration of the lung. The remaining dead animals did not show any gross pathological abnormality. At the termination of the post exposure observation period gross pathology revealed partly swelling in lungs of all males, or dark-red foci and sunken surface of the lung in all females.

Clinical signs of toxicity in animals exposed to 5.081 mg/L comprised accelerated and intermittent respiration, respiration labored, abdominal respiration, red discharge of the nose, poor general state, hypothermia, hunched posture, piloerection, diarrhea, black discolored feces, substance-contaminated and discolored (substance like) fur. Findings were observed from hour 2 of exposure until the end of the post-exposure observation period of 14 days. The mean body weights of the male animals decreased on the first post-exposure observation days but increased thereafter. The mean body weights of the surviving female animals decreased on the first post-exposure observation week but increased thereafter. During necropsy of the four dead female animals, only one of them showed macroscopic changes characterized by dark-red discoloration and partly sunken surface in all lobes of the lung, and additional black foci in the stomach. The other dead females did not show any gross pathological abnormality. Remaining male and female animals sacrificed at the end of the post-exposure observation period showed no gross pathological abnormalities.

 

Histopathological examination of the lungs was conducted in animals Nos. 172 and 178 (sacrificed on 14 days after exposure to 2.037 mg/L). Histopathology revealed an interstitial inflammation accompanied by alveolar fibrosis, interstitial and alveolar edema and additional histiocytosis (with black particles) in the lungs of both animals. 

 

Under the current study conditions, the LC50 value for female Wistar rats was 2.048 mg/L (calculated based on analytical concentration) after a 4-hour inhalation exposure to the dust aerosol of KDLNO. The LC50 value for male Wistar rats is considered to be > 2.037 mg/L (based on analytical concentration) after a 4-hour inhalation exposure to the dust aerosol of KDLNO although.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LC50

Value:

2.048 mg/L air

Physical form:

inhalation: dust

Quality of whole database:

LC50 value chosen for females as a worst case.

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 2017 to March 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Japan MAFF 8147, 2000

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1200 (Acute Dermal Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.3 (Acute Toxicity (Dermal))

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

no

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Wistar

Remarks:

Test species / strain / quality: Rat / Wistar / Crl:WI (Han) SPF Reasons for selection of the test species: Rats were selected since this rodent species is recommended in the respective test guidelines.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test species / strain / quality:
Rat / Wistar / Crl:WI (Han) SPF
Reasons for selection of the test species:
Rats were selected since this rodent species is recommended in the respective test guidelines. Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.
Age on day 0:
Young adult animals (male animals approx. 8 weeks, female animals approx. 12 weeks)
Sex:
Males and females
The female animals were nulliparous and non-pregnant
Supplier:
Charles River Wiga GmbH, Germany
Arrival in the testing facility:
Acclimatization period of at least 5 days before the beginning of the experimental phase; during the acclimatization period, the animals were accustomed to the environmental conditions of the study and to the diet.
Identification:
Individual identification by cage cards and tail marking.
Body weight on day 0:
Animals of comparable weight (± 20% of the mean weight, actual weights )

HOUSING AND DIET
Room temperature / relative humidity:
The animals were housed in fully air-conditioned rooms. Central air-conditioning guaranteed a range of 22°C ± 3°C for temperature and of 30 – 70% for relative humidity. There were no deviations from these ranges, which influenced the results of the study.
Air changes per hour
Approx. 10
Day / night rhythm:
12 h / 12 h (6.00 a.m. – 6.00 p.m. / 6.00 p.m. – 6.00 a.m.)
Type of cage:
Makrolon cage, type III
Number of animals per cage:
Single housing
Feeding:
VRF1(P); SDS Special Diets Services, 67122 Altrip, Germany, ad libitum
Drinking water:
Tap water ad libitum
Bedding:
H 15005-29; Ssniff, Spezialdiäten GmbH (Experimental Animal Diets Inc., 59494 Soest, Germany)
Enrichment:
Wooden gnawing blocks (Type NGM E-022); ABEDD® LAB & VET Service GmbH, Hasnerstraße 84/6; 1160 Wien – Austria

Type of coverage:

semiocclusive

Vehicle:

corn oil

Details on dermal exposure:

Conduct of the study
Clipping of the fur:
About 24 hours before application
Route of application:
Single application to the clipped skin (dorsal and dorsolateral parts of the trunk); covering of the application site with a semi-occlusive dressing* for 24 hours. Afterwards removal of the semi-occlusive dressing, rinsing of the application site with warm water**.
Application area:
About 40 cm² (corresponds to at least 10% of the body surface)
Time of day of application:
In the morning
Observation period:
14 days
Body weight determination:
Individual body weights shortly before application (day 0), weekly thereafter and on the last day of observation.
Clinical observations:
Clinical signs for each animal were recorded several times on the day of application and at least once during each workday thereafter.
Scoring of skin findings:
Individual readings 30 – 60 minutes after removal of the semi-occlusive dressing (day 1), weekly thereafter and on the last day of observation.
Mortality:
A check for any dead or moribund animals was made at least once each workday, these records are archived by Bioassay.
Pathology:
Necropsy with gross-pathology examination was performed on the last day of the observation period after sacrifice by CO2-inhalation in a chamber with gradually increasing concentrations.

* The test item was covered with an air-permeable dressing (4 layers of absorbent gauze (Ph. Eur. supplied by Lohmann GmbH & Co., KG) and stretch bandage (Fixomull® Stretch (adhesive fleece) supplied by Beiersdorf AG).
** The removal of the semi-occlusive dressing and the rinsing of the application site with warm water were documented together in a protocol by confirming the time of the removal of the dressing.

Assessment of skin reactions
The evaluation of skin reactions was performed according to Draize, J. H. "Dermal toxicity." Appraisal of the safety of chemicals in foods, drugs and cosmetics (1959): 46-59. Appraisal of the safety of chemicals in foods, drugs and cosmetics. The association of food and drug officials of the United States Austin, Texas:

Erythema and eschar formation
Grading
0
No erythema
1
Very slight erythema (barely perceptible)
2
Well- defined erythema
3
Moderate to severe erythema
4
Severe erythema (beet redness) to eschar formation preventing grading of erythema
Edema formation
Grading
0
No edema
1
Very slight edema (barely perceptible)
2
Slight edema (edges of area well- defined by definite raising)
3
Moderate edema (raised approx. 1 mm)
4
Severe edema (raised more than 1 mm and extending beyond area of exposure)
Descriptions of any dermal findings not covered by this scale were recorded.

Duration of exposure:

Single application to the clipped skin (dorsal and dorsolateral parts of the trunk); covering of the application site with a semi-occlusive dressing for 24 hours.

Doses:

In an acute dermal toxicity study (Limit Test), young adult Wistar rats (5 males and 5 females) were dermally exposed to a single dose of 2000 mg/kg bw of KDLNO (as suspension in corn oil Ph.Eur.). The clipped application site (dorsal and dorso-lateral parts of the trunk, comprising at least 10% of the total body surface) was covered by semi-occlusive dressing during the 24-hour exposure period. The animals were observed for 14 days.

No. of animals per sex per dose:

In an acute dermal toxicity study (Limit Test), young adult Wistar rats (5 males and 5 females) were dermally exposed to a single dose of 2000 mg/kg bw of KDLNO (as suspension in corn oil Ph.Eur.). The clipped application site (dorsal and dorso-lateral parts of the trunk, comprising at least 10% of the total body surface) was covered by semi-occlusive dressing during the 24-hour exposure period. The animals were observed for 14 days.

Details on study design:

In an acute dermal toxicity study (Limit Test), young adult Wistar rats (5 males and 5 females) were dermally exposed to a single dose of 2000 mg/kg bw of KDLNO (as suspension in corn oil Ph.Eur.). The clipped application site (dorsal and dorso-lateral parts of the trunk, comprising at least 10% of the total body surface) was covered by semi-occlusive dressing during the 24-hour exposure period. The animals were observed for 14 days.

Conduct of the study
Clipping of the fur:
About 24 hours before application
Route of application:
Single application to the clipped skin (dorsal and dorsolateral parts of the trunk); covering of the application site with a semi-occlusive dressing* for 24 hours. Afterwards removal of the semi-occlusive dressing, rinsing of the application site with warm water**.
Application area:
About 40 cm² (corresponds to at least 10% of the body surface)
Time of day of application:
In the morning
Observation period:
14 days
Body weight determination:
Individual body weights shortly before application (day 0), weekly thereafter and on the last day of observation.
Clinical observations:
Clinical signs for each animal were recorded several times on the day of application and at least once during each workday thereafter.
Scoring of skin findings:
Individual readings 30 – 60 minutes after removal of the semi-occlusive dressing (day 1), weekly thereafter and on the last day of observation.
Mortality:
A check for any dead or moribund animals was made at least once each workday, these records are archived by Bioassay.
Pathology:
Necropsy with gross-pathology examination was performed on the last day of the observation period after sacrifice by CO2-inhalation in a chamber with gradually increasing concentrations.

* The test item was covered with an air-permeable dressing (4 layers of absorbent gauze (Ph. Eur. supplied by Lohmann GmbH & Co., KG) and stretch bandage (Fixomull® Stretch (adhesive fleece) supplied by Beiersdorf AG).
** The removal of the semi-occlusive dressing and the rinsing of the application site with warm water were documented together in a protocol by confirming the time of the removal of the dressing.

Assessment of skin reactions
The evaluation of skin reactions was performed according to Draize, J. H. "Dermal toxicity." Appraisal of the safety of chemicals in foods, drugs and cosmetics (1959): 46-59. Appraisal of the safety of chemicals in foods, drugs and cosmetics. The association of food and drug officials of the United States Austin, Texas:

Erythema and eschar formation
Grading
0
No erythema
1
Very slight erythema (barely perceptible)
2
Well- defined erythema
3
Moderate to severe erythema
4
Severe erythema (beet redness) to eschar formation preventing grading of erythema
Edema formation
Grading
0
No edema
1
Very slight edema (barely perceptible)
2
Slight edema (edges of area well- defined by definite raising)
3
Moderate edema (raised approx. 1 mm)
4
Severe edema (raised more than 1 mm and extending beyond area of exposure)
Descriptions of any dermal findings not covered by this scale were recorded.

Key result

Sex:

male

Dose descriptor:

LD50

Effect level:

> 2 000 mL/kg bw

Based on:

test mat.

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mL/kg bw

Based on:

test mat.

Mortality:

No mortality occurred.

Clinical signs:

other: Systemic effects No systemic clinical signs were observed during clinical examination. Local effects Due to the black discoloration of the application site, no erythema could be determined in the animals from study day 1 until study day 6. Thereafte,r no

Gross pathology:

No macroscopic pathologic abnormalities were noted in any animal examined on the last day of observation (5 males and 5 females).

Interpretation of results:

GHS criteria not met

Executive summary:

In an acute dermal toxicity study (Limit Test), young adult Wistar rats (5 males and 5 females) were dermally exposed to a single dose of 2000 mg/kg bw of KDLNO (as suspension in corn oil Ph.Eur.). The clipped application site (dorsal and dorso-lateral parts of the trunk, comprising at least 10% of the total body surface) was covered by semi-occlusive dressing during the 24-hour exposure period. The animals were observed for 14 days.

 No mortality occurred.
 No signs of systemic toxicity were observed in the animals.
 Due to the black discoloration of the application site, no erythema could be determined from study day 1 until study day 6. Thereafter, no local findings were seen in all animals.
 The body weight in all male animals and in two out of five female animals increased within the normal range throughout the study period.
The body weight of two female animals slightly decreased or stagnated during the first week, but increased within the normal range during the second week, while another female showed stagnation of body weight throughout the study period. Due to the fact that stagnation or slight loss of body weight is commonly known for females after dermal applications, this stagnation is considered to be unspecific.
 No macroscopic pathologic abnormalities were noted in any animal examined at the end of the study (5 males and 5 females).

Accordingly, the acute dermal median lethal dose (LD50) was determined to be LD50, dermal, rat > 2000 mg/kg bw

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

> 2 000 mg/kg bw

Justification for classification or non-classification

In an acute oral toxicity study the median lethal dose of KDLNO after oral administration was found to be greater than 500 mg/kg bw and less than 2000 mg/kg bw in rats. Hence, the substance is considered harmful if swallowed.

 

The substance is not classified according to the acute inhalation and acute dermal toxicity studies.