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Diss Factsheets

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
4-31 december 2020
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.1300 (Acute inhalation toxicity)
Principles of method if other than guideline:
The animals were exposed to an atmosphere of the test item for a single, continuous four-hour period, generated according to the system and flow rates determined during the Technical Trials. The four-hour exposure period was not start until theoretical chamber concentration equilibration had been reached.
GLP compliance:
yes (incl. QA statement)
Test type:
fixed concentration procedure
Limit test:
yes

Test material

impurity 1
Reference substance name:
Total unspecified impurities
IUPAC Name:
Total unspecified impurities
Constituent 1
Reference substance name:
Cobalt Nickel Manganese Oxide
Cas Number:
37348-84-8
Molecular formula:
Ni1-x-yMnxCoyOz with Me=Ni+Mn+Co=1 and Ni/Me= 0.45-0.98 Mn/Me= 0.01-0.35 Co/Me= 0.01-0.35 O/Me= 1.00-1.33
IUPAC Name:
Cobalt Nickel Manganese Oxide
Test material form:
solid
Specific details on test material used for the study:
pNMC-oxide, 6:2:2 grade
Batch:Lot Number: PHX-418 PHXM10
Purity: considered 100% (Co: 11.13% ; Mn: 13.91% ; Ni: 48.99%)
Expiry date: 30 april 2021
Storage conditions: Controlled room temperature (15-25°C, ≤70% relative humidity). Protected from light and humidity (store in a tightly closed container).

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Male and female Crl:WI Wistar rats (from SPF colony) were obtained from Charles River Laboratories, Research Models and Services, Germany GmbH (Sandhofer Weg 7, D-97633 Sulzfeld, Germany). After arrival, the animals’ health was certified by the Veterinarian.
Hygienic level: SPF at arrival, standard housing conditions during the study

Number of animals was 2 animal/sex for the Sighting exposure, and 5 animals/sex for the Main Study. Animals were acclimated to laboratory conditions for 6 days (Sighting Study) and 7 days (Main Study) prior to involvement in the study. Animals were also acclimatised to the test apparatus (restrain procedures) for a short period prior to testing in order to lessen the stress during exposure.
Animals were randomly allocated to exposure groups before animal exposure based on the actual body weights using PROVANTIS v.9.

Age and body weight range of animals on the exposure day (Day 0) were 8 weeks old and 312-320g(male)/200-210g (female) (Sighting Exposure), and 8 weeks old, 320-338g(male)/198-222g(female)

Housing: was individual caging in the Sighting Study, and group caging (2 or 3 animals by sex/cage) in the main study
Cage type: Polycarbonate solid floor cages (type II or III) with stainless steel mesh lids
Light: 12 hours of continuous artificial light in each 24-hour period (from 6.00 a.m. to 6.00 p.m.); Temperature: 19.9-23.9°C; Relative humidity: 25-63%
Ventilation: At least 15 air exchanges per hour

The temperature and relative humidity were recorded twice daily during the study.

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
ca. 3.2 µm
Geometric standard deviation (GSD):
ca. 4.27
Remark on MMAD/GSD:
The particle size of the test atmosphere was determined three times during the exposure period using a 7-stage impactor of Mercer style (TSE Systems GmbH, Bad Homburg, Germany). Such devices employ an inertial separation technique to isolate particles in the discrete aerodynamic size ranges. Samples were taken from an unoccupied exposure port (representing the animal’s breathing zone).
The collection substrates and the backup filter were weighed before and after sampling and the weight of test item, collected at each stage, calculated by this difference.
The total amount collected for each stage was used to determine the cumulative amount below each cut-off point size. In this way, the proportion (%) of aerosol less than 0.550, 0.960, 1.550, 2.105, 3.555, 6.655 and 10.550 µm was calculated.
From these data, using the software supplied with the impactor (TSE Systems GmbH, Bad Homburg, Germany), the Mass Median Aerodynamic Diameter (MMAD), and Geometric Standard Deviation (GSD) were calculated. In addition, the proportion (%) of aerosol less than 4 µm (considered to be the Respirable portion) was determined.
Details on inhalation exposure:
Each rat was individually held in a tapered, polycarbonate restraining tube fitted onto a single tier of the exposure chamber. Only the nose of each animal was exposed to the test atmosphere.
Following an equilibration period of at least the theoretical chamber equilibration time (T99), each group of rats was exposed to an atmosphere of the test material for a period of 4 hours.

Analytical verification of test atmosphere concentrations:
yes
Remarks:
The test atmosphere was sampled at regular intervals during the exposure period. The difference in the pre and post sampling weights, and divided by the volume of atmosphere sampled, was equal to the actual achieved test atmosphere concentration.
Duration of exposure:
4 h
Concentrations:
Sighting Study: Target concentration 0.5 mg/L ; Mean achieved concentration: 0.52 mg/L (nominal concentration: 17.23 mg/L)
Main Study: arget concentration 0.50 mg/L ; Mean achieved concentration: 0.50 mg/L (nominal concentration: 15.99 mg/L)
No. of animals per sex per dose:
5 per sex/dose
Control animals:
no
Details on study design:
Prior to animal exposures, test material atmospheres were generated within the exposure chamber. During these Technical Trials, air-flow settings, test material input rates were varied to achieve the required aerosol concentration of particles with a mass median aerodynamic diameter (MMAD) between 1 to 4 µm and a geometric standard deviation (GSD) in the range of 1.5 to 3.0. Measurements of aerodynamic particle size were performed from the animal’s breathing zone using a cascade impactor. Animal exposure was only start after Technical Trials indicate that the agreed atmosphere concentrations achieved consistently.

The test item was aerosolised using Palas RBG1000 (TSE Systems GmbH, Bad Homburg, Germany; Serial Number: 1719) located at the top of the exposure chamber.

The animals were exposed, nose-only, to an atmosphere of the test item using a TSE Rodent Exposure System (TSE Systems GmbH, Bad Homburg, Germany). This system comprises of two concentric anodised aluminium chambers and a computer control system incorporating pressure detectors and mass flow controllers. The exposure unit was placed in closed hood in order to avoid cross-contamination and contamination of the laboratory environment.
Fresh aerosol from the generation system was constantly supplied to the inner plenum (distribution chamber) of the exposure system from where, under positive pressure, it was distributed to the individual exposure ports. The animals were held in polycarbonate restraint tubes located around the chamber which allowed only the animal’s nostrils to enter the exposure port. After passing through the animal’s breathing zone, used aerosol entered the outer cylinder from where it was exhausted through a suitable filter system. Atmosphere generation was therefore dynamic. Airflows and relative pressures within the system were constantly monitored and controlled by the computer system thus ensuring a uniform distribution and constant flow of fresh aerosol to each exposure port (breathing zone). The flow of air through each port was at least 0.5 L/min. This flow rate was considered adequate to minimize re-breathing of the test atmosphere as it is approximately twice the respiratory minute volume of a rat.
Homogeneity of the test atmosphere within the test chamber and amongst the exposure ports was not specifically determined during this study. However, considerably larger chambers of this design have been fully validated and have shown to produce evenly distributed atmospheres in the animals’ breathing zones.



Statistics:
Data were collected using the software PROVANTIS v.9 or were recorded on data collection sheets taken from the relevant SOPs, then tabulated using PROVANTIS v.9, Microsoft Office Word and/or Excel, as appropriate. Only a limit test was performed, the four-hour inhalation LC50 was not calculated.

Results and discussion

Preliminary study:
Sighting Exposure was performed in order to estimate the test item’s inhalation toxicity, identify sex differences in susceptibility and assist in selecting exposure concentration levels for the Main Study.
Effect levels
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 0.5 mg/L air
Based on:
test mat.
Exp. duration:
4 h
Remarks on result:
other: no adverse effect noted at maximum achievable concentration
Mortality:
No mortality occurred during the study.
Body weight:
Sighting Exposure (Group 0.1 – 0.50 mg/L)
In the male animals, slight body weight losses were noted on Day 0-3. The body weight gain was normal from Day 3.
In the female animals, slight body weight losses were noted on Day 1-3. The body weight gain was normal from Day 3.

Main Group (Group 1 – 0.52 mg/L)
In the male animals, slight to moderate body weight losses were recorded on Days 0-3. The body weight gains were normal between Days 3-14.
In the female animals, slight body weight losses were recorded on Days 0-3. The body weight gains were normal between Days 3-14.

Gross pathology:
No test item related finding was noted during the necropsy.
Other findings:
Sighting Exposure (Group 0.1 – 0.52 mg/L)
In the male animals, laboured respiration (slight), noisy respiration (slight), fur staining by test item (on the head) and wet fur (on the whole body) were recorded from Day 0 up to Day 3. Both animals were symptom-free from Day 4. In the female animals, laboured respiration (slight), noisy respiration (slight), fur staining by test item (on the head) and wet fur (on the whole body) were recorded from Day 0 up to Day 1. Both animals were symptom-free from Day 2.

Main Group (Group 1 – 0.50 mg/L)
In the male animals, laboured respiration (slight), noisy respiration (slight), fur staining by test item (on the head and nose) and wet fur (on the whole body) were observed from Day 0 up to Day 8. All male animals were symptom-free from Day 9. In the female animals, laboured respiration (slight), noisy respiration (slight), fur staining by test item (on the head and nose) and wet fur (on the whole body) were observed from Day 0 up to Day 8. All female animals were symptom-free from Day 9.
Fur staining by test item and wet fur in the animals were considered to be related to the restraint and exposure procedures or discomfort of the animals but not to be toxicologically significant.

Any other information on results incl. tables

Table: Mean achieved actual and nominal aerosol concentrations

Group Number

Target Concentration
(mg/L)

Mean Achieved Concentration
(mg/L)

Standard Deviation of Achieved Concentration (mg/L)

Nominal Concentration
(mg/L)

Sighting Exposure

0.1

0.50

0.52

0.06

17.23

Main Study

1

0.50

0.50

0.11

15.99

Table: Mean achieved particle size distribution data (MMAD and GSD)

Group Number

Mean Achieved Concentration
(mg/L)

Mean Mass Median Aerodynamic Diameter (MMAD) (mm)

(Automatic calculation)

Mean Mass Median Aerodynamic Diameter (MMAD) (mm)

(Manual Calculation)

Geometric Standard Deviation
(GSD)

(Automatic calculation)

Respirable Fraction
(% < 4mm)

(Automatic calculation)

 

Respirable Fraction
(% < 4mm)

(Manual calculation)

Sighting Exposure

0.1

0.52

3.45

3.55

6.80

53.0

51.83

Main Study

1

0.50

3.42

3.20

4.27

54.2

55.20

Note: The OECD guideline gives the normal GSD range as 1.5-3.0. This range was exceeded in this study, however the relatively high values as low as were practically achievable.

The particle size distribution data are presented graphically in Figure 3 and detailed in Appendix 2, both as Automatic and Manual calculations.

Note that automatic calculations are made from the system software, these are considered to be accurate when the particle size distribution approximates to a ‘normal’ distribution. In this study, the size distribution did not appear to approximate to ‘normal’; hence a manual calculation was made as a Log-Log plot, to estimate the 50% point for cumulative mass.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of this study, no mortality occurred in Group 1 (Main Study) when exposed to a test atmosphere concentration of 0.50 mg/L as a maximum achievable concentration for 4 hours. The acute inhalation median lethal concentration (LC50) of Nickel Cobalt Manganese Oxide (6:2:2) in male and female Crl:WI rats was therefore considered to be above 0.50 mg/L.
Executive summary:

Information on theacute inhalationtoxicity ofpNMC oxide (6:2:2) in male and female Crl:WI Wistar rats was generated ina single4h-nose-only exposure, following OECD N° 403, EU Method B.2 and EOP OPPTS 870.1300.

Based on the results of a Sighting Exposure with two animals of both sexes, the main study with five male and five female rats was performed at the target concentration of 0.5 mg/L. Animals were exposed to the test atmosphere for 4 hours using a nose-only exposure system. Aerosol concentration was measured gravimetrically 17 times in the Sighting Exposure and in the main study during each 4-hour exposure and the particle size distribution of the test aerosol was determined 3 times. The day of exposure was designated as Day 0 followed by a 14-day observation period. Clinical observations were performed on all animals during exposure at hourly intervals, following removal from restraint, approximately 1 hour after exposure, and daily for 14 days thereafter. Body weight was measured on Days 0 (before the exposure), 1, 3, 7 and 14. Gross necropsy was performed on all animals on Day 14.

Results: No mortality occurred during the study. Laboured respiration (slight), noisy respiration (slight), fur staining by test item (on the head and nose) and wet fur (on the whole body) were observed from Day 0 up to Day 8. All animals were symptom-free from Day 9. Slight (male, female) to moderate (male only) body weight losses were recorded on Days 0-3. The body weight gains were normal between Days 3-14. Necropsy revealed test item related grey diffuse/focal discolouration of all lobes of lungs in one out of five male and four out of five females of the main study.

Conclusion: Under the experimental conditions of this study, no mortality occurred in the main study when exposed to a test atmosphere concentration of 0.50 mg/L as a maximum achievable concentration for 4 hours. The acute inhalation median lethal concentration (LC50) of pNMC oxide (6:2:2) in male and female Crl:WI rats was therefore considered to be above 0.50 mg/L.

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