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

Endpoint summary

Description of key information

- Oral: LD50 > 2000 mg/kg bw, females, rat, according to OECD TG 425, Matting 2014

- Inhalation: LC50 > 2.6 mg/L, males/females, rat, according to OECD TG 403, Nagy 2014

- Dermal: LD50 > 2000 mg/kg bw, males/females, rat, according to OECD TG 402, Matting 2014

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Jan 2014 to 16 May 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 425 (Acute Oral Toxicity: Up-and-Down Procedure)

Version / remarks:

2008

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Version / remarks:

2002

GLP compliance:

yes (incl. QA statement)

Test type:

up-and-down procedure

Limit test:

yes

Species:

rat

Strain:

Wistar

Remarks:

RccHan:(WIST))

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Young adult rats, 9 - 11 weeks old
- Weight at study initiation: 171 - 181 g
- Fasting period before study: The night before treatment
- Housing: Individual caging, cage Type II, polypropylene/polycarbonat. Lignocel Bedding for Laboratory animals was available to animals during the study.
- Diet: Autoclavable complete diet for rats and mice – breeding and maintenance. Ad libitum
- Water: Tap water. Ad libitum
- Acclimatisation period: At least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.4 – 24.6
- Humidity (%): 32 – 70
- Air changes (/hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 21 Jan 2014 To: 18 Feb 2014

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % Carboxymethyl cellulose in water

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 200 mL/kg bw
- Amount of vehicle (if gavage): 10 mL/kg bw

Doses:

2000 mg/kg bw

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 observed individually after dosing at 30 minutes, then 1, 2, 3, 4 and 6 hours post treatment and once each day for 14 days thereafter. Body weight was measured at the start and on day 7 and 14.
- Necropsy of survivors performed: yes. All animals were euthanised at the end of the observation period by exsanguination under pentobarbital anaesthesia. All animals were subjected to gross macroscopic evaluation. The cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed. All gross pathological changes were recorded for each animal on the post mortem record sheets and the animals were discarded.

Statistics:

- From the body weights, the group means and their standard deviations were calculated.
- The LD50 was calculated using the AOT425StatPgm programme (version 1.0, 2001).

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Mortality:

There were no deaths during the study.

Clinical signs:

other: Decreased activity in one animal at 1 hour after the treatment. The animal was symptom free from 2 hours after the treatment.

Gross pathology:

There was no evidence of macroscopic observations in animals terminated on day 14.

Interpretation of results:

GHS criteria not met

Conclusions:

The acute oral median LD50 of the test material was estimated to be > 2000 mg/kg bw in female rats.

Executive summary:

Five female RccHan:(WIST) rats were given a single oral (gavage) dose of 2000 mg/kg bw of the test material formulated in 0.5% carboxymethyl cellulose in a study according to OECD TG 425 and GLP principles. Prior to dosing, the animals were fasted overnight. The animals were observed individually after dosing at 30 minutes, then 1, 2, 3, 4 and 6 hours post treatment and once each day for 14 days thereafter for any signs of systemic toxicity and their body weights were recorded. All animals were examined macroscopically at the end of the study.

No deaths occurred during the study. Decreased activity in one animal at 1 hour after the treatment was recorded. The animal was symptom free from 2 hours after the treatment. There were no treatment related body weight changes. There was no evidence of the macroscopic observations in animals terminated at day 14.

Upon an acute oral administration of the test material and a 14 day post treatment observation period, the median LD50 in female rats was found to be > 2000 mg/kg bw.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

discriminating dose

Value:

> 2 000 mg/kg bw

Quality of whole database:

GLP compliant OECD TG 425 study.

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:

13 Mar 2014 to 27 Mar 2014

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:

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 (incl. QA statement)

Test type:

traditional method

Limit test:

yes

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

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.

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

discriminating conc.

Value:

> 2.6 mg/L air

Physical form:

inhalation: aerosol

Quality of whole database:

GLP compliant OECD TG 403 study.

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:

21 Jan 2014 to 06 Feb 2014

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:

1987

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1200 (Acute Dermal Toxicity)

Version / remarks:

1998

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Wistar

Remarks:

RccHan:(WIST) (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Young adult rats
- Weight at study initiation: Between 208 g and 253 g
- Housing: Individual caging
- Diet: Autoclavable complete diet for rats and mice – breeding and maintenance. Ad libitum
- Water: Tap water. Ad libitum
- Acclimatisation period: 6 and 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19.9 – 22.8 °C
- Humidity : 27 – 55 %
- Air changes: 15-20 air exchanges per hour
- Photoperiod: 12/12 hours light/dark

IN-LIFE DATES: From: 21 Jan 2014 To: 06 Feb 2014

Type of coverage:

semiocclusive

Vehicle:

water

Details on dermal exposure:

TEST SITE
- Area of exposure: Back
- Pre-treatment: Approximately 24 hours before treatment an area on the back of the rat was shaven with an electric clipper.
- % coverage: Approximately 10 % area of the total body surface
- Type of wrap if used: Sterile gauze pads, a patch with adhesive hypoallergenic plaster and semi occlusive plastic wrap

REMOVAL OF TEST SUBSTANCE
- Washing: After 24 hours, the dressing was removed and the treated area was washed using body temperature water.
- Time after start of exposure: 24 hours

TEST MATERIAL
- Concentration: 2000 mg/kg bw
- Constant volume or concentration used: yes
- For solids, paste formed: yes

Duration of exposure:

24 hours

Doses:

2000 mg/kg bw

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: Clinical observations along with a check of viability and mortality were performed on all animals at 1 and 5 hours after dosing and daily for 14 days thereafter. Observations included the skin and fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous system, and somatomotor activity and behaviour pattern. Particular attention was directed to the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. Body weight was measured prior to dosing on Day 0 and on Days 7 and 14.
- Necropsy of survivors performed: yes. All animals were subjected to gross macroscopic examination. All animals were anaesthetised and exsanguinated. After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed. Any gross macroscopic findings were recorded.

Statistics:

- From the body weights, the group means and their standard deviations were calculated.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Mortality:

There were no deaths during the study.

Clinical signs:

other: There were no adverse clinical signs noted in any animals throughout the study.

Gross pathology:

There was no evidence of macroscopic observations in animals terminated on day 14.

Other findings:

No treatment related skin irritation was observed in any animal throughout the study.

Interpretation of results:

GHS criteria not met

Conclusions:

The median LD50 of the test material after a single dermal administration was > 2000 mg/kg bw in male and female RccHan:(WIST) rats.

Executive summary:

Ten (5 male and 5 female) RccHan:(WIST) rats were treated with a single semi-occlusive dermal application of test material at the limit dose of 2000 mg/kg bw in a study according to OECD TG 402 and GLP principles. Sufficient water was used to dampen the test material to ensure good contact with the skin. The application period was 24 hours, followed by a 14-day observation period. Clinical observations along with a check of viability and mortality were performed on all animals at 1 and 5 hours after dosing and daily for 14 days thereafter. Body weight was measured prior to dosing on Day 0 and on Days 7 and 14. All animals were examined macroscopically at the end of the study.

No mortality occurred during the study. No adverse clinical signs were observed after treatment with the test material or during the 14 day observation period and no effects were observed at the site of application. There were no treatment related effects on body weight or body weight gain during the observation period. There was no evidence of necropsy.

The median LD50 of test material after a single dermal administration was > 2000 mg/kg bw in male and female RccHan:(WIST) rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

discriminating dose

Value:

> 2 000 mg/kg bw

Quality of whole database:

GLP compliant OECD TG 402 study.

Additional information

All available data was assessed and the studies representing the worst-case effects were included as key studies. Other studies are included as supporting information. Dosing with the test material in rats results in low acute toxicity by the oral, dermal and inhalation routes of exposure. The key studies are considered to be worst-case and were selected for the CSA.

 

Acute oral toxicity

Four acute oral toxicity studies in rats are available. The key study was performed according to OECD TG 425 under GLP (Matting 2014). Five female RccHan:(WIST) rats were given a single oral (gavage) dose of 2000 mg/kg bw of the test material. Prior to dosing, the animals were fasted overnight. The animals were observed individually after dosing at 30 minutes, then 1, 2, 3, 4 and 6 hours post treatment and once each day for 14 days thereafter for any signs of systemic toxicity and their body weights were recorded. All animals were examined macroscopically at the end of the study.

No deaths occurred during the study. Decreased activity in one animal at 1 hour after the treatment was recorded. The animal was symptom free from 2 hours after the treatment. There were no treatment related body weight changes. There was no evidence of the macroscopic observations in animals terminated at day 14.

Upon an acute oral administration of the test material and a 14 day post treatment observation period, the median LD50 in female rats was found to be > 2000 mg/kg bw.

 

The other acute oral studies, all performed in rats and according to OECD TG 425 under GLP, are supporting studies performed with different syn:anti isomer ratios (Arcelin 2007, Simon 2008, Ott 2006). These studies support the conclusion that the test substance has a low acute oral toxic potential. Moreover, they provide evidence that the anti-isomer is more acutely toxic compared to the syn-isomer (Simon 2008 and Ott 2006, respectively). Overall, the available data on the substance identified as isopyrazam do not meet the criteria for classification.

 

Acute inhalation toxicity

Two acute inhalation toxicity studies in rats are available. The key study was performed according to OECD TG 403 under GLP (Nagy 2014). Ten (5 male and 5 female) CRL:(WI) Wistar rats, were exposed to 2.60 mg/L test material. 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. There was no evidence of necropsy on day 14.

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

 

The other acute inhalation study was also performed according to OECD TG 403 under GLP (Rattray 2006). A higher maximum exposure level was reached in this study, but no mortality occurred during the observation period of 14 days. Therefore, the LC50 was determined to be > 5.28 mg/L.

 

Acute dermal toxicity

Two acute dermal toxicity studies in rats are available. The key study was performed according to OECD TG 402 under GLP (Matting 2014). Ten (5 male and 5 female) RccHan:(WIST) rats were treated with a single semi-occlusive dermal application of test material at the limit dose of 2000 mg/kg bw. Sufficient water was used to dampen the test material to ensure good contact with the skin. The application period was 24 hours, followed by a 14-day observation period. Clinical observations along with a check of viability and mortality were performed on all animals at 1 and 5 hours after dosing and daily for 14 days thereafter. Body weight was measured prior to dosing on day 0 and on days 7 and 14. All animals were examined macroscopically at the end of the study.

No mortality occurred during the study. No adverse clinical signs were observed after treatment with the test material or during the 14 day observation period and no effects were observed at the site of application. There were no treatment related effects on body weight or body weight gain during the observation period. There was no evidence of necropsy.

The median LD50 of test material after a single dermal administration was > 2000 mg/kg bw in male and female RccHan:(WIST) rats.

 

The other acute dermal toxicity study was also performed according to OECD TG 402 under GLP (Arcelin 2007). In this study a dose of 5000 mg/kg was applied under semi-occlusive conditions. No mortality occurred during this study. Therefore, the LD50 was determined to be > 5000 mg/kg bw. 

Justification for classification or non-classification

Based on the result of the acute toxicity studies classification is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.