29H,31H-phthalocyaninato(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:

July - Sept 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Current OECD guidelines for acute inhalation toxicity assessments incorporate a limit test aerosol concentration for poorly soluble particles that are either known or expected to be virtually non-toxic (OECD, 2009). Depending upon regulatory requirements, the limit test concentration for aerosols can be as high as 5 mg/L (or the maximum attainable concentration) if the United Nations (UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS) is used.
During the acute dust exposure study (GLP, OECD 403), rats exposed to 5 mg/L of the test substance died of suffocation after agglutinated pigment blocked the airways, whereas all rats survived the 4h dust exposure at the low dose of 1 mg/L.
It is widely recognized that external exposure concentrations of airborne particulates are not directly equivalent to the amounts that are inhaled, deposited, and retained in airways of laboratory animals and humans due to major differences in airway anatomy and physiology. In line with this, with regard to dose adjustment, the ECHA R.8 document on “Characterisation of dose[concentration]-response for human health” (Version 2.1, 2012) suggests to account for differences in respiratory rates of the experimental animal (at rest) and the human as well as for the increased respiratory rate of a worker, when inhalation DNELs are derived.
Without factoring in these species differences, results from inhalation toxicity studies are not directly translatable to human health risk. Accordingly, the results from the acute inhalation study on rats alone are regarded as insufficient to derive a potential human health hazard posed by the test substance.
Therefore, additional computational approaches that incorporate species-specific anatomy, physiology, and the physics of aerosol transport and deposition were utilized to assess whether the results from the acute inhalation study is relevant for humans.
Focusing upon acute toxicity testing guidelines, the MPPD model was applied to provide a generic (not chemical-specific) respiratory dosimetry assessment of rats exposed in nose-only inhalation chambers for 4-hr to aerosols with a respirable particle size of 2.75 µm mass median aerosol diameter (MMAD) and a geometric standard deviation (GSD) of 1.0 at a limit test concentration of 5 mg/L – similar to the settings of the in vivo study and analog to the particle properties of the test item used. Particular emphasis was placed upon particle load and retention in individual airways to assess potential vulnerabilities for physical obstruction of bronchial and pulmonary airways in the rat due to high rates of local aerosol deposition. In addition, human simulations for a variety of hypothetical activity and breathing conditions for the same exposure were also conducted to highlight cross-species differences, although it is unlikely that such high aerosol exposures would be considered tolerable, even for non-toxic materials.
As a result, the MPPD simulations indicate that the results from the high dose group of the acute dust exposure study (GLP, OECD 403) in rats are not relevant for resting or light exercising humans, since deviating internal exposure patterns of rat and humans were observed.
The major difference between both species is that 50% of the material is deposited in the head-region in humans (only 30% in rats) and can be blown or spit out. Moreover, at tracheobronchial and bronchial level, the rat receives ~5-10-fold higher dose/cm2 airway surface (when cumulative deposited mass is normalized to respective airway surface areas) than the human under resting nasal breathing ventilation conditions. The pulmonary region of humans receives a greater fraction of inhaled aerosol than the rat, though when normalized to airway surface areas, both species receive a relatively low overall pulmonary regional dose.
As to the applicability of limit test concentrations for aerosols with presumed low toxicity in guideline acute inhalation toxicity studies with rats, the potential for airway obstruction which could lead to decreases in airflows and pulmonary function was assessed using a geometric analysis of MPPD simulations. Given the smaller airways in the rat and the potential for significant amounts of deposited and retained mass of aerosols during a 4-hr nose-only exposure to aerosols at 5 mg/L atmospheric concentrations, the size (diameter) of retained masses in each airway segment were compared with their corresponding airway diameter. In this analysis, which assumed that the total retained mass in each airway at the end of the 4-hr exposure formed one spherical particle, a significant a percentage of pulmonary airways and to a lesser extent bronchiolar airways may be at risk of occlusion and impairment of gas-exchange. Such a potential for physical impact on pulmonary function under limit test exposure conditions for certain materials that are presumed to be of low toxicity raises questions for assessing acute toxicity risks for humans.
Finally, in comparison to the rat, more material is deposited in the head regions in human. Second, the wider airways in human hamper fast agglutination of the test material and therefore obstruction of the airways. At last, a 4h-exposure of worker to such high concentrations is rather unlikely even in case of an accident. The suffocation at 5 mg/L as observed in rats is therefore not regarded as relevant for humans and a classification as acute cat. 4 H332 not justified.

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:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Low chlorinated copper, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, wherein the number of chlorines is more than or equal to 0 and less than or equal to 7

EC Number:

701-372-3

Molecular formula:

C32HxClyCuN8

IUPAC Name:

Low chlorinated copper, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, wherein the number of chlorines is more than or equal to 0 and less than or equal to 7

Test material form:

solid: nanoform

Details on test material:

- Heliogen Blau BGN
- Lot Number: 210014P050
- Date of production: 22 Feb 2021
- State of aggregation: solid
- Particle size distribution (TEM): = 14.9 nm (D50)
- Mass median aerodynamic diameter (MMAD): 1.93 – 2.98 µm
- Geometric standard deviation (GSD): 2.67 – 3.13
- Shape of particles: spherical
- Aspect ratio: 1.4:1
- Surface area of particles (BET): 106 m^2/g
- Crystal structure: crytalline
- Coating: no
- Surface properties: not applicable
- Density: 1726 kg/m^3 at 20°C
- Moisture content: not specified
- Residual solvent: not specified
- Activation: not applicable
- Stabilisation: not applicable

Specific details on test material used for the study:

purity > 99%
Expiry date: 22 Feb 2031
Storage: Room temperature
physical appearance: solid/blue

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 54 – 64 days, female animals 54 – 80 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: 45-65 %
- 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):

> 1.9 - < 3 µm

Geometric standard deviation (GSD):

> 2.6 - < 3.2

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 brush 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 velocity 1.25 m/s, 4 samples at about hourly intervals, Sampling Volume 15L (low dose) and 6L (high dose)
- 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:

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 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.

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:

> 1.084 - < 5.212 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: High dose animals suffocated because the particles blocked the airways.

Mortality:

5.212 mg/L: All of the male and female animals died. Lethality was observed either during exposure, after exposure on study day 0 or on study day 1 or study day 2.
1.084 mg/L: All animals survived.

Clinical signs:

irregular respiration

Body weight:

1.084 mg/L: The mean body weights of the animals decreased on the first post-exposure observation day but increased thereafter.
5.212 mg/L: The mean body weights of the surviving animals decreased during the first post-exposure observation days. No further body weight data were available for the animals because all animals died.

Gross pathology:

1.084 mg/L: No gross pathological findings were noted during the necropsy of the animals at the termination of the post-exposure observation period.
5.212 mg/L: During necropsy of the dead five male and five female animals, many black foci were seen in the lung lobes with sunken surface. Blue discoloration of content of the stomach was seen in four males and three females and blue depositions in the trachea were present in four males and two females.

Other findings:

low dose (1 mg/L)
Clinical signs of toxicity in animals exposed to 1.084 mg/L comprised accelerated respiration, intermittent respiration, abdominal respiration, respiration sounds, feces substance like discolored, piloerection, substance-discolored fur and substance-contaminated fur. Findings were observed in the males from hour 1 of exposure through study day 11. No findings were detected in the male animals during the post-exposure observation period from study day 12 onwards. Findings were observed in the females from hour 1 of exposure until the end of the post-exposure observation period.

high dose (5 mg/L)
Clinical signs of toxicity in animals exposed to 5.212 mg/L comprised accelerated respiration, depressed respiration, abdominal respiration, no feces, activity: attention reduced, piloerection, substance-discolored fur and substance-contaminated fur. Findings were observed from hour 1 of exposure until the death of the animals. The mean body weights of the surviving animals decreased during the first post-exposure observation days. For further evaluation, histopathological examinations of the respiratory tract (nasal cavity, larynx, pharynx, trachea, and lung) from three animals were performed. The lung showed large amount of blue pigment within the bronchi, bronchioles and terminal bronchioles, leading to obstruction of the airways in two animals with one of them also showing emphysema. The trachea of all examined animals was dilated and contained blue pigment. The larynx at level I - II showed obstruction by blue pigment and large amounts of blue pigment at level III in 2 animals. The third animal presented with large amounts of blue pigment at larynx level I-II and small amounts at the third level. The nasal cavity at level I - IV contained small to moderate amounts of blue pigment.
The histopathological findings in the lung, the trachea, and the larynx of animal Nos. 793, 798 and 800 indicate an airway obstruction caused by the inhaled blue pigment as cause of death.

Any other information on results incl. tables

Table 1: Clinical signs and duration in the low concentration group

Test group 1 (1.084 mg/L)	Male animals	Female animals
Feces, discolored feces,substance like	d1	d1
Fur, discolored, substance like	d2 – d11	d2 – d14
Fur, piloerection	d0 – d1	d0 – d1
Fur, substance-contaminated	d0 – d1	d0 – d1
Respiration, abdominal	-	d0 – d1
Respiration, accelerated	h1 – d4	h1 – d4
Respiration, intermittent	d5 – d6	d5 – d6
Respiration, sounds	d2 – d4	d2 – d4

No findings were detected in the male animals during the post-exposure observation period from study day 12 onwards.

Table 2: Clinical signs and duration, high concentration group

Test group 2 (5.212 mg/L)	Male animals	Female animals
Lethality (number of animals)	5 / 5	5 / 5
Activity/behavior, attention reduced	d0	d0 – d1
Feces, no feces	-	d1
Fur, discolored, substance like	d0	d0 – d1
Fur, piloerection	d0	d0 – d1
Fur, substance-contaminated	d0	d0
Respiration, abdominal	d0	d0 – d1
Respiration, accelerated	h1 – h2	h1 – h2
Respiration, depressed	h3 – h4	h3 – h4

Table 3: Necropsy findings in animals that died during the study period

Findings	high concentration
Number of animals	5 males + 5 females
Lung: many black foci in all lobes (Æ4 mm), surface sunken	1 male + 3 females
Lung: many black foci in all lobes (Æ8 mm), surface sunken	4 males + 2 females
Stomach: blue discoloration of the content	4 males + 2 females
Trachea: blue deposition	4 males + 2 females

Table 4: Necropsy findings of animals at termination of the post exposure period

Findings	low concentration
Number of animals	5 males + 5 females
Organs without particular findings	5 males + 5 females

Table 5: Histopathology findings

	Animal No.:
	793	798	800
Lung
Bronchi, bronchioles and terminal bronchioles contain large amount of blue pigment	x	-	-
Bronchi, bronchioles and terminal bronchioles contain large amount of blue pigment, which obstructs the lumen	-	x	-
Bronchi, bronchioles and terminal bronchioles contain large amount of blue pigment, which obstructs the lumen, emphysema	-	-	x
Trachea
Dilation, contains blue pigment	x	x	x
Larynx
Level I Obstructed by blue pigment	x	-	x
Level I Contains large amounts of blue pigment	-	x	-
Level II Obstructed by blue pigment	x	-	x
Level II Contains large amounts of blue pigment, edema	-	x	-
Level III Contains large amounts of blue pigment	x	-	x
Level III Contains small amounts of blue pigment	-	x	-
Nasalcavity
Level IContains small amounts of blue pigment	x	x	x
Level II Contains small amounts of blue pigment	x	-	x
Level II Contains moderate amounts of blue pigment		x
Level III Contains small amounts of blue pigment	x	x	x
Level IV Contains small amounts of blue pigment	x	x	x

Applicant's summary and conclusion

Interpretation of results:

Category 4 based on GHS criteria