Registration Dossier

Administrative data

mechanistic studies
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well-documented special investigation. A default reliability of 2 is assigned because according to ECHA guidance, a study on a read-across substance can have no higher reliability score than 2.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
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:
Type of method:
in vivo
Endpoint addressed:
acute toxicity: inhalation

Test material

Details on test material:
- 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.

Test animals

Details on test animals and environmental conditions:
- 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

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

Administration / exposure

Route of administration:
inhalation: aerosol
Details on exposure:
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.

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.

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:
Details on analytical verification of doses or concentrations:
Duration of treatment / exposure:
4 h
Frequency of treatment:
Post exposure period:
24 h
Doses / concentrationsopen allclose all
Doses / Concentrations:
5.3 mg/l
nominal conc.
Doses / Concentrations:
1.1 mg/l
analytical conc.
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.

Results and discussion

Details on results:
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.

Applicant's summary and conclusion