[[2,2',2''-[29H,31H-phthalocyaninetriyltris(methylene)]tris[1H-isoindole-1,3(2H)-dionato]](2-)-N29,N30,N31,N32]copper

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January - March 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Deviations:

yes

Remarks:

Low relative humidity in exposure chamber during 4h exposure

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

29H,31H-phthalocyanine

EC Number:

209-378-3

EC Name:

29H,31H-phthalocyanine

Cas Number:

574-93-6

Molecular formula:

C32H18N8

IUPAC Name:

29H,31H-phthalocyanine

Test material form:

solid: nanoform, no surface treatment

Details on test material:

- Particle size distribution: D50 = 28.9 nm (TEM)
- Shape of particles: elongated, rod
- Surface area of particles: BET = 100.3
- Crystal structure: crystalline
- Coating: none
- Density: Rel density = 1.442; Bulk density 125 kg/m3

Specific details on test material used for the study:

Pigment Blue 16
Lot. 130004P040
Solid / blue
Expiry date: 14 Feb 2023
Storage conditions:Room temperature

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH,
- Age at study initiation: on study day 0: (male animals 58 - 62 days, female animals 73 - 77 days)
- Weight at study initiation: animals of comparable weight (+/- 20% of the mean body weight)
- Fasting period before study: no
- Housing: single houing or up to 5 animals
- Diet: Kliba laboratory diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20–24 °C
- Humidity: 30-65 %
- Air changes: 15 changes per hour
- Photoperiod: 12 hours light / 12 hours dark

Administration / exposure

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 2.58 - <= 2.67 µm

Geometric standard deviation (GSD):

>= 3.12 - <= 3.49

Remark on MMAD/GSD:

Two samples were analyzed.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: For each test group the dusts were produced inside the inhalation system with a dust generator and compressed air and passed into the inhalation system. The concentrations were adjusted by varying the apertural width rotation of the dosing wheel of the dust generator.
- Exposure chamber volume: 34 L
- Method of holding animals in test chamber: restraining tubes
- Source and rate of air: central air conditioning system,1.5 m³/h
- Method of conditioning air: Central air conditioning system provided cold air of about 15°C. This cold air passed through an activated charcoal filter, adjusted to room temperature of 20 to 24°C and passed through a second particle filter (H13 (HEPA) Camfil Farr, Germany). The so generated conditioned air was used to generate inhalation atmospheres.
- System of generating particulates/aerosols: The test item was stirred in its container before a sample for dust generation was taken. The test item was desagglomerated in a mixer (mixing for 10 seconds) before introduction into the dust generator via the dosing wheel (Gericke/BASF).
- Method of particle size determination: Stack Sampler Mark III (Andersen). Before sampling, impactor stages were assembled with preweighed glass fiber collecting discs, and equipped with a backup particle filter. The impactor was connected to a vacuum pump, and for each test group samples were taken from the breathing zone of the animals. Sampling occurred 30 minutes (or later) after the beginning of the exposure.
- Treatment of exhaust air: The exhaust air was filtered and conducted into the exhaust air of the building.

TEST ATMOSPHERE
- Brief description of analytical method used: gravimetric, filtration equipment with probe, internal diameter: 7 mm, Sampling Volume 15L, 4 samples at about hourly intervals
- Samples taken from breathing zone: yes

VEHICLE
none apart from air


TEST ATMOSPHERE:
- Particle size distribution: Based on cascade impactor measurements. The calculation of particle size distribution was carried out by means of mathematical methods for evaluating particle measurements.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

7.3 mg/L (nominal)
The actual measured concentration was 2.046 mg/L

No. of animals per sex per dose:

5 rats

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Individual body weight was determined once during the acclimatization period, at the start of the exposure period (Day 0) and at least on Days 1, 3 and 7, and weekly thereafter, or before the sacrifice of the animals at the end of the observation period.
- Clinical observations were recorded for each animal before exposure, separately several times during exposure (usually hourly) and after exposure. At least once daily on the preexposure day and during the post exposure observation period.
- Necropsy of survivors performed: yes

Pathology: At the end of the observation period the surviving animals were sacrificed with CO2-inhalation in a chamber with increasing concentration over time and were subjected to gross pathological examination as well as the animal which died before. To clarify the gross pathological findings, selected organs of individual animals were examined histopathologically.


Selection of target concentration:
The target concentration of 2 mg/L was tested. The concentration of 2 mg/L was chosen following the recommendation of OECD guidance document No. 39 (OECD guidance document on inhalation toxicity studies. Series on testing and assessment No. 39 (second edtion). 6 July 2018)
1) Paragraph 8 “Testing in GHS category 5 is generally discouraged and should only be considered when there is a strong likelihood that results of such a test would have direct relevance for protecting human health (see APPENDIX II of the OECD guidance document No. 39).”
2) Paragraph 70 “Testing aerosols, for acute toxicity, the primary goal should be to achieve a respirable particle size (MMAD equal to or less than 2 micrometer). This is possible with most test chemicals at a
concentration of 2 mg/L.”
The measured MMAD was 2.58 - 2.67 µm.

Statistics:

LC50 values were calculated for males, females and both sexes combined using a binomial test.

Results and discussion

Effect levels

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 2.046 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: 1 of 10 animals died. Relative humidity 7.2%

Remarks:

Death was observed in the 2nd hour of exposure.

Mortality:

No lethality was observed in the five females. One of five male animals died on study day 0 during exposure

Clinical signs:

irregular respiration

Body weight:

The mean body weights of the animals decreased on the first post exposure observation days but increased thereafter.

Gross pathology:

Clinical signs of toxicity in animals comprised abdominal and intermittent respiration,
respiration sounds, hunched posture, piloerection, substance-contaminated and discolored fur
substance like. Findings were observed from hour 1 of exposure until the end of the 14-day
post-exposure observation period. The mean body weights of the surviving animals decreased
on the first post-exposure observation day but increased thereafter. This is a typical finding for
this test design. Gross necropsy of the male animal that died on study day 0 showed blue
discoloration of the fur on the entire body. During necropsy of the surviving animals at the
termination of the post-exposure observation period the following gross pathological findings
were noted: many blue foci in all lung lobes, grey/blue discoloration of the mediastinal lymph
nodes and blue discoloration of the fur of the tail and/or head region.

Other findings:

Clinical signs of toxicity in animals comprised abdominal and intermittent respiration, respiration sounds, hunched posture, piloerection, substance-contaminated and discolored fur substance like. Findings were observed from hour 1 of exposure until the end of the 14-day post-exposure observation period.

To further evaluate the macroscopic findings, histopathological examination was carried out of the respiration tract from the male animal No. 721 which died on study day 0. Minimal amount of green-blue crystals revealing the presence of the test substance were seen in the lumen of the larynx, pharynx and nasal cavity. Furthermore, in the larynx, minimal grey granular material in the lumen and a slight edema in the region of the ventral pouch were noted. All these findings are regarded as treatment-related, however they are not considered sufficiently severe to explain the death of this animal.

Any other information on results incl. tables

Sample	MMAD (µm)	Geometrical standard deviation
1	2.67	3.49
2	2.58	3.12

Test group 1 (2.046 mg/L)	Male animals	Female animals
Lethality (number of animals)	1 / 5	-
Fur, discolored, substance like	d1 – d14	d1 – d14
Fur, substance-contaminated	d0	d0
Respiration, abdominal	d0 – d2	d0 – d2
Respiration, intermittent	h1 – h4	h1 – h4
Respiration, sounds	d0 – d1	d0 – d1
Piloerection	d1 – d2	d1 – d2
Posture, hunched posture	d0	d0

 

Histopathological findings in the animal no. 721 that died on study day 0

Animal No.	Organ	Finding	Modifier
721	Tracheobronchial and mediastinal lymph nodes	No finding	-
	Larynx:	Edema, slight Crystals, minimal multifocal Granular, material, slight	ventral pouch green-blue, luminal   grey, luminal
	Pharynx	Crystals, minimal multifocal	green-blue
	Trachea	No finding	-
	Lungs	No finding	-
	Nasal cavity	Crystals, minimal multifocal

Applicant's summary and conclusion

Interpretation of results:

Category 5 based on GHS criteria

Executive summary:

To determine the acute inhalation toxicity (single 4-hour exposure, nose only) of 29H,31H-phthalocyanine as a dust, a study was performed in male and female Wistar rats according to OECD-Guideline method 403. The test was run with an actual measured concentration of 2.064 mg/L (analytical concentration). Cascade impactor measurements resulted in particle size distributions with mass median aerodynamic diameters (MMADs) of 2.67 and 2.58 µm, which are well within the respirable range.

One of the five males was died at 2.064 mg/L on study day 0 during exposure.

The death was determined in the 2nd hour of the exposure. N

o mortality was observed in female animals. Clinical signs of toxicity in animals comprised abdominal and intermittent respiration, respiration sounds, hunched posture, piloerection, substance-contaminated and discolored fur substance like. Findings were observed from hour 1 of exposure until the end of the 14-day post-exposure observation period. The mean body weights of the surviving animals decreased on the first post-exposure observation day but increased thereafter.