2-(4-tert-butylphenyl)-6-cyano-5-[bis(ethoxycarbonylmethyl)carbamoyloxy]-1H-pyrrolo[1,2-b][1,2,4] triazole-7-carboxylic acid 2,6-di-tert-butyl-4-methylcyclohexylester

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 August 2013 - 28 August 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to valid guidelines and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1300 (Acute inhalation toxicity)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

448-050-6

EC Name:

-

Cas Number:

444065-11-6

Molecular formula:

C41 H57 N5 O8

IUPAC Name:

2,6-di-tert-butyl-4-methylcyclohexyl 5-{[bis(2-ethoxy-2-oxoethyl)carbamoyl]oxy}-2-(4-tert-butylphenyl)-6-cyano-1H-pyrrolo[1,2-b][1,2,4]triazole-7-carboxylate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Substance type: Off-white, slightly yellow fine, fluffy powder
- Storage conditions of test material: Controlled room temperature

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Stain: CRL:(WI) rats (SPF).
- Age at study initiation: Young adult rats, 9 - 12 weeks old.
- Weight at study initiation: males: 350 - 458 g; females: 211 - 288 g.
- Housing: Grouped by sex; 5 for the main test and 2 for the sighting group. Cages were Type III solid floor cages with stainless steel mesh lids.
- Diet: Complete feed for rats and mice, ad libitum.
- Water: Tap water, ad libitum.
- Acclimation period: 35 days (sighting group), 13 days (main group).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 – 70 %
- Air changes (per hr): 15 – 20 air charges/hour
- Photoperiod (hrs dark / hrs light): 12 hours of light daily, from 6.00 a.m. to 6.00 p.m.

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: TSE Rodent Exposure System. This system comprises of two, 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. Atmosphere generation was therefore dynamic.
- Exposure chamber volume: 3.85 L (inner plenum)
- Equilibration period: Sighting test 16 minutes; main test 13 minutes.
- Method of holding animals in test chamber: 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.
- Rate of air: Mean airflow into the inner plenum measured 15.0 L/min. Air flow was at least 0.7 L/min through each port.
- Method of conditioning air: Compressed air was supplied by an oil-free compressor and passed through a suitable filter system prior to introduction to the dust generator.
- System of generating particulates/aerosols: Due to the granular nature of the material, prior to use it was ground using a Ball Mill (‘Mixer Mill MM 400’). The test material was aerosolised using a dust generator (Wright’s Dust Feed Systems) located at the top of the exposure chamber.
- Method of 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.
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. In addition, the proportion (%) of aerosol less than 4 μm (considered to be the inhalable portion) was determined.
- Treatment of exhaust air: After passing through the animals’ breathing zone, used aerosol entered the outer cylinder from where it was exhausted through a suitable filter system.
- Conditions in test chamber:
> Sighting Group: Mean temperature: 24.5 °C (range 24.3 – 24.7 °C); Mean relative humidity: 60.2 % (range 58.8 – 74.7 %).
> Main Group: Mean temperature: 24.7 °C (range 24.0 – 25.0 °C); Mean relative humidity: 62.4 % (range 61.5 – 70.8 %).

TEST ATMOSPHERE
- Brief description of analytical method used: Aerosol concentrations were measured gravimetrically.
The test atmosphere was sampled at regular intervals during each exposure period. Samples were taken from an unoccupied exposure port 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.
- Samples taken from breathing zone: yes
- See Table 1 for test atmosphere concentrations for the main test.

TEST ATMOSPHERE
- The percentage of inhalable fraction (% < 4 µm) in the sighting test was 61.2 % and 61.9 % in the main test.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.):
> Sighting test: MMAD = 3.11 µm; GSD = 2.42
> Main test: MMAD = 3.00; GSD = 2.56
- See Table 2 for particle size distribution for the main test.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

Mean achieved concentration sighting test: 5.07 ± 0.12 mg/L (nominal concentration 15.18 mg/L)
Mean achieved concentration main test: 5.05 ± 0.17 mg/L (nominal concentration 12.88 mg/L)

No. of animals per sex per dose:

Sighting test: 2 per sex per dose
Main test: 5 per sex per dose

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Morbidity/Mortality: Animals were observed hourly during exposure, one hour after exposure and then twice daily during the 14 day post exposure observation period.
- Clinical signs: All animals were observed hourly during exposure, as soon as practically possible following removal from restraint at the end of exposure, one hour after exposure and subsequently once daily for 14 days.
- Bodyweight: Individual bodyweights were recorded prior to treatment on the day of exposure (Day 0) and on Days 1, 3, 7 and 14.
- Necropsy of survivors performed: Yes. At the end of the 14 day observation period, the animals were euthanised 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.

Results and discussion

Effect levels

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5.05 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: Mean achieved concentration

Mortality:

No mortality was observed in either the sighting or main tests.

Clinical signs:

other: Wet fur and fur staining were commonly recorded mostly on the day of exposure and in some occasions several days after exposure. These observations were considered to be related to the restraint and exposure procedures and, in isolation, were considered n

Body weight:

Normal bodyweight gain was noted during the observation period for all animals.

Gross pathology:

No treatment related gross lesions were noted.

Applicant's summary and conclusion

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of the test, no death or overt signs of toxicity occurred in a group of five male and five female rats exposed to a mean achieved atmosphere of 5.05 mg/L for four hours. The LC50 was therefore determined to be greater than 5.05 mg/L.

Executive summary:

The acute toxicity of the test material was determined using Wistar strain rats in an acute inhalation toxicity study. The study was performed under GLP conditions and in accordance with the standardised guidelines OECD 403, EU Method B.2 and EPA OPPTS 870.1300.

A single sighting exposure was performed prior to the main study with 2 males and 2 females at the target concentration of 5 mg/L.

In the main study group, 10 (5 male and 5 female) rats were exposed to the mean achieved concentration of 5.05 mg/L. The animals were exposed for 4 hours using a nose-only exposure system, followed by a 14 day observation period. Aerosol concentrations were measured gravimetrically. The particle size distribution of the test aerosol was determined regularly during the exposure period. Clinical observations and bodyweights were recorded throughout the study, and at the end of the observation period the animals were euthanised and subjected to a gross examination post mortem.

In the main study the MMAD was 3.00 µm with a GSD of 2.56. The inhalable fraction (% < 4 µm) was 61.9 %.

Under the conditions of the test, no death or overt signs of toxicity occurred in a group of ten rats exposed to a mean achieved atmosphere of 5.05 mg/L for four hours. As no deaths occurred at 5.06 mg/L no further testing was required. The LC50 was therefore determined to be greater than 5.05 mg/L.