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Acute Toxicity: inhalation

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Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date: 06 February 2018, Experimental completion date: 08 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)
Deviations:
yes
Remarks:
Please see "Principles of method if other than guideline" for further details.
Qualifier:
according to guideline
Guideline:
EU Method B.52 (Acute Inhalation Toxicity - Acute Toxic Class Method)
Deviations:
yes
Remarks:
Please see "Principles of method if other than guideline" for further details.
Principles of method if other than guideline:
The following deviation from study plan occurred:
Deviation No 1
The relative humidity within the exposure chamber during the exposures was found to be lower than the range specified in the inhalation test guidelines (30 70 %). The decreased humidity was considered to be unavoidable due to the effect of the test item on the humidity.
This deviation is considered to have not affected the integrity or validity of the study.
GLP compliance:
yes (incl. QA statement)
Test type:
acute toxic class method
Limit test:
yes

Test material

Constituent 1
Reference substance name:
Slags, sponge iron production by coal reduction in retort
EC Number:
940-884-3
Molecular formula:
Not applicable for inorganic UVCB
IUPAC Name:
Slags, sponge iron production by coal reduction in retort
Test material form:
solid

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
Male and female RccHan™ : WIST strain rats were supplied by Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were randomly allocated to cages. After an acclimatization period of at least 5 days the animals were given a number unique within the study by ear punching and a number written on a color coded cage card. At the start of the study the animals were approximately 8 to 12 weeks old and within the weight range of 200 g to 350 g. The females were nulliparous and non pregnant.
The animals were housed in groups of up to three by sex in solid floor polypropylene cages with stainless steel lids, furnished with softwood flakes. With the exception of the exposure period, free access to mains drinking water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The diet, drinking water and bedding were routinely analyzed and were considered not to contain any contaminants that would reasonably be expected to affect the purpose or integrity of the study.
- Fasting period before study:

ENVIRONMENTAL CONDITIONS
The temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70% respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness. The animals were retained in this accommodation at all times except during the exposure period.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
3.76 µm
Geometric standard deviation (GSD):
2.92
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Prior to the start of the study, test item atmospheres were generated within the exposure chamber. During this characterization period test item input rates were varied in an attempt to achieve the required atmospheric conditions.
A dust atmosphere was produced from the test item using a SAG 410 Solid Aerosol Generator (TOPAS GmbH, Dresden, Germany) located adjacent to the exposure chamber. The SAG 410 was connected to a metered compressed air supply.
Compressed air was supplied by means of an oil free compressor and passed through a water trap and respiratory quality filters before it was introduced to the SAG 410.
The cylindrical exposure chamber had a volume of approximately 30 liters (dimensions: 28 cm diameter x 50 cm high). The concentration within the chamber was controlled by adjusting the test item feed rate from the SAG 410.
During the exposure period, each rat was individually held in a tapered, polycarbonate restraining tube fitted onto a single tier of the exposure chamber and sealed by means of a rubber ‘O’ ring. Only the nose of each animal was exposed to the test atmosphere.
The extract from the exposure chamber passed through a ‘scrubber’ trap and was connected with a high efficiency filter to a metered exhaust system. The chamber was maintained under negative pressure.
The temperature and relative humidity inside the exposure chamber were measured by an electronic thermometer/humidity meter (Hanna Instruments Ltd, Beds., UK) located in a vacant port in the animals’ breathing zone of the chamber and recorded every 30 minutes throughout the 4 Hour exposure period.
Oxygen levels within the exposure chamber were measured by an electronic oxygen analyzer (Servomex (UK) Ltd, Crowborough, East Sussex) located in a port in the animals breathing zone during the 4 Hour exposure period. The test atmosphere was generated to contain at least 19% oxygen.

The particle size of the generated atmosphere inside the exposure chamber was determined three times during the exposure period using a Marple Personal Cascade Impactor (Westech IS Ltd, Beds., UK). This device consisted of six impactor stages (10.4, 7.7, 4.1, 1.3, 0.9 and 0.56 µm cut points) with stainless steel collection substrates and a backup glass fiber filter, housed in an aluminum sampler. The sampler was temporarily sealed in a sampling port in the animals’ breathing zone and a suitable, known volume of exposure chamber air was drawn through it using a vacuum pump.
The collection substrates and backup filter were weighed before and after sampling and the weight of test item, collected at each stage, calculated by difference.
The mean amount 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 10.4, 7.7, 4.1, 1.3, 0.9 and 0.56 µm µm was calculated.
The resulting values were converted to probits and plotted against Log10 cut point size. From this plot, the Mass Median Aerodynamic Diameter (MMAD) was determined (as the 50% point) and the geometric standard deviation was calculated. In addition the proportion (percentage) of aerosol less than 4 µm (considered to be the inhalable fraction) was determined.

TEST ATMOSPHERE
The actual chamber concentration was measured at regular intervals during the exposure period. The gravimetric method used glass fiber filters placed in a filter holder. The holder was temporarily sealed in a vacant port in the exposure chamber in the animals’ breathing zone and a suitable, known volume of exposure chamber air was drawn through the filter using a vacuum pump.
Each filter was weighed before and after sampling in order to calculate the weight of collected test item. The difference in the two weights, divided by the volume of atmosphere sampled, gave the actual chamber concentration.
The nominal chamber concentration was calculated by dividing the mass of test item disseminated into the chamber by the total volume of air that flowed through the chamber during the exposure.

TEST ATMOSPHERE
Homogeneity of the test atmosphere within the chamber was not specifically determined during this study. Chambers of the same design (ADG Developments Ltd, Hitchin, Herts, UK) have been fully validated and shown to produce evenly distributed atmospheres in the animals’ breathing zone with a wide variety of test items (Green J D et al, 1984).

CLASS METHOD
As insufficient data was available on the expected inhalation toxicity of the test item, a sighting test was performed to determine the initial exposure concentration. A group of two animals (one male and one female) was exposed to an aerosol atmosphere of the test item at a target concentration of 2.0 mg/L.
Based on the results of the sighting test, a limit test was performed. A group of six animals (three males and three females) was exposed to an aerosol atmosphere of the test item at a target concentration of 5.0 mg/L.
Analytical verification of test atmosphere concentrations:
no
Duration of exposure:
4 h
Remarks on duration:
Following an appropriate equilibration period each group was exposed to an atmosphere of the test item for a period of 4 hours.
Concentrations:
5.07 mg/L
No. of animals per sex per dose:
3 male, 3 female
Control animals:
no
Details on study design:
All animals were observed for clinical signs at hourly intervals during exposure, immediately on removal from the restraining tubes at the end of exposure, 1 hour after termination of exposure and subsequently once daily for 14 days. Any evidence of overt toxicity was recorded at each observation.
Individual body weights were recorded on arrival, prior to treatment on the day of exposure (Day 0) and on Days 1, 3, 7 and 14.
At the end of the 14 observation period all animals were killed by intravenous overdose of sodium pentobarbitone. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded. The respiratory tract was subjected to a detailed macroscopic examination for signs of irritancy or local toxicity.

Results and discussion

Preliminary study:
During exposure both animals exhibited decreased respiratory rate and wet fur. On removal from the chamber both animals exhibited decreased respiratory rate, labored respiration, hunched posture, fur stained dark grey by test item and wet fur. One hour post-exposure labored respiration was no longer present. No significant abnormalities were noted on Day 1 post-exposure. Both animals showed body weight loss on Day 1 post exposure and expected gains in body weight from Days 1 to 3. The female showed body weight loss and the male showed expected gains in body weight from Days 3 to 6 post-exposure.The following macroscopic abnormalities were detected at necropsy:
Lungs - Dark patches.
The observed abnormalities were potentially due to irritancy or local toxicity; however this was not confirmed by histopathological examination. It is also possible that the observations were caused by a residue, or deposition, of the test item. As a mean achieved atmosphere concentration of 2.02 mg/L was tolerated in the sighting test a target concentration of 5.0 mg/L was selected for the limit test.
Effect levels
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
5.07 mg/L air
Based on:
test mat.
Exp. duration:
4 h
Mortality:
There were no deaths noted.
Clinical signs:
other: Signs of hunched posture and pilo-erection are commonly seen in animals for short periods on removal from the chamber following 4-hour inhalation studies. Wet fur is commonly recorded both during and for a short period after exposure and staining of anima
Body weight:
All animals showed body weight loss on Day 1 post-exposure. With the exception of one female that showed body weight loss from Days 1 to 3 post-exposure all animals showed expected gains in body weight during the remainder of the recovery period.
Gross pathology:
The following macroscopic abnormalities were detected at necropsy:
Lungs – Pale, abnormally red, dark patches.
No macroscopic abnormalities were detected in one animal.
The observed abnormalities were potentially due to irritancy or local toxicity; however this was not confirmed by histopathological examination. It is also possible that the observations were caused by a residue, or deposition, of the test item.

Any other information on results incl. tables

Nominal Concentration

Comparison of the nominal and the actual concentration gives an indication of the generation efficiency of the of the test system. A lower value is considered to indicate a higher efficiency and values above 1000% are considered to indicate a highly inefficient test system. The nominal concentration in comparison to the actual concentration shows that the test system was highly inefficient during the exposure.

1645% of the actual mean achieved atmosphere concentration.

Exposure Chamber Concentration

The test atmosphere was sampled nine times during the exposure period and the actual concentration of the test item calculated. The mean values obtained were as follows:

Group Number

Atmosphere Concentration

Mean Achieved (mg/L)

Standard Deviation

Nominal (mg/L)

1

5.07

0.18

83.39

The chamber flow rate was maintained at 40 L/min providing 80 air changes per hour.

The theoretical chamber equilibration time (T99) was 4 minutes*(Silver, 1946).

* =  The test atmosphere was generated for 20 minutes prior to animal insertion to ensure test item concentration was being achieved

Particle Size Distribution

The particle size analysis of the atmosphere drawn from the animals’ breathing zone was as follows:

Group Number

Mean Achieved Atmosphere Concentration (mg/L)

Mean Mass Median Aerodynamic Diameter (µm)

Inhalable Fraction

(% <4 µm)

Geometric Standard Deviation

1

5.07

3.76

52.3

2.92


Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
None of the animals died in a group of six rats exposed to mean achieved atmosphere concentration of 5.07 mg/L. It was therefore considered that the acute inhalation median lethal concentration (4 hour LC50) of the test item in the Wistar strain rat was greater than 5.07 mg/L.
The test item does not meet the criteria for classification according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.
The test item does not meet the criteria for classification according to the Globally Harmonized System of Classification and Labelling of Chemicals.
Executive summary:

Introduction

A study was performed to assess the acute inhalation toxicity of the test item. The method used was designed to be compatible with that described in the OECD Guideline for

Testing of Chemicals (2009) No. 436 “Acute Inhalation Toxicity – Acute Toxic Class Method” and Method B.52. Acute Inhalation Toxicity – Acute Toxic Class Method, 2014, of Commission Regulation (EC) No. 440/2008.

Methods

As insufficient data was available on the expected inhalation toxicity of the test item, a sighting test was performed to determine the initial exposure concentration. A group of two animals (one male and one female) was exposed to an aerosol atmosphere of the test item at a target concentration of 2.0 mg/L.

Based on the results of the sighting test, a limit test was performed. A group of six animals (three males and three females) was exposed to an aerosol atmosphere of the test item at a target concentration of 5.0 mg/L.

RccHanTM: WIST strain rats were exposed for 4 hours using a nose only exposure system followed by an observation period. The observation period was six days for the sighting test, sufficient to ensure recovery of the animals, and fourteen days for the limit test.

Results

The mean achieved atmosphere concentration was as follows:

Group Number

Atmosphere Concentration

Mean Achieved (mg/L)

Standard Deviation

Nominal (mg/L)

Sighting

2.02

0.07

30.70

1

5.07

0.18

83.39

The characteristics of the achieved atmosphere were as follows:

Group Number

Mean Achieved Atmosphere Concentration (mg/L)

Mean Mass Median Aerodynamic Diameter (µm)

Inhalable Fraction

(% <4 µm)

Geometric Standard Deviation

Sighting

2.02

3.33

56.7

3.01

1

5.07

3.76

52.3

2.92

The mortality data were summarized as follows:

Group Number

Mean Achieved Atmosphere Concentration

(mg/L)

Deaths

Male

Female

Total

Sighting

2.02

0/1

0/1

0/2

1

5.07

0/3

0/3

0/6

Clinical Observations. Sighting: Common abnormalities noted during the study included decreased respiratory rate, labored respiration, hunched posture, pilo-erection, wet fur and fur stained dark grey by test item. No significant abnormalities were noted on Day 1 post‑exposure. 

Group 1: Common abnormalities noted during the study included decreased respiratory rate, hunched posture, pilo-erection, wet fur, fur stained black or dark grey by test item and tail stained black by test item. No significant abnormalities were noted on Day 2 post-exposure. Black colored tail was noted in all animals on Days 5 to 7 post‑exposure and fur stained dark grey by test item was apparent in all animals up to  Day 10 post-exposure, persisting in all males up to Day 13 post-exposure.

Body Weight. Sighting: Both animals showed body weight loss on Day 1 post-exposure and the female showed body weight loss from Days 3 to 6 post-exposure. Expected gains in body weight were noted during the remainder of the recovery period.

Group 1: All animals showed body weight loss on Day 1 post-exposure. With the exception of one female that showed body weight loss from Days 1 to 3 post-exposure all animals showed expected gains in body weight during the remainder of the recovery period. 

Necropsy. Sighting: The following macroscopic abnormalities were detected at necropsy:

Lungs – Dark patches.

Group 1:The following macroscopic abnormalities were detected at necropsy:

Lungs – Pale, abnormally red, dark patches.

No macroscopic abnormalities were detected in one animal.

The observed abnormalities were potentially due to irritancy or local toxicity; however this was not confirmed by histopathological examination.


Conclusion

None of the animals died in a group of six rats exposed to mean achieved atmosphere concentration of 5.07mg/L. It was therefore considered that the acute inhalation median lethal concentration (4 hour LC50) of the test item in the Wistar strain rat was greater than 5.07mg/L.

The test item does not meet the criteria for classification according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

The test item does not meet the criteria for classification according to the Globally Harmonized System of Classification and Labelling of Chemicals.

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