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

Acute Toxicity: inhalation

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

mechanistic studies

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline available

Principles of method if other than guideline:

Asessment of acute inflammatory/cytotoxic responses in the rat lung following a short-term inhalation of an aerosol.

GLP compliance:

yes

Type of method:

in vivo

Endpoint addressed:

acute toxicity: inhalation

Specific details on test material used for the study:

- Physical state: red powder
- Purity: 100 %
- Lot/batch No.: 62792709
- Expiration date of the lot/batch: 2003-12-31
- Storage condition of test material: In the light at room temperature and in the original container.

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Manston Road, Margate, Kent, England
- Age at study initiation: 7 - 8 weeks
- Weight at study initiation: 219 - 225 g (male groups 1-4), 196 - 200 g (female groups 1-4)
- Housing: groups of 5/sex in stainless steel cages (35 cmx53cmx25cm)
- Diet (ad libitum): SDS rat and mouse diet (RM1)
- Water (ad libitum): tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2
- Humidity (%): 55 +/- 10
- Air changes (per hr): 12 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

inhalation: aerosol

Vehicle:

air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
A Wright dust feed mechanism according to Wright, B.M. (A new dust-feed mechanism, J. Sc. Instr. 27, 12, 1950) was designed to produce and maintain atmospheres containing dust with a concentration of total particulate close to 1 mg/l. Atmospheres were passed through an elutriation column to reduce the amount of non-respirable particulate. The snout-only exposure chambers used for the exposures were of cylindrical form (10 cm diameter, 60 cm height) and made of aluminium alloy. The chambers had an enclosed volume of approximately 5 liters. The rats were held for exposure in moulded polycarbonate restraining tubes which were attached at evenly spaced ports in the cylindrical section of the chamber, and were designed to allow only the snout to project into the chamber. Each rat was restrained in a forward position by an adjustable foamed plastic stopper which also provided a seal for the tube.

Six samples of air were removed from the exposure chamber of each treatment group during exposure. The concentration of the aerosol was determined gravimetrically and a mean chamber concentration was calculated from the individual data.
At 90 and 210 minutes of each exposure additional samples were taken, at a sampling rate of 2 litres per minute. The particle size distribution of the test atmosphere was assessed using linear regression analysis.
The nominal concentration was calculated from the total amount dispersed by the generator and the total volume of air flowing through the exposure system during the period of generation.
Temperature and humidity were recorded at the start of exposure and then at 30-minute intervals during the four-hour exposure.

TEST ATMOSPHERE
The mean chamber concentrations were:
Titanium dioxide 1.10, Sikron F600 1.09 and 3,6-bis(4-chlorophenyl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione 1.08 mg/l.
The nominal concentrations were:
Titanium dioxide 4.11, Sikron F600 8.05, 3,6-bis(4-chlorophenyl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione 5.29 mg/l.

PARTICLE SIZE DISTRIBUTION
The mass median aerodynamic diameters (MMAD's) were:
Titanium dioxide 2.5, Sikron F600 2.7 and 3,6-bis(4-chlorophenyl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione 2.0 µM
At least 90% of the particles were of a respirable size (less than 7 µM in aerodynamic diameter).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

gravimetrically

Duration of treatment / exposure:

4 h

Frequency of treatment:

single

Post exposure period:

24 h

Dose / conc.:

5.3 mg/L air (nominal)

Dose / conc.:

1.1 mg/L air (nominal)

No. of animals per sex per dose:

10 animals (5 females, 5 males)

Control animals:

other: negative control: exposure to air (see attached file)

Details on study design:

- In addition to the test group, 1 air control group, 1 negative control group (Titanium dioxide) and 1 positive control group (Sikron F600) were used.
- Duration of observation period following administration: 14 days
- Frequency of observations: Clinical signs were recorded at 0.25, 0.5 and 1.0 hour and then at hourly intervals during the exposure and 1, 2 and 24 hours post exposure.
- Frequency of weighing: All rats were weighed immediately before exposure and at sacrifice.
- Necropsy of survivors performed: yes, all animals were sacrificed and subjected to gross pathology.
- The lungs (including the larynx and trachea), were dissected free and weighed. Bronchoalveolar lavage was performed according to Lindenschmidt et al. (1990). Lungs were first lavaged with two separate washings. These two lavage samples were pooled and centrifuged. The resultant cell-free supernatant was analysed for various biochemical parameters. Additionally, lungs were lavaged three times and cell pellets from all washes were combined for cell counting and differentiation.

Details on results:

Mortality
None of the animals died.

Clinical signs
None indicative of a toxic or irritant effect. During exposure exaggerated respiratory movements were evident in a proportion of rats exposed from 15 minutes of exposure. This finding was also apparent in control group Titanium dioxide (2 h) and control group Sikron F600 (15 minutes), respectively, but not the air control.


Body weight
Mean bodyweight for all treated groups following exposure were similar to air control values.

Gross pathology
There were no treatment-related macroscopic findings following the 24 hour post exposure observation period.
No effects on lung weights for dust-treated animals were seen.

Other findings
Laboratory investigations of bronchoalveolar lavage samples: Differences from air control were evident in Group 2 (Titanium dioxide), Group 3 (Sikron F600) and Group 4 (test substance). Differences were generally more marked in Group 4 than Groups 2 or 3. Biochemical parameters examination showed that β-glucuronidase, N-acetyl-glucosaminidase and lactate dehydrogenase levels for Groups 2 (Titanium dioxide), 3 (Sikron F600) and 4 (test substance) were higher than air control values. Total protein values for Groups 2 and 4 were also higher than air controls. The total and viable cell counts for Groups 2 to 4 were higher than air control values. Total and viable cell counts for test article treated rats (group 4) were also higher than negative control (group 2). The proportion of neutrophils present in lavage samples of Groups 2 to 4 was higher than the air control with an associated decrease in the proportion of macrophages.
Red stained feces and staining of the skin/fur were noted in both sexes post exposure.

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Reference 2

Reference Type:

other: Amendment to study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Details on test material:

- Physical state: solid
- Analytical purity: approx. 99 %
- Lot/batch No.: MS 70037.62
- Expiration date of the lot/batch: 1995-December
- Storage condition of test material: room temperature

Test animals

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

Administration / exposure

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.

Results and discussion

Effect levels

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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.