Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: 09 October 2014 Experimental Completion Date: 27 November 2014
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Qualifier:
according to
Guideline:
EPA OPPTS 870.1300 (Acute inhalation toxicity)
Qualifier:
according to
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: white solid

Test animals

Species:
rat
Strain:
other: RccHanTM : WIST strain
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK
- Age at study initiation: eight to twelve weeks of age
- Weight at exposure start: 198-242 g
- Fasting period before study: With the exception of the exposure period, free access to mains drinking water and food (Harlan 2014C Rodent Diet, Harlan Laboratories UK Ltd, Oxon, UK) was allowed throughout the study.
- Housing: TThe animals were housed in groups of five by sex in solid-floor polypropylene cages with stainless steel lids, furnished with softwood flakes
- Diet (e.g. ad libitum): 2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) , ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): at least 15

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Details on inhalation exposure:
3.2 Inhalation Exposure
3.2.1 Atmosphere Generation
For Groups 1 and 2 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.
For Group 2, a particle separator was introduced after the SAG 410 before the aerosol entered the exposure chamber in order to remove large particles and thereby increase the inhalable portion of the generated aerosol.
For Group 3 the test item formulation was aerosolized using a glass concentric jet nebulizer (Radleys, Saffron Walden, Essex, UK) located at the top of the exposure chamber. The
* = One female animal (Number 20) was slightly outside the weight range specified in the Study Plan (200 – 350g)
Harlan Study Number: 41402912 Report Page 15
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 or 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 exposure chamber was controlled by adjusting the rate of the belt speed or 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. Diagrams of the dynamic (continuous flow) systems employed are shown in Figures 1 to 3.
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 input rates, generation systems and formulation details were varied in an attempt to achieve the required atmospheric conditions.
3.2.2 Exposure Procedure
Prior to the day of exposure each rat was acclimatized (for approximately 2 hours) to a tapered polycarbonate restraining tube. During each 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 three groups, each of ten rats (five males and five females), were subjected to a single exposure to the test item (Groups 1 and 2) or to an atmosphere of a formulation of the test item (Group 3) (TDA-sulfone : Acetone, 5 : 95 w/w) for a period of four hours. Based on the expected toxicity of the test item, a target concentration of 2.0 mg/L was used for the first exposure. Further concentrations were selected after consideration of the results of the previous exposure.
3.2.3 Exposure Chamber Temperature and Relative Humidity
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 thirty minutes throughout the four-hour exposure period. Individual values are given in Tables 19 and 20.
Harlan Study Number: 41402912 Report Page 16
3.2.4 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. Individual values are given in Table 21.
3.2.5 Exposure Chamber Atmosphere Concentration - Groups 1 and 2
The actual chamber concentration was measured at regular intervals during each 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 used by the total volume of air passed through the chamber.
The nominal concentrations were 272% and 753% of the actual mean achieved atmosphere concentration for Groups 1 and 2 respectively, these show that keeping the aerosol airborne was relatively straight forward for Group 1 but became difficult for group 2
3.2.6 Exposure Chamber Atmosphere Concentration - Group 3
As the test item was a solid in its’ original form and acetone is incredibly volatile, the non-volatile component of the batch of the formulated test item used during the final formal exposure was considered to be 100 %.
The test atmosphere was sampled at regular intervals during each exposure period. A weighed glass fiber 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 filters were then dried in a desiccator between 18 and 20 °C for approximately 24 hours and then weighed again. 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.
These figures were adjusted to obtain a true figure for the test item concentration in the chamber.
Harlan Study Number: 41402912 Report Page 17
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.
The nominal concentration was 445 % of the actual mean achieved atmosphere concentration and shows that keeping the aerosol airborne was moderately difficult.
3.2.7 Particle Size Distribution
The particle size of the generated atmosphere inside the exposure chamber was determined three times during each exposure period using a Marple Personal Cascade Impactor (Westech IS Ltd, Beds., UK). The devices consisted of six impactor stages (8.4, 7.3, 3.6, 1.3, 0.94 and 0.43 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 8.4, 7.3, 3.6, 1.3, 0.94 and 0.43 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
Duration of exposure:
ca. 4 h
Concentrations:
Group Number Mean Achieved Atmosphere Concentration (mg/L)
1 2.09
2 1.01
3 0.20
No. of animals per sex per dose:
10
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: observation hourly during exposure and one hour after exposure, twice daily for the following 14 days
- Necropsy of survivors performed: no survivors but necropsy performed. None of the Group 1 animals survived past the day of exposure, none of the Group 2 animals survived past Day 1 post-exposure and none of the Group 3 animals survived past Day 2 post-exposure

Results and discussion

Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 0.2 mg/L air
Based on:
test mat.
Mortality:
None of the Group 1 animals survived past the day of exposure, none of the Group 2 animals survived past Day 1 post-exposure and none of the Group 3 animals survived past Day 2 post-exposure
Clinical signs:
other: Common abnormalities noted during the study included decreased respiratory rate, labored respiration, noisy respiration, gasping respiration, ataxia, lethargy, hunched posture, pilo-erection and wet fur. Isolated occurrences of sneezing, prostration and t
Body weight:
Group 1 – No significant body weight data was collected as none of the animals survived past the day of exposure.
Group 2 – Two females exhibited body weight losses on the first day post-exposure. None of the animals survived past Day 1 post-exposure and therefore no further significant body weight data was collected.
Group 3 – One male and four females exhibited body weight losses on the first day post-exposure. None of the animals survived past Day 2 post-exposure and therefore no further significant body weight data was collected.
Gross pathology:
The following macroscopic abnormalities were detected at necropsy amongst all animals that died or were humanely killed during the course of the study:
Lungs – abnormally dark or dark patches;
Stomach – gaseous distension;
Small Intestine – gaseous distension.
Due to the observations noted during the study and at necropsy it is considered that deaths noted during the study may have been mainly attributable to local toxicity.

Applicant's summary and conclusion

Interpretation of results:
Toxicity Category I
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
No animals survived in three groups of ten rats exposed to mean achieved atmosphere concentrations of 2.09, 1.01 and 0.20 mg/L respectively. It was therefore considered that the acute inhalation median lethal concentration (4 hr LC50) of TDA-sulfone, in the RccHanTM : WIST strain rat, should be considered to be much less than 0.20 mg/L.