Pyrrolo[3,4-c]pyrrole-1,4-dione, 3,6-bis([1,1'-biphenyl]-4-yl)-2,5-dihydro-

Endpoint summary

Description of key information

Oral:

• according to OECD TG 401, GLP, rat, LD50 (m/f) >2000 mg/kg bw

 

Inhalation (inconclusive):

• read-across from CAS 84632-65-5 (bulk), according to OECD TG 403, GLP, rat, 4h, LC50 (m/f) >2.25 mg/L air


• read-across from EC 465-080-5 (nano), according to OECD TG 403, GLP, rat, 4h, LC0 (m/f) = 1 mg/L air, LC 100 (m/f) = 5 mg/L air

 

Dermal:

• according to OECD TG 402, GLP, rat, semi-occlusive, LD50 (m/f) >2000 mg/kg bw

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:

1993

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

GLP compliance:

yes

Test type:

acute toxic class method

Limit test:

yes

Specific details on test material used for the study:

- Substance type: Organic
- Physical state: Solid
- Lot/batch No.: Z-2281/1,2,4,5
- Expiration date of the lot/batch: April 01, 1997
- Storage condition of test material: at room temperature in the dark

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL Ltd., Basel, Switzerland
- Age at study initiation: Approx. 9 weeks
- Weight at study initiation: males: 222 - 243 g, females: 166 - 182 g
- Fasting period before study: overnight
- Housing: 5 animals per sex per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C
- Humidity (%): 55
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1993-01-13 To: 1993-01-27

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

VEHICLE
MAXIMUM DOSE VOLUME APPLIED: 10 ml/kg body weight.

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: Twice daily (mortality, viability), once daily (weighing)
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs (daily)

Sex:

male/female

Dose descriptor:

discriminating dose

Effect level:

2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: No mortality observed.

Mortality:

none

Clinical signs:

other: No clinical signs were observed during the study period. However, red discolouration of skin, tail and faeces were noted from day 3 onward.

Gross pathology:

Macroscopic post mortem examination of the surviving animals at termination did not reveal any abnormalities.

Interpretation of results:

GHS criteria not met

Conclusions:

The oral LD50 value of the test substance in Wistar rats of either sex was established as exceeding 2000 mg/kg body weight.

Executive summary:

The acute oral toxicity of the test substance (purity 97.5%) was examined in a reliable study according to OECD Guideline 401 and GLP. The test substance dissolved in polyethylene glycol was administered by single oral gavage to five Wistar rats of each sex at 2000 mg/kg body weight. Animals were subjected to daily observations and weekly determination of body weight. Macroscopic examination was performed after terminal sacrifice (day 15). No mortality or clinical signs were observed during the study period. However, red discolouration of skin, tail and faeces were noted from day 3 onward. The mean body weight gain shown by the animals over the study period was considered to be normal. No abnormalities were found at macroscopic post-mortem examination of the animals. Therefore, the oral LD50 was established to exceed 2000 mg/kg body weight. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

discriminating dose

Value:

2 000 mg/kg bw

Quality of whole database:

according to OECD TG 401, GLP, Klimisch 1

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16.08. - 27.11.1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

1981

Deviations:

no

GLP compliance:

yes

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

other: Tif:RAI f (SPF) hybrids of RII/1 x RII/2

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CIBA-GEIGY Limited Animal Production, Stein /Switzerland
- Weight at study initiation: 177 - 227 g
- Fasting period before study: at least 5 d
- Housing: Groups of 5 in Makrolon type-4 cages
- Diet (ad libitum): Rat diet Nafag 890
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/- 10
- 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):

0.4 µm

Geometric standard deviation (GSD):

>= 3.6 - <= 5.2

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The exposure apparatus was developed by Battelle Research Center (Geneve/Switzerland). The internal active volume was less than 1L and the flow in any individual aerosol delivery chamber was standardised to 2 L/min (velocity 1.25 m/s).
- Method of holding animals in test chamber: For inhalation period rats were placed in Macrolon animal holders.
- Treatment of exhaust air: The exhaust air was decontaminated by passage through a Pall HDC absolute filter.

VEHICLE
The test compound tended to form secondary agglomerates. Therefore, it was mixed with inert silica. A 10% mixture of Sipernat 50S with the test article was used in the animal exposure tests.

TEST ATMOSPHERE
The aerosol concentration was determined gravimetrically five times during exposure period. The Particle size determination was conducted four times during exposure using an eight-stage cascade impactor. In the same intervall temperature, relative humidity and oxygen content of the inhalation chambers were assessed.

The test substance was administered as an aerosol in a nose-only exposure system that ensures uniform exposure and avoids re-breathing of the aerosol. During exposure, the animals were placed in Macrolon animal holders positioned radially around the exposure chamber, so that only the snouts and nostrils were exposed. The aerosol was generated from the solid test material blended with 10 % Sipernat 50S (Degussa, Germany) by means of a brush-feed micronizing jet mill. A cyclone-type classifier ensures that only particles of the desired diameter leave the jet mill. The control animals were exposed to an inhalation atmosphere of Sipernat 50S at a nominal concentration of 0.48 mg/l under the same conditions as described above.

Analytical verification of test atmosphere concentrations:

yes

Remarks:

gravimetrically

Duration of exposure:

4 h

Concentrations:

2.25 mg/L
(due to the properties of the test material, it was not possible to generate higher concentrations of the test compound with the equipment used in this study.)

No. of animals per sex per dose:

10 (5 males, 5 females)

Control animals:

yes

Details on study design:

- The control animals were exposed to an inhalation atmosphere of Sipernat 50S at a nominal concentration of 0.48 mg/l under the same conditions as treated animals
- Duration of observation period following administration: 14 days
- Frequency of observations of clinical symptoms and mortality: During and after exposure, therafter daily
- Frequency of weighing: Body weights were recorded prior to treatment and on day 7 and 14
- Necropsy of survivors performed: yes, all animals were sacrificed and subjected to gross pathology

Statistics:

Body weights of treated and untreated animals were compared by analysis of variance.

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 2.25 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: No mortality observed.

Mortality:

None of the animals died.

Clinical signs:

other: Piloerection, hunched posture and dyspnea were seen in animals exposed to the test material. They recovered within 5 days.

Body weight:

Males exposed to the test substance showed a significantly higher body weight gain during the first and the second observation week as compared to control animals.

Gross pathology:

No macroscopic findings were observed at necropsy.

Other findings:

HISTOPATHOLOGY
- In all examined tissue samples, the alveolar lumen contained alveolar macrophages (phagocytic cells) filled with brown pigment, most likely representing the test article. This change was minimal in males and moderate in females. The pneumocytes type II in the alveolar epithelium of all animals were activated. This activation was minimal and multifocal in 3 males and one female, moderate and multifocal in 2 males and 4 females. The bronchial lymph node of one male and one female showed moderate brown pigmentation, regarded to represent the test article. In one male the bronchiolar epithelium was minimally and focally hyperplastic.
- The minimal congestion, the minimal emphysema and the minimal and multifocal bronchiolar dilatation seen in all animals are a common response in rats treated by inhalation. Therefore, it was considered not to be treatment-related.

Table 1: Mean body weights in grams (Dose level: 2.25 mg/l)

Test day	1*	7	14
Control males	212 +/-4	246 +/- 12	278 +/-19
Treated males	202 +/-6	254 +/- 7	297 +/- 9
Control females	187 +/- 5	199 +/- 4	215 +/- 8
Treated females	186 +/- 6	199 +/- 8	215 +/- 7

*body weights on day 1 were assessed before application of 2.25 mg/l.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study, the LC50 was above 2.25 mg/l for both sexes after 4-hours nose-only exposure of rats to the test substance.

Executive summary:

In an acute inhalation toxicity study according to OECD 403 and GLP, male and female Tif:RAI f (SPF) rats were exposed to an aerosol of the test substance for 4 hours, nose-only. Due to the properties of the test material, it was not possible to generate higher concentrations than 2250 mg/m^3 air. The exposure to the maximum attainable concentration was thus considered a limit test as stated in the OECD test guideline 403. The animals were observed for a post-dosing period of 14 days. No mortality and no macroscopic findings at necropsy were observed in males and females. Clinical symptoms were piloerection, hunched posture and dyspnea. From this, the animals recovered within 5 to 9 days. Histopathological examinations of the lungs revealed minimal congestion, minimal emphysema and minimal and multifocal bronchiolar dilatation in all animals. These changes are common response in rats treated by inhalation with a nuisance dust and was therefore regarded not to be treatment-related. From the absence of mortalities a LC50 >2250 mg/m^3 for both sexes can be assumed.

Reference 2

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

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

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Specific details on test material used for the study:

- Substance type: pigment
- Physical state: solid
- Purity test date: Feb 13, 2013
- Expiration date of the lot/batch: Oct 2014
- Stability under test conditions: stable
- Storage condition of test material: at room temperature

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., The Netherlands
- Age at study initiation: 8 to 9 weeks
- Weight at study initiation: Males: 256.8 to 285.6 g, Females: 170.5 to 219.3 g
- Fasting period before study: no
- Housing: groups of 5 of the same sex
- Diet: ad libitum
- Water: e.g. ad libitum
- Acclimation period: 5 - 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C,
- Humidity (%): 30 - 70%
- Photoperiod (hrs dark / hrs light): hour fluorescent light / 12 hour dark cycle.

IN-LIFE DATES: From: Jan 31, 2013 To: March 27, 2013

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: flow-past, nose-only exposure system
- Method of holding animals in test chamber: restraint tubes
- Source and rate of air: 1.0 L/min
- System of generating particulates/aerosols: A dust aerosol was generated from the test item using a CR3020 rotating brush aerosol generator
connected to an AirVac TD110M. The aerosol generated was then discharged into the exposure chamber through a 63Ni charge neutralizer.
- Method of particle size determination: Mercer 7 stage cascade Impactor
- Temperature, humidity, pressure in air chamber: The temperature and relative humidity of the test atmosphere was measured continuously during
exposure using a calibrated device. The results were recorded manually and are reported in 30 minute intervals from the start of exposure.

TEST ATMOSPHERE
- Brief description of analytical method used: Gravimetric determination of aerosol concentration was performed twice (group 1) or six times
(groups 2 and 3) during exposure. The samples were collected on a Millipore®durapore filter, Type HVLP loaded in a 47 mm in-line stainless steel filter sampling device. The filters were weighed before and immediately after sampling using a calibrated balance. The test aerosol concentration was calculated from the amount of test item present on the filter and the sample volume.
- Samples taken from breathing zone: yes

TEST ATMOSPHERE : see table

CLASS METHOD (if applicable)
- Rationale for the selection of the starting concentration: Limit dose

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

0.5, 1 and 5 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 weighing: Weighing on test days 1 (before exposure), 2, 4, 8 and 15 (before necropsy).
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight,organ weights, histopathology, other: Observations for viability were recorded once before exposure on the day of exposure (test day 1), three times during exposure, immediately and 1 h after exposure on test day 1 and twice daily during the observation period. Each animal was examined three times during exposure, immediately and 1 h after exposure on test day 1 and once daily during the observation period. Observations were detailed and carefully recorded using explicitly defined scales as appropriate. Only grossly abnormal signs were detectable during exposure as the animals were restrained in the exposure tubes.

Statistics:

No statistical analysis was performed.

Sex:

male/female

Dose descriptor:

LC0

Effect level:

1 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Sex:

male/female

Dose descriptor:

LC100

Effect level:

5 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Mortality:

All animals exposed to 5.0 mg/l air died within two hours after exposure start. All animals exposed to 0.52 or 1.0 mg/L air survived the scheduled observation period.

Clinical signs:

other: In one male and two females exposed to 5.0 mg/L air, clinical signs such as increased activity, tachypnea and reduced body temperature were recorded before their death during exposure; the remaining seven animals exposed to 5.0 mg/L air died during exposu

Body weight:

Between test days 1 and 2, slight body weight loss was noted in all animals exposed to 0.52 mg/L or 1.0 mg/L air. This effect persisted in two females exposed to 0.5 mg/L air as well as in two females exposed to 1.0 mg/L air and in up to day 4 of treatment. From test day 4 onwards, these females showed normal body weight gain. In the remaining animals exposed to 0.52 or 1.0 mg/L air, normal body weight development was noted from day 2 of treatment onwards.

Gross pathology:

After their spontaneous death during exposure, all animals exposed to 5.0 mg/L air showed reddish discolored and incompletely collapsed lungs at necropsy. Although a relation to the treatment with test item cannot be fully excluded (the substance is an orange pigment) - this finding is considered to be most likely
secondary to the spontaneous death of the animals. In animals exposed to 0.52 or 1.0 mg/L air there were no macroscopic findings at scheduled necropsy.

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

The LD50 for acute inhalation in rats is therefore higher than 1 mg/L, but lower than 5 mg/L.

Executive summary:

In a procedure following OECD guideline 403 and GLP, three groups of five male and five female albino rats were exposed by nose-only, flow-past inhalation for four hours to the test substance at gravimetrically determined mean concentrations of 5.0 mg/L air, 0.52 mg/L air and 1.0 mg/L air. All animals were observed for clinical signs and mortality during the inhalation exposure and the subsequent 14-day observation period or until they were found dead. Body weights were recorded prior to exposure on test day 1, and during the observation period on test days 2, 4, 8 and 15 before necropsy. On test day 15 all animals were sacrificed and necropsied. Animals which died were necropsied as soon as they were found dead. All animals exposed to 5.0 mg/l air died within two hours after exposure start. All animals exposed to 0.52 or 1.0 mg/L air survived the scheduled observation period. In one male and two females exposed to 5.0 mg/L air, clinical signs such as increased activity, tachypnea and reduced body temperature were recorded before their death during exposure; the remaining seven animals exposed to 5.0 mg/L air died during exposure without recording of clinical signs. In animals exposed to 0.52 or 1.0 mg/L air, clinical signs were mostly limited to the day of treatment and consisted of slightly decreased activity, slightly ruffled fur and tachypnea immediately and one hour after exposure. Animals exposed to 1.0 mg/L air additionally showed labored breathing. In single animals exposed to 0.52 mg/L air, slightly ruffled fur persisted until test days 2 or 3; in single animals exposed to 1.0 mg/L air, slightly ruffled fur and tachypnea persisted until test day 2. All animals exposed to 0.52 or 1.0 mg/L air did not show any clinical signs from test day 4 until the end of the observation period. Between test days 1 and 2, slight body weight loss was noted in all animals exposed to 0.52 mg/L or 1.0 mg/L air. This effect persisted in two females exposed to 0.52 mg/L air as well as in two females exposed to 1.0 mg/L air and in up to day 4 of treatment. From test day 4 onwards, these females showed normal body weight gain. In the remaining animals exposed to 0.52 or 1.0 mg/L air, normal body weight development was noted from day 2 of treatment onwards. After their spontaneous death during exposure, all animals exposed to 5.0 mg/L air showed reddish discolored and incompletely collapsed lungs at necropsy. Although a relation to the treatment with test item cannot be fully excluded, this finding is considered to be most likely secondary to lung deposition of the pigment rather than a substance specific toxicity. In animals exposed to 0.52 or 1.0 mg/L air there were no macroscopic findings at scheduled necropsy. The LD50 for acute inhalation in rats is therefore higher than 1 mg/L, but lower than 5 mg/L.

Reference 3

Endpoint:

acute toxicity: inhalation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Sex:

male/female

Dose descriptor:

LC50

Remarks:

(CAS 84632-65-5)

Effect level:

> 2.25 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: No mortality oberved. / The database was considered to be inconclusive for classification due to contradictory results.

Sex:

male/female

Dose descriptor:

LC50

Remarks:

(EC 465-080-5)

Effect level:

> 1 - < 5 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: The database was considered to be inconclusive for classification due to contradictory results.

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

The database was considered to be inconclusive for classification due to contradictory results.

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:

1993

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

GLP compliance:

yes

Test type:

fixed dose procedure

Limit test:

yes

Specific details on test material used for the study:

- Substance type: Organic
- Physical state: Solid
- Lot/batch No.: Z-2281/1,2,4,5
- Expiration date of the lot/batch: April 01, 1997
- Storage condition of test material: at room temperature in the dark

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL Ltd., Basel, Switzerland
- Age at study initiation: Approx. 9 weeks
- Weight at study initiation: males: 235 to 287 g, females: 181 to 213 g
- Fasting period before study: overnight
- Housing: individual
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C
- Humidity (%): 55
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1993-01-14 To: 1993-01-28

Type of coverage:

semiocclusive

Vehicle:

polyethylene glycol

Details on dermal exposure:

TEST SITE
- Area of exposure: on the back of the animal
- coverage: 25 cm^2 (5x5 cm) for males and 18 cm^2 (3.5x5 cm) for females
- Amount(s) applied (volume or weight with unit): 10 ml/kg body weight
- Type of wrap if used: application on a gauze patch fixed successively to aluminium foil and flexible bandage

REMOVAL OF TEST SUBSTANCE
- Washing (if done): substance was removed with tissue moistened with tap-water
- Time after start of exposure: 24h

TEST MATERIAL
- Concentration (if solution): 10 ml/kg body weight
- Constant volume or concentration used: yes
- For solids, paste formed: yes

Duration of exposure:

24h

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

5

Control animals:

not required

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Twice daily (mortality, viability), once daily (weighing)
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs (daily)

Sex:

male/female

Dose descriptor:

discriminating dose

Effect level:

2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: No mortality observed.

Mortality:

No mortality occured.

Clinical signs:

other: No clinical signs of ill health or behavioural changes were observed during the study period.

Gross pathology:

Macroscopic post mortem examination of the aniraals at termination did not reveal any abnormalities.

Other findings:

The treated skin area of all animals was discoloured red by the test substance during the study period.

Interpretation of results:

GHS criteria not met

Conclusions:

The dermal LD50 value of the test substance in Wistar rats of either sex was established as exceeding 2000 mg/kg body weight.

Executive summary:

A GLP conform study was performed with the test substance (purity 97.5%) to assess the acute dermal toxicity in rats according to OECD guideline 402. The test substance dissolved in polyethylene glycol was administered to the skin of five Wistar rats of each sex by semi-occlusive application at 2000 mg/kg body weight for 24 hours. Animals were subjected to daily observations and weekly determination of body weight. Macroscopic examination was performed after terminal sacrifice (day 15). No mortality occurred. Red staining of the treated skin and/or head, noted among all animals, were considered to be related to staining properties of the test substance. Therefore, the dermal LD50 value of the test substance in Wistar rats of either sex was established as exceeding 2000 mg/kg body weight.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

discriminating dose

Value:

2 000 mg/kg bw

Quality of whole database:

according to OECD TG 402, GLP, Klimisch 1

Additional information

Oral

A GLP conform study was performed to assess the acute oral toxicity in rats according to OECD guideline 401 (Acute Toxic Class Method). The test substance (purity 97.5%) was administered by oral gavage to five Wistar rats of each sex at 2000 mg/kg body weight. Animals were subjected to daily observations and weekly determination of body weight. Macroscopic examination was performed after terminal sacrifice (day 15). No mortality occurred. No clinical signs were observed during the study period. However, red discolouration of skin, tail and faeces were noted from day 3 onward. The mean body weight gain shown by the animals over the study period was considered to be normal. No abnormalities were found at macroscopic post mortem examination of the animals. The oral LD50 value of the test substance in Wistar rats was established to exceed 2000 mg/kg body weight. 

 

 

Inhalation

No studies on acute inhalation toxicity were available for the test substance. Therefore, read across on reliable data from analogue substances was performed. Data on the structural analogues CAS 84632-65-5 (bulk) and EC 465-080-5 (nano) are available.

 

An acute inhalation toxicity test was performed with the read-across substance CAS 84632-65-5 (bulk) according to OECD TG 403 and GLP. A nose inhalation system was used to expose 5 Wistar rats per sex to an aerosol at a concentration of 2.25 mg/L for 4 hours. It was not possible to generate higher concentrations of the test compound. The exposure to the maximum attainable concentration was thus considered a limit test as stated in the OECD TG 403. The animals were observed for a post-dosing period of 14 days. No mortality and no macroscopic findings at necropsy were observed in males and females. Clinical symptoms were piloerection, hunched posture and dyspnea. From this, the animals recovered within 5 to 9 days. Histopathological examinations of the lungs revealed minimal congestion, minimal emphysema and minimal and multifocal bronchiolar dilatation in all animals. These changes are common response in rats treated by inhalation with a nuisance dust.

 

In a mechanistic follow-up study the acute lung response to CAS 84632-65-5 (bulk), especially acute inflammatory/cytotoxic responses in the rat lung following a single administration by inhalation of an aerosol for 4 hours was assessed by examination of various biochemical and cellular parameters in bronchoalveolar lavage fluid obtained 24 hours after exposure. The investigation was based on a publication by Lindenschmidt et al. (“The comparison of a fibrogenic and two nonfibrogenic dusts by bronchoalveolar lavage.” Toxicology and Applied Pharmacology, 102, 268 – 281 (1990)). As it is a non-standard study, historical control data was not available. Measured concentrations were 1.1 mg/L for the test item, the negative control titanium dioxide (nuisance dust) and the positive control Sikron F600 ((also known as HSE Standard Quartz, fibrinogenic agent). The MMAD was 2.5, 2.0 and 2.7 µm for titanium dioxide, Sikron F600 and the test item, respectively. At least 90% of the particles were had a diameter of less than 7 µm. Another control group was exposed to air only. There were no signs indicative of a toxic or irritant effect following exposure to the test compound. Red stained feces and staining of the skin/fur were noted in both sexes post exposure to the test item. Exposure exaggerated respiratory movements were evident in a proportion of rats exposed to the test compound from 15 minutes of exposure. This finding was also apparent after inhalation of titanium dioxide (2 h) and Sikron F600 (15 minutes), respectively, but not the air control. There were no treatment-related macroscopic findings following the 24 hour post exposure observation period. No effects on lungs weights after exposure to 3,6-bis(4-chlorophenyl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione were seen. After laboratory investigations of bronchoalveolar lavage samples, differences to air control samples were evident in animals treated with the test compound, titanium dioxide and Sikron F600. Biochemical examinations showed thatβ-glucuronidase, N-acetyl-glucosaminidase and lactate dehydrogenase levels in animals exposed to aerosols of the three dust samples were higher than in air control values. Total protein values as well as the total and viable cell counts were also higher than in air controls. The order of magnitude of effects was similar for both control dusts and the test item, with the responses to the test item being generally higher than those to both dust controls. The experimental design was set up to test equal particle load in regard to number and size, but the density and the surface area of the particles were not taken into account. Therefore, a quantitative interpretation of the results is not possible. This acute response is typical of the lung response to inert and biologically active particles and it is only several weeks after exposure that the cellular response to inert and biologically active particles differs (Lindenschmidt 1990).

 

In a procedure following OECD guideline 403 and GLP, three groups of five male and five female albino rats were exposed by nose-only, flow-past inhalation for four hours to the structural analogue EC 465-080-5 (nano) at gravimetrically determined mean concentrations of 5.0 mg/L air, 0.52 mg/L air and 1.0 mg/L air. All animals were observed for clinical signs and mortality during the inhalation exposure and the subsequent 14-day observation period or until they were found dead. Body weights were recorded prior to exposure on test day 1, and during the observation period on test days 2, 4, 8 and 15 before necropsy. On test day 15 all animals were sacrificed and necropsied. Animals which died were necropsied as soon as they were found dead. All animals exposed to 5.0 mg/l air died within two hours after exposure start. All animals exposed to 0.52 or 1.0 mg/L air survived the scheduled observation period. In one male and two females exposed to 5.0 mg/L air, clinical signs such as increased activity, tachypnea and reduced body temperature were recorded before their death during exposure; the remaining seven animals exposed to 5.0 mg/L air died during exposure without recording of clinical signs. In animals exposed to 0.52 or 1.0 mg/L air, clinical signs were mostly limited to the day of treatment and consisted of slightly decreased activity, slightly ruffled fur and tachypnea immediately and one hour after exposure. Animals exposed to 1.0 mg/L air additionally showed labored breathing. In single animals exposed to 0.52 mg/L air, slightly ruffled fur persisted until test days 2 or 3; in single animals exposed to 1.0 mg/L air, slightly ruffled fur and tachypnea persisted until test day 2. All animals exposed to 0.52 or 1.0 mg/L air did not show any clinical signs from test day 4 until the end of the observation period. Between test days 1 and 2, slight body weight loss was noted in all animals exposed to 0.52 mg/L or 1.0 mg/L air. This effect persisted in two females exposed to 0.52 mg/L air as well as in two females exposed to 1.0 mg/L air and in up to day 4 of treatment. From test day 4 onwards, these females showed normal body weight gain. In the remaining animals exposed to 0.52 or 1.0 mg/L air, normal body weight development was noted from day 2 of treatment onwards. After their spontaneous death during exposure, all animals exposed to 5.0 mg/L air showed reddish discolored and incompletely collapsed lungs at necropsy. Although a relation to the treatment with test item cannot be fully excluded, this finding is considered to be most likely secondary to lung deposition of the pigment rather than a substance specific toxicity. In animals exposed to 0.52 or 1.0 mg/L air there were no macroscopic findings at scheduled necropsy. The LD50 for acute inhalation in rats is therefore higher than 1 mg/L, but lower than 5 mg/L.

 

 

Dermal

A GLP conform study was performed with the test substance (purity 97.5%) to assess the acute dermal toxicity in the rat according to OECD guideline 402. The test substance was administered to five Wistar rats of each sex by dermal application at 2000 mg/kg body weight for 24 hours. Animals were subjected to daily observations and weekly determination of body weight. Macroscopic examination was performed after terminal sacrifice (day 15). No mortality occurred. Red staining of the treated skin and/or head, noted among all animals, were considered to be related to staining properties of the test substance.

 

 

References:

Lindenschmidt, R. C., Driscoll, K. E., Perkins, M. A., Higgins, J. M., Maurer, J. K., & Belfiore, K. A. (1990). The comparison of a fibrogenic and two nonfibrogenic dusts by bronchoalveolar lavage. Toxicology and applied pharmacology, 102(2), 268-281

Justification for classification or non-classification

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

The available experimental test data on acute oral and dermal toxicity are reliable and suitable for classification purposes under Regulation 1272/2008. As a result, the substance is not considered to be classified for acute oral or acute dermal toxicity under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2020/217.

No studies on acute inhalation toxicity were available for the test substance. Due to contradictory Data on acute inhalation toxicity of the structural analogues CAS 84632-65-5 (bulk) and EC 465-080-5 (nano), the database was considered to be inconclusive for classification for the endpoint acute inhalation toxicity under Regulation (EC) No. 1272/2008.