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Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 Jul 2016 to 17 Aug 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Version / remarks:
2009
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.1300 (Acute inhalation toxicity)
Version / remarks:
1998
Qualifier:
according to guideline
Guideline:
EU Method B.2 (Acute Toxicity (Inhalation))
Version / remarks:
2008
GLP compliance:
yes
Test type:
traditional method
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
4-(5-(3,5-dichloro-4-fluorophenyl)-5-(trifluoromethyl)-4,5-dihydro-1,2-oxazol-3-yl)-N-(2-ethyl-3-oxo-1,2-oxazolidin-4-yl)-2-methylbenzamide
Cas Number:
2061933-85-3
Molecular formula:
C23H19Cl2F4N3O4
IUPAC Name:
4-(5-(3,5-dichloro-4-fluorophenyl)-5-(trifluoromethyl)-4,5-dihydro-1,2-oxazol-3-yl)-N-(2-ethyl-3-oxo-1,2-oxazolidin-4-yl)-2-methylbenzamide
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:(WI)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 12 weeks old
- Body weight at study initiation: males: 396 - 420 g; females: 244 - 269 g
- Diet: Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenance, ad libitum
- Water: Tap water, ad libitum
- Housing: Group caging in Type III solid floor cages with stainless steel mesh lids. Rodents were housed with deep wood sawdust bedding to allow digging and other normal rodent activities
- Acclimatisation period: At least 5 days

ENVIRONMENTAL CONDITION
- Temperature (°C): 19.0 – 24.3
- Humidity (%): 34 – 70
- Air changes (per hour): 15 - 20
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 20 Jul 2016 To:17 Aug 2016

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
clean air
Mass median aerodynamic diameter (MMAD):
>= 3.11 - <= 3.38 µm
Geometric standard deviation (GSD):
>= 2.34 - <= 2.37
Details on inhalation exposure:
EXPOSURE CONDITIONS
Prior to animal exposures, test material atmospheres were generated within the exposure chamber. During these technical trials, air-flow settings, test substance input rates were varied to achieve the required aerosol concentration of particles Measurements of aerodynamic particle size were performed from the animal’s breathing zone using a cascade impactor. Due to the physicochemical properties of the test substance, it was not possible to achieve the maximum (limit) concentration of 5 mg/L with an MMAD of 1 - 4 µm. More information is provided in a table in the section "Any other information on materials and methods incl tables"

GENERATION OF THE TEST ATMOSPHERE / CHAMBER DESCRIPTION
The test substance was aerosolised using a Wright’s Dust Feed System located at the top of the exposure chamber. Compressed air was supplied by means of an oil-free compressor and passed through a suitable filter system prior to introduction to the dust generator. The animals were exposed, nose-only, to an atmosphere of the test substance using a TSE Rodent Exposure System. This system comprised of 2 concentric anodised aluminium chambers and a computer control system incorporating pressure detectors and mass flow controllers. 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 nares 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 0.7 L/min. This flow rate was considered adequate to minimise re-breathing of the test atmosphere as it is about 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, chambers of this design have been fully validated and have shown to produce evenly distributed atmospheres in the animals’ breathing zones (Pauluhn, 1994).

TEST ATMOSPHERE CONCENTRATION
The test atmosphere was sampled at regular intervals during each exposure period. Samples were taken from an unoccupied exposure port (representing the animal’s breathing zone) by pulling a suitable, known volume of test atmosphere through weighed GF10 glass fibre filters. The difference in the pre and post sampling weights, divided by the volume of atmosphere sampled, was equal to the actual achieved test atmosphere concentration. The nominal concentration was calculated by dividing the mass of test material disseminated into the chamber by the total volume of air that through the chamber during the same period. Particle size determination: The particle size of the test atmosphere was determined three times during the exposure period using a 7-stage impactor of Mercer style. 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 0.55, 0.96, 1.55, 2.11, 3.56, 6.66 and 10.55 μm was calculated. From these data, using software supplied with the impactor , 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 inhalable portion) was determined.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
4.76 mg/L
No. of animals per sex per dose:
Sighting exposure: 2
Main group: 5
Control animals:
no
Details on study design:
ANIMAL ASSIGNMENT AND TREATMENT
Prior to the start of the study the animals were examined to ensure that they were physically normal and exhibited normal activity. All animals were observed for mortality during the exposure period. The animals were examined for signs of gross toxicity, and behavioural changes upon removal from the exposure tube and at least once daily thereafter for 14 days. Observations included gross evaluation of skin and fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea and coma. In group 1, one female rat was found dead on Day 7 and was cannibalized. A specific cause of death was not determined for this animal. At necropsy collapsed, dark/red discoloured lungs were observed. No clinical observations were observed in this animal for several days prior to death and whilst the animal lost body weight following treatment, these losses were no greater than those observed in other animals in this treatment group. It is therefore uncertain that the death of this animal was a result of test item administration. Individual body weights were recorded 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, the surviving animals were sacrificed by exsanguination under anaesthesia (Euthanimal 40 % (pentobarbital sodium) injection and gross macroscopic examination was performed. All animals were subject to a gross necropsy which included a detailed examination of the abdominal and thoracic cavities. Special attention was given to the respiratory tract for macroscopic signs of irritancy or local toxicity.
Statistics:
The acute inhalation LC50 was not estimated.

Results and discussion

Preliminary study:
During the sighting study, laboured respiration (slight) was observed. The animals were symptom free from Day 1
Effect levels
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
4.62 mg/L air (analytical)
Exp. duration:
4 h
Remarks on result:
other: Highest attainable concentration
Mortality:
In group 1, one female rat was found dead on Day 7 and was cannibalized. A specific cause of death was not determined for this animal
Clinical signs:
other: Ruffled fur or red-brown staining (as chromodacryorrhea) and fur staining by the test substance occurring in study animals from Day 0 - 2 were considered to be related to the restraint and exposure procedures not to be toxicologically significant.
Remarks:
During the sighting and the main study, laboured respiration (slight) was observed. The animals were symptom free from Day 1
Body weight:
In Group 0.1, slight and moderate body weight loss were observed in both sexes between Day 0 and 3 (males: 5.9 - 7.3 %, females: 13.7 - 17 % respectively) of the observation period. The body weight gain of male and female rats normalised by the end of the observation period. In Group 1, all male and all female animals showed slight or moderate body weight loss (males: 4.8 - 10 %, females: 11.7 - 13.9 % respectively) in the first 3 days of the observation period. In case of surviving animals, the body weight gain returned to the normal range, thereafter (Day 14).

Gross pathology:
A single four hour nose-only exposure of the test substance to Crl: WI Wistar strain rats exposed to the concentration of 4.38 and 4.62 mg/L during Sighting exposure and Main study, respectively, did not produce any test substance-related gross findings on Day 14.

Any other information on results incl. tables

Table 3: Mortality overview

Group
Number

Mean Achieved
(mg/L)

Male Deaths

Female Deaths

Total Deaths

1

(Maximum attainable)

0/5

1/5

1/10

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of this study, one female was found dead in a group of 10 rats exposed to the mean achieved concentration of 4.62 mg/L (maximum attainable concentration) for 4 hours. The acute inhalation median lethal concentration of the test substance, in CRL:WI Wistar strain rats is therefore considered to be greater than 4.62 mg/L
Executive summary:

This OECD 403 study performed in compliance with GLP to assess the acute inhalation toxicity of the test substance following a 4 hour exposure at the maximum achievable concentration for the test substance was approximately 4.76.mg/L to 5 male and 5 female rats. A sighting exposure was performed prior to the main study with 2 males and 2 females at a mean achieved concentration of 4.38 mg/L. The main study group, 10 (5 male and 5 female) CRL:WI Wistar strain rats, was exposed to the mean achieved concentration of 4.62 mg/L the test substance The animals were exposed for 4 hours using a nose-only exposure system, followed by a 14 day observation period. The day of exposure was designated Day 0. Aerosol concentrations were measured gravimetrically. The particle size distribution of the test aerosol was determined regularly during the exposure period. Clinical observations and body weights were recorded throughout the study and at the end of the scheduled period the animals were euthanised and subjected to a gross examination post mortem.

In group 1, one female rat was found dead on Day 7 and was cannibalized.  A specific cause of death was not determined for this animal.  At necropsy collapsed, dark/red discoloured lungs were observed. Ruffled fur or red-brown staining (as chromodacryorrhea) and fur staining by the test substance occurring in study animals from Day 0 up to Day 2 were considered to be related to the restraint and exposure procedures not to be toxicologically significant. During the sighting and the main study, laboured respiration (slight) was observed. The animals were symptom free from Day 1. In Group 0.1, slight and moderate body weight loss were observed in both sexes between Day 0 and 3 (males: 5.9 - 7.3 %, females: 13.7 - 17 % respectively) of the observation period. The body weight gain of male and female rats normalised by the end of the observation period.

In Group 1, all male and all female animals showed slight or moderate body weight loss (males: 4.8 - 10 %, females: 11.7 - 13.9 % respectively) in the first 3 days of the observation period. In case of surviving animals, the bodyweight gain returned to the normal range, thereafter (Day 14). A single four hour nose-only exposure of the test substance to Crl: WI Wistar strain rats exposed to the concentration of 4.38 and 4.62 mg/L during Sighting exposure and Main study, respectively, did not produce any test substance-related gross findings on Day 14. Under the experimental conditions of this study, one female was found dead in a group of 10 rats exposed to the mean achieved concentration of 4.62 mg/L (maximum attainable concentration) for 4 hours. The acute inhalation median lethal concentration of the test substance, in CRL:WI Wistar strain rats is therefore considered to be greater than 4.62 mg/L.