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Toxicological information

Acute Toxicity: inhalation

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Principles of method if other than guideline:
A study on the acute inhalation toxicity of Macrolex Orange 3G on rats has been conducted in accordance with OECD Test Guideline no. 403 (2009). One group of rats consisting of 3 male and 3 female rats was nose-only exposed to the solid
aerosol of the test item at 157.4 mg/m³. This procedure is in accordance with the limit test of the OECD Test Guideline no. 403 (2009).
GLP compliance:
yes
Test type:
traditional method
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
12H-phthaloperin-12-one
EC Number:
230-049-5
EC Name:
12H-phthaloperin-12-one
Cas Number:
6925-69-5
Molecular formula:
C18H10N2O
IUPAC Name:
12H-isoindolo[2,1-a]perimidin-12-one
Specific details on test material used for the study:
Test item: Macrolex Orange 3G
Purity: 99.9%
CAS name: 12H-Phthaloperin-12-one
CAS number: 6925-69-5
Molecular weight: approx. 270.3 g/mol
Aggregation state: solid, orange-yellow powder
Empirical formula: C18H10N2O

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Species and species justification: The study was carried out in rats, a rodent species recommended in the test guidelines.
Healthy young adult SPF bred Wistar rats, strain HsdCpb:WU were used. Historical data on their physiology, diseases and spontaneous alterations are available. T

Acclimatization: The animals were acclimatized to the animal room conditions for at least 5 days before use. During this period, rats were also acclimatized to the restraining tubes.

Identification: Animals were identified by both individual color-marking and cagelabels. All animals from this study were located on one cage-rack.

Randomization: Before the start of the study the health status of each animal was assessed. Animals were subsequently assigned to exposure groups at random.

Health status: Only healthy rats free of signs were used for this study. The animals were not vaccinated or treated with anti-infective agents either before their arrival or during the acclimatization or study periods. The females were nulliparous and not pregnant.

Age and weight: At the study start the variation of individual weights did not exceed ± 10 per cent of the mean for each sex. Animals of the weight class used are approximately 6-7 weeks old and hence fulfill the criterion for young adults.

Animal housing: During the acclimatization and study periods the animals were housed singly in conventional Makrolon® Type IIIH cages.

Bedding: Bedding consisted of certified low-dust spruce wood granulates, particle size (main part): 1.25-2.50 mm.

Environmental Conditions in the Animal Room
The animal room environment was as follows:
Room temperature: 22 ± 3°C
Relative humidity: 40-60%
Dark/light cycle: 12 h/12 h; artificial light from 6.00 a.m. to 6.00 p.m.
Light intensity: approximately 150 Lux
Ventilation: approximately 10 air changes per hour

The room humidity and temperature were continuously monitored and documented using a calibrated thermohygrograph.

Cleaning, disinfection, and pest control: The animal room was regularly cleaned and disinfected once a week.

Feeding: Ration consisted of a standard fixed-formula diet (ssniff® R/M-H pellets maintenance diet for rats and mice and tap water (drinking bottles). Both food and water were available ad libitum.

Water: Drinking quality municipality tap-water.

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
2.73 µm
Geometric standard deviation (GSD):
2.12
Remark on MMAD/GSD:
The mean mass median aerodynamic diameter (MMAD) was 2.73 µm and the mean geometric standard deviation (GSD) was 2.12.
Details on inhalation exposure:
Atmosphere generation:
Pre-tests and technical feasibility investigations were performed without animals. Different technical setups and dust generators were utilized in order to achieve the limit test atmosphere concentration according to the OECD TG No. 403 (2009). The primary goal according to the OECD Test Guideline 403 (2009) is the generation of an aerosol with a MMAD between 1-4 µm and a GSD in the range of 1-3.
In conclusion, an adequate inhalable test atmosphere according to the recommendation of the OECD TG 403 (MMAD 1-4 j.Jm and GSD 1.5-3) was, from a technically point of view, only achievable at concentrations below 2000 mg/m³.

Final setup used for animal exposure: To generate a stable test atmosphere a Wright-Dust-Feeder II (CH Technologies, USA) was used (2.0 rounds per minute). The test item was filled into a large cartridge and then compressed (1 metric ton)
using a Carver Laboratory Press (Carver Inc., USA). The primary air flow through the generator was 28 L/min. A metal cyclone was utilized to optimize the respirability.

Inhalation chamber: An aluminum inhalation chamber with the following dimensions was used: inner diameter = 14 cm, outer diameter = 35 cm (two-chamber system), height = 25 cm (internal volume = about 3.8 l).
Inhalation chamber equilibrium concentration: The test atmosphere generation conditions provide an adequate number of air exchanges per hour [28 Llmin x 60 min/(3.8 L) = 442, continuous generation of test atmosphere].

Conditioning the compressed air: Compressed air was supplied by Boge compressors and was conditioned (i.e. freed from water, dust, and oil) automatically. Adequate control devices were employed.

Air flows: During the exposure period air flows were monitored continuously by flow meters and, if necessary, readjusted to the conditions required. Measured air-flows were calibrated with precision flow-meters and/or specialized flow-calibration devices.

Inhalation Chamber Temperature and Humidity
Temperature and humidity measurements were performed by a computerized Data Acquisition and Control System. The position of the probe was at the exposure location of rats.



Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
One group of rats was nose-only exposed to the solid aerosol of the test item at 157.4 mg/m³.
No. of animals per sex per dose:
Three male and three female rats were simultaneously exposed under nose-only conditions for 4 h.
Control animals:
yes
Details on study design:
Body Weights and Duration of Observation Period
Body weights were measured before exposure (day 0), on days 1, 3, 7, and 14. The period of observation was for 2 weeks.

Clinical Signs
The appearance and behavior of each rat were examined carefully at least two times on the day of exposure and at least once daily thereafter. Weekend assessments were made once a day (morning).

Rectal Temperatures
The rectal temperatures were measured shortly after cessation of exposure (approximately within 1/2 hour after the end of exposure) using a digital thermometer with a rectal probe for rats.

Necropsy
All surviving rats were sacrificed at the end of the observation period. All rats, irrespective of the day of death, were given a gross-pathological examination. Consideration was given to performing a gross necropsy on animals as indicated by the nature of toxic effects, with particular reference to changes related to the respiratory tract. All gross pathological changes were recorded and evaluated.
Statistics:
A statistical analysis was performed.

Results and discussion

Effect levels
Key result
Sex:
male/female
Dose descriptor:
discriminating conc.
Effect level:
157.4 mg/m³ air
Based on:
test mat.
Exp. duration:
4 h
Remarks on result:
other: 157.4 mg/m³ was the highest technically feasible concentration
Mortality:
Mortality did not occur at 157.4 mg/m³.
Clinical signs:
other: One female rat exposed to 157.4 mg/m³ test item revealed piloerection (low severity) on day 1 and 2 during the recovery period. Test item-dependent orange discoloration of the fur was seen at the head, forelegs, neck and thorax. No findings were seen at t
Body weight:
No toxicological relevant test item-related changes in incremental body weight gain were observed.
Gross pathology:
One male and one female rat exposed to the test item revealed light colored lungs at the necropsy after the 2 week recovery period.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
A limit test according the OECD Test Guideline was performed. The LC50 is >157.4 mg/m³ (maximum attainable concentration with guideline compliant MMAD in the range of 1-4 µm and GSD in the range of 1-3).
Executive summary:

A study on the acute inhalation toxicity of Macrolex Orange 3G on rats has been conducted in accordance with OECD Test Guideline no. 403 (2009). One group of rats consisting of 3 male and 3 female rats was nose-only exposed to the solid aerosol of the test item at 157.4 mg/m³. This procedure is in accordance with the limit test of the OECD Test Guideline no. 403 (2009). The LC50 is >157.4 mg/m³ (maximum attainable concentration with guideline compliant MMAD in the range of 1-4 µm and GSD in the range of 1-3).