diethyl 1-(2,4-dichlorophenyl)-5-methyl-4,5-dihydro-1H-pyrazole-3,5-dicarboxylate

Endpoint summary

Description of key information

Acute oral toxicity (OECD 401), rat: LD50 > 5000 mg/kg bw

Acute oral toxicity (OECD 401), mouse: LD50 > 5000 mg/kg bw

Acute dermal toxicity (OECD 402), rat: LD50 > 4000 mg/kg bw

Acute inhalation toxicity (OECD 403), rat (4 h exposure): LC50 > 1.32 mg/L air

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:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 81-1 (Acute Oral Toxicity)

Deviations:

no

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: HOECHST AG, Kastengrund, SPF breeding colony
- Age at study initiation: approx. 4 weeks
- Weight at study initiation: mean: males 23 g, females 20 g
- Fasting period before study: yes
- Housing: in fully air-conditioned rooms in Makrolon cages (Type 3) on soft wood granulate in groups of 5 animals
- Diet: Altromin 1324 rat diet (Altromin GmbH, Lage/Lippe), ad libitum
- Water: tap water in plastic bottles, ad libitum
- Acclimation period: approx. 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 50±20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 2% starch mucilage

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 25% w/v
- Amount of vehicle (if gavage): 20 mL/kg bw


MAXIMUM DOSE VOLUME APPLIED: 20 mL/kg bw

Doses:

5000 mg/kg bw

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

- Duration of observation period following administration: 15 days
- Frequency of observations and weighing: twice daily up to day 5, once daily thereafter (no observations on day 6), weighing weekly
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 5 000 mg/kg bw

Based on:

test mat.

Mortality:

During the 15-day observation period, no mortality occurred among either males or females after treatment with 5000 mg/kg bw.

Clinical signs:

other: The clinical signs of intoxication indicated no sex-specific differences. Clinical signs such as decreased spontaneous activity and squatting position began to emerge immediately after administration, followed by irregular respiration, contracted flanks a

Gross pathology:

The animals killed at the end of the observation period were free of macroscopically visible changes.

Interpretation of results:

other: CLP/EU 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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 81-1 (Acute Oral Toxicity)

Deviations:

no

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: HOECHST AG, Kastengrund, SPF breeding colony
- Age at study initiation: males approx. 6 weeks, females approx. 7 weeks
- Weight at study initiation: mean males 156 g, mean females 159 g
- Fasting period before study: from about 16 hours before to 3 - 4 hours after treatment
- Housing: in fully air-conditioned rooms in Makrolon cages (Type 4) on soft wood granulate in groups of 5 animals
- Diet (e.g. ad libitum): Altromin 1324 rat diet (Altromin GmbH, Lage/Lippe), ad libitum
- Water (e.g. ad libitum): tap water in plastic bottles, ad libitum
- Acclimation period: approx. 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 50±20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 2% starch mucilage

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 50% w/v
- Amount of vehicle (if gavage): 10 mL/kg bw


MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg bw


DOSAGE PREPARATION (if unusual):
Hoe 107892 00 ZC99 0001 was dissolved in the stated concentrations in 2% starch mucilage (potato starch in deionised water) with a mortar and pestle and a Turrax and distributed homogeneously by means of a magnetic stirrer.

Doses:

5000 mg/kg bw

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

- Duration of observation period following administration: 15 days
- Frequency of observations and weighing: Observations over a period of 6 hours on application day, otherwise twice daily up to day 6 and once daily thereafter, weighing of females at day 9 and of males at day 10
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 5 000 mg/kg bw

Based on:

test mat.

Mortality:

During the 15-day observation period, no mortality occurred among males or females after treatment with 5000 mg/kg bw.

Clinical signs:

other: The clinical signs of intoxication indicated sex-specific differences. The female animals were free of clinical signs during the whole study. Clinical signs such as decreased spontaneous activity, flanks drawn in, squatting posture and increased respirato

Gross pathology:

The animals killed at the end of the observation period were free of macroscopically visible changes.

Interpretation of results:

other: CLP/EU 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

Quality of whole database:

The available information comprise adequate and reliable (Klimisch score 1) studies performed with the registered substance, and is thus sufficient to fulfil the standard information requirements set out in Annex VII-X, 8.6, 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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 81-3 (Acute inhalation toxicity)

Deviations:

no

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: HOECHST AG, Kastengrund, SPF breeding colony
- Age at study initiation: 8-10 weeks
- Weight at study initiation: mean males 192 g, females 188 g
- Housing: in fully air-conditioned rooms in Makrolon cages (Type 4) on soft wood granulate, in groups of 5 animals
- Diet: Altromin 1324 rat diet (Altromin GmbH, Lage/Lippe), ad libitum
- Water: tap water in plastic bottles, ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 50 ± 20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: polyethylene glycol/ethanol

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Stainless-steel and glass cylinder, standing in a vent pipe with a volume of approx. 4 m³. The chambers have proved to yield a uniform distribution of aerosols at the different breathing zones of the animals.
- Exposure chamber volume: 60 L
- Method of holding animals in test chamber: individually in cylindrical plastic cages
- Source and rate of air: air was pumped at a pressure of 4 bar into a special nozzle with a welded-in supply tube for the test substance. The air supply at the nozzle was maintained at 800 L/h by means of an air-calibrated rotameter (Rota, Wehr). A suction device at the bottom of the exposure chamber drew off the aerosol at a rate of 1100 L/h through a cotton-wool filter, a Buehler filter, a washing flask filled with 10% sodium hydroxide solution and a calcium chloride flask. The difference in rate between the introduction of air at 800 L/h through the nozzle and its extraction at 1100 L/h is necessary to maintain the kinetic energy of the aerosol particles and to ensure a laminar flow of the aerosol from the top to the bottom of the exposure chamber.
- Method of conditioning air: passing through an oil separation filter and an absolute filter. In order to ensure the higher atmospheric humidity required by the current guidelines, air was passed via a porous stone at a rate of 100 L/h through a 1000 ml vacuum filter flask filled with water. The moisturised air was then conducted straight into the exposure chamber.
- System of generating particulates/aerosols: Hoe 107892 00 ZC97 0001 was injected into a nozzle at a constant speed by means of a continuous infusion apparatus. The primary aerosol was formed in a separator. Smaller aerosol particles (secondary aerosol) passed through a rising tube into the exposure chamber.
- Method of particle size determination:
- Treatment of exhaust air: Particles of test substance escaping from the exposure chamber into the vent pipe were drawn off and neutralised by gas-cleaning equipment.
- Temperature, humidity, pressure in air chamber: 20 - 22°C, 58.6 - 63.7%, no data on pressure


TEST ATMOSPHERE
- Brief description of analytical method used: Gravimetric analysis of the aerosol-concentration in the breathing zone of the animals was performed at intervalls of about 15 minutes. For this purpose, the test atmosphere was drawn off through a rotary gas counter, then through a glass fiber filter and a membrane filter with a pore width of 0.65 µm. The extraction rate was 3 L/minute, resulting in a flow rate of 1.25 m/sec. Before use, the filters were stored in an exsiccator filled with calcium chloride for 24 hours. The filters were weighed before and after sampling by means of an electronic balance. In order to determine the exact quantity of Hoe 107892 00 ZC97 0001 in the exposure chamber, chemical-analytical examinations were carried out. For this purpose, 31 L were drawn from the inhalation chamber over a period of 60 minutes and passed successively through three gas-washing flasks filled with Acetonitril and kept standing in a cold bath. Sampling was carried out within the breathing zone of the animals. Additionally two of the filters used for gravimetric measurements were floated with 30 ml Acetonitril and also analysed. Hoe 107892 00 ZC97 0001 was isolated by HPLC and determined spectrophotometrically.
Determination of the aerosol particle size distribution was performed with an Anderson 7 stage cascade impactor (Model Mark III, Anderson Samples Inc., Atlanta, U.S.A.). The test atmosphere was impacted at each stage onto steel discs which were weighed before and after sampling. Sampling was performed at a volume rate of 9.5 L/minute, resulting in a flow velocity of 1.25 m/s. Mass median aerodynamic diameter, standard deviation and other parameters of the particle size distribution were calculated using linear regression (probit values versus the logarithm of the particle size).
- Samples taken from breathing zone: yes


VEHICLE
- Composition of vehicle (if applicable): Hoe 107892 00 ZC97 0001 was tested as solution in polyethylene glycol 400/ Ethanol p.a.
- Concentration of test material in vehicle (if applicable): 15%
- Justification of choice of vehicle: substance could not be dispersed in its original form, stability and homogeneity of the solution for a period of 6 hours was verified before start of the study.


TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: < 0.6 to > 10.3 µm
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): 1.18 µm/3.36 µm (1st measurement), 1.02 µm/2.65 µm (2nd measurement)

Analytical verification of test atmosphere concentrations:

yes

Remarks:

gravimetric and chemical-analytical

Duration of exposure:

4 h

Concentrations:

1.32 mg/L air

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: behaviour continuously during exposure, thereafter twice daily; weighing on day 8 and day 15
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight

Statistics:

Means and standard deviations of body weights and body weight gains, linear regression and probit analysis for calculation of particle size distribution parameters.

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 1.32 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: Highest technically administrable dose.

Mortality:

No deaths occurred.

Clinical signs:

other: Irregular breathing, stilted gait, uncoordinated gait, ataxic gait and squatting posture. Additionally, ruffled coat was observed. The animals treated with the vehicle alone also showed irregular breathing, uncoordinated gait, ataxic gait and squatting p

Body weight:

Body weight development was not impaired.

Gross pathology:

The animals killed at the end of the observation period showed no macroscopically visible abnormalities.

Interpretation of results:

other: CLP/EU 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:

LC50

Value:

1.32 mg/m³ air

Quality of whole database:

The available information comprises an adequate and reliable study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-X, 8.6, 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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 81-2 (Acute Dermal Toxicity)

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: HOECHST AG, Kastengrund, SPF breeding colony
- Age at study initiation: males approx. 8 weeks, females approx. 9 weeks
- Weight at study initiation: males 221 g mean, females 193 g mean
- Fasting period before study:
- Housing: in fully air-conditioned rooms in Makrolon cages (Type 3) on soft wood granulate, one animal per cage
- Diet (e.g. ad libitum): Altromin 1324 rat diet (Altromin GmbH, Lage/Lippe), ad libitum
- Water (e.g. ad libitum): tap water in plastic bottles, ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 50±20
- Photoperiod (hrs dark / hrs light): 12/12

Type of coverage:

occlusive

Vehicle:

other: sesame oil

Details on dermal exposure:

TEST SITE
- Area of exposure: 30 cm².
- Type of wrap if used: The treated skin area was covered with an aluminium foil (6x8 cm), which was held in place with an elastic plaster bandage fixed around the animal's body (Fixomull, and ElastoplastR , 8 cm in width, both manufactured by Beiersdorf).

REMOVAL OF TEST SUBSTANCE
- Washing (if done): warm water
- Time after start of exposure: 24 h


TEST MATERIAL
- Constant volume or concentration used: yes
- For solids, paste formed: yes


VEHICLE
- Amount(s) applied (volume or weight with unit): preparation of 1 g test substance per 0.67 ml vehicle

Duration of exposure:

24 hours

Doses:

4000 mg/kg bw

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

- Duration of observation period following administration: 15 days
- Frequency of observations and weighing: observations twice daily until d6, thereafter once daily, weighing weekly
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 4 000 mg/kg bw

Based on:

test mat.

Mortality:

During the 15-day observation period, no mortality occurred among either males or females following treatment with 4000 mg/kg bw.

Clinical signs:

other: The clinical signs of intoxication indicated no sex-specific differences. The clinical signs began to emerge 1 day after administration and persisted in some cases up to day 6 of the study. Clinical signs such as irregular breathing and squatting position

Gross pathology:

The animals killed at the end of the observation period were free of macroscopically visible changes.

Other findings:

- Other observations: Signs of skin irritation such as dry and rough skin surface covered with fine scales were observed in most of the animals. They emerged on day 4 and persisted in some cases up to day 8 of the study.

Interpretation of results:

other: CLP/EU 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:

4 000 mg/kg bw

Quality of whole database:

The available information comprise an adequate and reliable (Klimisch score 1) study performed with the registered substance, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-X, 8.6, of Regulation (EC) No. 1907/2006.

Additional information

Acute oral toxicity

There are 2 acute oral toxicity GLP guideline studies avilable, one in rat and mouse each. Both were designed as limit tests with administration of a single dose of 5000 mg/kg bw. No mortality occurred during the course of the studies. Clinical signs comprised decreased spontaneous activity, retracted flanks, squatting posture, increased or irregular respiration, stilted gait and swollen abdomen, but not all animals were concerned, and in rats the symptoms were limited to the males. Clinical signs emerged immediately or shortly after administration and persisted in some cases up to day 4 (rats) or 5 (mice) of the observation period. There was no effect on body weight gains, and the animals did not show any macroscopically visible changes at necropsy.

 

Acute dermal toxicity

Acute dermal toxicity was tested in rats in a limit test, in which a single dose of 4000 mg/kg bw was applied to the skin under occlusive dressing for 24 hours. No mortality occurred during the course of the study. Clinical signs comprised irregular breathing and squatting position in almost all animals and to a smaller extent reduced spontaneous activity and increased fright reaction. There was no effect on body weight gains, and the animals were free of macroscopically visible changes at necropsy.

 

Acute inhalation toxicity

Acute toxicity by inhalation was addressed in a single study in rats in which an aerosol of the test substance was administered for 4 h at the highest technically administrable concentration of 1.32 mg/L. No mortality occurred during the course of the study. Clinical signs were comparable to oral administration, comprising irregular breathing, stilted, uncoordinated, ataxic gait and squatting posture, which were reversible by day 7. Remarkably, the animals of the control group demonstrated similar symptoms which abated by day 3, therefore, the symptoms seemed to be related to exposure itself. Body weight development was not impaired, and the animals demonstrated no macroscopically visible abnormalities at necropsy.

 

Taken together, the test substance-related findings were comparable regardless of the route of application, and no mortality occurred either after a single administration of doses up to 4000 and 5000 mg/kg bw, respectively, nor inhalation of 1.32 mg/L (the highest technically administrable dose) for 4 hours.

Justification for classification or non-classification

The available data on acute oral, dermal and inhalation toxicity do not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.