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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 Starting Date: 10 March 2014; Experimental Completion Date: 04 August 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)
Deviations:
yes
Remarks:
The first exposure was repeated due to a high achieved concentration. Animals from Group 3 were acclimatized to the restraint tubes on the day of exposure. These deviations are considered not to affect the purpose or validity of the study.
GLP compliance:
yes (incl. QA statement)
Test type:
acute toxic class method
Limit test:
no

Test material

Constituent 1
Reference substance name:
Hydrocarbons, C18-C24, iso-alkanes, <2% aromatics
IUPAC Name:
Hydrocarbons, C18-C24, iso-alkanes, <2% aromatics
Constituent 2
Chemical structure
Reference substance name:
Hydrocarbons, C18-C24, iso-alkanes <2% aromatics
EC Number:
940-734-7
Molecular formula:
C18H38, C19H40, C20H42, C21H44, C22H46, C23H48, C24H50
IUPAC Name:
Hydrocarbons, C18-C24, iso-alkanes <2% aromatics
Test material form:
liquid
Details on test material:
Identification: Shell GTL Solvent GS310
CAS number: Not available
Physical state/appearance: Clear colorless liquid
Batch: Not supplied
Purity: 100.00 %
Expiry date: Not supplied
Storage conditions: Room temperature, in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Male and female RccHan™ : WIST strain rats were supplied by Harlan Laboratories UK Ltd. On receipt the animals were randomly allocated to cages. After an acclimatization period of at least five 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 eight to twelve weeks old and within the weight range of 200g to 350g. 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 and provided with environmental enrichment items: wooden chew blocks and cardboard “fun tunnels”. With the exception of the exposure period, free access to mains drinking water and food (Harlan 2014C Rodent Diet) was allowed throughout the study. The diet, drinking water, bedding and chew blocks are routinely analyzed and are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

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 twelve hours continuous light (06:00 to 18:00) and twelve hours darkness. The animals were retained in this accommodation at all times except during the exposure period.



Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
ATOMOSPHERE GENERATION:
The test item was aerosolized using a glass concentric jet nebulizer located at the top of the exposure chamber. The nebulizer was connected to a plastic syringe attached to an infusion pump, which provided a continuous supply of test item under pressure, and 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 nebulizer.

The cylindrical exposure chamber had a volume of approximately 30 liters (dimensions: 28 cm diameter x 50 cm high). The concentration within the exposure chamber was controlled by adjusting the rate of the infusion pump. 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.

Homogeneity of the test atmosphere within the chamber was not specifically determined during this study. Chambers of the same design have been fully validated and shown to produce evenly distributed atmospheres in the animals’ breathing zone with a wide variety of test items.

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 to achieve the required atmospheric conditions.

EXPOSURE PROCEDURE:
On the day of exposure (Group 3) and prior to the day of exposure (Group 2) each rat was acclimatized (for approximately 2 hours) to a tapered polycarbonate restraining tube. 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.

Following an appropriate equilibration period two groups, each of six rats (three males and three females), were subjected to a single exposure to the test item for a period of four hours*. Based on the expected toxicity of the test item, a target concentration of 5.0 mg/L was used for the first exposure. The second concentration was selected after consideration of the results of the first exposure.

*Previously to this another group of animals (Group 1) had been exposed to an atmosphere of the test item. However, this exposure was not suitable for the purposes of the study due to the achieved atmosphere concentration being significantly higher than the target concentration of 5mg/L.

EXPOSURE CHAMBER TEMPERATURE AND RELATIVE HUMIDITY:
The temperature and relative humidity inside the exposure chamber were measured by an electronic thermometer/humidity meter located in a vacant port in the animals’ breathing zone of the chamber and recorded every thirty minutes throughout the four-hour exposure period.

EXPOSURE CHAMBER OXYGEN CONCENTRATION:
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 four-hour exposure period. The test atmosphere was generated to contain at least 19% oxygen.

EXPOSURE CHAMBER ATMOSPHERE CONCENTRATION:
During the characterization phase of the study the test atmosphere was sampled twice and filter samples were then submitted for chemical analysis to determine if the original test item was similar to the composition of the airborne test item. The standard and sample solutions were analyzed by GC.

Prior to the inhalation phase of the study, the non-volatile component of the test item was determined by adding a small, known amount of test item to glass fibre filters and recording their weights. The filters were then dried in a desiccator between 20 and 21°C for approximately 24 hours and then weighed again. The difference in the two weights was taken as the volatile content of the test item and the non-volatile component was calculated as a percentage. The mean non-volatile component of the batch used during the formal exposure was found to be 99.42 % (n=10).

The test atmosphere was sampled at regular intervals during the exposure period. A weighed glass fibre filter was placed in a filter holder and temporarily sealed in a vacant port of the exposure chamber in the animals’ breathing zone. A known quantity of the exposure chamber atmosphere was drawn through the filter using a vacuum pump.

After sampling, the filter was dried, under similar conditions as those previously described, and weighed again 24 hours later. The difference in the pre and post sampling weights, divided by the volume of atmosphere sampled, was the chamber concentration in terms of non-volatile component.

Based on the results of the preliminary work, these figures were adjusted to obtain a true figure for the test item concentration in the chamber.

The nominal chamber concentration was calculated by dividing the mass of test item used by the total volume of air passed through the chamber.

The nominal concentration for each group was found to be 250 % or 227 % (Groups 2 and 3 respectively) of the actual mean achieved atmosphere concentration and shows that keeping the aerosol airborne was relatively straightforward at each target concentration.

PARTICLE SIZE DISTRIBUTION:
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. The device consisted of six impactor stages (8.9, 6.2, 3.6, 1.6, 0.93 and 0.37 µm cut points) with stainless steel collection substrates and a back up 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 8.9, 6.2, 3.6, 1.6, 0.93 and 0.37 µm cut points 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 (%) of aerosol less than 4 µm (considered to be the inhalable fraction) was determined.












Analytical verification of test atmosphere concentrations:
yes
Remarks:
It was considered appropriate to utilize gravimetric analysis to determine test atmosphere concentrations during the formal exposure (see exposure chamber atmosphere concentration)
Duration of exposure:
4 h
Concentrations:
Group 2:
Mean Achieved Atmosphere Concentration (mg/L): 4.98

Group 3:
Mean Achieved Atmosphere Concentration (mg/L): 1.14
No. of animals per sex per dose:
3 males and females per dose group.
Control animals:
no
Details on study design:
Observations:
Clinical signs: All animals were observed for clinical signs at hourly intervals during exposure, immediately on removal from the restraining tubes at the end of exposure, one hour after termination of exposure and subsequently once daily for up to fourteen days. Any evidence of overt toxicity was recorded at each observation.

Body Weight: Individual body weights were recorded on arrival, prior to treatment on the day of exposure and on Days 1, 3, 7 and 14.

Necropsy: At the end of the fourteen day observation period the 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.
Statistics:
Data evaluations included the relationship, if any, between the animals’ exposure to the test item and the incidence and severity of all abnormalities including behavioral and clinical observations, necropsy findings, body weight changes, mortality and any other toxicological effects.

Using the mortality data obtained, an estimate of the acute inhalation median lethal concentration (LC50) of the test item was made.

Results and discussion

Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 1 - 5 mg/L air
Based on:
test mat.
Exp. duration:
4 h
Mortality:
The mortality data is summarised as follows:
Group 2:
Mean Acheived Atmosphere Concentration (mg/L): 4.98
Deaths:
Male: 2/3 (deaths on Days 1 and 2 of observation)
Female: 1/3 (death on Day 1 of observation)
Total: 3/6

Group 3:
Mean Acheived Atmosphere Concentration (mg/L): 1.14
Deaths:
Male: 0/3
Female: 1/3 (death on Day 2 of observation)
Total: 1/6
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. These observations a
Body weight:
Group 2 – All animals exhibited body weight losses on Day 1 post-exposure. All surviving animals exhibited body weight loses or showed no body weight gain from Days 1 to 3 post-exposure, body weight gains were noted in all surviving animals during the remainder of the recovery period.

Group 3 – All males and two female animals exhibited body weight losses on Day 1 post-exposure. Body weight gains were noted in all surviving animals during the remainder of the recovery period.
Gross pathology:
Necrospy:
Dark patches on the lungs were noted at necropsy amongst all three surviving animals from Group 2 and in one out of five surviving animals from Group 3, a further surviving female from Group 3 exhibited a dark liver at the end of the fourteen day recovery period.

The following macroscopic abnormalities were detected in the animals that died during the course of the study at necropsy:

Lungs – abnormally dark or dark patches;
Liver – dark;
Large Intestine – gaseous distension.

Due to the observations noted it is considered that the deaths noted during the study may have been mainly attributable to local toxicity.

Dark patches in the lungs and abnormally dark lungs in a proportion of animals in both treatment groups were associated with congestion at histopathological examination, and were considered to be treatment-related.

Other findings:
Histopathology:
The following treatment-related findings were present in animals surviving to terminal sacrifice:

Lungs:
- minimal to slight congestion
- minimal to slight hemorrhage
- moderate edema
- minimal to slight alveolar macrophages
- slight acute alveolar inflammation
- minimal to slight acute perivascular inflammation

Trachea:
- minimal intraepithelial inflammation

The following treatment-related findings were present in the lungs of premature decedent animals:
- slight to moderate congestion
- slight hemorrhage
- minimal to moderate edema
- minimal to slight alveolar macrophages
- minimal to slight acute alveolar inflammation
- minimal to moderate acute perivascular inflammation

There were no findings in the trachea.











Any other information on results incl. tables

Exposure Chamber Concentration:

Test atmospheres were sampled seventeen times during each exposure period and the actual concentration of the test item calculated. 

The mean values obtained for each group were:

Group Number

Atmosphere Concentration

Mean Achieved (mg/L)

Standard Deviation

Nominal (mg/L)

2

4.98

0.17

12.4

3

1.14

0.12

2.59

The chamber flow rate was maintained at 60 L/min providing 120 air changes per hour.

 

The theoretical chamber equilibration time (T99) was 3 minutes*(Silver, 1946).


*= The test atmosphere was generated for 20 minutes and 11 minutes prior to animal insertion (for Groups 2 and 3 respectively) to ensure test item concentration was being achieved.

It is noted that one sample from Group 3 was lower than 20% of the mean achieved atmosphere concentration. As the majority of samples taken during the exposure were above the target concentration of 1mg/L this deviation to the test guideline was considered not to have affected the purpose or validity of the study.

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

2

4.98

3.17

60.9

2.34

3

1.14

2.45

78.1

1.88

Exposure Chamber Temperature:

Temperature maintained at 19 - 20 °C for Groups 2 and 3.

Exposure Chamber Relative Humidity:

25 - 26% Group 2 and 26 - 27% Group 3.

The relative humidity within the exposure chamber during each exposure was found to be slightly lower than the range specified in the inhalation test guidelines (30 – 70 %). The humidity probe was removed from the test chamber and was found to be working correctly. The reduced humidity may well have been due to the nature of the test item, the reduced humidity was therefore considered to be an accurate representation within the exposure chamber and is considered not to have affected the purpose or validity of this study

Exposure Chamber Oygen Concentration:

20.8 - 20.9 %.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Three deaths occurred in a group of six rats exposed to a mean achieved atmosphere concentration of 4.98 mg/L, whereas, one death occurred at a mean achieved atmosphere concentration of 1.14 mg/L. It was therefore considered that the acute inhalation median lethal concentration (4 hr LC50) of Shell GTL Solvent GS310, in the RccHanTM : WIST strain rat, was in the range of >1 - 5 mg/L.

The microscopic findings observed in the lungs (alveolar and perivascular inflammation, edema, congestion and hemorrhage) were all consistent with hydrocarbon aspiration induced inflammation (i.e. a chemical pneumonitis). The substance is therefore classified as Aspiration Toxicity Category 1.
Executive summary:

Introduction:

A study was performed to assess the acute inhalation toxicity of the test item. The method used was compatible with that described in the OECD Guidelines for Testing of Chemicals (2009) No. 436 “Acute Inhalation Toxicity – Acute Toxic Class Method”.

Methods:

Two groups of six RccHan™ : WIST strain rats (three males and three females) were exposed to an aerosol atmosphere. The animals were exposed for four hours using a nose only exposure system, followed by a fourteen day observation period.

Results:

The mean achieved atmosphere concentration was as follows:

 

Group Number

Atmosphere Concentration

Mean Achieved (mg/L)

Standard Deviation

Nominal (mg/L)

2

4.98

0.17

12.4

3

1.14

0.12

2.59

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

2

4.98

3.17

60.9

2.34

3

1.14

2.45

78.1

1.88

The mortality data were summarized as follows:

 

Group Number

Mean Achieved Atmosphere Concentration

(mg/L)

Deaths

Male

Female

Total

2

4.98

2/3

1/3

3/6

3

1.14

0/3

1/3

1/6

Clinical Observations:

Common abnormalities noted during the study included increased respiratory rate, hunched posture, pilo-erection and wet fur, occasional instances of tip-toe gait were also noted. Surviving Group 2 animals recovered to appear normal on Day 8 post-exposure. Surviving Group 3 animals recovered to appear normal from Days 6 to 7 post-exposure. 

Body Weight:

Group 2 – All animals exhibited body weight losses on Day 1 post-exposure. All surviving animals exhibited body weight loses or showed no body weight gain from Days 1 to 3 post-exposure, body weight gains were noted in all surviving animals during the remainder of the recovery period.

 

Group 3 – All males and two female animals exhibited body weight losses on Day 1 post-exposure. Body weight gains were noted in all surviving animals during the remainder of the recovery period.

Necropsy:

Dark patches on the lungs were noted at necropsy amongst all three surviving animals from Group 2 and in one out of five surviving animals from Group 3, a further surviving female from Group 3 exhibited a dark liver at the end of the fourteen day recovery period.

 

The following macroscopic abnormalities were detected in the animals that died during the course of the study at necropsy:

 

Lungs – abnormally dark or dark patches;

Liver – dark;

Large Intestine – gaseous distension.

 

Due to the observations noted it is considered that the deaths noted during the study may have been mainly attributable to local toxicity.

 

Dark patches in the lungs and abnormally dark lungs in a proportion of animals in both treatment groups were associated with congestion at histopathological examination, and were considered to be treatment-related.

Histopathology:

The following treatment-related findings were present in animals surviving to terminal sacrifice:

 

Lungs:

-          minimal to slight congestion

-          minimal to slight hemorrhage

-          moderate edema

-          minimal to slight alveolar macrophages

-          slight acute alveolar inflammation

-          minimal to slight acute perivascular inflammation

Trachea:

-          minimal intraepithelial inflammation

The following treatment-related findings were present in the lungs of premature decedent animals:

-          slight to moderate congestion

-          slight hemorrhage

-          minimal to moderate edema

-          minimal to slight alveolar macrophages

-          minimal to slight acute alveolar inflammation

-          minimal to moderate acute perivascular inflammation

 

There were no findings in the trachea.

Conclusion:

Three deaths occurred in a group of six rats exposed to a mean achieved atmosphere concentration of 4.98 mg/L, whereas, one death occurred at a mean achieved atmosphere concentration of 1.14 mg/L. It was therefore considered that the acute inhalation median lethal concentration (4 hr LC50) of Shell GTL Solvent GS310, in the RccHan: WIST strain rat, was in the range of >1 - 5 mg/L.

The microscopic findings observed in the lungs (alveolar and perivascular inflammation, edema, congestion and hemorrhage) were all consistent with hydrocarbon aspiration induced inflammation (i.e. a chemical pneumonitis).