2-cyano-2-[2,3-dihydro-3-(tetrahydro-2,4,6-trioxo-5(2H)-pyrimidinylidene)-1H-isoindol-1-ylidene]-N-methylacetamide

Endpoint summary

Description of key information

Key: oral, rat: LD50 > 5000 mg/kg bw; signs of toxicity: no clinical signs observed (non-GLP, OECD 401, 1982)

Key: inhalative, rat: LC50 >5.42 mg dust/L air; signs of toxicity: no mortality and no toxicity observed (non-GLP, similar to OECD 403, 1982)

Sup: intraperitoneal, rat: LD50 > 2000 mg/kg bw; signs of toxicity: no mortality; dyspnea, apathy, staggering, tonus with stretching, yellow/orange colored urine, piloerection in females, poor general state in males and femals (non-GLP, non-guideline, 1982)

 

Read-across from CAS 36888-99-0: Key: dermal, rat: LD50 > 2500 mg/kg bw; transient signs of toxicity: no abnormality detected (non-GLP, similar to OECD 402, 1973)

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Version / remarks:

adopted 12th May 1981

Deviations:

no

GLP compliance:

no

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: Dr. K. Thomae GMBH, Biberach, Germany
- Age at study initiation: 12 weeks
- Mean weight at study initiation: 170 g (males), 180 g (females)
- Fasting period before study: 16 hours before administration, but water is available ad libitum.
- Housing: 5 per cage, Stainless steel wire meeh cages, type DK-III
- Diet (e.g. ad libitum): Ssniff R; FA. SSNIFF, Soest, Germany
- Water: Demineralized water each workday, tap water on holidays; ad libitum
- Acclimation period: at least 1 week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26
- Humidity (%): 45-75
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 25 % in 0.5% aqueous carboxymethyl cellulose

MAXIMUM DOSE VOLUME APPLIED: 20 mL/kg

Doses:

5000 mg/kg

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: weighing: day of application, then days 3, 7 and 13; cageside observations: < 15', 15', 30', 1 h, 2 h, 4 h and 5 h after administration of the test substance and then once each workday. Check for moribund and dead animale twice each workday and once on holidays.
- Necropsy of survivors performed: yes, withdrawal of food 16 houre before sacrifice; necropsy with grose-pathological examination.
- Other examinations performed: body weight

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 5 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: No mortality, no toxicity observed.

Mortality:

No mortality observed (0/10 animals).

Clinical signs:

other: No clinical signs observed.

Gross pathology:

No abnormalities detected.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study the median lethal dose of the test substance after oral application was found to be greater than 5000 mg/kg body weight for male and female rats.

Executive summary:

A study was performed to assess the acute toxicity following oral administration of the test substance in rats according to OECD guideline 401. To a group of five animals per sex and per dose a single oral dose of the test material preparation (dose volume: 20 ml/kg bw) in an aqueous solution of carboxymethyl cellulose at a dose of 5000 mg/kg bw was given.

The animals were examined for clinical signs and dead animals twice each workday and once on holidays. The observation period was 14 days following administration. Surviving animals were necropsied at the end of the study.

No clinical signs were observed during the study. All animals survived until the end of the study period. At necropsy no abnormalities were observed.

Under the conditions of this study the median lethal dose of the test substance after oral application was found to be greater than 5000 mg/kg body weight for male and female rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

discriminating dose

Value:

5 000 mg/kg bw

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:

03.11.1981 - 17.11.1981

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

comparable to Guideline 403, non GLP, but comparable reporting details

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

GLP compliance:

no

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: WIGA Versuchstierzuchtanstalt, Sulzfeld, Germany, outbred strain: CAW-ICO-Wiga
- Age at study initiation: about 8 - 10 weeks
- Weight at study initiation: male animais 318 +/- 27 g, female animals 228 +/- 19 g
- Housing: five per cage, cages of Becker, type D III, without bedding.
- Diet: SSNIFF R complete diet for rats and mice, manufacturer: SSNIFF-Versuchstierdiaeten GmbH, Soest, Germany, ad libitum
- Water: tap water ad libitum during the observation period

ENVIRONMENTAL CONDITIONS
- Temperature (°C): temperature 22 +/- 2
- Humidity (%): 55 +/- 5
- Photoperiod (hrs dark / hrs light): 12 h/12 h

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose/head only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

1.7 µm

Geometric standard deviation (GSD):

4

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Read-nose inhalation system INA 20 (glass-steel construction, BASF AG)
- Exposure chamber volume: 55 L
- Method of holding animals in test chamber: The animals are restrained in tubes and their snouts project into the inhalation chamber.
- Source and rate of air: 1500 L/h compressed air by the injector and 1500 L/h ventilator air as dilution air.
- System of generating particulates/aerosols: A mixture of dust and air was generated by means of dust metering equipment (BASF).
By means of a dust generator the substance to be tested was generated into a dust aerosol, which was passed into the inhalation system.
An automatic vibrator was used for dust generation. The concentration was adjusted by varying the apertural width and varying the amplitude of oscillations of the metering beaker.
- Method of particle size determination: Andersen Stack Sampler Mark III Millipore vacuum compressed air pump XX 60 220 50, limiting orifice 3 L/min, Millipore sampling probe, internal diameter 6.9 mm, 30 minutes after the beginning of the test at the earliest, one sample was taken for the particle size analysis. Before the sampling, the impactor was equipped with glass-fiber collecting discs and a backup particle filter.
The impactor was connected to the pump and the test apparatus, and one sample (9 L) was removed.
The impactor was taken apart, and the collecting discs and the backup particle filter were weighed. The contents of the pre-impactor were determined gravimetrically.
- Pressure in air chamber: 1500 L/h compressed air by the injector and 1500 L/h ventilator air as dilution air
- To ensure, that the mixture of the test substance and air was not diluted with fresh air, the air suction system was reduced about 10 % compared to the supply air system (excess pressure).

TEST ATMOSPHERE
- Brief description of analytical method used:
• Vacuum compressed air pump (Millipore) XX 60 220 50,
• Filtration equipment with probe (Millipore) (internal diameter: 4 mm),
• Filter: MN 85/90 Bf (d = 4.7 cm),
• Balance: Cahn 26
Gravimetric determination of the concentration: the preweighed filter was placed into a filtration equipment. By means of a vaccuum compressed air pump a volume of the dust aerosol characterized by a limiting orifice was drawn through the filter.
The dust concentration in mg/L was calculated from the difference between the preweight of the filter and the weight of the filter after sampling with reference to the sample volume.
- Samples taken from breathing zone: yes,
• Sampling: Velocity: 1.25 m/s, amount: 1 L, probe diameter: 4 mmm, frequency: half-hourly, in the immediate vicinity of the animal noses


- Particle size distribution: EACD 50% (effective aerodynamic cutoff diameter 50%);
Stage: Pre-impactor / EACD 50 % (µm): 26.6
Stage: Cascade impactor: Backup filter / EACD 50 % (µm): <1.2 / Percentage distribution (%): 41.14
Stage: Cascade impactor: 7 / EACD 50 % (µm): 1.2 / Percentage distribution (%): 18.69 / Cumulative distribution (%): 41.14
Stage: Cascade impactor: 5 / EACD 50 % (µm): 2.8 / Percentage distribution (%): 22.28 / Cumulative distribution (%): 59.83
Stage: Cascade impactor: 4 / EACD 50 % (µm): 5.5 / Percentage distribution (%): 8.40 / Cumulative distribution (%): 82.11
Stage: Cascade impactor: 3 / EACD 50 % (µm): 8.5 / Percentage distribution (%): 5.22 / Cumulative distribution (%): 90.51
Stage: Cascade impactor: 1 / EACD 50 % (µm): 18.2 / Percentage distribution (%): 2.14 / Cumulative distribution (%): 95.73
Stage: Cascade impactor: 0 / EACD 50 % (µm): 29.5 / Percentage distribution (%): 2.13 / Cumulative distribution (%): 97.87

A respirable dust fraction of 90.5% was obtained from the results of the particle size analysis.

- MMAD (Mass median aerodynamic diameter) 50 % = 1.7 µm/ GSD (Geometric st. dev.): 4.0

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

5.42 mg/l (analytical), 19.7 mg/l (nominal)

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: daily observation, weighing; before start of application, then on day 7 and day 14,
- Necropsy of survivors performed: yes, with grosspathological examination.
- Other examinations performed: body weight

Statistics:

The statistical evaluation of the concentrations-response relationship was carried out in accordance with the binomial test (Wittig, H.: Mathematische Statistik 1974, pp.32 - 35) using tables of the BASF Computer Center.
The particle size was determined in accordance with mathematical and graphical methods of evaluating particle measurements (Silverman, L.: Particle Size Analysis in Industrial Hygiene, 1971, pp. 235-259).

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5.42 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: No mortality and no toxicity observed.

Mortality:

no mortality

Clinical signs:

other: During exposure: no abnormalities; After exposure: fur discolored by the test substance, particularly the region of the head; otherwise no abnormalities.

Body weight:

The body weight of the animals showed no adverse effects in comparison with that of the control.
Mean body weight (male / female)
- Before the study
Treatment group: 318 / 228
Control: 307 / 230
- After 7 days
Treatment group: 345 / 242
Control: 340 / 240
- After 14 days
Treatment group: 368 / 251
Control: 367 / 249

Gross pathology:

No abnormalities detected.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study the median lethal concentration of the test substance after inhalative exposure was found to be greater than 5.42 mg dust/L air for male and female animals.

Executive summary:

In an Industrial Hygiene Orientating Investigation study evaluating acute toxicity following inhalative administration, Sprague-Dawley rats were exposed to the test substance similar to OECD guideline 403. Five male and female animals were exposed to 5.42 mg/l (analytical) test substance in a read-nose inhalation system for 4 hours. The MMAD 50% (mass median aerodynamic diameter 50%) is 1.7 µm, calculated from the results of the particle size analysis and the respirable dust fraction was calculated to be 90.5%. During the observation period of 14 days, the animals were examined for clinical signs daily and at the end of the study necropsied with gross pathological examination. All animals survived until the end of the study period. No clinical signs were observed during the study period. Only the fur was discolored by the test substance, particularly the region of the head.

Under the conditions of this study the median lethal concentration of the test substance after inhalative exposure was found to be greater than 5.42 mg dust/L air for male and female animals.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

discriminating conc.

Value:

5 420 mg/m³ air

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

acceptable restrictions: occlusive treatment, 24 h exposure, incomplete documentation

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

yes

Remarks:

(occlusive treatment, 24 h treatment)

GLP compliance:

no

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Wiga, Ottobrunn, Germany
- Weight at study initiation: mean weight: males 151 g, females 129 g

ENVIRONMENTAL CONDITIONS: not reported

Type of coverage:

occlusive

Vehicle:

water

Details on dermal exposure:

TEST SITE
- Area of exposure: dorsal, p.c., 50 cm²
- % coverage: > 10 %

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2.5 g/kg animal
- Concentration (if solution): 50 % aequous solution
- For solids, paste formed: yes

Duration of exposure:

24 h

Doses:

2.5 g/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: No weighing was done. Observation was several times at the day of exposure and daily on working days.
- Necropsy of survivors performed: yes

Statistics:

None

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 500 mg/kg bw

Based on:

test mat.

Remarks on result:

other: No mortality and no clinical signs were observed.

Mortality:

0/10 animals died after the exposure

Clinical signs:

other: no abnormalities detected

Gross pathology:

9/10 no abnormalities detected
1/10 right peak pulmonary lobe with chronic Bronchopneumonia

Other findings:

- Other observations: after 24 h local orange substance residues

Interpretation of results:

GHS criteria not met

Conclusions:

After the end of the observation period (14 days), no mortality was observed. Therefore, the LD50 was determined to be the limit dose of 2500 mg/kg bw in male and female rats.

Executive summary:

This acute dermal toxicity test was conducted similar to OECD test guideline 402 and was performed according to the recommendations by Noakes and Sanderson (1969). 2.5 g/kg of the test substance was applied in a 50 % aequous solution to the back on a 50 cm² test site. No weighing of the animals was done. Observations occurred several times at the day of exposure and daily on working days.

14 days after application, no mortality occured. The LD50 was set to be 2500 mg/kg bw. For 9 out of 10 animals, no abnormalities in gross pathology were detected. One animal had a right peak pulmonary lobe with chronic Bronchopneumonia.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Additional information

There are reliable studies available to assess the acute oral and inhalative toxicity of the test substance. 

 

Key study: Acute oral toxicity

An oral acute toxicity study was performed with the test substance in Wistar rats according to OECD guideline 401 (BASF, 1982). A group of five animals per sex were administered a single oral dose of 5000 mg/kg bw test material preparation by gavage (dose volume: 20 ml/kg bw of an aqueous solution of the test substance in 0.5% carboxymethyl cellulose). The animals were examined for clinical signs several times on the day of administration and once on workdays. The observation period was 14 days. No clinical signs were observed during the study. When the tested animals were necropsied at the end of the study no abnormalities were observed.

Under the conditions of this study the median lethal dose of the test substance after oral application was found to be greater than 5000 mg/kg body weight for male and female rats.

 

Key study: Acute inhalative toxicity

In an Industrial Hygiene Orientating Investigation study evaluating acute toxicity following inhalative administration, Sprague-Dawley rats were exposed to the test substance similar to OECD guideline 403 with deviations (BASF, 1982). Five male and female animals were exposed to 5.42 mg/l (analytical) test substance in a read-nose inhalation system for 4 hours. The MMAD 50% (mass median aerodynamic diameter 50%) is 1.7 µm, calculated from the results of the particle size analysis and the respirable dust fraction was calculated to be 90.5%. During the observation period of 14 days, the animals were examined for clinical signs daily and at the end of the study necropsied with gross pathological examination. All animals survived until the end of the study period. No clinical signs were observed during the study period. Only the fur was discolored by the test substance, particularly the region of the head.

Under the conditions of this study the median lethal concentration of the test substance after inhalative exposure was found to be greater than 5.42 mg dust/L air for male and female animals.

 

 

Supporting study: Acute intraperitoneal toxicity

Absence of acute oral and inhalative toxicity is supported by absence of mortality in rats observed in the study with intraperitoneal injection at the dose level of 2000 mg/kg bw (BASF, 1982). For this route of exposure, 100% systemic availability is given.

In this non-guideline study, 5 rats per sex were administered a single dose (2000 mg/kg bw in 0.5% aqueous CMC-solution) of the test substance via intraperitoneal injection (BASF, 1982). Clinical signs were monitored and necropsy was carried out at termination or after death if an animal died during the study. The observation period was until day 14 after application.

No mortality was observed. Clinical signs were dyspnea, apathy, staggering, tonus with stretching, yellow/orange colored urine, poor general state in males and femals. Additionally, females also showed piloerection. The body weight of the animals showed no adverse effects in comparison with that of the control. After sacrifice, abdominal organs, particularly Glisson's capsule, were severely stained by the test substance in both males and females. No other findings were recorded. 

In conclusion, the median lethal dose (LD50) after intraperitoneal injection in rats was determined to be > 2000 mg/kg bw. 

 

Read-across from CAS 36888-99-0: Key: Acute dermal toxicity

A study was performed to assess the acute toxicity following dermal administration of the test substance in Sprague-Dawley rats similar to OECD guideline 402 with deviations (BASF SE, 1973). Five animals per sex were treated with the test substance at 2500 mg/kg bw by dermal occlusive application. The test item was diluted in vehicle (an aqueous solution of carboxymethyl cellulose) at a concentration of 50%. The application period was 24 hours. During the observation period of 14 days, the animals were examined for clinical signs several times on the day of exposure and daily afterwards on working days. All animals survived and no clinical signs were observed during the study period. In nine of ten animals no abnormalities were detected at necropsy. In only one of ten animals right peak pulmonary lobe with chronic Bronchopneumonia was observed in gross pathology. Under the conditions of this study the median lethal dose of the test substance after dermal application was found to be greater than 2500 mg/kg body weight for male and female animals.

 

 

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No mortality occurred at the limit dose of 5000 mg/kg bw. As a result, the substance is not considered to be classified for acute oral or inhalative toxicity under Regulation (EC) No. 1272/2008, as amended for the seventeenth time in Regulation (EC) No. 2021/849.