1-(3-chloropyridin-2-yl)-N-[4-cyano-2-methyl-6-(methylcarbamoyl)phenyl]-3-{[5-(trifluoromethyl)-2H-tetrazol-2-yl]methyl}-1H-pyrazole-5-carboxamide

Endpoint summary

Description of key information

Oral (OECD 423), rat: LD50 > 2000 mg/kg bw

Inhalation (OECD 403), rat: LC50 > 5.01 mg/L air

Dermal (OECD 402), rat: LD50 > 2000 mg/kg bw

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Mar - 11 Apr 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Version / remarks:

adopted in 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Version / remarks:

adopted in 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Version / remarks:

adopted in 2008

GLP compliance:

yes (incl. QA statement)

Remarks:

GYEMSZI, National Institute for Quality- and Organizational Development in Healthcare and Medicine, Budapest, Hungary

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

other: CRL:(WI)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9 - 10 weeks
- Weight at study initiation: 192 - 209 g
- Fasting period before study: animals were fasted overnight prior to administration
- Housing: 3 animals of the same sex per cage in type II polypropylene/polycarbonate cages, Lignocel Bedding
- Diet: ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" (ssniff Spezialdiäten GmbH, Soest, Germany) ad libitum
- Water: tap water from the municipal supply ad libitum
- Acclimation period: at least 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.2 - 24
- Humidity (%): 30 - 68
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: polyethylene glycol 400

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 200 mg/mL
- Amount of vehicle: 10 mL/kg bw
- Lot/batch no.: BCBH5068V

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg bw

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

3

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: animals were observed at 30 min, 1, 2, 3, 4, and 6 h after dosing and daily thereafter; body weight was recorded on the day before treatment, on the day of the treatment and weekly after.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight, macroscopic appearance of organs and tissues

Statistics:

Mean and standard deviation of the body weight and body weight gain were calculated.

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality occured during the study period.

Clinical signs:

other: No clinical signs of toxicity were observed up to the end of the 14-day observation period.

Gross pathology:

Necropsy examination revealed no substance-related findings.

Interpretation of results:

other: CLP/GHS criteria not met; no classification required according to Regulation (EC) No. 1272/2008

Conclusions:

CLP: not classified

Reference 2

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 Jun - 25 Jun 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Version / remarks:

adopted in 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Version / remarks:

adopted in 1998

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Version / remarks:

adopted in 2008

GLP compliance:

yes (incl. QA statement)

Remarks:

GYEMSZI, National Institute for Quality- and Organizational Development in H ealthcare and Medicine, Budapest, Hungary

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

other: CRL:(WI)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: 300 - 329 g
- Fasting period before study: animals were fasted overnight prior to administration
- Housing: 3 animals of the same sex per cage in type II polypropylene/polycarbonate cages, Lignocel Bedding
- Diet: ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" (ssniff Spezialdiäten GmbH, Soest, Germany) ad libitum
- Water: tap water from the municipal supply ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.8 - 25
- Humidity (%): 33 - 68
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: polyethylene glycol 400

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 200 mg/mL
- Amount of vehicle: 10 mL/kg bw
- Lot/batch no.: BCBK9981V

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg bw

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

3

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: animals were observed at 30 min, 1, 2, 3, 4, and 6 h after dosing and daily thereafter; body weight was recorded on the day before treatment, on the day of the treatment and weekly after.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight, macroscopic appearance of organs and tissues

Statistics:

Mean and standard deviation of the body weight and body weight gain were calculated.

Key result

Sex:

male

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality occured during the study period.

Clinical signs:

other: No clinical signs of toxicity were observed up to the end of the 14-day observation period.

Gross pathology:

Necropsy examination revealed no substance-related findings.

Interpretation of results:

other: CLP/GHS criteria not met; no classification required according to Regulation (EC) No. 1272/2008

Conclusions:

CLP: not classified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex VII, 8.5, of Regulation (EC) No 1907/2006.

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 May - 18 Jun 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

adopted in 2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1300 (Acute inhalation toxicity)

Version / remarks:

adopted in 1998

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2008

GLP compliance:

yes (incl. QA statement)

Remarks:

GYEMSZI National Institute for Quality- and Organizational Development in Healthcare and Medicines, Budapest, Hungary

Test type:

traditional method

Limit test:

yes

Species:

rat

Strain:

other: CRL:(WI) rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9 - 11 weeks
- Weight at study initiation: 324 - 435 g (males), 213 - 234 g (females)
- Housing: 5 animals of the same sex per cage (individual in case of sighting group) in type III solid floor cages with stainless stell mesh lids, deep wood sawdust bedding (Lignocel® Hygienic Animal Bedding, J. Rettenmaier & Söhne GmbH+Co. KG, Rosenberg, Germany)
- Diet: ssniff SM R/M-Z+H (ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

3.64 µm

Geometric standard deviation (GSD):

2.4

Remark on MMAD/GSD:

The mean mass median aerodynamic diameter was 3.72 µm in the sighting group and 3.64 µm in the main study. The geometric standard deviation was 2.47 in the sighting group and 2.40 in the main study.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: concentric anodised aluminium chambers with a computer control system incorporating pressure detectors and mass flow controllers (TSE Rodent Exposure System; TSE Systems GmbH, Bad Homburg, Germany)
- Exposure chamber volume: 3.85 L (Theoretical Volume)
- 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 chamber.
- Source and rate of air: filtered air at 0.7 L/min
- System of generating particulates/aerosols: generation of an aerosol using a Dust Generator Budiman (TSE Systems GmbH, Bad Homburg, Germany) at the top of the chamber under dynamic conditions
- 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 (TSE Systems GmbH, Bad Homburg, Germany) isolating particles in the discrete aerodynamic size ranges. Samples were taken from an unoccupied exposure port and the weight of the test item was determined gravimetrically.
- Treatment of exhaust air: Used aerosol entered the outer cylinder from where it was exhausted through a suitable filter system.
- Temperature, humidity in air chamber: 25.0 - 27.1°C, 25.0 - 50.0%,

TEST ATMOSPHERE
- Brief description of analytical method used: The test substance concentration was determined by gravimetric analysis (glass-fibre filter, Whatman GmbH, Dassel, Germany)
- Samples taken from breathing zone: yes

TEST ATMOSPHERE
- Particle size distribution: During the study the test atmosphere was respirable to the animals. see table 1
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): see table 1

Analytical verification of test atmosphere concentrations:

yes

Remarks:

gravimetric

Duration of exposure:

4 h

Concentrations:

4.83 mg/L (sighting group, analytical)
5.01 mg/L (main study, analytical, maximum attainable concentration)

No. of animals per sex per dose:

1 (sighting group)
5 (main study)

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: animals were observed hourly during exposure, 1 h after exposure and twice daily thereafter for mortality/ moribundity; hourly during exposure, twice on the day of exposure and once daily thereafter for clinical signs. Bodyweights were determined prior to treatment and on Days 1, 3, 7 and 14.
- Necropsy of survivors performed: yes

Preliminary study:

In the preliminary sighting study 1 male and 1 female were exposed to test substance target concentration of 5.0 mg/L. No mortality occured during the study.

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5.01 other: mg/L air (gravimetrical)

Based on:

test mat.

Remarks:

maximum attainable concentration

Exp. duration:

4 h

Mortality:

1/5 male died shortly after the exposure

Clinical signs:

other: Wet fur and fur staining were observed in most animals on the day of exposure and one day after exposure. These observations were considered to be related to the restraint and exposure procedures and therefore, not considered to be treatment-related. Slig

Body weight:

Bodyweight loss was observed in the majority of animals, which is due to the restrained procedure during the exposure. However, the mean bodyweight and bodyweight gain of the animals at termination of the study was in the normal range, compared to untreated animals of the same age and strain.

Gross pathology:

No macroscopic changes were noted. However, dark/red discoloration of the non-collapsed lungs was macroscopically found in the dead animal.

Table 2 Mortality and clinical signs

Group	Mortality	Clinical Signs
Males
Sighting group (4.83 mg/L)	0/1	1/1
Main study group (5.01 mg/L)	1/5	5/5
Females
Sighting group (4.83 mg/L)	0/1	1/1
Main study group (5.01 mg/L)	0/5	5/5

Interpretation of results:

other: CLP/GHS criteria not met; no classification required according to Regulation (EC) No. 1272/2008

Conclusions:

CLP: not classified

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LC50

Value:

5.01 mg/m³ air

Quality of whole database:

The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex VII, 8.5, of Regulation (EC) No 1907/2006.

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 Apr - 17 Apr 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Version / remarks:

adopted in 1987

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1200 (Acute Dermal Toxicity)

Version / remarks:

adopted in 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.3 (Acute Toxicity (Dermal))

Version / remarks:

adopted in 2008

GLP compliance:

yes (incl. QA statement)

Remarks:

GYEMSZI, National Institute for Quality- and Organizational Development in H ealthcare and Medicine, Budapest, Hungary

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

other: CRL:(WI)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: young adults
- Weight at study initiation: 214 - 258 g (males and females)
- Housing: individual caging in type II polypropylene/polycarbonate cages, "Lignocel Hygienic Animal Bedding" (J. Rettenmaier & Söhne GmbH+Co.KG, Rosenberg, Germany)
- Diet: ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" (ssniff Spezialdiäten GmbH, Soest, Germany) ad libitum
- Water: tap water from the municipal supply ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 - 25
- Humidity (%): 32 - 50
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

TEST SITE
- Area of exposure: 5 x 5 cm² shorn skin of the dorsal area of the trunk
- % coverage: 10%
- Type of wrap: the test material was held in contact with the skin by gauze pads dampened with water to ensure good contact with the skin. An adhesive hypoallergenic plaster kept the gauze pads in contact with the skin. The entire trunk of the animals was then wrapped with semiocclusive plastic wrap for 24 h.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): residual test material was removed with water at body temperature.
- Time after start of exposure: 24 h

TEST MATERIAL
- Amount applied: 2000 mg/kg bw
- Constant concentration used: yes
- For solids, paste formed: no

Duration of exposure:

24 h

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

5 males per male dose group and 5 females per female dose group

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: animals were observed at 1 and 5 hours after the application of the test item and daily thereafter; body weight was recorded at the beginning of the experiment (day 0) and on days 7 and 14.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight, macroscopic appearance of organs and tissues

Statistics:

Mean and standard deviation of the body weight and body weight gain were calculated.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality occured during the study period.

Clinical signs:

other: No clinical signs of toxicity were observed up to the end of the 14-day observation period.

Gross pathology:

2000 mg/kg bw: necropsy examination revealed no substance-related findings.

Interpretation of results:

other: CLP/GHS criteria not met; no classification required according to Regulation (EC) No. 1272/2008

Conclusions:

CLP: not classified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex VII, 8.5, of Regulation (EC) No 1907/2006.

Additional information

Reliable studies regarding acute oral, inhalation and dermal toxicity are available for the test substance.

Oral:     

The acute oral toxicity of the test substance was assessed in a study performed according to OECD Guideline 423 and in compliance with GLP (M-462506-01-1). A group of three fasted female CRL:(WI)rats was treated with the test material at a dose level of 2000 mg/kg bw. The test material was administered by gavage formulated in polyethylene glycol 400 at a dosing volume of 10 mL/kg bw. The animals were observed for 14 days and body weights were measured. All animals were subjected to a necropsy and a macroscopic examination. As no mortality was observed, a confirmatory group of three fasted females was treated at the same dose level. No mortality was observed in the confirmatory group; therefore no further testing was required according to OECD Guideline 423. Treatment with the test substance did not cause any clinical signs during the 14 day observation period. Body weight and body weight gain of treated animals showed no indication of a treatment-related effect. There was no evidence of macroscopic observations at a dose level of 2000 mg/kg bw. In addition, under the same conditions, the acute oral toxicity of the test substance was assessed in male CRL:(WI)rats according to OECD Guideline 423 and in compliance with GLP (M-496837-01-1). The test substance did not cause mortality at a dose level of 2000 mg/kg bw. Treatment did not cause any clinical signs during the 14 day observation period. Body weight and body weight gain of treated animals showed no indication of a treatment-related effect. There was no evidence of the macroscopic observations in surviving animals dosed at 2000 mg/kg bw and subjected to the necropsy on Day 14. Thus, under the conditions of this study, the acute oral LD50 value of the test substance was found to be > 2000 mg/kg bw in female and male CRL:(WI) rats.

Inhalation:

The acute inhalation toxicity of the test substance was assessed in a study performed according to OECD Guideline 403 and in compliance with GLP (M-461520-01-1). Five male and five female CRL:(WI)rats were exposed to the milled test material at the mean achieved concentration of 5.01 mg/L for 4 h using a nose-only exposure system followed by a 14-day observation period. A single sighting exposure was performed prior to the main study with one male and one female at a target concentration of 5 mg/L. 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 the animals were sacrificed at the end of the scheduled period and subjected to a gross examination. For the sighting exposure the mean achieved atmosphere concentration was 4.83 mg/L. The MMAD (Mean Mass Aerodynamic Diameter) was 3.72mm ± 2.47 (GSD [Geometric Standard Deviation]). For the main study the mean achieved atmosphere concentration was 5.01 mg/L. The MMAD was 3.64mm ± 2.40 GSD. There was one death in the main study during the course of the study. Due to the restraint and exposure procedures wet fur and fur staining were commonly recorded on the day of exposure and several days after exposure. Slight laboured respiration was recorded in exposed animals on the day of exposure and one day after exposure. In addition, sneezing, noisy respiration, decreased activity and hunched posture were recorded in animals several days after exposure. However, all clinical signs ceased and no clinical signs were noted from Day 3, with the exception of one female where fur loss around the eyes was recorded until Day 11. Following exposure, bodyweight loss was observed in the majority of animals, which is due to the restraint procedure during the exposure. Normal bodyweight gain was noted for all animals from Day 1 of the observation period. The mean bodyweight and bodyweight gain of the animals at termination of the study was in the normal range, compared to untreated animals of the same age and strain. In surviving animals, no macroscopic changes were noted during either sighting exposure or main study. However, dark/red discoloration of the non-collapsed lungs was macroscopically found in the dead animal. Thus, under the conditions of this study, the acute inhalation LC50 value of the test substance was found to be > 5.01 mg/L in male and female CRL:(WI) rats.

Dermal:

The acute dermal toxicity of the test substance was assessed in a study performed according to OECD Guideline 402 and in compliance with GLP (M-462507-01-1). Five male and five female CRL:(WI)rats were treated with the test substance by a single semi-occlusive dermal application at 2000 mg/kg bw (limit test). The animals were observed for 14 days and body weights were measured. All animals were subjected to a necropsy and a macroscopic examination. No mortality occurred and no clinical signs or local dermal signs were observed. There were no treatment related effects on body weight or body weight gain. There was no evidence of any observations at a dose level of 2000 mg/kg bw at necropsy. Thus, under the conditions of this study, the acute dermal LD50 value of the test substance was found to be > 2000 mg/kg bw in male and female CRL:(WI) rats.

Justification for classification or non-classification

The available data on acute oral, inhalation and dermal toxicity do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.