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Toxicological information

Acute Toxicity: inhalation

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Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)
Deviations:
no
GLP compliance:
yes
Test type:
acute toxic class method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
All formulations were prepared with ethanol (Pivalic Acid : Ethanol, 50 : 50 w/w) to improve the aerosolization properties of the test item.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
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.

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: aerosol
Type of inhalation exposure:
nose only
Vehicle:
clean air
Mass median aerodynamic diameter (MMAD):
ca. 0.26 µm
Geometric standard deviation (GSD):
ca. 5.74
Remark on MMAD/GSD:
The results of the particle size distribution should be viewed with caution and are included only as a guide to the aerosol/vapor ratio of the generated atmosphere, as the volatility of a test item will be over-emphasized by the high air velocities and low pressures within a cascade impactor.
Details on inhalation exposure:
The test item formulation was aerosolized using a metal concentric jet nebulizer (Envigo CRS Limited, UK) 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 formulation 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 and air flow settings through the chamber. 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 (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).

Prior to the start of the study, test item atmospheres were generated within the exposure chamber. During this characterization period test item formulation input rates were varied in an attempt to achieve the required atmospheric conditions.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
5.30 mg/L
No. of animals per sex per dose:
3
Control animals:
no
Details on study design:
One day prior to the day of each exposure, 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 ofthe 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 one group, of five males and five females, were subjected to a single exposure of an atmosphere of the formulated test item Pivalic Acid : ethanol, 50:50 w/w for a period of four hours. As the mean achieved concentration was 106% of target and no deaths occurred, no further levels were required.

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 fiber filters and recording their weights. The filters were then dried in a desiccator at room temperature 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 non-volatile component of the batch used during the study was found to be approximately 1.66 % (n=3). Due to the low levels of non-volatiles contained within this test item it was considered appropriate to use chemical analysis in order to determine test atmosphere concentrations throughout the exposure.

The test atmosphere was sampled nine times during the exposure period. The sampling procedure involved 2 liters of test atmosphere being drawn through a glass impinger containing RO water (made up to 80mL). The samples were then submitted for chemical analysis.

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 was 267% of the actual mean achieved atmosphere concentration and shows that keeping the aerosol airborne was relatively straight forward.

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 day 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.

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
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.3 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
none
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:
All male animals and two female animals exhibited body weight losses or showed no body weight gain on the first day post-exposure. One male animal and two female animals exhibited a body weight loss or showed no body weight gain from Days 1 to 3 post-exposure. With the exception of one male animal and two female animals which showed no body weight gain from Days 7 to 14, body weight gains were noted for all animals during the remainder of the recovery period.
Gross pathology:
The following macroscopic abnormalities were detected at necropsy in one female animal:
Lungs – dark patches.
No macroscopic abnormalities were detected at necropsy of three males and two females.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
No deaths occurred in a group of six rats exposed to a mean achieved atmosphere concentration of 5.30 mg/L for 4 hours. 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 5.30 mg/L.
Executive summary:

It is noted that the mean mass median aerodynamic diameter (MMAD) was lower than the range given in test guidelines (1-4 μm) and the Geometric Standard Deviation (GSD) was higher than the generally accepted range (1.5 – 3). These deviations are considered to be due to the physical characteristics of the test item (low levels of non-volatiles contained within the test item). However, as the volatility of a test item will be over-emphasised by the high air velocities and low pressures within a cascade impactor (as shown by a small amount of non-volatiles being retained by the impactor), such results should be viewed with caution and will be included in the formal report only as a guide to the aerosol/vapour ratio and particle size of the generated atmosphere in the aerosol form that could be retained by the cascade impactor. The mean proportion of test atmosphere found to be in the vapour phase during this exposure was found to be approximately 58% and as such it can be concluded that the majority of the generated atmosphere would have been respirable to the test animals.

Re-running this group because of these deviations to the OECD test guideline is considered to be inappropriate and unethical as the results obtained (in terms of animal observations) would be considered to be similar other groups exposed at the same target concentration which inevitably would contain a similar proportion of the test atmosphere in the vapour phase.