Reaction mass of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9RS)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide and of 3-(difluoromethyl)-1-methyl-N-[(1RS,4SR,9SR)-1,2,3,4-tetrahydro-9-isopropyl-1,4-methanonaphthalen-5-yl]pyrazole-4-carboxamide; isopyrazam

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Mar 2014 to 27 Mar 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

2009

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1300 (Acute inhalation toxicity)

Version / remarks:

1998

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.2 (Acute Toxicity (Inhalation))

Version / remarks:

2008

GLP compliance:

yes (incl. QA statement)

Test type:

traditional method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

632-619-2

EC Number:

632-619-2

Cas Number:

881685-58-1

Molecular formula:

C20 H23 F2 N3 O

IUPAC Name:

632-619-2

Test animals

Species:

rat

Strain:

Wistar

Remarks:

CRL: (WI) (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Sex: 5 male and 5 female, nulliparous and non-pregnant
- Age at study initiation: Young adult rats, 9 weeks old
- Weight at study initiation: 189 to 309 g (males: 276-309 g; females: 189-217g)
- Housing: Standard housing conditions
- Diet: Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenancen. Ad libitum
- Water: Tap water. Ad libitum
- Acclimatisation period: 14 days
- Method of randomisation in assigning animals to test and control groups: Selected based on body weight prior to the exposure

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30-70%
- Air changes: 15-20 air exchanges/hour
- Photoperiod: 12/12 hours light/dark

IN-LIFE DATES: From: 13 Jan 2014 To: 27 May 2014

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

5 µm

Geometric standard deviation (GSD):

2.4

Remark on MMAD/GSD:

Generally, the MMAD was higher as the exposure concentration increased.
The inhalable fraction (% < 4μm) was 39.9%.

Details on inhalation exposure:

TECHNICAL TRIALS
Prior to animal exposures, test material atmospheres were generated within the exposure chamber. During these technical trials, air-flow settings and test material input rates were adjusted to achieve the required atmospheric characteristics.

ATMOSPHERE GENERATION
The test material 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.

ANIMAL EXPOSURE SYSTEM
The animals were exposed, nose-only, to an atmosphere of the test item 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. The chamber volume was 3.85 L.
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.

EXPOSURE PROCEDURE
Each rat was individually held in a tapered, polycarbonate restraining tube fitted onto a single tier of the exposure chamber. Only the nose of each animal was exposed to the test atmosphere.
Following an equilibration period of at least the theoretical chamber equilibration time (T99), a group of 10 rats (5 male and 5 female) was exposed to the target concentration of 2.5 mg/L for a period 4 hours.

EXPOSURE MONITORING
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 went through the chamber during the same period.

PARTICLE SIZE ANALYSIS
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 material, 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. The proportion (%) of aerosol less than 4 μm (considered to be the inhalable portion) was determined.
Due to the nature of the test material the optimum attainable MMAD (~5.0 μm) during the technical trials was higher than 4 μm (upper limit) specified in the test guidelines. Generally, the MMAD was higher as the exposure concentration increased. Based on the objectives of the study in terms of balancing test guideline requirements and the need to assign an appropriate hazard classification for the test material, a test concentration of approximately 2.6 mg/l was selected for the main study.

CHAMBER ENVIRONMENTAL CONDITIONS
- Airflow in/ out (L/ min): 30.2/ 27.5
- Temperature (°C): 24.7
- Oxygen concentration (%): 20.4
- Carbon Dioxide (%): 0

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

2.60 mg/L

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: animals were checked for morbidity/mortality 1 hour after exposure and twice daily; clinical signs were checked at hourly intervals during exposure and 1 hour after exposure and subsequently once daily for 14 days; body weights were recorded prior to treatment on Day 0, 1, 3, 7 and 14.
- Necropsy of survivors performed: yes. At the end of the 14 day observation period, the surviving animals were sacrificed by exsanguination under anaesthesia 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:

- From the body weights, the group means and their standard deviations were calculated.
- The Mass Median Aerodynamic Diameter (MMAD) and Geometric Standard Deviation (GSD) were calculated.

Results and discussion

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 2.6 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

not determinable due to absence of adverse toxic effects

Mortality:

There were no deaths in the study.

Clinical signs:

other: Wet fur, and/or fur staining; Ruffled fur; Slight to severe laboured respiration; Decreased activity.

Remarks:

No clinical signs were recorded from Day 2 of the observation period.

Body weight:

Normal body weight gain was noted for all exposed animals during the observation period.

Gross pathology:

No macroscopic findings were observed during necropsy on Day 14.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The acute inhalation median LC50 of the test material in male and female rats was estimated to be > 2.60 mg/L.

Executive summary:

Ten (5 male and 5 female) CRL: (WI) Wistar rats, were exposed to 2.60 mg/L test material in a study according to OECD TG 403 and GLP principle. The animals were exposed for 4 hours using a nose-only exposure system, followed by a 14 day observation period. Clinical observations and body weights were recorded throughout the study and at the end of the scheduled period the animals were subjected to a gross examination post mortem.

The mean achieved atmosphere concentration of the test material was 2.60 mg/L. The MMAD was 5.00 μm ± 2.40 (GSD). There was no mortality in the study. Wet fur, and/or fur staining were recorded in all animals on the day of exposure and day following exposure. Ruffled fur was recorded in one male and one female on the day following exposure. These observations were considered not to be of toxicological relevance. Slight to moderate laboured respiration was noted for the exposed animals mostly on the day of exposure and in one occasion on day following exposure. Decreased activity was only seen in one male 4 and 5 hours after exposure. All clinical signs ceased from Day 2 of the observation period. Normal body weight gain was noted for all exposed animals during the observation period. No macroscopic findings were observed during necropsy on Day 14

The acute inhalation median LC50 of the test material, in CRL: (WI) Wistar strain rats was estimated to be > 2.60 mg/L.