Perylene-3,4:9,10-tetracarboxydiimide

Endpoint summary

Description of key information

Oral: Read-across, NOAEL (28d) >= 1000 mg/kg bw/d, rat, according to OECD 407, GLP

Inhalation: Read-across, NOAEC systemic (90d) = 20 mg/m³ air, NOAEC local (90d) = 1 mg/m³ air, rat, according to OECD 413, GLP

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Justification for type of information:

see attached justification

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 November 2005 - 04 March 2006 (experimental)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

(1995)

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Physical state: Solid, black powder
- Analytical purity: > 99%
- Storage condition of test material: Room temperature
- Lot/batch No.: Partie053001
- Expiration date of the lot/batch: 18 November 2020
- CAS No. Cis: 55034-81-6 Trans: 55034-79-2

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Wistar Crl : (WI) BR (outbred, SPF-Quality)
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 6 weeks.
- Weight at study initiation: no data
- Housing: 5 animals per sex in Macrolon plastic cages
- Diet (ad libitum): standard pelleted laboratory animal diet (from Altromin (code VRF- 1, Lage, Germany).
- Water (ad libitum): tap water
- Acclimation period: at least 5 days before start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0°C (actual range : 19.2 - 22.6°C)
- Humidity (%): 30-70% (actual range : 23 - 94%)
- Air changes (per hr): approximately 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkness per day.

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

(Milli-U) (Millipore Corporation, Bedford, USA)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Dose volume: 5 mL/kg bw/day

VEHICLE
- Justification for use and choice of vehicle: Based on trial formulations performed at the testing laboratory and on information from the sponsor.
- Purity: (Milli-U) (Millipore Corporation , Bedford, USA).
- Method of formulation: Formulations (w/w) were prepared daily within 4 hours prior to dosing and were homogenised to a visually acceptable level.
- Storage conditions: At ambient temperature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test substance formulations in water (Milli-U) were noted as stable for at least 5 hours and formed a homogeneous suspension at the concentrations tested. Analysis of the accuracy of dose preparations revealed values within the range of 88% to 99% of nominal. The accuracy level of 88% obtained for one group 3 sample was slightly outside the acceptable range of 90 - 110% of nominal. However, since all other accuracy values were within this range and the accuracy results were in line with the procedural recovery results, the overall accuracy for formulations was considered to be acceptable.

Duration of treatment / exposure:

28 days followed by a 14 day recovery period (control and high dose only)

Frequency of treatment:

daily for at least 28 days, 7 days per week

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 (low and intermediate dose groups)
10 (vehicle control and high dose groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Based on the results of a 5-day range finding study and in consultation with the sponsor, the dose levels for the 28-day toxicity study were selected to be 0, 100, 300 and 1000 mg/kg bw/day.
- Rationale for selecting satellite groups: There were two satellite (recovery) groups: 0 and 1000 mg/kg bw/day
- Post-exposure recovery period in satellite groups: 14 days

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A check for mortality was made at least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily, detailed clinical observations were made in all animals. Detailed clinical observations were also performed outside the home cage in a standard arena on a weekly basis.

BODY WEIGHT: Yes
- Time schedule for examinations: Treatment period: on days 1, 8, 15, 22 and 28. Recovery period: on days 1, 8 and 14.

FOOD CONSUMPTION:
- Food consumption for each animal determined: Yes (Weekly)

WATER CONSUMPTION: Yes (but no quantification)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination at the end of the treatment period.
- Anaesthetic used for blood collection: Yes: iso-flurane (Abbott Laboratories Ltd., Zwolle, The Netherlands)
- Animals fasted: Yes: overnight
- How many animals: all
- Parameters examined: White blood cells (WBC), differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), red blood cells, red blood cell distribution width (RDW), haemoglobin, haematocrit, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration, platelets. Clotting Potential: Prothrombin time (PT), activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination at the end of the treatment period.
- Animals fasted: Yes: overnight
- How many animals: all
- Parameters examined: Alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (ALP), total protein, albumin, total bilirubin, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate (Inorg. Phos.).

URINALYSIS: Yes
- Time schedule for collection of urine: overnight (approximately 16 hrs) from all animals at the end of the treatment period.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: Volume, colour score, Clarity, specific gravity, pH, protein, glucose, ketone, bilirubin, occult blood, leucocytes, nitrite, urobilinogen, sodium, potassium, calcium, sediment (white blood cells (WBC-SED), red blood cells (RBC), casts, epithelial cells, crystals, bacteria.

NEUROBEHAVIOURAL EXAMINATION: Yes (Functional Observations)
- Time schedule for examinations: during week 4 of treatment
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity:
- hearing ability (HEARING), pupillary reflex (PUPIL L/R), static righting reflex (STATIC R) and grip strength (GRIP) (Score 0 = normal/present, score 1 = abnormal/absent).
- motor activity test (recording period : 12 hours during overnight for individual animals, using a computerised monitoring system, Pearson Technical Services, Debenham, Stowmarket, England).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Samples of the following tissues and organs were collected from all animals at necropsy and fixed in a neutral phosphate buffered 4% formaldehyde solution:
Adrenal glands, Aorta, Brain [cerebellum, mid-brain, cortex], Caecum, Cervix, (Clitoral gland), Colon, Duodenum, Epididymides, (Eyes with optic nerve [if detectable] and Harderian gland), (Female mammary gland area), (Femur including joint), Heart, Ileum, Jejunum, Kidneys, (Larynx), (Lacrimal gland, exorbital), Liver, Lung infused with formalin, Lymph nodes - mandibular, mesenteric, (Nasopharynx), Oesophagus, Ovaries, Pancreas, Peyer's patches [jejunum, ileum] if detectable, Pituitary gland, (Preputial gland), Prostate gland, Rectum, (Salivary glands - mandibular, sublingual), Sciatic nerve, Seminal vesicles, (Skeletal muscle), (Skin), Spinal cord -cervical, midthoracic, lumbar, Spleen, Sternum with bone marrow, Stomach, Testes, Thymus, Thyroid including parathyroid [if detectable], (Tongue), Trachea, Urinary bladder, Uterus, Vagina, All gross lesions.

Tissues mentioned within brackets were not examined mlcroscopically as there were no signs of toxicity or target organ involvement. Histological examinations were performed on organs and tissues from all Main Group 1 and 4 animals (0 and 1000 mg/kg bw/d), and all gross lesions. All organ and tissue samples were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin.

The following organ weights (and terminal body weight) were recorded from the animals on the scheduled day of necropsy:
Adrenal glands, Heart, Epididymides, Liver, Kidneys, Spleen, Testes, Thymus, Brain.

In addition, a histopathological investigation of liver, kidneys and adrenal glands of the animals of test groups 2 and 3 (100 and 300 mg/kg bw/day) was performed to meet the additional requirements by the Japanese authority (MHLW) (BASF SE, 99P0617/04P001, 30 Apr 2010).

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. No statistical analysis was performed on histopathology findings.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No toxicologically relevant clinical signs were observed.
Black faeces observed at 1000 mg/kg bw/day from week 3 of treatment onwards and incidental cases of black staining of parts of the fur were considered to be due to staining properties of the test substance (a black powder). These findings had resolved during the recovery phase. Incidental findings that were noted in single animals during the treatment or recovery phase included alopecia, scabs and a broken tail apex. These findings are occasionally noted in rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance. No clinical signs were noted in control animals and in males at 300 mg/kg bw/day and females at 100 and 300 mg/kg bw/day.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights and body weight gain were considered to have been unaffected by treatment with the test substance.
The statistically significant lower body weight gain of females at 100 mg/kg bw/day (day 15, 24% compared to 30% in the control) was absent at higher dosages, and was therefore considered to be of no toxicological significance.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption before or after allowance for body weight was considered to have been unaffected by treatment with the test substance.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes occurred in haematological parameters of treated rats.
The statistically significant lower prothrombin time (PT) of females at 1000 mg/kg bw/day at the end of the treatment phase was well within the historical control range. Considering the direction (i.e. a decrease) and slight degree of change, this was considered to be of no toxicological significance (17.4 s compared to 18.2 s in the control). The lower mean corpuscular haemoglobin (MCH) level of males at 300 mg/kg bw/day achieving a level of statistical significance occurred in the absence of a treatment-related distribution and was therefore also considered to be of no toxicological significance (1.15 fmol compared to 1.19 fmol in the control).

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats.
Any statistically significant deviations in clinical biochemistry parameters were within the historical control range and occurred in the absence of a (clear) dose-related response. Therefore these changes were considered to be without toxicological significance. These changes comprised higher albumin (33.8 and 33.7 g/L as compared to 32.5 g/L in the control) and chloride levels (102, 103, and 102 mmol/L as compared to 101 mmol/L in the control) in females at 100, 300 and/or 1000 mg/kg bw/day, lower aspartate aminotransferase activity levels (ASAT) in males at 100 mg/kg bw/day (71.2 U/L as compared to 85.6 U/L in the control) and higher sodium levels in females at 300 mg/kg bw/day (138.3 mmol/L as compared to 135.9 mmol/L in the control).

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes occurred in urinary parameters of treated rats.
Any statistically significant changes in urinary parameters of females at 300 mg/kg bw/day were absent at 1000 mg/kg bw/day and were therefore considered to be of no toxicological relevance. These changes comprised a lower urinary volume, a higher specific gravity, and a higher sodium and potassium concentration (absent when corrected for urinary volume).

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant changes occurred in organ weights of treated rats.
The lower heart to body weight ratio of males at 100 mg/kg bw/day occurred in the absence of a dose-related distribution (0.319 % as compared to 0.347% in the control) and the mean was well within the historical control range. This change was therefore considered to be of no toxicological relevance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically relevant necropsy findings were observed.
Black contents of the gastro-intestinal tract (or parts thereof, i.e. stomach, caecum and/or colon) in most animals at 1000 mg/kg bw/day and in one male at 300 mg/kg bw/day at the end of the treatment phase were considered to represent test substance (a black powder). These findings had resolved at the end of the recovery phase and occurred in the absence of any correlating histopathological tissue reaction. Therefore, these observations were considered to be of no toxicological relevance. Incidental findings among control and/or treated animals included pelvic dilation of the kidneys, red foci on the kidneys, red discolouration of the thymus, enlarged mandibular lymph node, fluid in the uterus, and tail apex fracture. These findings are occasionally seen among rats used in these types of studies. In the absence of a treatment-related distribution they were considered changes of no toxicological significance.

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals. The variation in motor activity did not indicate a relation with treatment. Variations noted in individual motor activity data between treated and control animals occurred in the absence of a dose-related response, similar changes of the low or high sensors, and/or supportive clinical signs. Therefore, they were considered to be of no toxicological relevance.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No histopathological abnormalities were observed that were considered to be of toxicological significance.
Black pigment deposits in the lumen or on the mucosal surface of the gastrointestinal tract in 5/5 males and 4/5 females at 1000 mg/kg bw/day occurred in the absence of any histopathological tissue reaction and were absent at the end of the recovery period. Therefore, these observations were considered to be of no toxicological relevance. The range of other microscopic observations recorded in this study was within the normal range of physiological changes and background alterations that may be seen in untreated animals of this age and strain.

In addition, liver, kidney and adrenal glands of all male and female animals of test group 2 and 3 (100 and 300 mg/kg bw/day) were investigated.
Livers of all animals revealed minimal to slight multifocal lymphoid infiltrates, characterized by a randomly scattered distribution of aggregates of lymphoid cells and minimal Kupffer cell granuloma. In addition, two males (1 of test group 2, 1 of test group 3) revealed in the area of lymphoid infiltration/Kupffer cell granuloma minimal single cell necrosis (3-5 hepatocytes) as accompanying finding. In two females of test group 3 an extramedullary hematopoiesis was seen.
One male of test group 2 revealed in the kidney a focal basophilic tubule. Additionally, two females of test group 3 showed a minimal unilateral pyelitis.
Accessory cortical tissue (accessory nodule) was found in the adrenal gland of 2 males and 1 female of test group 3.
All these findings noted were single observations and/or were biologically equally distributed in both test groups. They were all considered to be incidental and spontaneous in nature and not related to treatment.

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Conclusions:

From the results presented in this report a definitive No Observed Adverse Effect Level (NOAEL) for the test article of 1000 mg/kg/day was established.

Executive summary:

The repeated dose toxicity of the test article was investigated in guideline and GLP compliant 28-day oral toxicity study by daily gavage in the rat, followed by a 14-day recovery period. Based on the resutls of a 5-day range finding study and in consultation with the sponsor, the dose levels for the 28-day toxicity study were selected to be 0, 100, 300 and 1000 mg/kg/day. Each group consisted of 5 males and 5 females. An extra 5 animals per sex in the control and high dose group were allowed 14 days of recovery. Formulation analyses were conducted once during treatment to assess accuracy, homogeneity and stability of formulations. The following parameters were evaluated: clinical signs daily; functional observation tests; body weight and food consumption weekly; clinical pathology at the end of treatment; macroscopy at termination; organ weights and histopathology on a selection of tissues. Accuracy, homogeneity and stability over 5 hours of formulations of test substance in Milli-U water were demonstrated by analyses. No toxicologically relevant changes were observed in any of the parameters determined in this study. No evidence for neurotoxicity or immunotoxicity was obtained in this study. NOAEL was therefore determined to be 1000 mg/kg.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Justification for type of information:

see attached justification

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

20 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse treatment-related systemic effects observed up to the highest tested dose.

Dose descriptor:

LOAEC

Remarks:

local

Effect level:

5 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

other: BAL parameters

Dose descriptor:

NOAEC

Remarks:

local

Effect level:

1 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse treatment-related effects observed at this dose.

Critical effects observed:

yes

Lowest effective dose / conc.:

5 mg/L air (analytical)

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

presumably yes

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 Jul 2021 - 27 Apr 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (90-Day (Subchronic) Inhalation Toxicity Study

Version / remarks:

25 June 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

24 January 2014

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)

Version / remarks:

August 1998

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Kaiser-Friedrich-Straße 7, 55116 Mainz

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier): BASF Colors & Effects
- Lot/batch number of test material: P 110014
- Purity: ≥ 98 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Expiry date: 18 Feb 2023
- The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Homogeneity: Homogenous


TREATMENT OF TEST MATERIAL PRIOR TO TESTING:
- See section "Details on inhalation exposure"

FORM AS APPLIED IN THE TEST:
- dust aerosol

OTHER SPECIFICS
- physical state: solid
- color: red

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

Reason for selection of the test species: Rats were selected since this rodent species is recommended in the respective test guidelines. Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.

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; Sandhofer Weg 7, 97633 Sulzfeld
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Arrival of the animals/experimental starting date (age: 7 weeks), Start of exposure (14-15 weeks)
- Weight at study initiation: 249.4 - 250.5 (test group mean)
- Fasting period before study: no
- Housing: together (5 animals per cage)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 weeks


DETAILS OF FOOD AND WATER QUALITY:
- The food used in the study was assayed for chemical as well as for microbiological contaminants. On the basis of the duration of use and the analytical findings with respect to chemical and microbiological contaminants, the food was found to be suitable.
- The drinking water is regularly assayed for chemical contaminants. On the basis of the analytical findings the drinking water was found to be suitable.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 45 - 65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 06 Jul 2021 To: 13 Dec 21

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 0.59 - <= 0.62 µm

Geometric standard deviation (GSD):

2.84

Remarks on MMAD:

The cascade impactor measurement showed comparable MMADs and GSDs in all 3 concentration groups. The calculated mass fractions of particles below 3 µm aerodynamic size ranged between 88.8 and 97.3 %. The generated aerosols were highly respirable for rats.


By APS the measured MMADs were generally higher than those measured by cascade impactor. Especially at the high concentration of 20 mg/m³, the measured MMAD by APS is almost 10 times that measured by cascade impactor.

According to the OECD Test Guideline 413 and the Guidance Document 39, multistage cascade impactors should be given preference. Other devices or physical principles may be used if equivalence to the cascade impactor can be shown (with regard to MMAD and GSD, including the mass concentration sensed). In this study, equivalence to the cascade impactor cannot be shown. Thus, the more reliable cascade impactor data are used to appraise the technical validity of this study. APS measurement stopped after first 7 weeks.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Generator systems: Solid particle generators (brush-generator), Aerosol mixing tube (stainless steel), Glass connecting pipe, Glass cyclonic separators
- Generation procedure: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage mixed with conditioned dilution air and passed via the cyclonic separator into the inhalation system. The concentration was adjusted by varying the piston feed and by varying the brush rotation speed.
- Test substance flow and air flows: see table 11
- Exposure apparatus: The inhalation atmosphere was maintained inside aerodynamic exposure systems (INA 60, volume V = ca. 90 L) consisting of a cylindrical inhalation chamber made of stainless steel sheeting and cone shaped outlets and inlets.
- Method of holding animals in test chamber: The rats were restrained in glass exposure tubes. Their snouts projected into the inhalation chamber and thus they inhaled the aerosol.
- Method of conditioning air: The central air conditioning system provides cold air of about 15°C. This cold air passes through an activated charcoal filter, is adjusted to room temperature of 20 to 24°C and passes through a second particle filter (H13 (HEPA) Camfil Farr, Germany). The so generated conditioned air was used to generate inhalation atmospheres.
- Temperature, humidity, pressure in air chamber: 20.6 - 23.8°C, 37.3 and 48.3%. Both relative humidity and chamber temperature were within the required range of the guideline.
- Air flow rate: 5.0 - 7.0 m3/h
- Air change rate: 67 times per hour
- Method of particle size determination: The particle size analysis was carried out with a cascade impactor. Particle Size distribution of the test atmosphere were determined also with the Aerodynamic Particle Spectrometer APS 3321 (TSI, USA). MMAD and GSD is obtained directly by the piece of equipment used APS 3321. Frequency: Once weekly during the first 7 weeks, with 3 repeats on each day. Particle size distribution measurements with scanning mobility particle sizer: To determine the particle size distribution in the submicrometer range, each test atmosphere was measured with the Scanning Mobility Particle Sizer (SMPS; Grimm Aerosol Technik GmbH & Co KG, Ainring, Germany). The SMPS system comprises an Electrostatic Classifier (Model Vienna U-DMA) which separates the particles into known size fractions, and a Condensation Particle Counter (CPC) which measures particle count concentrations. The DMA was equipped with Am-241 neutralizer. The sampling duration was about 7 minutes. As a rule, 10 repeats were measured for each exposure concentration.
- 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: The concentrations of the inhalation atmospheres were determined by gravimetry. Real time monitoring of constancy of concentrations via Scattered light photometers (VisGuard (Sigrist).
- Samples taken from breathing zone: yes

VEHICLE: The test substance was used unchanged.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Gravimetrical method of analyses:
The concentrations of the inhalation atmospheres were determined by gravimetry.
A preweighed filter was placed into the filtration equipment. By means of a vacuum compressed air pump a defined volume of the dust/aerosol was drawn through the filter.
The dust concentration in mg/m³ was calculated from the difference between the weight of the pre-weighed filter and the weight of the filter after sampling, with reference to the sample volume of the inhalation atmosphere.
See table 11.

Real time monitoring of constancy of concentrations:
Scattered light photometers (VisGuard (Sigrist) were used to continuously monitor the constancy of concentrations of test substance aerosols in the inhalation systems. To this end the inhalation atmosphere was continuously sampled by the measuring devices. The measurements were recorded using line recorders and transferred to the automated measuring system.
Real time surveillance of the inhalation atmospheres with scattered light photometers generally proved the constancy of each concentration throughout the daily exposures

Duration of treatment / exposure:

90 days (+ 60 days recovery)

Frequency of treatment:

6 hours/day, 5 days/week

Dose / conc.:

20 mg/m³ air (analytical)

Dose / conc.:

5 mg/m³ air (analytical)

Dose / conc.:

1 mg/m³ air (analytical)

Dose / conc.:

0 mg/m³ air (analytical)

No. of animals per sex per dose:

exposure period: 10 animals/sex/dose
recovery group: 5 animals/sex/dose

Control animals:

yes, concurrent vehicle

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) on working days and once a day (in the morning) on Saturdays, Sundays and public holidays.


CLINICAL OBSERVATIONS: Yes
- Time schedule: The clinical observation was performed on each animal at least three times (before, during and after exposure) on exposure days and once a day during pre-exposure and post exposure observation days. On exposure-free weekends and post exposure observation weekends, no clinical observation was performed. Signs and findings were recorded for each animal.
- During exposure only a group wise examination was possible.


BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed prior to the pre-exposure period, at the start of the exposure period (day 0) and twice weekly thereafter until termination of the exposure. The body weight was determined once weekly during the post-exposure observation period.
- During the exposure period, the body weight change of the respective week was calculated as the difference of Friday to the previous Monday. Those of the weekends was calculated as the difference of Monday to the previous Friday.
- During the recovery period, the body weight change was determined weekly as the difference to the last weighing.


FOOD CONSUMPTION: Yes
- Time schedule for examinations: Food consumption was determined twice weekly (Monday and Friday) During post-exposure period, food consumption was determined once weekly.
- The animals were maintained in social-housing cages, with 5 animals per cage, during the whole study period. Therefore, the food consumption was determined cage-wise. The food consumption per animal and day was calculated by dividing food consumption of the day of a respective cage by the 5 animals per cage. As the animals of each test group were housed in only two cages per sex, no statistical evaluation of food consumption is possible.


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before the start of the exposure period (day -3 for males and day -4 for females) the eyes of all animals, and at the end of the study the eyes of the animals (study day 88 and 89 for males, and day85 for females) were examined for any changes in the refracting media with an ophthalmoscope (HEINE Optotechnik, Herrsching, Germany) after administration of a mydriatic (Mydrum, Chauvin ankerpharm GmbH)


HAEMATOLOGY: Yes
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table 4-5 were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table 4-5 were examined.


BRONCHOALVEOLAR LAVAGE FLUID (BAL): Yes
- Time schedule for analysis: main groups (day 90/91), recovery groups (day 150)
- Number of animals: all
- Dose groups that were examined: all groups
- Parameters checked in table 17 - 20 were examined.
- The animals designated for lung lavage were killed by exsanguination from aorta abdominalis and vena cava under Narcoren® anesthesia. The lung was lavaged by two instillations of physiologic saline.
- The following examinations were carried out:
-- Cytology in BAL: Total cell counts were determined using a haematology analyzer (Advia 120 Siemens Diagnostics, Fernwald, Germany). Cytocentrifuge preparations were stained according to Wright and evaluated microscopically. Parameters checked in table 6 were examined.
-- Total Protein and enzymes in BAL: An automatic analyzer (Cobas c501; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters. Parameters checked in table 7 were examined.


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No


IMMUNOLOGY: No


LUNG BURDEN: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 8 for organ weights)
HISTOPATHOLOGY: Yes (see table 8 and 9)

Statistics:

Means, medians and standard deviations of each test group were calculated for several parameters.
Table 10 contains the statistical analyses used in this report.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During the pre-exposure period the animals showed no clinical signs and findings different from normal.

During the exposure period, all animals of the high concentration male and female animals showed substance contaminated fur and discoloration of the fur. This finding was also observed in a few male and female animals of the mid concentration (5 mg/m³), as well as in individuals of female animals of the low concentration group (1 mg/m³). This finding was considered treatment-related but not adverse, because the substance was a pigment.

In one male animal of the mid concentration (No. 28), swelling right ear was observed from study day 49 onward. The restrainer tube was likely to press on the swelling ear and cause pain. For animal welfare reason, no exposure was performed from study day 53 onward. Because the swelling persisted, due to the missing exposures, this animal was not comparable with the other animals of this group. On study day 56, the animals was sacrificed prematurely. This finding was only observed in this one individual; therefore, it was considered incidental.

During recovery period, a small injury was noted in the head region of one control male animal. This finding was considered not treatment-related.

Mortality:

no mortality observed

Description (incidence):

No deaths were recorded throughout the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight of the main group animals:

The body weight of the main group male and female animals of the low concentration (1 mg/m³) and mid concentration (5 mg/m³) were not statistically significantly different from the control group 0, as well as male animals of the high concentration group (20 mg/m³).

The body weight of the female animals of the high concentration (20 mg/m³) was statistically significantly increased about 5.9 % and 5.3 % on day 13 and 17, respectively.

Body weight of the recovery group animals:
The mean body weight of the male recovery group animals was statistically not different to the control throughout the pre-exposure, exposure and post-exposure period. There were no female animals used for recovery period.

Body weight change:

The body weight change of the male animals of the low concentration (1 mg/m³) and female animals of all exposed groups was comparable with the control group throughout the study period.

The body weight change of the male animals of the main group mid concentration (5 mg/m³, test group 2) was statistically different on the following time point:

• Week 8 (Mo – Fr): -4.9 g (p≤ 0.05, concurrent control was -10.6 g)

The body weight change of the male animals of the recovery group mid concentration (5 mg/m³, test group 12,) was statistically different on the following time point:

• Day 31 to 35: -7.3 g (p≤ 0.01, concurrent control was +0.2 g)
• Day 45 to 49: -7.1 g (p≤ 0.01, concurrent control was -1.7 g)
• Day 91 to 98: +24.4 g (p≤ 0.05, concurrent control was +20.2 g)

The body weight change of the male animals of the main group high concentration (20 mg/m³) was statistically different on the following time point:

• Week 8 (Mo – Fr) test group 3: -4.8 g (p≤ 0.05, concurrent control was -10.6 g)
• Week 8 (Fr – Mo) test group 3: +10.0 g (p≤ 0.01, concurrent control was +13.5 g)
• Week 10 (Fr – Mo) test group 3: +3.9 g (p≤ 0.05, concurrent control was +9.4 g)
• Week 11 (Mo – Fr) test group 3: +0.5 g (p≤ 0.05, concurrent control was -6.9 g)
• Week 12 (Mo – Fr) test group 3: -5.0 g (p≤ 0.05, concurrent control was -9.6 g)

The body weight change of the male animals of the recovery group high concentration (20 mg/m³, test group 13,) was statistically different on the following time point:

• Day 91 to 98: +25.9 g (p≤ 0.05, concurrent control was +20.2 g)

The above listed mean body weight changes differed only a few grams from the concurrent control group and were of transient nature. None of them led to significantly lower mean body weight. Therefore, they were considered not biologically relevant.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No substance-related changes of food consumption were observed during the whole study period.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Spontaneous findings such as remainders of the pupillary membrane or corneal stippling were observed in several animals of all test groups and the control group without any concentration-response relationship.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related, adverse changes among hematological parameters were observed.

After the administration period, in males and females of test group 3 (20 mg/m³) absolute neutrophil counts were significantly increased. This was also true for absolute neutrophil counts in males of test group 2 (5 mg/m³). However, neutrophil counts in males of both test groups were within the historical control range, those of females of test group 3 slightly above this range (absolute neutrophil, males 0.77-1.31 Giga/L, females 0.40-0.82 Giga/L). This was the only changed differential blood cell counts among these individuals. Therefore, the alteration in males was regarded as incidental and not treatment related, that one in females as non-adverse if it was treatment related at all (ECETOC Technical Report No. 85, 2002).

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among clinical chemistry parameters were observed.

After the administration period, in females of test group 3 (20 mg/m3) total bilirubin values were significantly decreased, but the values were within the historical control range (females, total bilirubin 1.38-2.71 µmol/L). Therefore, this alteration was regarded as incidental and not treatment related.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute weights (main groups):
- When compared with control group 0 (=100%), the mean absolute weights of lungs (male and female), liver (female) and spleen (female) were significantly increased in test group 3 (see table 21). All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights (main groups):
- When compared with control group 0 (=100%), the following mean relative organ weights of lungs (male/female) and spleen (female) were significantly increased in test group 3 (table 22).
- The significantly increased absolute and relative lung weights of males (30.5%; +27%) and females (+ 37.8%; + 31.2%) of test group 3 (20 mg/m³) was assumed to be treatment related.
- The significantly increased absolute liver weight in females of test group 3 (5.182 g; +10.9%) was within the historical control range (4.886g – 5.200g) and without histological correlate and was regarded as incidental and not treatment-related.
- The absolute and relative spleen weight in females of test group 3 (0.441g; 0.221%) was significantly increased. The absolute spleen weight was within the historical control range (0.417g – 0.460g), the relative spleen weight was slightly above the historical control range (0.206% - 0.22%). No histological correlate was detected and therefore the weight increase was regarded as incidental.
- All other mean relative weight parameters did not show significant differences when compared to the control group 0.

Absolute weights (recovery groups):
- When compared with control group 10 (=100%), the mean absolute weights of the lungs (males) were significantly increased in test group 13 (see table 27).
All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights (recovery groups):
- When compared with control group 10 (=100%), the mean relative organ weights of the lungs (males) were significantly increased in test group 3 (see table 28). The significant increase of absolute and relative weight of the lungs of test group 13 (+ 41.1%; + 37.0%) was regarded as treatment related. All other mean relative weight parameters did not show significant differences when compared to the control group 0.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross lesions (main groups):
- See table 23
- A red discoloration was observed in the lungs of 8 out of 10 males of test group 2, in all males of test group 3 and in all females of test group 2 and 3. This finding correlated with the histologically diagnosed alveolar histiocytosis with particles and was regarded as treatment related.
- Additionally, a red discoloration was noted in mediastinal lymph nodes of 9 out of 10 males of test group 2, all males of test group 3, 1 out of 10 females of test group 1 and all females of test group 2 and 3. This finding correlated with macrophage aggregates with particles detected histologically in the mediastinal lymph node and was regarded as treatment related.
- All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Gross lesions (recovery groups):
- See table 29
- A red discoloration was observed in lungs of all males of test groups 12 and 13. This finding correlated with the histologically diagnosed alveolar histiocytosis with particles and was regarded as treatment related.
- A red discoloration was noted in mediastinal and tracheobronchial lymph nodes of males of test group 12 and 13 with additionally enlargement of the mediastinal lymph node of males of test group 13. The described findings correlated with macrophage aggregated with particles detected histologically and was regarded as treatment related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathology (main groups)
- The treatment-related findings were observed in the lungs, the mediastinal and tracheobronchial lymph nodes and the nasal cavity (level III and IV, only) with incidences and grading according to table 24
- In the lung, macrophages, which contained dark red to black particles (presumably inhaled test compound) were multifocally distributed within the lumen of alveoli and terminal bronchioles, with a concentration-dependent increase in number of macrophages and amount of particles storage within each macrophage. The severity was minimal in test group 1 (1 mg/m³) up to moderate to marked in test group 3 (20 mg/m³) and histiocytosis was present in lungs of all treated animals.
- In test groups 2 (5 mg/m³) and 3 (20 mg/m³), aggregation of particle-laden macrophages was detected in the terminal bronchioles. The affected terminal bronchioles displayed a minimal to moderate hyperplasia/hypertrophy and the surrounding alveoli a minimal to moderate hyperplasia of type II pneumocytes. A lymphohistiocytic infiltrate with particle-laden macrophages, which was mostly located perivascular, was noted interstitially in those areas.
- Cellular debris, including free particles (presumably originating from remnants of alveolar macrophages which have undergone necrosis), accompanied by infiltrating neutrophils were detected in alveoli of males and females of test groups 2 and 3.
- In test group 1, the number of alveolar macrophages containing particles was only minimally increased. The described epithelial (hyperplasia/hypertrophy of terminal bronchioles, hyperplasia of type II pneumocytes) or inflammatory changes (lymphohistiocytic or neutrophilic infiltrate) were not detected.
- In the BALT of males and females of all test groups, macrophages containing comparable particles as described within the alveoli, were detected. Aggregation of macrophages was noted in males and females of test group 2 and 3.
- Additionally intravascular macrophages containing intracytoplasmic particles were noted in in males and females of all test groups. All described findings were regarded as treatment related.
- Tracheobronchial and mediastinal lymph nodes: Within the lymph nodes of animals of test groups 1, 2 and 3 (1, 5 and 20 mg/m³), histiocytes containing comparable red to black particles within their cytoplasm as observed in the lungs, were present. Macrophages were either individually distributed within the lymph nodes or formed aggregates. The findings were regarded to be treatment related (table 25).
- Nasal cavity (level III and level IV): Within the nasal associated lymphatic tissue (NALT) of males and females of test groups 1, 2 and 3, few macrophages containing red to black particles within their cytoplasm were detected. This finding was regarded as treatment related (table 26). All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.


Histopathology (recovery groups)
- The treatment-related findings were observed in the lungs, the mediastinal and tracheobronchial lymph nodes and the nasal cavity (level III and IV only) with incidences and grading according to tables 30.
- In general, histopathological findings in the lungs of males of the recovery test group 12 (5 mg/m3) and 13 (20 mg/m3) were comparable to those observed in the corresponding main test groups 2 (5 mg/m3) and 3 (20 mg/m3).
- The number of alveolar histiocytes was comparable in main and recovery groups, however the number of intracytoplasmic dark red to black particles was slightly reduced in recovery groups. Slight differences were also detected in the distribution of alveolar particle-laden macrophages: The formation of macrophage aggregates in terminal bronchioles and surrounding alveoli was slightly increased, whereas the number of individual particle-laden macrophages within alveoli was slightly reduced. Macrophage aggregates were accompanied by hypertrophy/hyperplasia of terminal bronchioles, hyperplasia of type II pneumocytes and an interstitial, often perivascular located, lymphohistiocytic infiltrate with particle-containing macrophages.
- Cellular debris, neutrophilic infiltrate within alveoli, intravascular macrophages with particles and macrophages/macrophages aggregates containing particles within the bronchus associated lymphatic tissue (BALT) was comparable in animals of test group 12 and 13 to animals of the corresponding main test group 2 and 3.
- The described findings were regarded to be treatment-related.
- Tracheobronchial and mediastinal lymph nodes: In the mediastinal and tracheobronchial lymph nodes of test groups 12 (5 mg/m3) and 13 (20 mg/m3) comparable findings (macrophages with particles and macrophage aggregates with particles) with a comparable severity to those observed in the corresponding main test groups 2 (5 mg/m3) and 3 (20 mg/m3) were observed (table 31).
- Nasal cavity (level III and level IV): See table 32. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BRONCHOALVEOLAR LAVAGE FLUID (BAL)
See table 17 - 20.
After the administration period in BAL of males and females in test group 3 (20 mg/m3) total cell counts as well as absolute and relative lymphocyte, neutrophil and monocyte counts were significantly increased. Absolute macrophage counts were also significantly higher compared to controls only in males whereas relative macrophage counts were significantly lower in both sexes. Absolute lymphocyte, neutrophil and monocyte counts were already significantly higher in BAL of males and females of test group 2 (5 mg/m3). This was also true for relative lymphocyte counts in BAL of females as well as relative neutrophil and monocyte counts in BAL of both sexes in this test group. Relative macrophage counts were significantly decreased in BAL of males and females of this test group. These alterations were regarded as treatment related and adverse.

After the eight-week recovery period, total cell counts in males of test group 13 20 (mg/m3) were as high increased as after the administration period. Absolute and relative lymphocyte and monocyte in males of test groups 12 and 13 (5 and 20 mg/m3) were also higher increased compared to those after the administration period (monocytes not statistically significantly). In contrast absolute and relative neutrophil counts were statistically significantly increased in males of test groups 12 and 13, but not as great as in the correspondent groups after the administration. Absolute eosinophil counts were also relevantly increased in males of test groups 12 and 13, although not statistically significantly. Relative macrophage counts were significantly lower in males of test groups 12 and 13 compared to study controls.

After the administration period, in BAL of males and females of test group 3 (20 mg/m3) total protein levels as well as gamma-glutamyl-transferase (GGT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and -N-acetyl glucosaminidase (NAG) activities were significantly increased with the most prominent activity increase of LDH. After an eight-week recovery period, in males of test group 13 (20 mg/m3) total protein levels as well as LDH and NAG activities were higher compared to the administration period. ALP and GGT activities were also still significantly increased. These alterations were regarded as treatment related and adverse.

After the administration period, in BAL of males and females in test group 2 (5 mg/m3) LDH, ALP and GGT activities were already significantly higher compared to controls. This was also true for total protein levels in females of this test group as well as LDH activity in males of test group 1 (1 mg/m3). However, the increases were marginal (≤ 2-fold increase) and therefore were regarded as treatment related but non-adverse. After the recovery period, in males of test group 12 (5mg/m3) LDH and ALP activity as well as total protein levels were significantly increased. ALP activity and total protein level changes were marginal (≤ 2-fold increase). The mean LDH activity was the same as in males of test group 1 after the administration period, but the LDH activity in the study controls was very low after the recovery period. Therefore, the fold-increase of LDH in males of test group 2 after the recovery would have been also below 2 if study control LDH activity was as high as after the administration period. In conclusion, the increase of LDH after the recovery period in males of test group 12 was regarded as marginal and therefore treatment related, but non-adverse.

Details on results:

During the exposure period, the target concentrations were maintained as constant and stable as could be provided with dust aerosol generation techniques in the concentration range tested. Particle size distribution measurement demonstrated that the aerosols were respirable for rats.

During the exposure period, all animals of the high concentration male and female animals showed substance contaminated fur and discoloration of the fur. This finding was also observed in a few male and female animals of the mid concentration (5 mg/m³), as well as in individuals of female animals of the low concentration group (1 mg/m³). This finding was considered treatment-related but not adverse, because the substance was a pigment. During the recovery period, no clinical signs of toxicity was observed. There were no biologically relevant changes of body weight development. There were no substance-related changes noted in food consumption and ophthalmology.

Regarding clinical pathology, no treatment-related adverse effects in systemic clinical pathology blood parameters were observed up to a dose of 20 mg/m³.
After the administration period, in the bronchoalveolar lavage (BAL) of rats of both sexes in test group 3 (20 mg/m3) an acute inflammation was observed by increases of total cell counts as well as absolute macrophage, lymphocyte, neutrophil and monocyte counts. In consequence also total protein levels in BAL as well as -glutamyl-transferase (GGT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and -N-acetyl glucosaminidase (NAG) activities in BAL of both sexes of this test grouped were significantly increased. In test group 2 (5 mg/m³) absolute neutrophil, lymphocyte and monocyte counts in BAL of both sexes were also significantly increased whereas total cell counts as well as total protein and enzyme activities were not relevantly (>2-fold) increased.
After the eight-week recovery period, in males of test group 13 (20 mg/m³) total protein levels as well as LDH and NAG activities were higher compared to the administration period. ALP and GGT activities were also still significantly increased. Regarding BAL cytology, higher absolute lymphocyte and monocyte counts but lower neutrophil counts indicated the change from a granulocytic to lymphocytic-monocytic inflammation. In males of test group 12 (5 mg/m³) total protein level, LDH and ALP activity were not relevantly increased anymore, but total cell counts as well as absolute neutrophil, lymphocyte and monocyte counts were already significantly increased.

Regarding pathology, the target organs were lungs, mediastinal and tracheobronchial lymph nodes and the nasal cavity.

Main groups

The absolute and relative lung weights of males and females of test group 3 (20 mg/m³) were significantly increased (males: + 30.5%; + 27%; females + 37.8%; + 31.2%). The weight increases correlated to a macroscopically detected red discoloration of the lungs in all males and females of test group 3 (20 mg/m³) and all females and 8 males of test group 2 (5 mg/m³). The discoloration was correlated to a slightly to marked increase in numbers of alveolar macrophages (histiocytosis) which contained dark red to black pigment (presumably the inhaled test substance). In animals of test group 2 and 3 the macrophages were partly randomly distributed within the alveoli and partly formed aggregates in the terminal bronchioles and adjacent alveoli. The surrounding epithelial structures (terminal bronchioles and alveolar epithelium) showed a minimal to moderate hyperplasia/hypertrophy of terminal bronchioles and hyperplasia of pneumocytes type II. Interstitial thickening by a minimal to moderate lymphohistiocytic infiltrate, with particle-laden macrophages was frequently observed in affected areas. Infiltration of neutrophils and presence of cellular debris and free particles (presumably due to alveolar macrophages which have previously undergone cell death) were seen in males and females of test groups 2 and 3 and were interpreted as an ongoing inflammatory process. The combination of all above-described findings were regarded as treatment related and adverse.
Additionally, in the bronchus associated lymphatic tissue (BALT) in animals of test group 2 and 3 a minimal to mild number of macrophages with particles and a minimal to moderate number of macrophage aggregates with particles were detected. Macrophages with particles were seen in vessels in the lungs. Those findings were regarded as sign of the physiologically clearance of the inhaled test substance and were regarded as treatment related but not adverse.

In lungs of all males and females of test group 1 (1 mg/m³), a minimal increased number of alveolar histiocytes (histiocytosis), which contained dark red to black particles was detected. The histiocytes were randomly distributed within the alveoli. Aggregation of histiocytes was not detected. Individual particle-laden macrophages were detected in the BALT and intravascularly in the lungs. Hyperplasia/hypertrophy of terminal bronchioles or hyperplasia of type II pneumocytes or any other signs of inflammation were not observed in animals of test group 1. This led to the interpretation of described changes as treatment related but not adverse.

A minimal to slight number of particle-laden macrophages with minimal to marked formation of macrophage aggregates were present in tracheobronchial and mediastinal lymph nodes of animals of all test groups.

Additionally, in the nasal cavity (level III and level IV) of males and females of all test groups few individual macrophages with intracytoplasmic particles were detected in the nasal associated lymphatic tissue (NALT).
The findings in mediastinal and tracheobronchial lymph nodes, as well as the described findings in the nasal cavity were considered to be treatment related and not adverse, since they are not associated with inflammation or cell injury and only reflect an increased clearance of the inhaled pigment.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Recovery groups

The absolute and relative lung weights was significantly increased in males test group 13 (+ 41.1%; + 37.0%). This was, as in the main group, accompanied by a macroscopically detected red discoloration of the lungs and histologically detected minimal to marked increase in numbers of alveolar macrophages which contain particles in males of test groups 12 (5 mg/m³) and 13 (20 mg/m³). The number of alveolar macrophages in the main test groups 2 and 3 and the corresponding recovery test groups 12 and 13 was comparable, but the distribution of alveolar macrophages varied slightly: The number of individual macrophages within alveoli was slightly decreased and the number of aggregates within terminal bronchioles and adjacent alveoli was more pronounced in the recovery test groups. The morphology and severity of the epithelial (hypertrophy/hyperplasia of terminal bronchioles and type II hyperplasia) and inflammatory changes (cellular debris, neutrophilic infiltrate within alveoli, lymphohistiocytic interstitial infiltrate) was comparable to the lesions described in the main test groups. All above mentioned lesions were regarded as treatment related and adverse.

Additionally, in males of test group 12 and 13 a minimal to mild number of macrophages with particles and a minimal to moderate number of macrophage aggregates with particles in the bronchus associated lymphatic tissue (BALT), as well as intravascular macrophages with particles were observed. Those findings were regarded as signs of the physiologically clearance of the inhaled test substance and were regarded as treatment related but not adverse.

A minimal to slight number of particle-laden macrophages and a minimal to marked number of particle- laden macrophage aggregates were present in tracheobronchial and mediastinal lymphnodes of animals of all test groups. Those findings are considered to be treatment related and not adverse, since they are not associated with inflammation or cell injury and only reflect an increased clearance of the inhaled pigment.

Additionally in the nasal cavity (level III and leven IV) of males and females of all test groups few individual macrophages with intracytoplasmatic particles were detected in the nasal associated lymphatic tissue (NALT). No signs of inflammation or cell injury was present and therefore this finding is as well regarded as treatment related but not adverse.

In general, findings observed in the lungs, the tracheobronchial and mediastinal lymph nodes and the nasal cavity of the recovery test groups 12 and 13 were comparable to those of the corresponding main groups 2 and 3.

All other mean relative weight parameters did not show significant differences when compared to the control group 0.

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

20 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse treatment-related systemic effects observed up to the highest tested dose.

Dose descriptor:

LOAEC

Remarks:

local

Effect level:

5 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

other: BAL parameters

Dose descriptor:

NOAEC

Remarks:

local

Effect level:

1 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse treatment-related effects observed at this dose.

Critical effects observed:

yes

Lowest effective dose / conc.:

5 mg/m³ air (analytical)

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

presumably yes

Table 11: Study means and standard deviations of test substance concentrations

 

Test group	Target concentration (mg/m³)	Measured concentration (mg/m³)		Nominal concentration (mg/m³)	Effectiveness of dust generation (%)
		Mean	SD
1	1	1.0	0.1	1.6	62.5
2	5	5.0	0.2	7.7	64.9
3	20	20.0	1.0	27.8	71.9

 

 

Table 12: Mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) measured by cascade impactors during the exposure period

Target concentration	Measurement	MMAD (µm)	GSD	Fraction < 3 µm
1 mg/m³   (Test group 1)	1	0.68	3.40	88.8 %
	2	0.70	2.81	92.1 %
	3	0.64	2.89	92.7 %
	4	0.59	2.88	93.7 %
	5	0.57	2.79	94.8 %
	6	0.58	2.57	95.9 %
	7	0.58	2.50	96.3 %
	8	0.57	2.48	96.6 %
	9	0.60	2.29	97.3 %
	10	0.59	2.50	96.1 %
	11	0.56	2.70	95.5 %
	12	0.54	3.19	93.0 %
	13	0.53	3.02	94.1 %
	Mean	0.59	2.77	94.4 %
	SD	0.05	0.31	2.3 %
	Median	0.58	2.79	94.8 %
5 mg/m³ (Test groups 2 and 12)	1	0.68	3.31	89.2 %
	2	0.73	2.83	91.3 %
	3	0.65	2.70	93.8 %
	4	0.61	3.02	92.5 %
	5	0.58	3.41	91.0 %
	6		2.96	93.1 %
	7	0.64	2.81	93.2 %
	8	0.63	2.49	95.7 %
	9	0.61	2.44	96.3 %
	10	0.58	2.77	94.8 %
	11	0.56	3.48	91.1 %
	12	0.59	2.87	93.9 %
	13	0.57	3.31	91.8 %
	Mean	0.62	2.95	92.9 %
	SD	0.05	0.34	2.0 %
	Median	0.61	2.87	93.1 %
20 mg/m³   (Test groups 3 and 13)	1	0.63	3.32	90.4 %
	2	0.62	2.63	94.9 %
	3	0.59	2.53	96.0 %
	4	0.75	2.42	94.2 %
	5	0.60	2.82	94.0 %
	6	0.58	3.10	92.6 %
	7	0.55	3.07	93.5 %
	8	0.62	2.57	95.3 %
	9	0.60	2.51	96.0 %
	10	0.59	2.71	94.8 %
	11	0.54	2.87	94.7 %
	12	0.50	2.92	95.2 %
	13	0.58	2.93	93.6 %
	Mean	0.60	2.80	94.2 %
	SD	0.06	0.27	1.5 %
	Median	0.59	2.82	94.7 %

 

 

Table 13: Mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) measured by APS during the exposure period at low concentration of 1 mg/m³

Target concentration	Measurement Day	Measurement No.	MMAD (µm)	GSD
1 mg/m³   (Test group 1)	20 Jul 2021	1	1.04	2.04
		2	1.34	3.40
		3	1.21	2.58
	27 Jul 2021	1	1.32	2.49
		2	1.32	2.46
		3	1.43	2.84
	04 Aug 2021	1	1.04	1.96
		2	1.34	3.26
		3	1.07	1.94
	11 Aug 2021	1	1.09	2.45
		2	1.24	2.78
		3	1.14	2.54
	18 Aug 2021	1	1.30	3.17
		2	1.44	3.81
		3	1.32	3.11
	24 Aug 2021	1	1.52	3.16
		2	1.28	2.29
		3	1.46	3.15
	01 Sep 2021	1	1.38	2.93
		2	1.43	2.96
		3	1.48	3.03
	Mean ± SD		1.29 ± 0.15	2.78 ± 0.49
	Median		1.32	2.84

 

 

Table 14: Mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) measured by APS during the exposure period at mid concentration of 5 mg/m³

Target concentration	Measurement Day	Measurement No.	MMAD (µm)	GSD
5 mg/m³ (Test groups 2 and 12)	19 Jul 2021	1	1.60	2.96
		2	1.56	2.78
		3	1.69	3.10
	26 Jul 2021	1	1.58	2.73
		2	1.72	2.98
		3	1.61	2.80
	02 Aug 2021	1	1.58	3.24
		2	1.41	2.71
		3	1.65	3.23
	12 Aug 2021	1	1.57	3.01
		2	1.56	3.03
		3	1.69	3.22
	16 Aug 2021	1	1.84	3.26
		2	1.68	3.13
		3	1.71	3.06
	23 Aug 2021	1	1.74	3.17
		2	1.96	3.26
		3	1.93	3.35
	30 Aug 2021	1	1.92	3.13
		2	1.91	3.10
		3	2.00	3.24
	Mean ± SD		1.71 ± 0.16	3.07 ± 0.19
	Median		1.69	3.10

 

 

Table 15: Mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) measured by APS during the exposure period at high concentration of 20 mg/m³

Target concentration	Measurement Day	Measurement No.	MMAD (µm)	GSD
20 mg/m³ (Test groups 3 and 13)	19 Jul 2021	1	3.02	3.21
		2	3.04	3.30
		3	2.92	3.28
	26 Jul 2021	1	5.90	3.20
		2	4.92	3.30
		3	5.20	3.29
	02 Aug 2021	1	6.16	3.09
		2	6.82	3.19
		3	6.13	3.14
	12 Aug 2021	1	6.46	3.23
		2	5.46	3.19
		3	4.86	3.27
	16 Aug 2021	1	6.30	3.35
		2	6.66	3.22
		3	6.28	3.25
	23 Aug 2021	1	5.80	3.09
		2	6.80	3.03
		3	7.05	3.10
	30 Aug 2021	1	6.51	3.13
		2	6.57	3.09
		3	6.31	3.13
	Mean ± SD		5.67 ± 1.27	3.19 ± 0.09
	Median		6.16	3.20

 

 

Table 16: Particle count distribution measured by SMPS

Target concentration	Measurement	Total count concentration (N/cm³)	Geometric mean diameter
1 mg/m³	1	12489	368
	2	13523	348
	3	15636	359
	4	14403	348
	5	13092	351
	6	16317	355
	7	15024	347
	8	16132	341
	9	16898	337
	10	15550	339
	11	22499	349
	12	13790	337
	13	18160	338
	Mean ± SD	15655 ± 2613	347 ± 9
	Median	15550	348
5 mg/m³	1	64553	362
	2	66474	354
	3	69855	359
	4	68419	352
	5	69180	345
	6	70556	349
	7	76433	340
	8	89304	346
	9	81272	344
	10	91225	330
	11	80258	345
	12	86530	338
	13	94320	339
	Mean ± SD	77568 ± 10258	346 ± 9
	Median	76433	345
Target concentration 20 mg/m³	1	167993	354
	2	220310	344
	3	242891	333
	4	271284	324
	5	221537	343
	6	214915	344
	7	206028	310
	8	167409	330
	9	242184	319
	10	244227	319
	11	215953	335
	12	232199	325
	13	281324	328
	Mean ± SD	225250 ± 33534	331 ± 12
	Median	221537	330

 

 

Table 17: Changes in mean absolute cell counts in BAL (x-fold of concurrent control) in male rats on study days 91 (main groups, 1 day after last exposure, N = 10) and after an 8-week recovery period on study day 150 (N = 5).

Analyte	Study day 90/91			Study day 150
	Gr. 1 1 mg/m^3	Gr. 2 5 mg/m^3	Gr. 3 20 mg/m^3	Gr. 12 0.5 mg/m^3	Gr.13 20 mg/m^3
Total Cells	1.2	1.3	8.6**	1.6*	7.0**
Macrophages	1.3	1.0	1.6**	0.9	1.4
Lymphocytes	1.0	1.8*	20.7**	5.9**	38.3**
Neutrophils	1.2	17.5**	332.4**	8.3**	58.6**
Monocytes	2.2	4.8**	52.7**	8.7	49.4**
Eosinophils	0.3	0.0	1.1	4.0	8.9
Epithelial cells	1.0	1.2	1.1	1.3	0.0

One-sided Wilcoxon-test: * : p <= 0.05; ** : p <= 0.01

++ increase could not be calculated because of zero activity in controls

 

 

Table 18: Changes in mean absolute cell counts in BAL (x-fold of concurrent control) in female rats on study day 90/91 (main groups, 1 day after last exposure, N = 10).

Analyte	Study day 90
	Gr. 1 1 mg/m^3	Gr. 2 5 mg/m^3	Gr.3 20 mg/m^3
Total Cells	0.8	1.1	8.3**
Macrophages	0.8	0.7	1.3
Lymphocytes	0.9	8.0**	74.7**
Neutrophils	1.8	6.5**	116.3**
Monocytes	++	++**	++**
Eosinophils	++	++	++
Epithelial cells	0.9	0.4	0.0

One-sided Wilcoxon-test: * : p <= 0.05; ** : p <= 0.01

++ increase could not be calculated because of zero activity in controls

 

 

Table 19: Changes in mean total protein and enzyme levels in BAL (x-fold of concurrent control) in male rats on study day 90 (main groups, 1 day after last exposure) and after an 8-week recovery period on study day 150.

Analyte	Study day 90/91			Study day 150
	Gr. 1 1 mg/m^3	Gr.2 5 mg/m^3	Gr.3 20 mg/m^3	Gr. 12 5 mg/m^3	Gr. 13 20 mg/m^3
Total Protein	0.8	0.7	3.0**	1.5*	6.1**
LDH	1.6*	1.5**	9.8**	2.6*	14.4**
ALP	1.1	1.3**	3.1**	1.5**	3.0**
NAG	1.1	1.0	1.8**	1.2	3.2**
GGT	1.2	1.3*	4.7**	1.2	2.8**

GGT = g-Glutamyl-transferase; LDH = Lactate dehydrogenase; ALP = Alkaline phosphatase;

NAG = b-N-Acetyl glucosaminidase, One-sided Wilcoxon-test: * : p <= 0.05; ** : p <= 0.01

 

 

Table 20: Changes in mean total protein and enzyme levels in BAL (x-fold of concurrent control) in female rats on study day 90/91 (main groups, 1 day after last exposure).

Analyte	Study day 90
	Gr. 1 1 mg/m^3	Gr. 2 5 mg/m^3	Gr. 3 20 mg/m^3
Total Protein	1.1	1.6**	6.6**
LDH	0.9	2.0**	12.5**
ALP	0.9	1.4**	4.2**
NAG	1.0	1.2	4.3**
GGT	0.9	1.5**	5.5**

GGT = g-Glutamyl-transferase; LDH = Lactate dehydrogenase; ALP = Alkaline phosphatase;

NAG = b-N-Acetyl glucosaminidase, One-sided Wilcoxon-test: * : p <= 0.05; ** : p <= 0.01

 

 

Table 21: Relative increase of absolute organ weights in main group animals

	Male animals			Female animals
Test group (mg/m³)	1 (1)	2 (5)	3 (20)	1 (1)	2 (5)	3 (20)
Lungs	+1.2%	+5.1%	+30.5%**	+2.3%	+12.1%	+37.8%**
Liver				+5.2%	+4.8%	+10.9%**
Spleen				+2.1%	+8.8%	+17.0%**

* : p <= 0.05, **: p <= 0.01

 

 

Table 22: Relative increase of relative organ weights in main group animals

	Male animals			Female animals
Test group (mg/m³)	1 (1)	2 (5)	3 (20)	1 (1)	2 (5)	3 (20)
Lungs	-0.8%	+4.1%	+27.0%	-0.8%	+11.0%	+31.2%
Spleen				-1.1%	+7.4%	+11.4%

* : p <= 0.05, **: p <= 0.01

 

 

Table 23: Incidence of gross lesions in main group animals

	Male animals				Female animals
Test group (mg/m³)	0 (0)	1 (1)	2 (5)	3 (20)	0 (0)	1 (1)	2 (5)	3 (20)
No. of animals	10	10	10	10	10	10	10	10
Lungs	10	10	10	10	10	10	10	10
·         Discoloration	0	0	8	10	0	0	10	10
Mediastinal lymph node	10	10	10	10	10	10	10	10
·         Discoloration	0	0	9	10	0	1	10	10

 

 

Table 24: Incidence and severity of histological findings in lungs of main group animals.

	Male animals				Female animals
Test group (mg/m³)	0 (0)	1 (1)	2 (5)	3 (20)	0 (0)	1 (1)	2 (5)	3 (20)
No. of animals	10	10	10	10	10	10	10	10
Lungs	10	10	10	10	10	10	10	10
Histiocytosis, alveolar, with particles	0	10	10	10	0	10	10	10
Grade 1	0	10	0	0	0	10	0	0
Grade 2	0	0	8	0	0	0	9	0
·         Grade 3	0	0	2	8	0	0	1	5
·         Grade 4	0	0	0	2	0	0	0	5
Hyperplasia, type II pneumocytes	0	0	10	10	0	0	10	10
·         Grade 1	0	0	7	2	0	0	4	2
·         Grade 2	0	0	3	7	0	0	6	7
·         Grade 3	0	0	0	1	0	0	0	1
Hypertrophy/ hyperplasia, terminal bronchioles	0	0	7	10	0	0	10	10
·         Grade 1	0	0	7	5	0	0	9	9
·         Grade 2	0	0	0	4	0	0	1	1
·         Grade 3	0	0	0	1	0	0	0	0
Infiltrate, interstitial, lymphohistiocytic, with particles	0	3	9	10	0	0	10	10
·         Grade 1	0	3	9	1	0	0	7	0
·         Grade 2	0	0	0	9	0	0	3	7
·         Grade 3	0	0	0	0	0	0	0	3
Debris, cellular	0	0	6	10	0	0	10	10
·         Grade 1	0	0	5	0	0	0	7	0
·         Grade 2	0	0	1	7	0	0	3	3
·         Grade 3	0	0	0	3	0	0	0	7
Infiltrate, neutrophilic	0	0	10	10	0	0	7	10
·         Grade 1	0	0	9	0	0	0	7	0
·         Grade 2	0	0	1	10	0	0	0	8
·         Grade 3	0	0	0	0	0	0	0	2
Balt: macrophages with particles	0	10	10	10	0	10	10	10
·         Grade 1	0	10	10	1	0	10	10	10
·         Grade 2	0	0	0	8	0	0	0	0
·         Grade 3	0	0	0	1	0	0	0	0
Balt: macrophage aggregates with particles	0	0	2	10	0	0	7	9
·         Grade 1	0	0	2	0	0	0	5	5
·         Grade 2	0	0	0	8	0	0	2	2
·         Grade 3	0	0	0	2	0	0	0	2
Macrophages with particles, intravascular	0	9	9	9	0	8	9	10

 

 

Table 25: Incidence and severity of histological findings in tracheobronchial and mediastinal lymph nodes of main group animals.

	Male animals				Female animals
Test group (mg/m³)	0 (0)	1 (1)	2 (5)	3 (20)	0 (0)	1 (1)	2 (5)	3 (20)
No. of animals	10	10	10	10	10	10	10	10
Mediastinal lymph node	10	10	10	10	9	10	10	10
Macrophages with particles	0	9	10	10	0	7	10	10
Grade 1	0	9	7	1	0	7	9	6
Grade 2	0	0	3	9	0	0	1	4
Macrophage aggregates with particles	0	4	9	10	0	3	9	10
·         Grade 1	0	4	0	0	0	3	3	0
·         Grade 2	0	0	9	3	0	0	6	5
·         Grade 3	0	0	0	3	0	0	0	5
·         Grade 4	0	0	0	4	0	0	0	0

 

 

Table 26: Incidence and severity of histological findings in nasal cavity of main group animals.

	Male animals				Female animals
Test group (mg/m³)	0 (0)	1 (1)	2 (5)	3 (20)	0 (0)	1 (1)	2 (5)	3 (20)
No. of animals	10	10	10	10	10	10	10	10
Nasal cavity level III	10	10	10	10	10	10	10	10
NALT: Macrophages with particles	0	0	7	7	0	1	7	8
Grade 1	0	0	7	7	0	1	7	8
Nasal cavity level IV	10	10	10	10	10	10	10	10
NALT: Macrophages with particles	0	1	2	5	0	0	2	6
·         Grade 1	0	1	2	5	0	0	2	6

 

 

Table 27: Relative increase of absolute lung weight in recovery group animals

	Male animals
Test group (mg/m³)	12 (5)	13 (20)
Lungs	+4.4%	+41.1%**

* : p <= 0.05, **: p <= 0.01

 

 

Table 28: Relative increase of relative lung weight in recovery group animals

	Male animals
Test group (mg/m³)	12 (5)	13 (20)
Lungs	+8.5%	+37.0%**

* : p <= 0.05, **: p <= 0.01

 

 

Table 29: Incidence of gross lesions in recovery group animals

	Male animals
Test group (mg/m³)	10 (0)	12 (5)	13 (20)
No. of animals	5	5	5
Lungs	5	5	5
·         Discoloration	0	5	5
Mediastinal lymph node	5	5	5
·         Discoloration	0	5	5
·         Size enlarged	0	0	2
Tracheobronchial lymph node	5	5	5
·         Discoloration	0	4	5

 

 

Table 30: Incidence and severity of histological findings in lungs of recovery group animals

	Male animals
Test group (mg/m³)	10 (0)	12 (5)	13 (20)
No. of animals	5	5	5
Histiocytosis, alveolar, with particles	0	5	5
Grade 1	0	1	0
Grade 2	0	5	0
·         Grade 3	0	0	3
·         Grade 4	0	0	2
Hyperplasia, type II pneumocytes	0	5	5
·         Grade 1	0	3	2
·         Grade 2	0	2	3
Hypertrophy/hyperplasia, terminal bronchioles	0	5	5
·         Grade 1	0	5	5
Infiltrate, interstitial, lymphohistiocytic, with particles	0	5	5
·         Grade 1	0	4	0
·         Grade 2	0	1	5
Debris, cellular	0	5	5
·         Grade 1	0	4	1
·         Grade 2	0	1	2
·         Grade 3	0	0	2
Infiltrate, neutrophilic	0	4	4
·         Grade 1	0	2	0
·         Grade 2	0	2	2
·         Grade 3	0	0	2
Balt: macrophages with particles	0	5	5
·         Grade 1	0	3	4
·         Grade 2	0	2	1
Balt: macrophage aggregates with particles	0	5	5
·         Grade 1	0	3	0
·         Grade 2	0	2	3
·         Grade 3	0	0	2
Macrophages with particles, intravascular	0	4	5

 

 

Table 31: Incidence and severity of histological findings in tracheobronchial and mediastinal lymph nodes of recovery group animals

	Male animals
Test group (mg/m³)	10 (0)	12 (5)	13 (20)
No. of animals	5	5	5
Macrophages with particles	0	3	5
Grade 1	0	1	0
Grade 2	0	2	5
Macrophage aggregates with particles	0	3	5
·         Grade 1	0	0	0
·         Grade 2	0	3	2
·         Grade 3	0	0	2
·         Grade 4	0	0	1

 

 

 

Table 32: Incidence and severity of histological findings in nasal cavity of recovery group animals

	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)
No. of animals	5	5	5
Nasal cavity level III	5	5	5
Nalt: Macrophages with particles	0	4	4
Grade 1	0	4	4
Nasal cavity level IV	5	5	5
Nalt: Macrophages with particles	0	1	4
·         Grade 1	0	1	4

 

 

Conclusions:

Inhalation exposure of the test substance for 90 days (65 exposures) caused concentration and clearly treatment-related changes of several lavage parameters, which correlated with the significantly increased lung weight and several histological changes in lungs. A No Observed Adverse Effect Concentration (NOAEC) for local effect was determined 1 mg/m³ under the current study conditions. All the above-mentioned local effects were not reversible within 8 weeks recovery period. There was not any indication for systemic effect as shown by clinical chemistry and hematology and histological examinations. A No Observed Adverse Effect Concentration (NOAEC) for systemic effect was the highest tested concentration of 20 mg/m³ under the current study conditions.

Executive summary:

Groups of male and female Wistar rats were exposed nose-only to the dust aerosol of the test item for 6 hours per day on 5 consecutive days a week for 90 days. The target concentrations were 1, 5 and 20 mg/m³. A concurrent control group was exposed to clean air. Daily clinical observations, body weights, food consumption, ophthalmology. Additional assessments including hematology, clinical chemistry of the blood, bronchoalveolar lavage, and histopathology according to the referenced guidelines were carried out at the termination of the study. A recovery period of 60 days was included in recovery groups of male animals.

The following treatment-related, adverse effects were observed:

 

Test group 3 (20 mg/m³, main group)

• Increased absolute and relative lymphocyte, neutrophil and monocyte counts in BAL of both sexes


• Increased absolute macrophage counts in BAL of males


• Decreased relative macrophage counts in BAL of both sexes


• Increased total protein levels as well as g-glutamyl-transferase (GGT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and b-N-acetyl glucosaminidase (NAG) activities in BAL of both sexes


• Increase of absolute and relative lung weights in males (+30.5%; +27%) and females (+ 37.8%; + 31.2%).


• Red discoloration of lungs of all males and all females.


• Moderate to marked alveolar histiocytosis with particles in all males and all females.


• Minimal to moderate hyperplasia of type II pneumocytes in all males and females.


• Hypertrophy/hyperplasia of terminal bronchioles in all males (minimal to moderate) and all females (minimal to slight).


• Slight to moderate cellular debris in all males and all females


• Neutrophilic infiltrates in all males (slight) and all females (slight to moderate)

 

Test group 13 (20 mg/m³, males only, recovery group)

• Increased total protein levels as well as g-glutamyl-transferase (GGT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and b-N-acetyl glucosaminidase (NAG) activities in BAL of males


• Increased total cell as well as absolute and relative neutrophil, monocyte and lymphocyte counts in males


• Increased ablute eosinophil counts in males


• Decreased relative macrophage counts in males


• Increase of absolute and relative lung weights in males (+30.5% ; +27%) and females (+ 41.1%; + 37.0%).


• Red discoloration the lungs of all males.


• Moderate to marked alveolar histiocytosis with particles in all males.


• Minimal to slight hyperplasia of type II pneumocytes in all males.


• Minimal hypertrophy/hyperplasia of terminal bronchioles in all males.


• Minimal to moderate cellular debris in all males.


• Minimal to moderate neutrophilic infiltrates in all males.

 

Test group 2 (5 mg/m³, main group)

• Increased absolute and relative neutrophil and monocyte counts in BAL of both sexes


• Decreased relative macrophage counts in BAL of both sexes


• Increased absolute lymphocyte counts in BAL of both sexes


• Increased relative lymphocyte counts in BAL of females


• Increased total protein levels as well as g-glutamyl-transferase (GGT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and b-N-acetyl glucosaminidase (NAG) activities in BAL of both sexes


• Red discoloration of lungs of 8 out of 10 males and all females.


• Slight to moderate alveolar histiocytosis with particles in all males and all females.


• Minimal to slight hyperplasia of type II pneumocytes.


• Hypertrophy/hyperplasia of terminal bronchioles in 7 out of 10 males (minimal) and all females (minimal to slight).


• Minimal to slight cellular debris in all males and all females.


• Neutrophilic infiltrates in all males (minimal to slight) and 7 out of 10 females (minimal).

 

Test group 12 (5 mg/m³, males only, recovery group)

• Increased total cell as well as absolute and relative neutrophil, monocyte and lymphocyte counts in males


• Increased ablute eosinophil counts in males


• Decreased relative macrophage counts in males


• Red discoloration the lungs of all males


• Minimal to slight alveolar histiocytosis with particles in all males.


• Minimal to slight hyperplasia of type II pneumocytes in all males.


• Minimal hypertrophy/hyperplasia of terminal bronchioles in all males.


• Minimal to slight cellular debris in all males.


• Minimal to slight neutrophilic infiltrates in all males.

 

Test group 1 (1 mg/m³)

No treatment-related, adverse effects.

 

Conclusion: Inhalation exposure of the test substance for 90 days (65 exposures) caused concentration and clearly treatment-related changes of several lavage parameters, which correlated with the significantly increased lung weight and several histological changes in lungs. A No Observed Adverse Effect Concentration (NOAEC) for local effect was determined 1 mg/m³ under the current study conditions. All the above-mentioned local effects were not reversible within 8 weeks recovery period. There was not any indication for systemic effect as shown by clinical chemistry and hematology and histological examinations. A No Observed Adverse Effect Concentration (NOAEC) for systemic effect was the highest tested concentration of 20 mg/m³ under the current study conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

20 mg/m³

Study duration:

subchronic

Experimental exposure time per week (hours/week):

30

Species:

rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Justification for type of information:

see attached justification

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

20 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse treatment-related systemic effects observed up to the highest tested dose.

Dose descriptor:

LOAEC

Remarks:

local

Effect level:

5 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

other: BAL parameters

Dose descriptor:

NOAEC

Remarks:

local

Effect level:

1 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse treatment-related effects observed at this dose.

Critical effects observed:

yes

Lowest effective dose / conc.:

5 mg/L air (analytical)

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

presumably yes

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 Jul 2021 - 27 Apr 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (90-Day (Subchronic) Inhalation Toxicity Study

Version / remarks:

25 June 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

24 January 2014

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)

Version / remarks:

August 1998

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Kaiser-Friedrich-Straße 7, 55116 Mainz

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier): BASF Colors & Effects
- Lot/batch number of test material: P 110014
- Purity: ≥ 98 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Expiry date: 18 Feb 2023
- The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Homogeneity: Homogenous


TREATMENT OF TEST MATERIAL PRIOR TO TESTING:
- See section "Details on inhalation exposure"

FORM AS APPLIED IN THE TEST:
- dust aerosol

OTHER SPECIFICS
- physical state: solid
- color: red

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

Reason for selection of the test species: Rats were selected since this rodent species is recommended in the respective test guidelines. Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.

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; Sandhofer Weg 7, 97633 Sulzfeld
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Arrival of the animals/experimental starting date (age: 7 weeks), Start of exposure (14-15 weeks)
- Weight at study initiation: 249.4 - 250.5 (test group mean)
- Fasting period before study: no
- Housing: together (5 animals per cage)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 weeks


DETAILS OF FOOD AND WATER QUALITY:
- The food used in the study was assayed for chemical as well as for microbiological contaminants. On the basis of the duration of use and the analytical findings with respect to chemical and microbiological contaminants, the food was found to be suitable.
- The drinking water is regularly assayed for chemical contaminants. On the basis of the analytical findings the drinking water was found to be suitable.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 45 - 65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 06 Jul 2021 To: 13 Dec 21

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 0.59 - <= 0.62 µm

Geometric standard deviation (GSD):

2.84

Remarks on MMAD:

The cascade impactor measurement showed comparable MMADs and GSDs in all 3 concentration groups. The calculated mass fractions of particles below 3 µm aerodynamic size ranged between 88.8 and 97.3 %. The generated aerosols were highly respirable for rats.


By APS the measured MMADs were generally higher than those measured by cascade impactor. Especially at the high concentration of 20 mg/m³, the measured MMAD by APS is almost 10 times that measured by cascade impactor.

According to the OECD Test Guideline 413 and the Guidance Document 39, multistage cascade impactors should be given preference. Other devices or physical principles may be used if equivalence to the cascade impactor can be shown (with regard to MMAD and GSD, including the mass concentration sensed). In this study, equivalence to the cascade impactor cannot be shown. Thus, the more reliable cascade impactor data are used to appraise the technical validity of this study. APS measurement stopped after first 7 weeks.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Generator systems: Solid particle generators (brush-generator), Aerosol mixing tube (stainless steel), Glass connecting pipe, Glass cyclonic separators
- Generation procedure: For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage mixed with conditioned dilution air and passed via the cyclonic separator into the inhalation system. The concentration was adjusted by varying the piston feed and by varying the brush rotation speed.
- Test substance flow and air flows: see table 11
- Exposure apparatus: The inhalation atmosphere was maintained inside aerodynamic exposure systems (INA 60, volume V = ca. 90 L) consisting of a cylindrical inhalation chamber made of stainless steel sheeting and cone shaped outlets and inlets.
- Method of holding animals in test chamber: The rats were restrained in glass exposure tubes. Their snouts projected into the inhalation chamber and thus they inhaled the aerosol.
- Method of conditioning air: The central air conditioning system provides cold air of about 15°C. This cold air passes through an activated charcoal filter, is adjusted to room temperature of 20 to 24°C and passes through a second particle filter (H13 (HEPA) Camfil Farr, Germany). The so generated conditioned air was used to generate inhalation atmospheres.
- Temperature, humidity, pressure in air chamber: 20.6 - 23.8°C, 37.3 and 48.3%. Both relative humidity and chamber temperature were within the required range of the guideline.
- Air flow rate: 5.0 - 7.0 m3/h
- Air change rate: 67 times per hour
- Method of particle size determination: The particle size analysis was carried out with a cascade impactor. Particle Size distribution of the test atmosphere were determined also with the Aerodynamic Particle Spectrometer APS 3321 (TSI, USA). MMAD and GSD is obtained directly by the piece of equipment used APS 3321. Frequency: Once weekly during the first 7 weeks, with 3 repeats on each day. Particle size distribution measurements with scanning mobility particle sizer: To determine the particle size distribution in the submicrometer range, each test atmosphere was measured with the Scanning Mobility Particle Sizer (SMPS; Grimm Aerosol Technik GmbH & Co KG, Ainring, Germany). The SMPS system comprises an Electrostatic Classifier (Model Vienna U-DMA) which separates the particles into known size fractions, and a Condensation Particle Counter (CPC) which measures particle count concentrations. The DMA was equipped with Am-241 neutralizer. The sampling duration was about 7 minutes. As a rule, 10 repeats were measured for each exposure concentration.
- 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: The concentrations of the inhalation atmospheres were determined by gravimetry. Real time monitoring of constancy of concentrations via Scattered light photometers (VisGuard (Sigrist).
- Samples taken from breathing zone: yes

VEHICLE: The test substance was used unchanged.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Gravimetrical method of analyses:
The concentrations of the inhalation atmospheres were determined by gravimetry.
A preweighed filter was placed into the filtration equipment. By means of a vacuum compressed air pump a defined volume of the dust/aerosol was drawn through the filter.
The dust concentration in mg/m³ was calculated from the difference between the weight of the pre-weighed filter and the weight of the filter after sampling, with reference to the sample volume of the inhalation atmosphere.
See table 11.

Real time monitoring of constancy of concentrations:
Scattered light photometers (VisGuard (Sigrist) were used to continuously monitor the constancy of concentrations of test substance aerosols in the inhalation systems. To this end the inhalation atmosphere was continuously sampled by the measuring devices. The measurements were recorded using line recorders and transferred to the automated measuring system.
Real time surveillance of the inhalation atmospheres with scattered light photometers generally proved the constancy of each concentration throughout the daily exposures

Duration of treatment / exposure:

90 days (+ 60 days recovery)

Frequency of treatment:

6 hours/day, 5 days/week

Dose / conc.:

20 mg/m³ air (analytical)

Dose / conc.:

5 mg/m³ air (analytical)

Dose / conc.:

1 mg/m³ air (analytical)

Dose / conc.:

0 mg/m³ air (analytical)

No. of animals per sex per dose:

exposure period: 10 animals/sex/dose
recovery group: 5 animals/sex/dose

Control animals:

yes, concurrent vehicle

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) on working days and once a day (in the morning) on Saturdays, Sundays and public holidays.


CLINICAL OBSERVATIONS: Yes
- Time schedule: The clinical observation was performed on each animal at least three times (before, during and after exposure) on exposure days and once a day during pre-exposure and post exposure observation days. On exposure-free weekends and post exposure observation weekends, no clinical observation was performed. Signs and findings were recorded for each animal.
- During exposure only a group wise examination was possible.


BODY WEIGHT: Yes
- Time schedule for examinations: The animals were weighed prior to the pre-exposure period, at the start of the exposure period (day 0) and twice weekly thereafter until termination of the exposure. The body weight was determined once weekly during the post-exposure observation period.
- During the exposure period, the body weight change of the respective week was calculated as the difference of Friday to the previous Monday. Those of the weekends was calculated as the difference of Monday to the previous Friday.
- During the recovery period, the body weight change was determined weekly as the difference to the last weighing.


FOOD CONSUMPTION: Yes
- Time schedule for examinations: Food consumption was determined twice weekly (Monday and Friday) During post-exposure period, food consumption was determined once weekly.
- The animals were maintained in social-housing cages, with 5 animals per cage, during the whole study period. Therefore, the food consumption was determined cage-wise. The food consumption per animal and day was calculated by dividing food consumption of the day of a respective cage by the 5 animals per cage. As the animals of each test group were housed in only two cages per sex, no statistical evaluation of food consumption is possible.


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / Not specified


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before the start of the exposure period (day -3 for males and day -4 for females) the eyes of all animals, and at the end of the study the eyes of the animals (study day 88 and 89 for males, and day85 for females) were examined for any changes in the refracting media with an ophthalmoscope (HEINE Optotechnik, Herrsching, Germany) after administration of a mydriatic (Mydrum, Chauvin ankerpharm GmbH)


HAEMATOLOGY: Yes
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table 4-5 were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table 4-5 were examined.


BRONCHOALVEOLAR LAVAGE FLUID (BAL): Yes
- Time schedule for analysis: main groups (day 90/91), recovery groups (day 150)
- Number of animals: all
- Dose groups that were examined: all groups
- Parameters checked in table 17 - 20 were examined.
- The animals designated for lung lavage were killed by exsanguination from aorta abdominalis and vena cava under Narcoren® anesthesia. The lung was lavaged by two instillations of physiologic saline.
- The following examinations were carried out:
-- Cytology in BAL: Total cell counts were determined using a haematology analyzer (Advia 120 Siemens Diagnostics, Fernwald, Germany). Cytocentrifuge preparations were stained according to Wright and evaluated microscopically. Parameters checked in table 6 were examined.
-- Total Protein and enzymes in BAL: An automatic analyzer (Cobas c501; Roche, Mannheim, Germany) was used to examine the clinicochemical parameters. Parameters checked in table 7 were examined.


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No


IMMUNOLOGY: No


LUNG BURDEN: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 8 for organ weights)
HISTOPATHOLOGY: Yes (see table 8 and 9)

Statistics:

Means, medians and standard deviations of each test group were calculated for several parameters.
Table 10 contains the statistical analyses used in this report.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During the pre-exposure period the animals showed no clinical signs and findings different from normal.

During the exposure period, all animals of the high concentration male and female animals showed substance contaminated fur and discoloration of the fur. This finding was also observed in a few male and female animals of the mid concentration (5 mg/m³), as well as in individuals of female animals of the low concentration group (1 mg/m³). This finding was considered treatment-related but not adverse, because the substance was a pigment.

In one male animal of the mid concentration (No. 28), swelling right ear was observed from study day 49 onward. The restrainer tube was likely to press on the swelling ear and cause pain. For animal welfare reason, no exposure was performed from study day 53 onward. Because the swelling persisted, due to the missing exposures, this animal was not comparable with the other animals of this group. On study day 56, the animals was sacrificed prematurely. This finding was only observed in this one individual; therefore, it was considered incidental.

During recovery period, a small injury was noted in the head region of one control male animal. This finding was considered not treatment-related.

Mortality:

no mortality observed

Description (incidence):

No deaths were recorded throughout the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight of the main group animals:

The body weight of the main group male and female animals of the low concentration (1 mg/m³) and mid concentration (5 mg/m³) were not statistically significantly different from the control group 0, as well as male animals of the high concentration group (20 mg/m³).

The body weight of the female animals of the high concentration (20 mg/m³) was statistically significantly increased about 5.9 % and 5.3 % on day 13 and 17, respectively.

Body weight of the recovery group animals:
The mean body weight of the male recovery group animals was statistically not different to the control throughout the pre-exposure, exposure and post-exposure period. There were no female animals used for recovery period.

Body weight change:

The body weight change of the male animals of the low concentration (1 mg/m³) and female animals of all exposed groups was comparable with the control group throughout the study period.

The body weight change of the male animals of the main group mid concentration (5 mg/m³, test group 2) was statistically different on the following time point:

• Week 8 (Mo – Fr): -4.9 g (p≤ 0.05, concurrent control was -10.6 g)

The body weight change of the male animals of the recovery group mid concentration (5 mg/m³, test group 12,) was statistically different on the following time point:

• Day 31 to 35: -7.3 g (p≤ 0.01, concurrent control was +0.2 g)
• Day 45 to 49: -7.1 g (p≤ 0.01, concurrent control was -1.7 g)
• Day 91 to 98: +24.4 g (p≤ 0.05, concurrent control was +20.2 g)

The body weight change of the male animals of the main group high concentration (20 mg/m³) was statistically different on the following time point:

• Week 8 (Mo – Fr) test group 3: -4.8 g (p≤ 0.05, concurrent control was -10.6 g)
• Week 8 (Fr – Mo) test group 3: +10.0 g (p≤ 0.01, concurrent control was +13.5 g)
• Week 10 (Fr – Mo) test group 3: +3.9 g (p≤ 0.05, concurrent control was +9.4 g)
• Week 11 (Mo – Fr) test group 3: +0.5 g (p≤ 0.05, concurrent control was -6.9 g)
• Week 12 (Mo – Fr) test group 3: -5.0 g (p≤ 0.05, concurrent control was -9.6 g)

The body weight change of the male animals of the recovery group high concentration (20 mg/m³, test group 13,) was statistically different on the following time point:

• Day 91 to 98: +25.9 g (p≤ 0.05, concurrent control was +20.2 g)

The above listed mean body weight changes differed only a few grams from the concurrent control group and were of transient nature. None of them led to significantly lower mean body weight. Therefore, they were considered not biologically relevant.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No substance-related changes of food consumption were observed during the whole study period.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Spontaneous findings such as remainders of the pupillary membrane or corneal stippling were observed in several animals of all test groups and the control group without any concentration-response relationship.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related, adverse changes among hematological parameters were observed.

After the administration period, in males and females of test group 3 (20 mg/m³) absolute neutrophil counts were significantly increased. This was also true for absolute neutrophil counts in males of test group 2 (5 mg/m³). However, neutrophil counts in males of both test groups were within the historical control range, those of females of test group 3 slightly above this range (absolute neutrophil, males 0.77-1.31 Giga/L, females 0.40-0.82 Giga/L). This was the only changed differential blood cell counts among these individuals. Therefore, the alteration in males was regarded as incidental and not treatment related, that one in females as non-adverse if it was treatment related at all (ECETOC Technical Report No. 85, 2002).

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among clinical chemistry parameters were observed.

After the administration period, in females of test group 3 (20 mg/m3) total bilirubin values were significantly decreased, but the values were within the historical control range (females, total bilirubin 1.38-2.71 µmol/L). Therefore, this alteration was regarded as incidental and not treatment related.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute weights (main groups):
- When compared with control group 0 (=100%), the mean absolute weights of lungs (male and female), liver (female) and spleen (female) were significantly increased in test group 3 (see table 21). All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights (main groups):
- When compared with control group 0 (=100%), the following mean relative organ weights of lungs (male/female) and spleen (female) were significantly increased in test group 3 (table 22).
- The significantly increased absolute and relative lung weights of males (30.5%; +27%) and females (+ 37.8%; + 31.2%) of test group 3 (20 mg/m³) was assumed to be treatment related.
- The significantly increased absolute liver weight in females of test group 3 (5.182 g; +10.9%) was within the historical control range (4.886g – 5.200g) and without histological correlate and was regarded as incidental and not treatment-related.
- The absolute and relative spleen weight in females of test group 3 (0.441g; 0.221%) was significantly increased. The absolute spleen weight was within the historical control range (0.417g – 0.460g), the relative spleen weight was slightly above the historical control range (0.206% - 0.22%). No histological correlate was detected and therefore the weight increase was regarded as incidental.
- All other mean relative weight parameters did not show significant differences when compared to the control group 0.

Absolute weights (recovery groups):
- When compared with control group 10 (=100%), the mean absolute weights of the lungs (males) were significantly increased in test group 13 (see table 27).
All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights (recovery groups):
- When compared with control group 10 (=100%), the mean relative organ weights of the lungs (males) were significantly increased in test group 3 (see table 28). The significant increase of absolute and relative weight of the lungs of test group 13 (+ 41.1%; + 37.0%) was regarded as treatment related. All other mean relative weight parameters did not show significant differences when compared to the control group 0.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross lesions (main groups):
- See table 23
- A red discoloration was observed in the lungs of 8 out of 10 males of test group 2, in all males of test group 3 and in all females of test group 2 and 3. This finding correlated with the histologically diagnosed alveolar histiocytosis with particles and was regarded as treatment related.
- Additionally, a red discoloration was noted in mediastinal lymph nodes of 9 out of 10 males of test group 2, all males of test group 3, 1 out of 10 females of test group 1 and all females of test group 2 and 3. This finding correlated with macrophage aggregates with particles detected histologically in the mediastinal lymph node and was regarded as treatment related.
- All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Gross lesions (recovery groups):
- See table 29
- A red discoloration was observed in lungs of all males of test groups 12 and 13. This finding correlated with the histologically diagnosed alveolar histiocytosis with particles and was regarded as treatment related.
- A red discoloration was noted in mediastinal and tracheobronchial lymph nodes of males of test group 12 and 13 with additionally enlargement of the mediastinal lymph node of males of test group 13. The described findings correlated with macrophage aggregated with particles detected histologically and was regarded as treatment related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathology (main groups)
- The treatment-related findings were observed in the lungs, the mediastinal and tracheobronchial lymph nodes and the nasal cavity (level III and IV, only) with incidences and grading according to table 24
- In the lung, macrophages, which contained dark red to black particles (presumably inhaled test compound) were multifocally distributed within the lumen of alveoli and terminal bronchioles, with a concentration-dependent increase in number of macrophages and amount of particles storage within each macrophage. The severity was minimal in test group 1 (1 mg/m³) up to moderate to marked in test group 3 (20 mg/m³) and histiocytosis was present in lungs of all treated animals.
- In test groups 2 (5 mg/m³) and 3 (20 mg/m³), aggregation of particle-laden macrophages was detected in the terminal bronchioles. The affected terminal bronchioles displayed a minimal to moderate hyperplasia/hypertrophy and the surrounding alveoli a minimal to moderate hyperplasia of type II pneumocytes. A lymphohistiocytic infiltrate with particle-laden macrophages, which was mostly located perivascular, was noted interstitially in those areas.
- Cellular debris, including free particles (presumably originating from remnants of alveolar macrophages which have undergone necrosis), accompanied by infiltrating neutrophils were detected in alveoli of males and females of test groups 2 and 3.
- In test group 1, the number of alveolar macrophages containing particles was only minimally increased. The described epithelial (hyperplasia/hypertrophy of terminal bronchioles, hyperplasia of type II pneumocytes) or inflammatory changes (lymphohistiocytic or neutrophilic infiltrate) were not detected.
- In the BALT of males and females of all test groups, macrophages containing comparable particles as described within the alveoli, were detected. Aggregation of macrophages was noted in males and females of test group 2 and 3.
- Additionally intravascular macrophages containing intracytoplasmic particles were noted in in males and females of all test groups. All described findings were regarded as treatment related.
- Tracheobronchial and mediastinal lymph nodes: Within the lymph nodes of animals of test groups 1, 2 and 3 (1, 5 and 20 mg/m³), histiocytes containing comparable red to black particles within their cytoplasm as observed in the lungs, were present. Macrophages were either individually distributed within the lymph nodes or formed aggregates. The findings were regarded to be treatment related (table 25).
- Nasal cavity (level III and level IV): Within the nasal associated lymphatic tissue (NALT) of males and females of test groups 1, 2 and 3, few macrophages containing red to black particles within their cytoplasm were detected. This finding was regarded as treatment related (table 26). All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.


Histopathology (recovery groups)
- The treatment-related findings were observed in the lungs, the mediastinal and tracheobronchial lymph nodes and the nasal cavity (level III and IV only) with incidences and grading according to tables 30.
- In general, histopathological findings in the lungs of males of the recovery test group 12 (5 mg/m3) and 13 (20 mg/m3) were comparable to those observed in the corresponding main test groups 2 (5 mg/m3) and 3 (20 mg/m3).
- The number of alveolar histiocytes was comparable in main and recovery groups, however the number of intracytoplasmic dark red to black particles was slightly reduced in recovery groups. Slight differences were also detected in the distribution of alveolar particle-laden macrophages: The formation of macrophage aggregates in terminal bronchioles and surrounding alveoli was slightly increased, whereas the number of individual particle-laden macrophages within alveoli was slightly reduced. Macrophage aggregates were accompanied by hypertrophy/hyperplasia of terminal bronchioles, hyperplasia of type II pneumocytes and an interstitial, often perivascular located, lymphohistiocytic infiltrate with particle-containing macrophages.
- Cellular debris, neutrophilic infiltrate within alveoli, intravascular macrophages with particles and macrophages/macrophages aggregates containing particles within the bronchus associated lymphatic tissue (BALT) was comparable in animals of test group 12 and 13 to animals of the corresponding main test group 2 and 3.
- The described findings were regarded to be treatment-related.
- Tracheobronchial and mediastinal lymph nodes: In the mediastinal and tracheobronchial lymph nodes of test groups 12 (5 mg/m3) and 13 (20 mg/m3) comparable findings (macrophages with particles and macrophage aggregates with particles) with a comparable severity to those observed in the corresponding main test groups 2 (5 mg/m3) and 3 (20 mg/m3) were observed (table 31).
- Nasal cavity (level III and level IV): See table 32. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

BRONCHOALVEOLAR LAVAGE FLUID (BAL)
See table 17 - 20.
After the administration period in BAL of males and females in test group 3 (20 mg/m3) total cell counts as well as absolute and relative lymphocyte, neutrophil and monocyte counts were significantly increased. Absolute macrophage counts were also significantly higher compared to controls only in males whereas relative macrophage counts were significantly lower in both sexes. Absolute lymphocyte, neutrophil and monocyte counts were already significantly higher in BAL of males and females of test group 2 (5 mg/m3). This was also true for relative lymphocyte counts in BAL of females as well as relative neutrophil and monocyte counts in BAL of both sexes in this test group. Relative macrophage counts were significantly decreased in BAL of males and females of this test group. These alterations were regarded as treatment related and adverse.

After the eight-week recovery period, total cell counts in males of test group 13 20 (mg/m3) were as high increased as after the administration period. Absolute and relative lymphocyte and monocyte in males of test groups 12 and 13 (5 and 20 mg/m3) were also higher increased compared to those after the administration period (monocytes not statistically significantly). In contrast absolute and relative neutrophil counts were statistically significantly increased in males of test groups 12 and 13, but not as great as in the correspondent groups after the administration. Absolute eosinophil counts were also relevantly increased in males of test groups 12 and 13, although not statistically significantly. Relative macrophage counts were significantly lower in males of test groups 12 and 13 compared to study controls.

After the administration period, in BAL of males and females of test group 3 (20 mg/m3) total protein levels as well as gamma-glutamyl-transferase (GGT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and -N-acetyl glucosaminidase (NAG) activities were significantly increased with the most prominent activity increase of LDH. After an eight-week recovery period, in males of test group 13 (20 mg/m3) total protein levels as well as LDH and NAG activities were higher compared to the administration period. ALP and GGT activities were also still significantly increased. These alterations were regarded as treatment related and adverse.

After the administration period, in BAL of males and females in test group 2 (5 mg/m3) LDH, ALP and GGT activities were already significantly higher compared to controls. This was also true for total protein levels in females of this test group as well as LDH activity in males of test group 1 (1 mg/m3). However, the increases were marginal (≤ 2-fold increase) and therefore were regarded as treatment related but non-adverse. After the recovery period, in males of test group 12 (5mg/m3) LDH and ALP activity as well as total protein levels were significantly increased. ALP activity and total protein level changes were marginal (≤ 2-fold increase). The mean LDH activity was the same as in males of test group 1 after the administration period, but the LDH activity in the study controls was very low after the recovery period. Therefore, the fold-increase of LDH in males of test group 2 after the recovery would have been also below 2 if study control LDH activity was as high as after the administration period. In conclusion, the increase of LDH after the recovery period in males of test group 12 was regarded as marginal and therefore treatment related, but non-adverse.

Details on results:

During the exposure period, the target concentrations were maintained as constant and stable as could be provided with dust aerosol generation techniques in the concentration range tested. Particle size distribution measurement demonstrated that the aerosols were respirable for rats.

During the exposure period, all animals of the high concentration male and female animals showed substance contaminated fur and discoloration of the fur. This finding was also observed in a few male and female animals of the mid concentration (5 mg/m³), as well as in individuals of female animals of the low concentration group (1 mg/m³). This finding was considered treatment-related but not adverse, because the substance was a pigment. During the recovery period, no clinical signs of toxicity was observed. There were no biologically relevant changes of body weight development. There were no substance-related changes noted in food consumption and ophthalmology.

Regarding clinical pathology, no treatment-related adverse effects in systemic clinical pathology blood parameters were observed up to a dose of 20 mg/m³.
After the administration period, in the bronchoalveolar lavage (BAL) of rats of both sexes in test group 3 (20 mg/m3) an acute inflammation was observed by increases of total cell counts as well as absolute macrophage, lymphocyte, neutrophil and monocyte counts. In consequence also total protein levels in BAL as well as -glutamyl-transferase (GGT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and -N-acetyl glucosaminidase (NAG) activities in BAL of both sexes of this test grouped were significantly increased. In test group 2 (5 mg/m³) absolute neutrophil, lymphocyte and monocyte counts in BAL of both sexes were also significantly increased whereas total cell counts as well as total protein and enzyme activities were not relevantly (>2-fold) increased.
After the eight-week recovery period, in males of test group 13 (20 mg/m³) total protein levels as well as LDH and NAG activities were higher compared to the administration period. ALP and GGT activities were also still significantly increased. Regarding BAL cytology, higher absolute lymphocyte and monocyte counts but lower neutrophil counts indicated the change from a granulocytic to lymphocytic-monocytic inflammation. In males of test group 12 (5 mg/m³) total protein level, LDH and ALP activity were not relevantly increased anymore, but total cell counts as well as absolute neutrophil, lymphocyte and monocyte counts were already significantly increased.

Regarding pathology, the target organs were lungs, mediastinal and tracheobronchial lymph nodes and the nasal cavity.

Main groups

The absolute and relative lung weights of males and females of test group 3 (20 mg/m³) were significantly increased (males: + 30.5%; + 27%; females + 37.8%; + 31.2%). The weight increases correlated to a macroscopically detected red discoloration of the lungs in all males and females of test group 3 (20 mg/m³) and all females and 8 males of test group 2 (5 mg/m³). The discoloration was correlated to a slightly to marked increase in numbers of alveolar macrophages (histiocytosis) which contained dark red to black pigment (presumably the inhaled test substance). In animals of test group 2 and 3 the macrophages were partly randomly distributed within the alveoli and partly formed aggregates in the terminal bronchioles and adjacent alveoli. The surrounding epithelial structures (terminal bronchioles and alveolar epithelium) showed a minimal to moderate hyperplasia/hypertrophy of terminal bronchioles and hyperplasia of pneumocytes type II. Interstitial thickening by a minimal to moderate lymphohistiocytic infiltrate, with particle-laden macrophages was frequently observed in affected areas. Infiltration of neutrophils and presence of cellular debris and free particles (presumably due to alveolar macrophages which have previously undergone cell death) were seen in males and females of test groups 2 and 3 and were interpreted as an ongoing inflammatory process. The combination of all above-described findings were regarded as treatment related and adverse.
Additionally, in the bronchus associated lymphatic tissue (BALT) in animals of test group 2 and 3 a minimal to mild number of macrophages with particles and a minimal to moderate number of macrophage aggregates with particles were detected. Macrophages with particles were seen in vessels in the lungs. Those findings were regarded as sign of the physiologically clearance of the inhaled test substance and were regarded as treatment related but not adverse.

In lungs of all males and females of test group 1 (1 mg/m³), a minimal increased number of alveolar histiocytes (histiocytosis), which contained dark red to black particles was detected. The histiocytes were randomly distributed within the alveoli. Aggregation of histiocytes was not detected. Individual particle-laden macrophages were detected in the BALT and intravascularly in the lungs. Hyperplasia/hypertrophy of terminal bronchioles or hyperplasia of type II pneumocytes or any other signs of inflammation were not observed in animals of test group 1. This led to the interpretation of described changes as treatment related but not adverse.

A minimal to slight number of particle-laden macrophages with minimal to marked formation of macrophage aggregates were present in tracheobronchial and mediastinal lymph nodes of animals of all test groups.

Additionally, in the nasal cavity (level III and level IV) of males and females of all test groups few individual macrophages with intracytoplasmic particles were detected in the nasal associated lymphatic tissue (NALT).
The findings in mediastinal and tracheobronchial lymph nodes, as well as the described findings in the nasal cavity were considered to be treatment related and not adverse, since they are not associated with inflammation or cell injury and only reflect an increased clearance of the inhaled pigment.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Recovery groups

The absolute and relative lung weights was significantly increased in males test group 13 (+ 41.1%; + 37.0%). This was, as in the main group, accompanied by a macroscopically detected red discoloration of the lungs and histologically detected minimal to marked increase in numbers of alveolar macrophages which contain particles in males of test groups 12 (5 mg/m³) and 13 (20 mg/m³). The number of alveolar macrophages in the main test groups 2 and 3 and the corresponding recovery test groups 12 and 13 was comparable, but the distribution of alveolar macrophages varied slightly: The number of individual macrophages within alveoli was slightly decreased and the number of aggregates within terminal bronchioles and adjacent alveoli was more pronounced in the recovery test groups. The morphology and severity of the epithelial (hypertrophy/hyperplasia of terminal bronchioles and type II hyperplasia) and inflammatory changes (cellular debris, neutrophilic infiltrate within alveoli, lymphohistiocytic interstitial infiltrate) was comparable to the lesions described in the main test groups. All above mentioned lesions were regarded as treatment related and adverse.

Additionally, in males of test group 12 and 13 a minimal to mild number of macrophages with particles and a minimal to moderate number of macrophage aggregates with particles in the bronchus associated lymphatic tissue (BALT), as well as intravascular macrophages with particles were observed. Those findings were regarded as signs of the physiologically clearance of the inhaled test substance and were regarded as treatment related but not adverse.

A minimal to slight number of particle-laden macrophages and a minimal to marked number of particle- laden macrophage aggregates were present in tracheobronchial and mediastinal lymphnodes of animals of all test groups. Those findings are considered to be treatment related and not adverse, since they are not associated with inflammation or cell injury and only reflect an increased clearance of the inhaled pigment.

Additionally in the nasal cavity (level III and leven IV) of males and females of all test groups few individual macrophages with intracytoplasmatic particles were detected in the nasal associated lymphatic tissue (NALT). No signs of inflammation or cell injury was present and therefore this finding is as well regarded as treatment related but not adverse.

In general, findings observed in the lungs, the tracheobronchial and mediastinal lymph nodes and the nasal cavity of the recovery test groups 12 and 13 were comparable to those of the corresponding main groups 2 and 3.

All other mean relative weight parameters did not show significant differences when compared to the control group 0.

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

20 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse treatment-related systemic effects observed up to the highest tested dose.

Dose descriptor:

LOAEC

Remarks:

local

Effect level:

5 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

other: BAL parameters

Dose descriptor:

NOAEC

Remarks:

local

Effect level:

1 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse treatment-related effects observed at this dose.

Critical effects observed:

yes

Lowest effective dose / conc.:

5 mg/m³ air (analytical)

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

presumably yes

Table 11: Study means and standard deviations of test substance concentrations

 

Test group	Target concentration (mg/m³)	Measured concentration (mg/m³)		Nominal concentration (mg/m³)	Effectiveness of dust generation (%)
		Mean	SD
1	1	1.0	0.1	1.6	62.5
2	5	5.0	0.2	7.7	64.9
3	20	20.0	1.0	27.8	71.9

 

 

Table 12: Mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) measured by cascade impactors during the exposure period

Target concentration	Measurement	MMAD (µm)	GSD	Fraction < 3 µm
1 mg/m³   (Test group 1)	1	0.68	3.40	88.8 %
	2	0.70	2.81	92.1 %
	3	0.64	2.89	92.7 %
	4	0.59	2.88	93.7 %
	5	0.57	2.79	94.8 %
	6	0.58	2.57	95.9 %
	7	0.58	2.50	96.3 %
	8	0.57	2.48	96.6 %
	9	0.60	2.29	97.3 %
	10	0.59	2.50	96.1 %
	11	0.56	2.70	95.5 %
	12	0.54	3.19	93.0 %
	13	0.53	3.02	94.1 %
	Mean	0.59	2.77	94.4 %
	SD	0.05	0.31	2.3 %
	Median	0.58	2.79	94.8 %
5 mg/m³ (Test groups 2 and 12)	1	0.68	3.31	89.2 %
	2	0.73	2.83	91.3 %
	3	0.65	2.70	93.8 %
	4	0.61	3.02	92.5 %
	5	0.58	3.41	91.0 %
	6		2.96	93.1 %
	7	0.64	2.81	93.2 %
	8	0.63	2.49	95.7 %
	9	0.61	2.44	96.3 %
	10	0.58	2.77	94.8 %
	11	0.56	3.48	91.1 %
	12	0.59	2.87	93.9 %
	13	0.57	3.31	91.8 %
	Mean	0.62	2.95	92.9 %
	SD	0.05	0.34	2.0 %
	Median	0.61	2.87	93.1 %
20 mg/m³   (Test groups 3 and 13)	1	0.63	3.32	90.4 %
	2	0.62	2.63	94.9 %
	3	0.59	2.53	96.0 %
	4	0.75	2.42	94.2 %
	5	0.60	2.82	94.0 %
	6	0.58	3.10	92.6 %
	7	0.55	3.07	93.5 %
	8	0.62	2.57	95.3 %
	9	0.60	2.51	96.0 %
	10	0.59	2.71	94.8 %
	11	0.54	2.87	94.7 %
	12	0.50	2.92	95.2 %
	13	0.58	2.93	93.6 %
	Mean	0.60	2.80	94.2 %
	SD	0.06	0.27	1.5 %
	Median	0.59	2.82	94.7 %

 

 

Table 13: Mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) measured by APS during the exposure period at low concentration of 1 mg/m³

Target concentration	Measurement Day	Measurement No.	MMAD (µm)	GSD
1 mg/m³   (Test group 1)	20 Jul 2021	1	1.04	2.04
		2	1.34	3.40
		3	1.21	2.58
	27 Jul 2021	1	1.32	2.49
		2	1.32	2.46
		3	1.43	2.84
	04 Aug 2021	1	1.04	1.96
		2	1.34	3.26
		3	1.07	1.94
	11 Aug 2021	1	1.09	2.45
		2	1.24	2.78
		3	1.14	2.54
	18 Aug 2021	1	1.30	3.17
		2	1.44	3.81
		3	1.32	3.11
	24 Aug 2021	1	1.52	3.16
		2	1.28	2.29
		3	1.46	3.15
	01 Sep 2021	1	1.38	2.93
		2	1.43	2.96
		3	1.48	3.03
	Mean ± SD		1.29 ± 0.15	2.78 ± 0.49
	Median		1.32	2.84

 

 

Table 14: Mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) measured by APS during the exposure period at mid concentration of 5 mg/m³

Target concentration	Measurement Day	Measurement No.	MMAD (µm)	GSD
5 mg/m³ (Test groups 2 and 12)	19 Jul 2021	1	1.60	2.96
		2	1.56	2.78
		3	1.69	3.10
	26 Jul 2021	1	1.58	2.73
		2	1.72	2.98
		3	1.61	2.80
	02 Aug 2021	1	1.58	3.24
		2	1.41	2.71
		3	1.65	3.23
	12 Aug 2021	1	1.57	3.01
		2	1.56	3.03
		3	1.69	3.22
	16 Aug 2021	1	1.84	3.26
		2	1.68	3.13
		3	1.71	3.06
	23 Aug 2021	1	1.74	3.17
		2	1.96	3.26
		3	1.93	3.35
	30 Aug 2021	1	1.92	3.13
		2	1.91	3.10
		3	2.00	3.24
	Mean ± SD		1.71 ± 0.16	3.07 ± 0.19
	Median		1.69	3.10

 

 

Table 15: Mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) measured by APS during the exposure period at high concentration of 20 mg/m³

Target concentration	Measurement Day	Measurement No.	MMAD (µm)	GSD
20 mg/m³ (Test groups 3 and 13)	19 Jul 2021	1	3.02	3.21
		2	3.04	3.30
		3	2.92	3.28
	26 Jul 2021	1	5.90	3.20
		2	4.92	3.30
		3	5.20	3.29
	02 Aug 2021	1	6.16	3.09
		2	6.82	3.19
		3	6.13	3.14
	12 Aug 2021	1	6.46	3.23
		2	5.46	3.19
		3	4.86	3.27
	16 Aug 2021	1	6.30	3.35
		2	6.66	3.22
		3	6.28	3.25
	23 Aug 2021	1	5.80	3.09
		2	6.80	3.03
		3	7.05	3.10
	30 Aug 2021	1	6.51	3.13
		2	6.57	3.09
		3	6.31	3.13
	Mean ± SD		5.67 ± 1.27	3.19 ± 0.09
	Median		6.16	3.20

 

 

Table 16: Particle count distribution measured by SMPS

Target concentration	Measurement	Total count concentration (N/cm³)	Geometric mean diameter
1 mg/m³	1	12489	368
	2	13523	348
	3	15636	359
	4	14403	348
	5	13092	351
	6	16317	355
	7	15024	347
	8	16132	341
	9	16898	337
	10	15550	339
	11	22499	349
	12	13790	337
	13	18160	338
	Mean ± SD	15655 ± 2613	347 ± 9
	Median	15550	348
5 mg/m³	1	64553	362
	2	66474	354
	3	69855	359
	4	68419	352
	5	69180	345
	6	70556	349
	7	76433	340
	8	89304	346
	9	81272	344
	10	91225	330
	11	80258	345
	12	86530	338
	13	94320	339
	Mean ± SD	77568 ± 10258	346 ± 9
	Median	76433	345
Target concentration 20 mg/m³	1	167993	354
	2	220310	344
	3	242891	333
	4	271284	324
	5	221537	343
	6	214915	344
	7	206028	310
	8	167409	330
	9	242184	319
	10	244227	319
	11	215953	335
	12	232199	325
	13	281324	328
	Mean ± SD	225250 ± 33534	331 ± 12
	Median	221537	330

 

 

Table 17: Changes in mean absolute cell counts in BAL (x-fold of concurrent control) in male rats on study days 91 (main groups, 1 day after last exposure, N = 10) and after an 8-week recovery period on study day 150 (N = 5).

Analyte	Study day 90/91			Study day 150
	Gr. 1 1 mg/m^3	Gr. 2 5 mg/m^3	Gr. 3 20 mg/m^3	Gr. 12 0.5 mg/m^3	Gr.13 20 mg/m^3
Total Cells	1.2	1.3	8.6**	1.6*	7.0**
Macrophages	1.3	1.0	1.6**	0.9	1.4
Lymphocytes	1.0	1.8*	20.7**	5.9**	38.3**
Neutrophils	1.2	17.5**	332.4**	8.3**	58.6**
Monocytes	2.2	4.8**	52.7**	8.7	49.4**
Eosinophils	0.3	0.0	1.1	4.0	8.9
Epithelial cells	1.0	1.2	1.1	1.3	0.0

One-sided Wilcoxon-test: * : p <= 0.05; ** : p <= 0.01

++ increase could not be calculated because of zero activity in controls

 

 

Table 18: Changes in mean absolute cell counts in BAL (x-fold of concurrent control) in female rats on study day 90/91 (main groups, 1 day after last exposure, N = 10).

Analyte	Study day 90
	Gr. 1 1 mg/m^3	Gr. 2 5 mg/m^3	Gr.3 20 mg/m^3
Total Cells	0.8	1.1	8.3**
Macrophages	0.8	0.7	1.3
Lymphocytes	0.9	8.0**	74.7**
Neutrophils	1.8	6.5**	116.3**
Monocytes	++	++**	++**
Eosinophils	++	++	++
Epithelial cells	0.9	0.4	0.0

One-sided Wilcoxon-test: * : p <= 0.05; ** : p <= 0.01

++ increase could not be calculated because of zero activity in controls

 

 

Table 19: Changes in mean total protein and enzyme levels in BAL (x-fold of concurrent control) in male rats on study day 90 (main groups, 1 day after last exposure) and after an 8-week recovery period on study day 150.

Analyte	Study day 90/91			Study day 150
	Gr. 1 1 mg/m^3	Gr.2 5 mg/m^3	Gr.3 20 mg/m^3	Gr. 12 5 mg/m^3	Gr. 13 20 mg/m^3
Total Protein	0.8	0.7	3.0**	1.5*	6.1**
LDH	1.6*	1.5**	9.8**	2.6*	14.4**
ALP	1.1	1.3**	3.1**	1.5**	3.0**
NAG	1.1	1.0	1.8**	1.2	3.2**
GGT	1.2	1.3*	4.7**	1.2	2.8**

GGT = g-Glutamyl-transferase; LDH = Lactate dehydrogenase; ALP = Alkaline phosphatase;

NAG = b-N-Acetyl glucosaminidase, One-sided Wilcoxon-test: * : p <= 0.05; ** : p <= 0.01

 

 

Table 20: Changes in mean total protein and enzyme levels in BAL (x-fold of concurrent control) in female rats on study day 90/91 (main groups, 1 day after last exposure).

Analyte	Study day 90
	Gr. 1 1 mg/m^3	Gr. 2 5 mg/m^3	Gr. 3 20 mg/m^3
Total Protein	1.1	1.6**	6.6**
LDH	0.9	2.0**	12.5**
ALP	0.9	1.4**	4.2**
NAG	1.0	1.2	4.3**
GGT	0.9	1.5**	5.5**

GGT = g-Glutamyl-transferase; LDH = Lactate dehydrogenase; ALP = Alkaline phosphatase;

NAG = b-N-Acetyl glucosaminidase, One-sided Wilcoxon-test: * : p <= 0.05; ** : p <= 0.01

 

 

Table 21: Relative increase of absolute organ weights in main group animals

	Male animals			Female animals
Test group (mg/m³)	1 (1)	2 (5)	3 (20)	1 (1)	2 (5)	3 (20)
Lungs	+1.2%	+5.1%	+30.5%**	+2.3%	+12.1%	+37.8%**
Liver				+5.2%	+4.8%	+10.9%**
Spleen				+2.1%	+8.8%	+17.0%**

* : p <= 0.05, **: p <= 0.01

 

 

Table 22: Relative increase of relative organ weights in main group animals

	Male animals			Female animals
Test group (mg/m³)	1 (1)	2 (5)	3 (20)	1 (1)	2 (5)	3 (20)
Lungs	-0.8%	+4.1%	+27.0%	-0.8%	+11.0%	+31.2%
Spleen				-1.1%	+7.4%	+11.4%

* : p <= 0.05, **: p <= 0.01

 

 

Table 23: Incidence of gross lesions in main group animals

	Male animals				Female animals
Test group (mg/m³)	0 (0)	1 (1)	2 (5)	3 (20)	0 (0)	1 (1)	2 (5)	3 (20)
No. of animals	10	10	10	10	10	10	10	10
Lungs	10	10	10	10	10	10	10	10
·         Discoloration	0	0	8	10	0	0	10	10
Mediastinal lymph node	10	10	10	10	10	10	10	10
·         Discoloration	0	0	9	10	0	1	10	10

 

 

Table 24: Incidence and severity of histological findings in lungs of main group animals.

	Male animals				Female animals
Test group (mg/m³)	0 (0)	1 (1)	2 (5)	3 (20)	0 (0)	1 (1)	2 (5)	3 (20)
No. of animals	10	10	10	10	10	10	10	10
Lungs	10	10	10	10	10	10	10	10
Histiocytosis, alveolar, with particles	0	10	10	10	0	10	10	10
Grade 1	0	10	0	0	0	10	0	0
Grade 2	0	0	8	0	0	0	9	0
·         Grade 3	0	0	2	8	0	0	1	5
·         Grade 4	0	0	0	2	0	0	0	5
Hyperplasia, type II pneumocytes	0	0	10	10	0	0	10	10
·         Grade 1	0	0	7	2	0	0	4	2
·         Grade 2	0	0	3	7	0	0	6	7
·         Grade 3	0	0	0	1	0	0	0	1
Hypertrophy/ hyperplasia, terminal bronchioles	0	0	7	10	0	0	10	10
·         Grade 1	0	0	7	5	0	0	9	9
·         Grade 2	0	0	0	4	0	0	1	1
·         Grade 3	0	0	0	1	0	0	0	0
Infiltrate, interstitial, lymphohistiocytic, with particles	0	3	9	10	0	0	10	10
·         Grade 1	0	3	9	1	0	0	7	0
·         Grade 2	0	0	0	9	0	0	3	7
·         Grade 3	0	0	0	0	0	0	0	3
Debris, cellular	0	0	6	10	0	0	10	10
·         Grade 1	0	0	5	0	0	0	7	0
·         Grade 2	0	0	1	7	0	0	3	3
·         Grade 3	0	0	0	3	0	0	0	7
Infiltrate, neutrophilic	0	0	10	10	0	0	7	10
·         Grade 1	0	0	9	0	0	0	7	0
·         Grade 2	0	0	1	10	0	0	0	8
·         Grade 3	0	0	0	0	0	0	0	2
Balt: macrophages with particles	0	10	10	10	0	10	10	10
·         Grade 1	0	10	10	1	0	10	10	10
·         Grade 2	0	0	0	8	0	0	0	0
·         Grade 3	0	0	0	1	0	0	0	0
Balt: macrophage aggregates with particles	0	0	2	10	0	0	7	9
·         Grade 1	0	0	2	0	0	0	5	5
·         Grade 2	0	0	0	8	0	0	2	2
·         Grade 3	0	0	0	2	0	0	0	2
Macrophages with particles, intravascular	0	9	9	9	0	8	9	10

 

 

Table 25: Incidence and severity of histological findings in tracheobronchial and mediastinal lymph nodes of main group animals.

	Male animals				Female animals
Test group (mg/m³)	0 (0)	1 (1)	2 (5)	3 (20)	0 (0)	1 (1)	2 (5)	3 (20)
No. of animals	10	10	10	10	10	10	10	10
Mediastinal lymph node	10	10	10	10	9	10	10	10
Macrophages with particles	0	9	10	10	0	7	10	10
Grade 1	0	9	7	1	0	7	9	6
Grade 2	0	0	3	9	0	0	1	4
Macrophage aggregates with particles	0	4	9	10	0	3	9	10
·         Grade 1	0	4	0	0	0	3	3	0
·         Grade 2	0	0	9	3	0	0	6	5
·         Grade 3	0	0	0	3	0	0	0	5
·         Grade 4	0	0	0	4	0	0	0	0

 

 

Table 26: Incidence and severity of histological findings in nasal cavity of main group animals.

	Male animals				Female animals
Test group (mg/m³)	0 (0)	1 (1)	2 (5)	3 (20)	0 (0)	1 (1)	2 (5)	3 (20)
No. of animals	10	10	10	10	10	10	10	10
Nasal cavity level III	10	10	10	10	10	10	10	10
NALT: Macrophages with particles	0	0	7	7	0	1	7	8
Grade 1	0	0	7	7	0	1	7	8
Nasal cavity level IV	10	10	10	10	10	10	10	10
NALT: Macrophages with particles	0	1	2	5	0	0	2	6
·         Grade 1	0	1	2	5	0	0	2	6

 

 

Table 27: Relative increase of absolute lung weight in recovery group animals

	Male animals
Test group (mg/m³)	12 (5)	13 (20)
Lungs	+4.4%	+41.1%**

* : p <= 0.05, **: p <= 0.01

 

 

Table 28: Relative increase of relative lung weight in recovery group animals

	Male animals
Test group (mg/m³)	12 (5)	13 (20)
Lungs	+8.5%	+37.0%**

* : p <= 0.05, **: p <= 0.01

 

 

Table 29: Incidence of gross lesions in recovery group animals

	Male animals
Test group (mg/m³)	10 (0)	12 (5)	13 (20)
No. of animals	5	5	5
Lungs	5	5	5
·         Discoloration	0	5	5
Mediastinal lymph node	5	5	5
·         Discoloration	0	5	5
·         Size enlarged	0	0	2
Tracheobronchial lymph node	5	5	5
·         Discoloration	0	4	5

 

 

Table 30: Incidence and severity of histological findings in lungs of recovery group animals

	Male animals
Test group (mg/m³)	10 (0)	12 (5)	13 (20)
No. of animals	5	5	5
Histiocytosis, alveolar, with particles	0	5	5
Grade 1	0	1	0
Grade 2	0	5	0
·         Grade 3	0	0	3
·         Grade 4	0	0	2
Hyperplasia, type II pneumocytes	0	5	5
·         Grade 1	0	3	2
·         Grade 2	0	2	3
Hypertrophy/hyperplasia, terminal bronchioles	0	5	5
·         Grade 1	0	5	5
Infiltrate, interstitial, lymphohistiocytic, with particles	0	5	5
·         Grade 1	0	4	0
·         Grade 2	0	1	5
Debris, cellular	0	5	5
·         Grade 1	0	4	1
·         Grade 2	0	1	2
·         Grade 3	0	0	2
Infiltrate, neutrophilic	0	4	4
·         Grade 1	0	2	0
·         Grade 2	0	2	2
·         Grade 3	0	0	2
Balt: macrophages with particles	0	5	5
·         Grade 1	0	3	4
·         Grade 2	0	2	1
Balt: macrophage aggregates with particles	0	5	5
·         Grade 1	0	3	0
·         Grade 2	0	2	3
·         Grade 3	0	0	2
Macrophages with particles, intravascular	0	4	5

 

 

Table 31: Incidence and severity of histological findings in tracheobronchial and mediastinal lymph nodes of recovery group animals

	Male animals
Test group (mg/m³)	10 (0)	12 (5)	13 (20)
No. of animals	5	5	5
Macrophages with particles	0	3	5
Grade 1	0	1	0
Grade 2	0	2	5
Macrophage aggregates with particles	0	3	5
·         Grade 1	0	0	0
·         Grade 2	0	3	2
·         Grade 3	0	0	2
·         Grade 4	0	0	1

 

 

 

Table 32: Incidence and severity of histological findings in nasal cavity of recovery group animals

	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)
No. of animals	5	5	5
Nasal cavity level III	5	5	5
Nalt: Macrophages with particles	0	4	4
Grade 1	0	4	4
Nasal cavity level IV	5	5	5
Nalt: Macrophages with particles	0	1	4
·         Grade 1	0	1	4

 

 

Conclusions:

Inhalation exposure of the test substance for 90 days (65 exposures) caused concentration and clearly treatment-related changes of several lavage parameters, which correlated with the significantly increased lung weight and several histological changes in lungs. A No Observed Adverse Effect Concentration (NOAEC) for local effect was determined 1 mg/m³ under the current study conditions. All the above-mentioned local effects were not reversible within 8 weeks recovery period. There was not any indication for systemic effect as shown by clinical chemistry and hematology and histological examinations. A No Observed Adverse Effect Concentration (NOAEC) for systemic effect was the highest tested concentration of 20 mg/m³ under the current study conditions.

Executive summary:

Groups of male and female Wistar rats were exposed nose-only to the dust aerosol of the test item for 6 hours per day on 5 consecutive days a week for 90 days. The target concentrations were 1, 5 and 20 mg/m³. A concurrent control group was exposed to clean air. Daily clinical observations, body weights, food consumption, ophthalmology. Additional assessments including hematology, clinical chemistry of the blood, bronchoalveolar lavage, and histopathology according to the referenced guidelines were carried out at the termination of the study. A recovery period of 60 days was included in recovery groups of male animals.

The following treatment-related, adverse effects were observed:

 

Test group 3 (20 mg/m³, main group)

• Increased absolute and relative lymphocyte, neutrophil and monocyte counts in BAL of both sexes


• Increased absolute macrophage counts in BAL of males


• Decreased relative macrophage counts in BAL of both sexes


• Increased total protein levels as well as g-glutamyl-transferase (GGT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and b-N-acetyl glucosaminidase (NAG) activities in BAL of both sexes


• Increase of absolute and relative lung weights in males (+30.5%; +27%) and females (+ 37.8%; + 31.2%).


• Red discoloration of lungs of all males and all females.


• Moderate to marked alveolar histiocytosis with particles in all males and all females.


• Minimal to moderate hyperplasia of type II pneumocytes in all males and females.


• Hypertrophy/hyperplasia of terminal bronchioles in all males (minimal to moderate) and all females (minimal to slight).


• Slight to moderate cellular debris in all males and all females


• Neutrophilic infiltrates in all males (slight) and all females (slight to moderate)

 

Test group 13 (20 mg/m³, males only, recovery group)

• Increased total protein levels as well as g-glutamyl-transferase (GGT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and b-N-acetyl glucosaminidase (NAG) activities in BAL of males


• Increased total cell as well as absolute and relative neutrophil, monocyte and lymphocyte counts in males


• Increased ablute eosinophil counts in males


• Decreased relative macrophage counts in males


• Increase of absolute and relative lung weights in males (+30.5% ; +27%) and females (+ 41.1%; + 37.0%).


• Red discoloration the lungs of all males.


• Moderate to marked alveolar histiocytosis with particles in all males.


• Minimal to slight hyperplasia of type II pneumocytes in all males.


• Minimal hypertrophy/hyperplasia of terminal bronchioles in all males.


• Minimal to moderate cellular debris in all males.


• Minimal to moderate neutrophilic infiltrates in all males.

 

Test group 2 (5 mg/m³, main group)

• Increased absolute and relative neutrophil and monocyte counts in BAL of both sexes


• Decreased relative macrophage counts in BAL of both sexes


• Increased absolute lymphocyte counts in BAL of both sexes


• Increased relative lymphocyte counts in BAL of females


• Increased total protein levels as well as g-glutamyl-transferase (GGT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and b-N-acetyl glucosaminidase (NAG) activities in BAL of both sexes


• Red discoloration of lungs of 8 out of 10 males and all females.


• Slight to moderate alveolar histiocytosis with particles in all males and all females.


• Minimal to slight hyperplasia of type II pneumocytes.


• Hypertrophy/hyperplasia of terminal bronchioles in 7 out of 10 males (minimal) and all females (minimal to slight).


• Minimal to slight cellular debris in all males and all females.


• Neutrophilic infiltrates in all males (minimal to slight) and 7 out of 10 females (minimal).

 

Test group 12 (5 mg/m³, males only, recovery group)

• Increased total cell as well as absolute and relative neutrophil, monocyte and lymphocyte counts in males


• Increased ablute eosinophil counts in males


• Decreased relative macrophage counts in males


• Red discoloration the lungs of all males


• Minimal to slight alveolar histiocytosis with particles in all males.


• Minimal to slight hyperplasia of type II pneumocytes in all males.


• Minimal hypertrophy/hyperplasia of terminal bronchioles in all males.


• Minimal to slight cellular debris in all males.


• Minimal to slight neutrophilic infiltrates in all males.

 

Test group 1 (1 mg/m³)

No treatment-related, adverse effects.

 

Conclusion: Inhalation exposure of the test substance for 90 days (65 exposures) caused concentration and clearly treatment-related changes of several lavage parameters, which correlated with the significantly increased lung weight and several histological changes in lungs. A No Observed Adverse Effect Concentration (NOAEC) for local effect was determined 1 mg/m³ under the current study conditions. All the above-mentioned local effects were not reversible within 8 weeks recovery period. There was not any indication for systemic effect as shown by clinical chemistry and hematology and histological examinations. A No Observed Adverse Effect Concentration (NOAEC) for systemic effect was the highest tested concentration of 20 mg/m³ under the current study conditions.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

1 mg/m³

Study duration:

subchronic

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral toxicity

Regarding reapeated oral toxicity, no data is available for the test substance. To fill this data gap, a read-across to the category member EC 479-300-2 was performed.

 

In a repeated dose 28-day oral toxicity study conducted in accordance with OECD TG 407 and in compliance with GLP regulations, the test substance was administered daily for 28 days by oral gavage to SPF-bred Wistar rats (BASF AG, 2006). One vehicle control group and three treated groups were tested, each consisting of 5 males and 5 females. An extra 5 animals per sex in the control and high dose group were allowed 14 days of recovery. The following parameters were evaluated: clinical signs daily, functional observation tests, body weight and food consumption weekly, clinical pathology at the end of treatment, macroscopy at termination, organ weights and histopathology on a selection of tissues. In addition, a histopathological investigation of liver, kidneys and adrenal glands of the animals of test groups 2 and 3 (100 and 300 mg/kg bw/day) was performed. Accuracy, homogeneity and stability over 5 hours of formulations of test substance in Milli-U water were demonstrated by analyses. No toxicologically relevant changes were observed in any of the parameters determined in this study. No evidence for neurotoxicity or immunotoxicity was obtained in this study. The additional histopathological investigation of liver, kidneys and adrenal glands of the animals of test groups 2 and 3 (100 and 300 mg/kg bw/day) did not reveal any findings related to treatment with the test substance. All the findings noted were single observations and/or were biologically equally distributed in both test groups. They were all considered to be incidental and spontaneous in nature. From the results presented in the report a definitive No Observed Adverse Effect Level (NOAEL) for the substance of 1000 mg/kg bw/day was established.

 

Inhalation toxicity

Regarding subchronic reapeated inhalation toxicity, no data is available for the test substance. To fill this data gap, a read-across to the category member CAS 3049-71-6 was performed.

In the GLP-compliant key study according to OECD TG 413 (BASF, 2022), the inhalation exposure of the test material up to 20 mg/m³ for 90 days (65 exposures, 6 h/day on 5 consecutive days) caused concentration and clearly treatment-related changes of several lavage parameters, which correlated with the significantly increased lung weight and several histological changes in lungs. A No Observed Adverse Effect Concentration (NOAEC) for local effects was determined 1 mg/m³ under the current study conditions. All the above-mentioned local effects were not reversible within 8 weeks recovery period. There was not any indication for systemic effect as shown by clinical chemistry and hematology and histological examinations. A No Observed Adverse Effect Concentration (NOAEC) for systemic effect was the highest tested concentration of 20 mg/m³ under the current study conditions.

 

Further toxicological data of category members:

The test article belongs to the "perylene based organic pigments" category (see attached document for details on category members and for read across justification). According to the category approach, missing toxicity endpoints can be addressed with data available for other category members. Regarding repeated dose toxicity, reliable data are available for other members of the "Perylene based pigments category". All of these data are taken into account for the evaluation and assessment of the repeated dose toxicity of the test article.

Additional data is available for the oral and the inhalation route of exposure. After the oral exposure in further 28-day and/or 90-day repeated dose toxicity studies no adverse effects could be observed at the highest dose tested. In additional studies for the inhalation route of exposure local effects in the lung could be observed. After 5 and/or 90-day exposure inflammation processes were induced in the lung that were not fully reversible in two of the studies. Systemic effects after inhalation exposure could not be observed, even at the highest dose tested.

 

 

Statement on the Human Health Hazard Assessment of the Members of the Perylene Pigment Category including the Justification for Classification or Non-Classification

In the following statement the human health hazard of the Perylene pigments is assessed in the broader context of inhalation toxicity of poorly soluble particles. Therefore, a joint evaluation of particle specific properties (solubility, surface activity) and toxicological data of the perylene pigments was performed. Based on expert judgment, a classification or non-classification is derived for the individual members of the Perylene pigment category.

 

Particle specific investigations: 

Solubility:

The available data consistently demonstrate that Perylene pigments are poorly soluble substances.  All category members have a low water solubility (solubility < 0.1 mg/L for all substances) and low solubility in n-octanol (< 10 mg/l for all substances). While this indicates poor solubility, it is not sufficient to conclude the assessment, as biosolubility may differ significantly from the solubility in water. Therefore, the OECD ‘Guidance document on inhalation toxicity studies’ suggests assessing the solubility of a solid material by measuring solubility in a simulated biofluid (OECD, 2018). The OECD guidance document further defines poor solubility if a material has a solubility of less than 0.1 g dissolved in 100 ml dissolvent within 24 hours. A test on biosolubility (static) and on dissolution kinetics (dynamic) in phagolysosomal simulant fluids was performed with all pigments of the category, except the intermediate product Pigment Red 224 (CAS 128-69-8), to determine the persistence after uptake in cells, e.g., alveolar macrophages. All substances tested were insoluble in phagolysosomal simulant fluid at pH 4.5 in the static and dynamic dissolution assay. 

Surface Reactivity:

The surface reactivity of the pigment particles was investigated in chemico as well as in vitro using the FRAS (Ferric Reduction Ability of Serum) in combination with the EPR (Electron Paramagnetic Resonance) method and the in vitro macrophage assay. The assays were performed with all pigments of the category, except the intermediate Pigment Red 224 (CAS 128-69-8). None of the substances induced pro-inflammatory effects or cytotoxicity in rat alveolar macrophages according to the classification criteria of Wiemann et al. (2016.). The ability to induce biological oxidative damage in chemico was analyzed using the Ferric Reduction Ability of Serum (FRAS) assay and Electron Paramagnetic Resonance spectroscopy (EPR). The majority of the perylene pigments tested were classified as “passive” in both assays. Pigment Red 149 (CAS 4948-15-6) and 178 (CAS 3049-71-6), however, were classified as “passive” in the FRAS assay but “active” in EPR assay. Of note, the results of the red and violet Perylenes were consistently higher than those of the black Perylenes in the EPR assay. The black pigments appear to be significantly less surface active than the red and violet ones.

 

Toxicological in vivo data:

The available experimental data show, that the pigments of this category are not acutely toxic nor toxic after repeated oral exposure, not irritating to skin or eyes, do not cause skin sensitization, and are not genotoxic. In addition, no hazard concerning reproductive and developmental toxicity is concluded for members based on the currently available data. The only adverse effects observed were local effects after short-term and sub-chronic exposure via the inhalation route, whereas systemic effects could not be observed after inhalation exposure.

Up to now, three studies on inhalation toxicity following repeated exposure are available, two 5-day short-term inhalation toxicity studies (STIS) according to OECD guideline 412 on Pigment Red 178 (CAS 3049-71-6) and 179 (CAS 5521-31-3) and a 90-day subchronic toxicity study according to OECD guideline 413 with Pigment Red 179.

Both pigments caused inflammation in the lung tissue at concentrations of 20mg/m³ and above in the STIS, with the effects being reversible within 3 weeks for Pigment Red 179. At the top dose of 60 mg/m³, Pigment Red 178 and 179 caused increased total cell count, increased absolute and relative lymphocytes, neutrophils and monocyte counts in bronchioalveolar lavage fluid. Moreover, several biochemical parameters (protein concentration, enzyme activities and cytokine concentrations) were significantly increased in lavage fluid. Consistently, minimal infiltration of neutrophils within bronchiolar epithelium was observed. Most of these findings were also observed at 20 mg/m³ with reduced severity. For both pigments a NOAEC of 5 mg/m³ for local effects and a systemic NOAEC of above 60 mg/m³ was determined. The lung and the tracheobronchial lymph nodes were identified as target organs.

Due to the irreversibility of the effects of Pigment Red 178, the 90-day repeated dose inhalation study was performed with Pigment Red 178 following a worst-case approach. In this study, inflammation was observed at 20 and 5 mg/m³ as shown by increased inflammatory factors and protein levels in the lavage fluid, migration of inflammatory cells, cell debris, which correlated with the significantly increased lung weight and several histological changes in lungs. In line with the STIS results, there was not any indication for systemic effect. The effects were not reversible within 60 days. A NOAEC of 1 mg/m³ for local effects and a systemic NOAEC of above 60 mg/m³ was determined. The lung was identified as target organ. The observed effects were regarded as adverse and are presumably relevant for humans.

Overall, the perylene-based pigments are characterized by very low systemic toxicity even after repeated exposures to high doses. The absence of systemic effects observed after exposure via the oral or inhalation route and the insolubility observed in the solubility studies, indicate that the perylenes have a very low bioavailability. Therefore, the induction of local inflammation processes in the lung reported in the short-term inhalation studies as well as in the 90-day inhalation study are most likely induced by the surface activity of the particles. The results of the activity assays are evidence for the generation of reactive oxygen species at the particle surface for some of the Perylene pigments.

 

Conclusion:

As described above, the local effects after inhalation exposure are based on particle specific abiotic surface activity. Since the subgroups of the perylene pigments were demonstrated to differ in this property, a different evaluation of the pigments based on their measured abiotic surface activity is justified in this case.

 

Justification for Classification of Pigment Red 178 (CAS 3049-71-6), Pigment Red 179 (CAS 5521-31-3), Pigment Red 149 (CAS 4948-15-6), Pigment Violet 29 (CAS 81‑33-4) and intermediate product Pigment Red 224 (CAS 128-69-8):

As mentioned above, repeated inhalation exposure to Pigment Red 178 was shown to induce local inflammation processes in the lung at concentrations of 5 mg/m³ and above, but no systemic toxicity was observed in this subchronic inhalation study or any other of the toxicological in vivo studies available. The local effects to the lung were regarded as adverse and are presumably relevant for humans. Therefore, classification for Specific target organ toxicity (STOT RE) is justified under Regulation (EC) No. 1272/2008. However, due to the nature of the toxic effect observed, the guidance values mentioned in paragraphs 3.9.2.9.6 (Cat. 1) and 3.9.2.9.7 (Cat. 2), which take into account the duration of exposure and the dose/concentration which produced the effect(s), are not suitable for subclassification into category 1 or 2 in this case. Based on the explanations in chapter 3.9.2.9.8 these guidance values “are not intended as strict demarcation values”. Rather, in chapter 2 article 9 as well as in Annex I chapter 1.1.1, it is expressively permitted to “carry out an evaluation by applying a weight of evidence determination using expert judgement […] where the criteria cannot be applied directly to available identified information” and that “Expert judgement may also be required in interpreting data for hazard classification”. As outlined in chapter 3.9.1.3, adverse health effects in experimental animals relevant for STOT RE are defined as “toxicologically significant changes which have affected the function or morphology of a tissue/organ, or have produced serious changes to the biochemistry or haematology of the organism and these changes are relevant for human health”. One important reason for the deviation from the guidance values is that the local effects observed with the perylene pigments are not comparable to the effects that should be considered to support classification for STOT RE exposure according to chapter 3.9.2.7.3 such as e.g. morbidity or death, significant changes in biochemistry, significant organ damage noted as necropsy, microscopic changes indicative of necrosis or fibrosis. None of the listed effects nor any other effects indicative of a severe impairment of organ function or morphology were observed in the subchronic inhalation study on Pigment Red 178. As already mentioned above, in contrast to substances typically classified for STOT RE 2, it has a low systemic toxicity resulting from its insolubility and associated low bioavailability and the local effects observed in the respiratory tract can be most likely attributed to the surface activity of the pigment particles. Therefore, the effects are regarded as less severe compared to a STOT RE 1 classification. In order to still consider the adverse local effects to the lung, a classification for STOT RE 2 (H373, <lung>) is justified.

There are no data available on subchronic inhalation toxicity of Pigment Red 179 (CAS 5521-31-3), Pigment Red 149 (CAS 4948-15-6) and Pigment Violet 29 (CAS 81 33-4). In order to fill this data gap, a read-across from Pigment Red 178 (CAS 3049-71-6) was performed. The read-across is supported by the comparable high values measured in the EPR assay for the red and violet pigments as well as by the results from the STIS for Pigment 179. Therefore, Pigment Red 179, Pigment Red 149 and Pigment Violet 29 are classified for STOT RE 2 (H373, <lung>) too.

 

Justification for Non-classification of Pigment Black 31 (CAS 67075-37-0), Pigment Black 32 (CAS 83524-75-8), Perylen Black I (EC 479-300-2) and Perylen Black II (EC 475-310-6):

The induction of local inflammation processes in the lung reported in the short-term inhalation studies as well as in the 90-day inhalation study are most likely induced by the surface activity of the respective particles, as indicated in the results of the EPR. However, the values of the red and violet Perylenes were significantly higher than those of the black Perylenes in this assay. This means that although all category members are enormously similar in structural and physicochemical and toxicological parameters, there are differences between the substances in this property. The black pigments appear to be significantly less surface active than the red and violet ones and therefore are expected not to be less toxic to the lung.

Since, the results of the red and violet Perylenes were visibly higher than those of the black Perylenes it is justified to treat and classify the perylenes differently based on their activity measured in the EPR assay. This approach will be supported by two future short term inhalation studies investigating Pigment Black 32 and another of the much less active black pigments of this category. It is expected that no inflammatory, local effects will occur in these studies, further supporting the arguments for non-classification.

As described above, due to the local, partly non-reversible effects of Pigment Red 178, the red and violet pyrelene pigments must be classified as STOT RE2 under Regulation (EC) No. 1272/2008. However, as these effects are triggered by the surface activity of the pigments, a classification for the black pigments (Pigment Black 31/32 and Perylene Black I/II) is not justified. Therefore, no read-across from Pigment red 178 is performed.

 

References

OECD, 2018. Guidance document on inhalation toxicity studies. Series on testing and assessment No.39 (Paris).

Wiemann, Martin, et al. "An in vitro alveolar macrophage assay for predicting the short-term inhalation toxicity of nanomaterials." Journal of Nanobiotechnology 14.1 (2016): 1-27.

Regulation. "1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of substances and mixtures, amending and repealing Directive 67/548/EEC and 1999/45/EC and amending Regulation (EC) No 1907/2006." Official J Eur Union 353 (2008): 1.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. No data on repeated inhalation toxicity is available for the test substance, but relevant data are available for the category member CAS 3049-71-6 (Pigment Red 178), which were transferred to the test substance via read-across. Repeated inhalation exposure to Pigment Red 178 was shown to induce local inflammation processes in the lung at concentrations of 5 mg/m³ and above, but no systemic toxicity was observed in this subchronic inhalation study or any other of the toxicological in vivo studies available. The local effects to the lung were regarded as adverse and are presumably relevant for humans. Therefore, classification for Specific target organ toxicity (STOT RE) is justified under Regulation (EC) No. 1272/2008. However, due to the nature of the toxic effect observed, the guidance values mentioned in paragraphs 3.9.2.9.6 (Cat. 1) and 3.9.2.9.7 (Cat. 2), which take into account the duration of exposure and the dose/concentration which produced the effect(s), are not suitable for subclassification into category 1 or 2 in this case. Based on the explanations in chapter 3.9.2.9.8 these guidance values “are not intended as strict demarcation values”. Rather, in chapter 2 article 9 as well as in Annex I chapter 1.1.1, it is expressively permitted to “carry out an evaluation by applying a weight of evidence determination using expert judgement […] where the criteria cannot be applied directly to available identified information” and that “Expert judgement may also be required in interpreting data for hazard classification”. As outlined in chapter 3.9.1.3, adverse health effects in experimental animals relevant for STOT RE are defined as “toxicologically significant changes which have affected the function or morphology of a tissue/organ, or have produced serious changes to the biochemistry or haematology of the organism and these changes are relevant for human health”. One important reason for the deviation from the guidance values is that the local effects observed with the perylene pigments are not comparable to the effects that should be considered to support classification for STOT RE exposure according to chapter 3.9.2.7.3 such as e.g. morbidity or death, significant changes in biochemistry, significant organ damage noted as necropsy, microscopic changes indicative of necrosis or fibrosis. None of the listed effects nor any other effects indicative of a severe impairment of organ function or morphology were observed in the subchronic inhalation study on Pigment Red 178. As already mentioned above, in contrast to substances typically classified for STOT RE 2, it has a low systemic toxicity resulting from its insolubility and associated low bioavailability and the local effects observed in the respiratory tract can be most likely attributed to the surface activity of the pigment particles. Therefore, the effects are regarded as less severe compared to a STOT RE 1 classification. In order to still consider the adverse local effects to the lung, a classification for STOT RE 2 (H373, <lung>) is justified.

As a result, the substance is classified for STOT RE Cat. 2 under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2020/217.