2-cyano-2-[2,3-dihydro-3-(tetrahydro-2,4,6-trioxo-5(2H)-pyrimidinylidene)-1H-isoindol-1-ylidene]-N-methylacetamide

Endpoint summary

Description of key information

Repeated dose toxicity: oral

Key: oral, rat, 28 day study: NOEL (male/female) = 1000 mg/kg bw/day (GLP, OECD 407 with restrictions, 2000)

 

Repeated dose toxicity: inhalative

Read-across to CAS 36888-99-0: Key: inhalative, rat, 5 day study with 3 weeks recovery: NOAEC (local) = 5 mg/m³ air, NOAEC (systemic) = 60 mg/m³ air (GLP, similar to OECD 412, 2022)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

acceptable restrictions: functional observation battery not included

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Functional observation battery missing

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines on Industrial Chemnicals (1986)

Deviations:

not specified

GLP compliance:

yes

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Inc.
- Age at study initiation: 5 weeks
- Weight at study initiation: males: 146-169 g, females 126-150 g
- Housing: Five rats of the same sex before grouping and two rats of the same sex after grouping were housed in each cage (polycarbonate cages (265W X 426D X 200H mm, Tokiwa Kagaku Kikai Co., Ltd.) with hard wood chip bedding (Beta chip, Charles River Japan, Inc.)).
- Diet: Pellet diet for experimental animals (Mi, Oriental Yeast Co., Ltd.), ad libitum
- Water: ordinary tap water ad libitum, renewed once a week, filtered through a 5 µm filter and disinfected with UV.
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +/- 2
- Humidity (%): 55 +/- 15
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12h/12h

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % CMC-Na aqueous solution mixed with 0.1% Tween 80

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The dosing solutions were prepared once a week, and were stored in a dark refrigerated place. They were used within 8 days. The dosing solutions were treated with ultrasonicator for about 10 minutes at the time of use and then were continuously stirred until administration.

VEHICLE
- Concentration in vehicle: 0.5%
- Amount of vehicle (if gavage): 10 mL/kg
- Lot/batch no. (if required): Tween 80: Tokyo Kasei Kogyo Co., Ltd., Lot No. 10546; CMC-Na: Iwai Chemicals Company, Lot No. 041210.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity and stability of the test substance in the dosing solution for 8 days stored in a refrigerated place were confirmed at the dose of 0.4 and 100 mg/mL by HPLC method before the first administration. They were within ±10 for each indicated concentration.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once a day in the morning

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

200 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:

6

Control animals:

yes

Details on study design:

- Dose selection rationale:
A dose-finding 7-day repeated dose toxicity study (the test substance was administered to three rats of both sexes at dose levels of 0, 100, 500, and 1000 mg/kg, respectively) was performed.
The study revealed yellowish stool which was considered to be related to defecation of the test substance in both sexes of all treatment groups, loose stool in males of the 500 mg/kg group, decreased MCV and MCH in females of the 1000 mg/kg group, an increased relative weight of the liver in males of the 100 and 1000 mg/kg groups.
There were no toxic changes considered to be related to the administration of the test substance in body weight or at necropsy. As there were no grave changes in the 1000 mg/kg group, the highest dose level was set at 1000 mg/kg; this is the upper limit as prescribed in the guideline.
- Post-exposure recovery period in satellite groups: Additionally 6 animals per sex in the control, 200, and 1000 mg/kg groups were subjected to a recovery period of 14 days.

Observations and examinations performed and frequency:

OBSERVATIONS:
The day of the first administration was designated as Day 1, and the period from Day 1-7 was designated as Week 1. The period of Day 29 and after was designated as the recovery period.

CAGE SIDE OBSERVATIONS/Clinical Sign: Yes
- Time schedule: All rats were observed twice a day (before and after administration) during the treatment period and once a day on every other day in the morning.

BODY WEIGHT: Yes
- Time schedule for examinations: once a week

FOOD CONSUMPTION: once a week

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 29 and 43 at the scheduled sacrifices
- Anaesthetic used for blood collection: Yes, blood samples were obtained from the inferior vena cava of the rat without fasting, under intraperitoneal thiopental sodium anesthesia.
- Parameters examined and the corresponding method:
(1) Erythrocyte count (RBC): Sheath flow DC impedance detection method
(2) Hemoglobin concentration (Hb): SLS hemoglobin method
(3) Hematocrit (Ht): Cumulative pulse height detection method
(4) Mean corpuscular volume (MCV): Calculated from (1) and (3)
(5) Mean corpuscular hemoglobin (MCH): Calculated from (1) and (2)
(6) Mean corpuscular hemoglobin concentration (MCHC): Calculated from (2) and (3)
(7) Reticulocyte count: Flow cytometry by argon laser method
(8) Platelet count (PLT): Sheath flow DC impedance detection method
(9) Prothrombin time: Modified Quick one method
(10) Activated partial thromboplastin time (APTT): Activated cephaloplastin method
(11) Leukocyte count (WBC): RF/DC impedance detection method
(12) Leukocyte count-differential: Counting after Wright's stain

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day 29 and 43 at the scheduled sacrifices
- Animals fasted: No
- Parameters examined and the corresponding method:
(1) ASAT (GOT): UV-rate method (modified JSCC method)
(2) ALAT (GPT): UV-rate method (modified JSCC method)
(3) γGT: γ-glutamyl-p-nitroanilide substrate-method (modified SSCC method)
(4) Alkaline phosphatase (ALP) p-nitrophenyl-phosphate substrate-method (modified JSCC method)
(5) Total bilirubin: Enzymatic method (BOD method)
(6) Urea nitrogen: Enzymatic-UV method (Urease-GLDH method)
(7) Creatinine: Jaffé method
(8) Glucose: Enzymatic-UV method (GlcK-G6PDH method)
(9) Total cholesterol: Enzymatic method (CES-CO-POD method)
(10) Triglycerides: Enzymatic method (LPL-GK-G3PO-POD method)
(11) Total protein: Biuret method
(12) Albumin: BCG method
(13) A/G ratio: Calculated from (11) and (12)
(14) Calcium: OCPC method
(15) Inorganic phosphorus: Enzymatic method (PNP-XOD-POD method)
(16) Sodium (Na): Ion-selective electrode method
(17) Potassium (K): Ion-selective electrode method
(18) Chloride (Cl): Ion-selective eleotrode method

URINALYSIS: Yes
- Time schedule for collection of urine:
Fresh urine was collected from six males and females of each group on Day 23, and examined. As the result, urinalysis during the recovery period was not performed, since no changes suspected to be the effect of administration of the test substance were found.
- Parameters examined and the corresponding method:
(1) pH Paper tests (Multistix, Bayer Medical Ltd.)
(2) Protein Paper tests (Multistix, Bayer Medical Ltd.)
(3) Glucose Paper tests (Multistix, Bayer Medical Ltd.)
(4) Ketone bodies Paper tests (Multistix, Bayer Medical Ltd.)
(5) Bilirubin Paper tests (Multistix, Bayer Medical Ltd.)
(6) Occult blood Paper tests (Multistix, Bayer Medical Ltd.)
(7) Urobilinogen Paper tests (Multistix, Bayer Medical Ltd.)

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Organ weight of the brain, liver, kidneys, adrenals, thymus, spleen, testes, and ovaries

HISTOPATHOLOGY: Yes
- Histological examination was performed on following organs and tissues of the control and 1000 mg/kg groups at the end of the treatment period, along with all gross lesions found in any group: heart, liver, spieen, kidney, and adrenal.
Histological specimens were stained with H.E. stain in accordance with routine methods and examined. Additionally, all testes in any group at the end of the treatment period were also examined histologically since small of bilateral testes was observed with one male of the 1000 mg/kg group at necropsy at the end of the treatment period.

Statistics:

The numerical data were analyzed by multiple comparison tests. They were first analyzed by Bartlett's test. If the group variance was determined to be homogeneous, all groups were compared by a one-way analysis of variance. If Bartlett's test indicated heterogeneous variance, the Kruskal-Wallis test was employed, and a Dunnett's test or Dunnett type multiple comparison was used when there was a significant difference between the groups. The categorical data were analyzed by R x C Chi-square test, and when there was significance, Armitage's Chi-square test was used to compare the difference between the control group and each treatment group.
Statistical analysis was performed on items listed below. The analysis was not performed on cinical sign, necropsy, or histological findings.
Multiple comparison test: body weight, food consumption, hematology, blood chemistry, and organ weight.
Chi-square test: urinalysis.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No abnormalities were observed in any group.
Yellowish stool was observed in all cages of the 200 and 1000 mg/kg groups during the treatment period. However, this change was considered to be related to the color of the test substance, and was not a biological reaction.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight of the treatment groups progressed in the same manner as that of the control group.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The food consumption of the treatment groups changed in the same manner as that of the control group.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No changes related to the administration of the test substance were observed at the end of the treatment or recovery periods.

Decreased MCV (Mean Corpuscular Volume) and MCH (Mean Corpuscular hemoglobin) were observed in females of the 200 mg/kg group and a decreased MCHC (Mean Corpuscular hemoglobin concentration) was observed in males of the 8 and 40 mg/kg groups at the end of the treatment period; however, these changes were considered unrelated to the administration of the test substance, since they were slight and not observed in the 1000 mg/kg group.

An increased ratio of monocyte in leukocyte count-differential was observed in females of the 200 and 1000 mg/kg groups at the end of the recovery period; however, this change was considered unrelated to the administration of the test substance since this change was within normal range and not observed at the end of the treatment period.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No changes related to the administration of the test substance were observed at the end of the treatment or recovery periods.
Decreased sodium was observed in females of the 200 mg/kg group at the end of the recovery period; however, this change was considered unrelated to the administration of the test substance, since this change was slight and not observed in the 1000 mg/kg group and at the end of the treatment period.

Endocrine findings:

not examined

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No changes related to the administration of the test substance were observed during the treatment period.
Decreased ketone bodies were observed in females of the 8 mg/kg group; however, this change was considered unrelated to the administration of the test substance, since this change was not observed in the 40 mg/kg or above groups.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No changes were observed at the end of the treatment and recovery periods.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No changes related to the administration of the test substance were observed at the end of the treatment or recovery periods.
Although thymic remnant in neck in the thymus, nodule in the spleen, hemorrhage in the lungs, hepatodiaphragmatic nodule in the liver, cyst in the kidneys, and bilateral small in the testes were observed in the treatment groups at the end of the treatment or recovery periods, all of them were considered unrelated to the administration of the test substance, because of their circumstances of occurrence.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No changes related to the administration of the test substance were observed.
Although focal inflammatory cell infiltration in the heart, nodular lymphoid hyperplasia in the spleen, focal hemorrhage in the lungs, microgranuloma in the liver, basophilic tubule, cyst, hyaline droplet in the proximal tubular epithelium, and focal interstitial lymphocytic infiltration in the kidneys were observed in the treatment groups at the end of the treatment or recovery periods, all of them were considered unrelated to the administration of the test substance, because of their circumstances of occurrence.
They were spontaneous changes frequently seen in this strain of rat.
And additionally, testes of all animals in all groups at the end of the treatment period were examined histologically since small of bilateral testes was observed with one male of the 1000 mg/kg group at the necropsy of the end of the treatment period. As the result, bilateral diffuse atrophy of seminiferous tubule was observed in the animals with small testes; however, no histological changes were observed in the testes of the other animals. According to the result, the change observed in the testes was considered to be a spontaneous change.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed up to and including the highest tested dose

Critical effects observed:

not specified

Conclusions:

Under the conditions of this test the no-observed-effect-level (NOEL) of the test substance was determined to be 1000 mg/kg bw in both sexes after oral application for 28 days.

Executive summary:

The 28 day repeated dose study with rats was performed according to the OECD guideline 407 following GLP requirements. The study deviated from the guideline in that a functional observation battery was not included. The test substance (purity: 99.3 weight-%) was administered by gavage to male and female SD rats for 28 days at doses of 0, 8, 40, 200 and 1000 mg/kg bw to investigate its toxicity and, if necessary possible reversibility. A control group was dosed with a vehicle (0.5 % CMC-Na aqueous solution mixed with 0.1% Tween 80) only.

No changes attributable to the administration of the test substance were observed in any observations or measurements, namely, clinical sign, body weight, food consumption, hematology, blood chemistry, urinalysis, organ weight, necropsy or histology. Therefore, under the conditions of this test the NOEL of the test substance was determined to be 1000 mg/kg bw in both sexes.

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

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 Nov 2021 - 12 May 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

25 June 2018

Deviations:

yes

Remarks:

5-day dust inhalation study in rats (with bronchoalveolar lavage, 3 weeks recovery period)

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

COMMISSION REGULATION (EU) No 260/2014 of 14 January 2014

Deviations:

not specified

Principles of method if other than guideline:

5-day dust inhalation study in rats (with bronchoalveolar lavage, 3 weeks recovery period)

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Limit test:

no

Specific details on test material used for the study:

- Name of the test substance: Pigment Yellow 139
- Batch: 200012P040
- Purity: > 99%
- Homogeneity: given
- Storage stability: 20 Jul 2030
- Storage conditions: ambient (RT)
- Appearance: solid/orange

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

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

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; 97633 Sulzfeld
- Age at study initiation: about 7 weeks (when supplied)
- Weight at study initiation (means): ca 255g
- Housing: The rats were housed together (up to 5 animals per cage) in Polysulfon cages (H-Temp [P SU]) supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm2). Bedding in the Polycarbonate cages were Type Lignocel fibres, dust-free bedding, supplied by SSNIFF, Soest,
Germany. Dust-free wooden bedding was used in this study. For enrichment wooden gnawing blocks and play tunnel were added.
- Diet: Mouse/rat laboratory diet “GLP”, 10 mm pellets (Provimi Kliba SA, Kaiseraugst, Basel Switzerland), ad libitum.
- Water: Tap water, ad libitum

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

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose/head only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

>= 0.82 - <= 1.28 µm

Geometric standard deviation (GSD):

3.5

Remarks on MMAD:

MMAD (cascade impactor): between 0.82 and 1.28 µm with GSDs between 3.18 and 4.45. The calculated mass fractions of particles below 3 µm aerodynamic size is greater than 75 %. Thus, the aerosols were highly respirable for rats and a very high proportion of the aerosol particles reached the lungs. SMPS showed different geometric mean concentration than those measured by cascade impactor measurement. Major reason is that this geometric mean referred to count distribution, while cascade impactor measurement measured mass-based aerodynamic diameter. The geometric mean count diameters were between 289 nm and 304 nm.

Details on inhalation exposure:

For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage, mixed with conditioned air, and passed via the cyclonic separator and glass tube into the inhalation system

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Generator systems: Solid particle generators (brush-generator), Aerosol mixing tube (Stainless steel), Glass cyclonic separators
- Generation procedure: The test substance was used unchanged. By means of dust generators the substance to be tested is generated into dust aerosols using compressed air in a mixing stage, mixed
with conditioned air and passed into the inhalation systems via cyclonic separators. For each concentration, a solid particle generator (brush-generator) wias used for generating the dust. The con
centration was adjusted by varying the piston feed and by varying the brush rotation. 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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of the inhalation atmospheres in test groups 1 - 3 were analyzed by gravimetry. This method was applicable because the test item possessed extremely low vapor pressure. Daily means were calculated based on 3 measured samples per concentration and exposure. From the daily mean values of each concentration, mean concentrations and standard deviations for the entire study were derived.In these groups, the constancy of concentrations in each chamber was continuously monitored using scattered light photometers.

The particle size analysis was carried out with a cascade impactor with the following equipment:
• Stack sampler Marple 298 (New Star Environmental, Inc., Roswell, Georgia 30075, USA)
• Vacuum compressed air pump (Millipore Corporation, Billerica, MA 01821, USA)
• Limiting orifice 3 L/min (Millipore Corporation, Billerica, MA 01821, USA)
• Sampling probe internal diameter 6.9 mm
• Balance Sartorius MSA 6.6S-000-DF (Sartorius AG, Göttingen, Germany)
Sampling for particle size analyses:Pre-weighed metal collecting discs and a backup particle filter were placed into the cascade impactor and two samples were taken in each concentration at a sampling velocity of 1.25 m/sec. from the breathing zones of the animals.
The amount of dust deposited by each stage in mg was calculated from the difference between the weight of the filter/metal collecting disc and backup filter before and after sampling.The deposits in the probe and the wall losses in the impactor were also determined as difference of the total mass increase of the impactor and the sum of masses on the collecting discs and backup filter.

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. During the exposure period, one measurement per concentration with 10 repeats each were performed.

Real time surveillance of the inhalation atmospheres with scattered light photometers generally proved the constancy of each concentration throughout the daily exposures.
The air flows were constantly maintained in the desired range. An air change of about 65 to 67 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system. Daily mean relative humidities in the inhalation systems ranged between 33.7 and 49.6 %. Daily mean temperatures in the inhalation systems ranged between 20.6 and 22.1 °C. These values were within guideline recommendations.

Duration of treatment / exposure:

6h for 5 days

Frequency of treatment:

daily

Dose / conc.:

5.2 mg/m³ air (analytical)

Remarks:

125548 particles per cm3 (geometric mean diameter 289 nm)

Dose / conc.:

20.4 mg/m³ air (analytical)

Remarks:

222788 particles per cm3 (geometric mean diameter 302 nm)

Dose / conc.:

59.4 mg/m³ air (analytical)

Remarks:

382709 particles per cm3 (geometric mean diameter 304 nm)

No. of animals per sex per dose:

10 (five for sacrifice after exposure and 5 for sacrifice after recovery)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: not specified
- Rationale for animal assignment: random
- Fasting period before blood sampling for clinical biochemical biochemistry: overnight
- Rationale for selecting satellite groups: Clearance of inert particles by lung macrophages is known to take time
- Post-exposure recovery period in satellite groups: 3 weeks

Positive control:

No

Observations and examinations performed and frequency:

MORTALITY: Yes
A check for moribund or dead animals was carried out twice per day on working days and on Saturday and Sunday. A check for moribund or dead animals was carried out once per day on weekends and holidays.

CLINICAL OBSERVATION: Yes
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
The animals were weighed prior to the pre-exposure period (study day -5), at the start of the exposure period (study day 0), at the end of the exposure period (study day 4), as well as on the study days 5, 12, 19 and 26.

FOOD CONSUMPTION: Yes
Food consumption was determined once over the exposure period (study day 0 – study day 4), during the post-exposure period weekly and calculated as mean food consumption in grams per animal and day.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

Sacrifice and pathology:

CLINICAL PATHOLOGY
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood samples were carried out in a randomized sequence (the list of randomization instructions was compiled with a computer).
The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results.
The results of clinical pathology examinations were expressed in International System (SI) units. The following parameters of the animals were examined:
- Hematology: leukocyte count, erythrocyte count, hemoglobin, hematocrit, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, differential blood count, reticulocytes
- Clinical chemistry: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase,-Glutamyltransferase, Inorganic phosphate, Calcium, Urea, Creatinine, Glucose, Total bilirubin, Total protein, Albumin, Globulins, Triglycerides, Cholesterol
- Bronchoalveolar lavage fluid (BAL): 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 parameters and methods of cytological examination in BAL was carried out: Total cell count, Macrophages, Polymorphonuclear neutrophils, Lymphocytes, Eosinophils, Monocytes, Epithelial, Gamma−Glutamyltransferase, Protein, Lactate dehydrogenase, Alkaline phosphatase, N-acetyl-Beta-Glucosaminidase. Cytokines in BAL: Rat monocyte chemoattractant protein-1 (rat MCP-1), Rat cytokine-induced neutrophil chemoattractant-1 level (rat CINC-1/IL-8), Rodent osteopontin

NECROPSY
The animals were sacrificed under pentobarbital anesthesia by exsanguination from the abdominal aorta and vena cava. Afterwards, the thorax was opened, the right lung lobes were lavaged, whereas the left lung lobe was ligated during lavage. Immediately after lung lavage, the animals were necropsied and assessed by gross pathology.

The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (terminal body weight)
2. Adrenal glands (fixed)
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Lungs
9. Spleen
10. Testes
11. Thymus (fixed)
12. Thyroid glands (with parathyroid glands) (fixed)
All paired organs were weighed together (left and right).

The following organs or tissues were fixed in 4% neutral buffered formaldehyde solution:
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain with olfactory bulb
5. Epididymides
6. Esophagus
7. Eyes with optic nerve
8. Heart
9. Kidneys
10. Larynx/pharynx
11. Liver
12. Lungs
13. Lymph nodes (tracheobronchial and mediastinal lymph nodes)
14. Nose (nasal cavity)
15. Seminal vesicles
16. Spinal cord (cervical, thoracic and lumbar cord)
17. Spleen
18. Stomach (forestomach and glandular stomach)
19. Testes
20. Thyroid glands
21. Thymus
22. Trachea
23. Urinary bladder


Extend of histological processing and subsequent microscopical examinations in main group animals: all gross lesions, larynx (3 level), lungs, lymph nodes (tracheobronchial, mediastinal), nasal cavity (4 levels), trachea and in recovery group animals: all gross lesions, larynx (3 level), lungs, lymph nodes (tracheobronchial, mediastinal)

Other examinations:

Lung lavage: The animals intended for lung lavage were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava. The right lung will be lavaged in situ with physiological saline, whereas the left lung was ligated during this procedure.

Statistics:

Body weight, body weight change: Comparison of each group with the control group was performed using DUNNETT test (two-sided) for the hypothesis of equal means
Blood parameters: For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting pvalue was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians
For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
BALF: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
Organ weights: Non-parametric one-way analysis using the Kruskal-Wallis test (two-sided). If the resulting p-value was equal to or less than 0.05, a pair-wise comparison of each dose group with the control group was performed using the Wilcoxon test (two-sided) for the hypothesis of equal medians.

Terminal body weight: Comparison of each group with the control group was performed using the Dunnett test (two-sided) for the hypothesis of equal means.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Immediately after exposure, one of the ten animals of the test group 1 (5 mg/m³) and all animals of test group 2 (20 mg/m³) and 3 (60 mg/m³) showed test item contaminated fur. In eight of the ten test group 3 animals (60 mg/m³) substance-like discoloration of the nose region was observed before and during exposure on study days 2 and 3. This clinical finding was substance-related, but not adverse because it simply shows the exposure to the solid coloured dusty test substance.

No adverse findings were noted.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of the administration period, in males of test group 3 (60 mg/m3) absolute and relative neutrophil counts were significantly increased, and in males of test groups 1 and 2 (5 and 20 mg/m3) relative neutrophil counts were increased (in test group 2 not statistically significantly. However, all values were within historical control ranges (males, absolute neutrophils 0.53- 1.01Giga/L, relative neutrophils 8.5-16.0 %). Therefore, these alterations were regarded as incidental and not treatment related.

Clinical biochemistry findings:

no effects observed

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:

no effects observed

Description (incidence and severity):

See attached tables in "Overall remarks and attachments".

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

3 out of 5 animals of test group 2 and all animals of test group 3 of the main group revealed an orange discoloration of the lungs (see Table 4). In addition, a yellow discoloration of the mediastinal and tracheobronchial lymph nodes was observed in 4 out of 5 animals in test group 3. These findings correlated with the histological presence of particle-containing macrophages in these organs and were considered treatment-related.

After three weeks of recovery, the following was observed:
A yellow to orange discoloration of the lungs, mediastinal and tracheobronchial lymph nodes was present in almost all animals of test group 3 of the recovery group. In test group 2, one animal showed a yellow discoloration of the mediastinal lymph node. These findings correlated with the histological presence of particle-containing macrophages in these organs and were considered treatment-related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In three animals of test group 1, macrophages in the lungs were present in similar numbers to controls, but contained yellowish to brownish particles in their cytoplasm (see Tables 5 and 6). In addition, two animals of test group 1 as well as all animals of test groups 2 and 3 showed a dose-dependent minimal to slight increase in the number of macrophages within bronchioli and alveoli (alveolar histiocytosis) with the cells also containing yellowish to brownish particles. In test groups 2 and 3, macrophages were either individually distributed or formed aggregates within bronchioli. The latter showed a minimal increase in cell size, cell number, and/or cell layers of the epithelium (hyperplasia/hypertrophy) in test group 3, only. In addition, particle-containing macrophages were also present in the bronchial-associated lymphoid tissue (BALT) in all treated test groups.
In the larynges at level I, a minimal flattening of epithelial cells, partly associated with an increase in cell layers was multifocally present. The tracheobronchial lymph nodes were chosen exemplarily, similar findings were observed in the mediastinal lymph node. Macrophages containing intracytoplasmic, yellowish to brownish particles were observed in these lymph nodes in minimal to slight numbers.


After three weeks of recovery, the following was observed:
A yellow to orange discoloration of the lungs, mediastinal and tracheobronchial lymph nodes was present in almost all animals of test group 3 of the recovery group. In test group 2, one animal showed a yellow discoloration of the mediastinal lymph node. These findings correlated with the histological presence of particle-containing macrophages in these organs and were considered treatment-related.
In all animals of test group 1, macrophages in the lungs were present in similar numbers to controls, but contained yellowish to brownish particles in their cytoplasm. In all animals of test groups 2 and 3, a dose-dependent minimal to moderate increase in the number of macrophages (alveolar histiocytosis) was observed. The macrophages contained brownish particles and mainly formed aggregates within bronchioli and alveoli. In test group 3, the affected bronchioli showed a minimal increase in cell size, cell number, and/or cell layers of the epithelium (hyperplasia/hypertrophy) and the number of type II pneumocytes was minimally increased in affected alveoli (hyperplasia). Further, lymphocytes and macrophages were also infiltrating the interstitium in minimal numbers in animals of test group 3. In addition, minimal to slight numbers of particle-containing macrophages were found in the bronchial-associated lymphoid tissue in all treated test groups.
The tracheobronchial lymph nodes were chosen exemplarily, similar findings were observed in the mediastinal lymph node. Macrophages containing intracytoplasmic, yellowish to brownish particles were either individually distributed throughout the lymph node or formed aggregates.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Bronchoalveolar lavage fluid (BAL): Table 1-3
After the administration period, in males of test group 3 (60 mg/m3) total cell counts were marginally, but significantly increased, whereas absolute and relative lymphocyte, monocyte and neutrophil counts were moderately increased (lymphocytes not statistically significantly).
Absolute and relative neutrophil and lymphocyte counts were already marginally, but significantly increased in males of test group 2 (20 mg/m3). Relative macrophage counts were significantly decreased in males of test group 2 and 3. These alterations were regarded as treatment related and adverse.
After the 3-week recovery period absolute and relative lymphocyte, monocyte and neutrophil counts were still marginally increased in males of test group 3 (monocyte counts not statistically significantly) whereas the significantly increased total cell counts among these individuals were regarded as not toxicologically relevant. Relative macrophage counts were also still significantly decreased in BAL of males in test group 3. Neutrophil counts were also significantly increased in BAL of males in test group 1 (5 mg/m3), but this change was not dose dependent and therefore it was regarded as incidental and not treatment related.
After the administration period, in males of test group 3 (60 mg/m3) -glutamyl-transferase (GGT) and alkaline phosphatase (ALP) activities in BAL were slightly but significantly increased. These alterations were regarded as treatment related and adverse. Total protein and -N-acetylglucosaminidase (NAG) activities were only marginally (≤ 2-fold) increased. These increases were regarded as treatment related but non-adverse.
After the recovery period total protein levels as well as GGT, LDH and NAG levels in BAL of males in test group 3 (60 mg/m3) were significantly increased. However, when regarding the absolute values, they were within historical control ranges (males, total protein 2-50 mg/L, GGT 25-57 nkat/L, LDH 0.21-0.65 μkat/L, NAG 18-56 nkat/L). Therefore, these alterations were regarded as incidental and not treatment related.
After the administration period, in males of test group 3 (60 mg/m3) cytokine-induced neutrophil chemoattractant-1 (CINC-1/IL8), monocyte chemoattractant protein-1 (MCP-1) and osteopontin
levels in BAL were significantly increased. These alterations were regarded as treatment related and adverse. CINC-1/IL-8 values were already significantly increased in males of test group 2 (20 mg/m3) but the increase was below 2-fold, and therefore, it was regarded as treatment
related but non-adverse.
After the three-week recovery period in males of test group 3 (60 mg/m3) MCP-1 and osteopontin levels in BAL were still more than 2-fold increased (osteopontin not statistically significantly)
whereas CINC-1/IL-8 levels in males of test groups 1 and 3 (5 and 60 mg/m3) as well as MCP1 values in males of test group 2 (20 mg/m3) were significantly but less than 2-fold increased. Therefore, only CINC-1/IL-8 and osteopontin level increases in males of test group 3 were
regarded as relevant.

Dose descriptor:

NOAEC

Remarks:

local effects

Effect level:

5.2 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: at 20 mg/m3: Increased absolute and relative neutrophil and lymphocyte and decreased relative macrophage counts in BAL after exposure

Dose descriptor:

NOAEC

Remarks:

systemic effects

Effect level:

>= 59.4 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: only findings in BALF

Critical effects observed:

yes

Lowest effective dose / conc.:

59.4 mg/m³ air (analytical)

System:

respiratory system: upper respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

No adverse effect was observed at 5 mg/m³. No systemic effect was observed up to the highest tested concentration of 60 mg/m³. Target organ was the lung. While parameters in lavage fluid were fully reversible within 3 weeks post-exposure period, the histological changes at 60 mg/m³ were not reversible within the 3 -week recovery period.

 

 

 

Table 1: Changes in mean absolute cell counts in BAL (x-fold of concurrent control) on study day 5 (1 day after last exposure) and study days 26 (3 weeks after last exposure). At study day 5, mean of differential cell counts in control group (test group 0) is based on only two individuals due to bad cell quality. Therefore, for these parameters, statistics could not be performed.

	Study day 5			Study day 26
	Gr. 1 5 mg/m3	Gr. 2 20 mg/m3	Gr. 3 60 mg/m3	Gr. 1 5 mg/m3	Gr. 2 20 mg/m3	Gr. 3 60 mg/m3
Total Cells	0.9	0.9	2.2*	1.1	1.0	1.4*
Eosinophils	0.6	1.1	0.7	+	+	+
Lymphocytes	1.6	2.2*	12.7	0.8	1.2	3.4*
Macrophages	0.9	0.8	0.8	1.0	1.0	1.0
Neutrophils	0.3	10.2**	127.9**	4.8*	2.4	13.5**
Monocytes	+	+	+**	0.4	0.7	7.4
Epithelial cells	0.9	0.7	0.0	3.3	0.2	1.5

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

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

 

 

Table 2: Changes in mean total protein and enzyme levels in BAL (x-fold of concurrent control) of males on study day 5 (1 day after last exposure) and study day 26 (3 weeks afterlast exposure)

	Study day 5			Study day 26
	Gr. 1 5 mg/m3	Gr. 2 20 mg/m3	Gr. 3 60 mg/m3	Gr. 1 5 mg/m3	Gr. 2 20 mg/m3	Gr. 3 60 mg/m3
Total Protein	0.9	1.1	1.6*	0.9	0.9	1.3*
GGT	1.1	1.3	2.6**	1.0	1.2	1.5**
LDH	1.1	0.9	1.8	1.2	1.1	1.8**
ALP	1.2	1.3*	2.3**	0.8	0.9	1.2
NAG	1.3	1.3	1.6**	1.8	0.9	2.1*

GGT = Gamma-Glutamyl-transferase; LDH = Lactate dehydrogenase; ALP = Alkaline phosphatase; NAG = Beta-N-Acetyl glucosaminidase, One-sided Wilcoxon-test: * : p <= 0.05; ** : p <= 0.01

 

 

 

Table 3: Changes in antigen levels in BAL (x-fold of concurrent control means) of males onstudy day 5 (1 day after last exposure) and study day 26 (3 weeks after last exposure)

	Study day 5			Study day 26
	Gr. 1 5 mg/m3	Gr. 2 20 mg/m3	Gr. 3 60 mg/m3	Gr. 1 5 mg/m3	Gr. 2 20 mg/m3	Gr. 3 60 mg/m3
MCP-1	0.9	1.8	7.2*	1.0	1.6*	4.4**
CINC-1/IL-8	1.1	1.5*	5.0**	1.5*	1.0	1.4*
Osteopontin	0.4	0.6	2.7*	1.2	1.2	2.7

 

BALF = Broncho-alveolar lavage fluid; CINC-1/IL-8 = cytokine-induced neutrophil chemoattractant-1; MCP-1 = monocyte chemoattractant protein-1

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

 

 

Table 4: Gross lesions in the main and recovery group animals

	main group				recovery group
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5	5	5	5	5
Lungs ·    Discoloration	0	0	3	5	0	0	0	1
Mediastinal lymph node ·    Discoloration	0	0	0	4	0	0	1	4
Tracheobronchial lymph node ·    Discoloration	0	0	0	4	0	0	0	3

 

 

 

Table 5: Histopathology findings in rats of the main group

Lungs	main group
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Hyperplasia/hypertrophy, bronchioli; (multi)focal	0	0	0	5
·    Grade 1	0	0	0	5
Macrophages with particles; (multi)focal	0	3	0	0
·    Grade 1	0	3	0	0
Macrophages with particles; bronchial-associated lymphoid tissue; (multi)focal	0	1	4	5
·    Grade 1	0	1	4	5
Histiocytosis, alveolar, with particles; (multi)focal	0	2	5	5
·    Grade 1	0	2	5	0
·    Grade 2	0	0	0	5
Larynx (level I)	main group
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Epithelial alteration, (multi)focal	2	3	4	4
·    Grade 1	2	3	4	4
Tracheobronchial lymph nodes	main group
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Macrophages with particles; (multi)focal	0	1	3	4
·    Grade 1	0	1	3	3
·    Grade 2	0	0	0	1

 

 

 

 

Table 6: Histopathology findings after 3 weeks of recovery

Lungs	recovery group
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Hyperplasia/hypertrophy, bronchioli; (multi)focal	0	0	0	5
·    Grade 1	0	0	0	5
Macrophages with particles; (multi)focal	0	5	0	0
·    Grade 1	0	5	0	0
Macrophages with particles; bronchial-associated lymphoid tissue; (multi)focal	0	4	5	5
·    Grade 1	0	4	4	4
·    Grade 2	0	0	1	1
Histiocytosis, alveolar, with particles; (multi)focal	0	0	5	5
·    Grade 1	0	0	2	0
·    Grade 2	0	0	3	1
·    Grade 3	0	0	0	4
Hyperplasia, type ll pneumocytes; (multi)focal	0	0	0	5
·    Grade 1	0	0	0	5
Infiltrate; interstitial; lymphohistiocytic, (multi)focal	0	0	0	5
·    Grade 1	0	0	0	5
Tracheobronchial lymph nodes	recovery group
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Macrophages with particles; (multi)focal	0	3	4	4
·    Grade 1	0	3	4	1
·    Grade 2	0	0	0	3
Macrophage aggregates with particles; (multi)focal	0	0	1	4
·    Grade 1	0	0	1	3
·    Grade 2	0	0	0	1

Conclusions:

The no observed adverse effect concentration (NOAEC) for local effects was 5 mg/m³ for Pigment Yellow 139. The systemic NOAEC is above 60 mg/m³ (high concentration group). Target organ was the lung.

Executive summary:

The purpose of this study was to determine the pulmonary toxicity in rats using a short-term bioassay including bronchoalveolar lavage with clinico-chemical and cytological evaluation of lavage fluid and pathological examination of the lung. The No Observed Adverse Effect Concentration (NOAEC) after 5 days inhalation exposure to dust of the test substance was determined. In addition, recovery group animals were examined after an exposure-free period of 3 weeks to detect any reversibility or progression of potential toxic effects.

For this purpose, nine-week-old male Wistar rats (10 rats per concentration group) were nose only exposed to fresh air (control group) or dust of the test substance at concentrations of 5, 20, and 60 mg/m³ (low, mid, and high concentration) for 6 hours per day and 5 days. Body weight, mortality, and clinical observations were determined during the study. One half of the rats was examined at the end of the exposure period, whereas the other half was examined at the end of a 3-week post-exposure period by determining clinical pathology parameters including bronchoalveolar lavage with clinico-chemical and cytological evaluation of lavage fluid, organ weights and all histopathological changes.

The particle size resulted in MMADs between 0.82 and 1.28 µm with GSDs between 3.18 and 4.45. The calculated mass fractions of particles below 3 µm aerodynamic size is greater than 75 %. Thus, the aerosols were highly respirable for rats and a very high proportion of the aerosol particles reached the lungs. During the exposure period, the target concentrations were maintained as constant and stable as could be provided with dust generation techniques in the concentration range tested.

 

The exposure did not cause any clinical signs of toxicity, and there was no impairment of body weight development.

Regarding clinical pathology, no treatment related, adverse change was observed with blood parameters. After a 5-day administration period, in males of test group 3 (60 mg/m3) a slight local inflammation in the lungs can be assumed due to significantly increased total cell counts as well as neutrophil, lymphocyte and monocyte counts in the bronchoalveolar lavage fluid (BALF). Slightly increased gamma-glutamyl-transferase (GGT) and alkaline phosphatase (ALP) activities in BALF confirmed the very slight inflammation. CC-chemokine (cytokine-induced neutrophil chemoattractant-1 (CINC-1/IL8)) and CXC-chemokine levels (monocyte chemoattractant protein-1 (MCP-1)) as well as osteopontin levels in BAL were significantly increased in males of test group 3 indicating an activation of neutrophils as well as monocytes/macrophages and lymphocytes in the lungs. Neutrophil and lymphocyte counts in BALF were already marginally increased in males of test group 2 (20 mg/m³) but enzyme activities and cytokine as well as protein levels were not altered.

After a three-week recovery period, increased cell counts as well as increased MCP-1 and osteopontin levels in BAL of males in test group 3 (20 mg/m³) recovered partially, whereas enzyme activities in BAL recovered totally in this test group. Increased neutrophil and lymphocyte counts in BAL of males in test group 2 (20 mg/m³) recovered completely.

 

Regarding pathology, the main target organ was the lung. In the main group, animals of test groups 2 and 3 showed increased numbers of macrophages within alveoli and bronchioli (alveolar histiocytosis) with the cells containing yellowish to brownish particles within their cytoplasm. This finding correlated to the macroscopically observed discoloration. The particles were interpreted as the test material being phagocytized by the macrophages. In test group 3, a hyperplasia/hypertrophy of the bronchioli was observed in association with aggregates of macrophages. This reactive change is indicative of an underlying damage. Taken together, the findings were interpreted as treatment-related and adverse in test group 3. As no signs of lung damage were noted in test group 2, the findings were considered treatment-related, but not adverse. In test group 1, macrophage numbers were mostly within normal limits, but the cells also contained the yellowish to brownish particles. Particle-containing macrophages were also present in the bronchial-associated lymphoid tissue in all treated test groups. Overall, these findings were considered treatment-related, but not adverse as phagocytosis of foreign material entering the lung is part of the normal lung clearance. In the larynx (level I), minimal epithelial alteration was observed all test groups with a dose-dependent increase in severity. This well-known finding in inhalation studies was reversible after the recovery period and interpreted as treatment-related, but not adverse (Kaufmann et al., 2009).

 

Like in the lungs, particle-laden macrophages were also present in the mediastinal and tracheobronchial lymph nodes in test groups 2 and 3, causing the macroscopically observed discoloration. These findings were also considered part of the normal lung clearance with macrophages migrating to the draining lymph nodes following phagocytosis of the particles. Thus, the findings were considered treatment-related, but not adverse. After the recovery period, animals of test groups 2 and 3 revealed increased numbers of particle-containing macrophages, but they mostly formed aggregates within the bronchioli and alveoli. In test group 3, a hyperplasia/hypertrophy of the bronchioli and a type II pneumocyte hyperplasia of the alveoli was present in association with the macrophage aggregates. Further, an interstitial infiltration with lymphocytes and macrophages was present in test group 3. Overall, the findings were considered treatment-related and adverse in test group 3. As no signs of lung damage were noted in test group 2, the findings were considered treatment-related, but not adverse. In test group 1, macrophage numbers were comparable to control animals, but the cells contained yellowish to brownish particles. Particle-containing macrophages were also present in the bronchial-associated lymphoid tissue in all treated test groups. All these findings were considered to be part of the normal lung clearance and interpreted as treatment-related, but not adverse. Particle-laden macrophages were also present in the mediastinal and tracheobronchial lymph nodes in all test groups. In one animal of test group 2 and all animals of test group 3, formation of macrophage aggregates was noted. This is probably related to the increased amount of time the cells had to migrate to and accumulate within the lymph nodes. These findings were regarded treatment-related, but not adverse.

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.

 

In conclusion, inhalation exposure of rats to 60 mg/m³ of the test substance on 5 consecutive days caused increased total cell count, increased absolute and relative lymphocytes, neutrophils and monocyte counts in bronchoalveolar lavage. Moreover, several biochemical parameters (protein concentration, enzyme activities and cytokine concentrations) were significantly increased in lavage fluid. Histological examinations revealed alveolar histiocytosis with particles and hyptrophy/hyperplasia of bronchiole of the lung. At 20 mg/m³ only cytological parameters in lavage were significantly increased without histological correlate. No adverse effect was observed at 5 mg/m³. No systemic effect was observed up to the highest tested concentration of 60 mg/m³. Target organ was the lung. While parameters in lavage fluid were fully reversible within 3 weeks post-exposure period, the histological changes at 60 mg/m³ were not reversible.
Thus, under current study conditions, the no observed adverse effect concentration (NOAEC) for local effects was 5 mg/m³ and the systemic NOAEC is above 60 mg/m³ (high concentration group).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

60 mg/m³

Study duration:

subacute

Experimental exposure time per week (hours/week):

30

Species:

rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 Nov 2021 - 12 May 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Version / remarks:

25 June 2018

Deviations:

yes

Remarks:

5-day dust inhalation study in rats (with bronchoalveolar lavage, 3 weeks recovery period)

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

COMMISSION REGULATION (EU) No 260/2014 of 14 January 2014

Deviations:

not specified

Principles of method if other than guideline:

5-day dust inhalation study in rats (with bronchoalveolar lavage, 3 weeks recovery period)

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Limit test:

no

Specific details on test material used for the study:

- Name of the test substance: Pigment Yellow 139
- Batch: 200012P040
- Purity: > 99%
- Homogeneity: given
- Storage stability: 20 Jul 2030
- Storage conditions: ambient (RT)
- Appearance: solid/orange

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

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

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; 97633 Sulzfeld
- Age at study initiation: about 7 weeks (when supplied)
- Weight at study initiation (means): ca 255g
- Housing: The rats were housed together (up to 5 animals per cage) in Polysulfon cages (H-Temp [P SU]) supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm2). Bedding in the Polycarbonate cages were Type Lignocel fibres, dust-free bedding, supplied by SSNIFF, Soest,
Germany. Dust-free wooden bedding was used in this study. For enrichment wooden gnawing blocks and play tunnel were added.
- Diet: Mouse/rat laboratory diet “GLP”, 10 mm pellets (Provimi Kliba SA, Kaiseraugst, Basel Switzerland), ad libitum.
- Water: Tap water, ad libitum

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

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose/head only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

>= 0.82 - <= 1.28 µm

Geometric standard deviation (GSD):

3.5

Remarks on MMAD:

MMAD (cascade impactor): between 0.82 and 1.28 µm with GSDs between 3.18 and 4.45. The calculated mass fractions of particles below 3 µm aerodynamic size is greater than 75 %. Thus, the aerosols were highly respirable for rats and a very high proportion of the aerosol particles reached the lungs. SMPS showed different geometric mean concentration than those measured by cascade impactor measurement. Major reason is that this geometric mean referred to count distribution, while cascade impactor measurement measured mass-based aerodynamic diameter. The geometric mean count diameters were between 289 nm and 304 nm.

Details on inhalation exposure:

For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage, mixed with conditioned air, and passed via the cyclonic separator and glass tube into the inhalation system

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Generator systems: Solid particle generators (brush-generator), Aerosol mixing tube (Stainless steel), Glass cyclonic separators
- Generation procedure: The test substance was used unchanged. By means of dust generators the substance to be tested is generated into dust aerosols using compressed air in a mixing stage, mixed
with conditioned air and passed into the inhalation systems via cyclonic separators. For each concentration, a solid particle generator (brush-generator) wias used for generating the dust. The con
centration was adjusted by varying the piston feed and by varying the brush rotation. 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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of the inhalation atmospheres in test groups 1 - 3 were analyzed by gravimetry. This method was applicable because the test item possessed extremely low vapor pressure. Daily means were calculated based on 3 measured samples per concentration and exposure. From the daily mean values of each concentration, mean concentrations and standard deviations for the entire study were derived.In these groups, the constancy of concentrations in each chamber was continuously monitored using scattered light photometers.

The particle size analysis was carried out with a cascade impactor with the following equipment:
• Stack sampler Marple 298 (New Star Environmental, Inc., Roswell, Georgia 30075, USA)
• Vacuum compressed air pump (Millipore Corporation, Billerica, MA 01821, USA)
• Limiting orifice 3 L/min (Millipore Corporation, Billerica, MA 01821, USA)
• Sampling probe internal diameter 6.9 mm
• Balance Sartorius MSA 6.6S-000-DF (Sartorius AG, Göttingen, Germany)
Sampling for particle size analyses:Pre-weighed metal collecting discs and a backup particle filter were placed into the cascade impactor and two samples were taken in each concentration at a sampling velocity of 1.25 m/sec. from the breathing zones of the animals.
The amount of dust deposited by each stage in mg was calculated from the difference between the weight of the filter/metal collecting disc and backup filter before and after sampling.The deposits in the probe and the wall losses in the impactor were also determined as difference of the total mass increase of the impactor and the sum of masses on the collecting discs and backup filter.

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. During the exposure period, one measurement per concentration with 10 repeats each were performed.

Real time surveillance of the inhalation atmospheres with scattered light photometers generally proved the constancy of each concentration throughout the daily exposures.
The air flows were constantly maintained in the desired range. An air change of about 65 to 67 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system. Daily mean relative humidities in the inhalation systems ranged between 33.7 and 49.6 %. Daily mean temperatures in the inhalation systems ranged between 20.6 and 22.1 °C. These values were within guideline recommendations.

Duration of treatment / exposure:

6h for 5 days

Frequency of treatment:

daily

Dose / conc.:

5.2 mg/m³ air (analytical)

Remarks:

125548 particles per cm3 (geometric mean diameter 289 nm)

Dose / conc.:

20.4 mg/m³ air (analytical)

Remarks:

222788 particles per cm3 (geometric mean diameter 302 nm)

Dose / conc.:

59.4 mg/m³ air (analytical)

Remarks:

382709 particles per cm3 (geometric mean diameter 304 nm)

No. of animals per sex per dose:

10 (five for sacrifice after exposure and 5 for sacrifice after recovery)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: not specified
- Rationale for animal assignment: random
- Fasting period before blood sampling for clinical biochemical biochemistry: overnight
- Rationale for selecting satellite groups: Clearance of inert particles by lung macrophages is known to take time
- Post-exposure recovery period in satellite groups: 3 weeks

Positive control:

No

Observations and examinations performed and frequency:

MORTALITY: Yes
A check for moribund or dead animals was carried out twice per day on working days and on Saturday and Sunday. A check for moribund or dead animals was carried out once per day on weekends and holidays.

CLINICAL OBSERVATION: Yes
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
The animals were weighed prior to the pre-exposure period (study day -5), at the start of the exposure period (study day 0), at the end of the exposure period (study day 4), as well as on the study days 5, 12, 19 and 26.

FOOD CONSUMPTION: Yes
Food consumption was determined once over the exposure period (study day 0 – study day 4), during the post-exposure period weekly and calculated as mean food consumption in grams per animal and day.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

Sacrifice and pathology:

CLINICAL PATHOLOGY
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood samples were carried out in a randomized sequence (the list of randomization instructions was compiled with a computer).
The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results.
The results of clinical pathology examinations were expressed in International System (SI) units. The following parameters of the animals were examined:
- Hematology: leukocyte count, erythrocyte count, hemoglobin, hematocrit, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, differential blood count, reticulocytes
- Clinical chemistry: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase,-Glutamyltransferase, Inorganic phosphate, Calcium, Urea, Creatinine, Glucose, Total bilirubin, Total protein, Albumin, Globulins, Triglycerides, Cholesterol
- Bronchoalveolar lavage fluid (BAL): 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 parameters and methods of cytological examination in BAL was carried out: Total cell count, Macrophages, Polymorphonuclear neutrophils, Lymphocytes, Eosinophils, Monocytes, Epithelial, Gamma−Glutamyltransferase, Protein, Lactate dehydrogenase, Alkaline phosphatase, N-acetyl-Beta-Glucosaminidase. Cytokines in BAL: Rat monocyte chemoattractant protein-1 (rat MCP-1), Rat cytokine-induced neutrophil chemoattractant-1 level (rat CINC-1/IL-8), Rodent osteopontin

NECROPSY
The animals were sacrificed under pentobarbital anesthesia by exsanguination from the abdominal aorta and vena cava. Afterwards, the thorax was opened, the right lung lobes were lavaged, whereas the left lung lobe was ligated during lavage. Immediately after lung lavage, the animals were necropsied and assessed by gross pathology.

The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (terminal body weight)
2. Adrenal glands (fixed)
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Lungs
9. Spleen
10. Testes
11. Thymus (fixed)
12. Thyroid glands (with parathyroid glands) (fixed)
All paired organs were weighed together (left and right).

The following organs or tissues were fixed in 4% neutral buffered formaldehyde solution:
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain with olfactory bulb
5. Epididymides
6. Esophagus
7. Eyes with optic nerve
8. Heart
9. Kidneys
10. Larynx/pharynx
11. Liver
12. Lungs
13. Lymph nodes (tracheobronchial and mediastinal lymph nodes)
14. Nose (nasal cavity)
15. Seminal vesicles
16. Spinal cord (cervical, thoracic and lumbar cord)
17. Spleen
18. Stomach (forestomach and glandular stomach)
19. Testes
20. Thyroid glands
21. Thymus
22. Trachea
23. Urinary bladder


Extend of histological processing and subsequent microscopical examinations in main group animals: all gross lesions, larynx (3 level), lungs, lymph nodes (tracheobronchial, mediastinal), nasal cavity (4 levels), trachea and in recovery group animals: all gross lesions, larynx (3 level), lungs, lymph nodes (tracheobronchial, mediastinal)

Other examinations:

Lung lavage: The animals intended for lung lavage were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava. The right lung will be lavaged in situ with physiological saline, whereas the left lung was ligated during this procedure.

Statistics:

Body weight, body weight change: Comparison of each group with the control group was performed using DUNNETT test (two-sided) for the hypothesis of equal means
Blood parameters: For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting pvalue was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians
For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
BALF: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
Organ weights: Non-parametric one-way analysis using the Kruskal-Wallis test (two-sided). If the resulting p-value was equal to or less than 0.05, a pair-wise comparison of each dose group with the control group was performed using the Wilcoxon test (two-sided) for the hypothesis of equal medians.

Terminal body weight: Comparison of each group with the control group was performed using the Dunnett test (two-sided) for the hypothesis of equal means.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Immediately after exposure, one of the ten animals of the test group 1 (5 mg/m³) and all animals of test group 2 (20 mg/m³) and 3 (60 mg/m³) showed test item contaminated fur. In eight of the ten test group 3 animals (60 mg/m³) substance-like discoloration of the nose region was observed before and during exposure on study days 2 and 3. This clinical finding was substance-related, but not adverse because it simply shows the exposure to the solid coloured dusty test substance.

No adverse findings were noted.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of the administration period, in males of test group 3 (60 mg/m3) absolute and relative neutrophil counts were significantly increased, and in males of test groups 1 and 2 (5 and 20 mg/m3) relative neutrophil counts were increased (in test group 2 not statistically significantly. However, all values were within historical control ranges (males, absolute neutrophils 0.53- 1.01Giga/L, relative neutrophils 8.5-16.0 %). Therefore, these alterations were regarded as incidental and not treatment related.

Clinical biochemistry findings:

no effects observed

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:

no effects observed

Description (incidence and severity):

See attached tables in "Overall remarks and attachments".

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

3 out of 5 animals of test group 2 and all animals of test group 3 of the main group revealed an orange discoloration of the lungs (see Table 4). In addition, a yellow discoloration of the mediastinal and tracheobronchial lymph nodes was observed in 4 out of 5 animals in test group 3. These findings correlated with the histological presence of particle-containing macrophages in these organs and were considered treatment-related.

After three weeks of recovery, the following was observed:
A yellow to orange discoloration of the lungs, mediastinal and tracheobronchial lymph nodes was present in almost all animals of test group 3 of the recovery group. In test group 2, one animal showed a yellow discoloration of the mediastinal lymph node. These findings correlated with the histological presence of particle-containing macrophages in these organs and were considered treatment-related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In three animals of test group 1, macrophages in the lungs were present in similar numbers to controls, but contained yellowish to brownish particles in their cytoplasm (see Tables 5 and 6). In addition, two animals of test group 1 as well as all animals of test groups 2 and 3 showed a dose-dependent minimal to slight increase in the number of macrophages within bronchioli and alveoli (alveolar histiocytosis) with the cells also containing yellowish to brownish particles. In test groups 2 and 3, macrophages were either individually distributed or formed aggregates within bronchioli. The latter showed a minimal increase in cell size, cell number, and/or cell layers of the epithelium (hyperplasia/hypertrophy) in test group 3, only. In addition, particle-containing macrophages were also present in the bronchial-associated lymphoid tissue (BALT) in all treated test groups.
In the larynges at level I, a minimal flattening of epithelial cells, partly associated with an increase in cell layers was multifocally present. The tracheobronchial lymph nodes were chosen exemplarily, similar findings were observed in the mediastinal lymph node. Macrophages containing intracytoplasmic, yellowish to brownish particles were observed in these lymph nodes in minimal to slight numbers.


After three weeks of recovery, the following was observed:
A yellow to orange discoloration of the lungs, mediastinal and tracheobronchial lymph nodes was present in almost all animals of test group 3 of the recovery group. In test group 2, one animal showed a yellow discoloration of the mediastinal lymph node. These findings correlated with the histological presence of particle-containing macrophages in these organs and were considered treatment-related.
In all animals of test group 1, macrophages in the lungs were present in similar numbers to controls, but contained yellowish to brownish particles in their cytoplasm. In all animals of test groups 2 and 3, a dose-dependent minimal to moderate increase in the number of macrophages (alveolar histiocytosis) was observed. The macrophages contained brownish particles and mainly formed aggregates within bronchioli and alveoli. In test group 3, the affected bronchioli showed a minimal increase in cell size, cell number, and/or cell layers of the epithelium (hyperplasia/hypertrophy) and the number of type II pneumocytes was minimally increased in affected alveoli (hyperplasia). Further, lymphocytes and macrophages were also infiltrating the interstitium in minimal numbers in animals of test group 3. In addition, minimal to slight numbers of particle-containing macrophages were found in the bronchial-associated lymphoid tissue in all treated test groups.
The tracheobronchial lymph nodes were chosen exemplarily, similar findings were observed in the mediastinal lymph node. Macrophages containing intracytoplasmic, yellowish to brownish particles were either individually distributed throughout the lymph node or formed aggregates.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Bronchoalveolar lavage fluid (BAL): Table 1-3
After the administration period, in males of test group 3 (60 mg/m3) total cell counts were marginally, but significantly increased, whereas absolute and relative lymphocyte, monocyte and neutrophil counts were moderately increased (lymphocytes not statistically significantly).
Absolute and relative neutrophil and lymphocyte counts were already marginally, but significantly increased in males of test group 2 (20 mg/m3). Relative macrophage counts were significantly decreased in males of test group 2 and 3. These alterations were regarded as treatment related and adverse.
After the 3-week recovery period absolute and relative lymphocyte, monocyte and neutrophil counts were still marginally increased in males of test group 3 (monocyte counts not statistically significantly) whereas the significantly increased total cell counts among these individuals were regarded as not toxicologically relevant. Relative macrophage counts were also still significantly decreased in BAL of males in test group 3. Neutrophil counts were also significantly increased in BAL of males in test group 1 (5 mg/m3), but this change was not dose dependent and therefore it was regarded as incidental and not treatment related.
After the administration period, in males of test group 3 (60 mg/m3) -glutamyl-transferase (GGT) and alkaline phosphatase (ALP) activities in BAL were slightly but significantly increased. These alterations were regarded as treatment related and adverse. Total protein and -N-acetylglucosaminidase (NAG) activities were only marginally (≤ 2-fold) increased. These increases were regarded as treatment related but non-adverse.
After the recovery period total protein levels as well as GGT, LDH and NAG levels in BAL of males in test group 3 (60 mg/m3) were significantly increased. However, when regarding the absolute values, they were within historical control ranges (males, total protein 2-50 mg/L, GGT 25-57 nkat/L, LDH 0.21-0.65 μkat/L, NAG 18-56 nkat/L). Therefore, these alterations were regarded as incidental and not treatment related.
After the administration period, in males of test group 3 (60 mg/m3) cytokine-induced neutrophil chemoattractant-1 (CINC-1/IL8), monocyte chemoattractant protein-1 (MCP-1) and osteopontin
levels in BAL were significantly increased. These alterations were regarded as treatment related and adverse. CINC-1/IL-8 values were already significantly increased in males of test group 2 (20 mg/m3) but the increase was below 2-fold, and therefore, it was regarded as treatment
related but non-adverse.
After the three-week recovery period in males of test group 3 (60 mg/m3) MCP-1 and osteopontin levels in BAL were still more than 2-fold increased (osteopontin not statistically significantly)
whereas CINC-1/IL-8 levels in males of test groups 1 and 3 (5 and 60 mg/m3) as well as MCP1 values in males of test group 2 (20 mg/m3) were significantly but less than 2-fold increased. Therefore, only CINC-1/IL-8 and osteopontin level increases in males of test group 3 were
regarded as relevant.

Dose descriptor:

NOAEC

Remarks:

local effects

Effect level:

5.2 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: at 20 mg/m3: Increased absolute and relative neutrophil and lymphocyte and decreased relative macrophage counts in BAL after exposure

Dose descriptor:

NOAEC

Remarks:

systemic effects

Effect level:

>= 59.4 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: only findings in BALF

Critical effects observed:

yes

Lowest effective dose / conc.:

59.4 mg/m³ air (analytical)

System:

respiratory system: upper respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

No adverse effect was observed at 5 mg/m³. No systemic effect was observed up to the highest tested concentration of 60 mg/m³. Target organ was the lung. While parameters in lavage fluid were fully reversible within 3 weeks post-exposure period, the histological changes at 60 mg/m³ were not reversible within the 3 -week recovery period.

 

 

 

Table 1: Changes in mean absolute cell counts in BAL (x-fold of concurrent control) on study day 5 (1 day after last exposure) and study days 26 (3 weeks after last exposure). At study day 5, mean of differential cell counts in control group (test group 0) is based on only two individuals due to bad cell quality. Therefore, for these parameters, statistics could not be performed.

	Study day 5			Study day 26
	Gr. 1 5 mg/m3	Gr. 2 20 mg/m3	Gr. 3 60 mg/m3	Gr. 1 5 mg/m3	Gr. 2 20 mg/m3	Gr. 3 60 mg/m3
Total Cells	0.9	0.9	2.2*	1.1	1.0	1.4*
Eosinophils	0.6	1.1	0.7	+	+	+
Lymphocytes	1.6	2.2*	12.7	0.8	1.2	3.4*
Macrophages	0.9	0.8	0.8	1.0	1.0	1.0
Neutrophils	0.3	10.2**	127.9**	4.8*	2.4	13.5**
Monocytes	+	+	+**	0.4	0.7	7.4
Epithelial cells	0.9	0.7	0.0	3.3	0.2	1.5

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

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

 

 

Table 2: Changes in mean total protein and enzyme levels in BAL (x-fold of concurrent control) of males on study day 5 (1 day after last exposure) and study day 26 (3 weeks afterlast exposure)

	Study day 5			Study day 26
	Gr. 1 5 mg/m3	Gr. 2 20 mg/m3	Gr. 3 60 mg/m3	Gr. 1 5 mg/m3	Gr. 2 20 mg/m3	Gr. 3 60 mg/m3
Total Protein	0.9	1.1	1.6*	0.9	0.9	1.3*
GGT	1.1	1.3	2.6**	1.0	1.2	1.5**
LDH	1.1	0.9	1.8	1.2	1.1	1.8**
ALP	1.2	1.3*	2.3**	0.8	0.9	1.2
NAG	1.3	1.3	1.6**	1.8	0.9	2.1*

GGT = Gamma-Glutamyl-transferase; LDH = Lactate dehydrogenase; ALP = Alkaline phosphatase; NAG = Beta-N-Acetyl glucosaminidase, One-sided Wilcoxon-test: * : p <= 0.05; ** : p <= 0.01

 

 

 

Table 3: Changes in antigen levels in BAL (x-fold of concurrent control means) of males onstudy day 5 (1 day after last exposure) and study day 26 (3 weeks after last exposure)

	Study day 5			Study day 26
	Gr. 1 5 mg/m3	Gr. 2 20 mg/m3	Gr. 3 60 mg/m3	Gr. 1 5 mg/m3	Gr. 2 20 mg/m3	Gr. 3 60 mg/m3
MCP-1	0.9	1.8	7.2*	1.0	1.6*	4.4**
CINC-1/IL-8	1.1	1.5*	5.0**	1.5*	1.0	1.4*
Osteopontin	0.4	0.6	2.7*	1.2	1.2	2.7

 

BALF = Broncho-alveolar lavage fluid; CINC-1/IL-8 = cytokine-induced neutrophil chemoattractant-1; MCP-1 = monocyte chemoattractant protein-1

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

 

 

Table 4: Gross lesions in the main and recovery group animals

	main group				recovery group
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5	5	5	5	5
Lungs ·    Discoloration	0	0	3	5	0	0	0	1
Mediastinal lymph node ·    Discoloration	0	0	0	4	0	0	1	4
Tracheobronchial lymph node ·    Discoloration	0	0	0	4	0	0	0	3

 

 

 

Table 5: Histopathology findings in rats of the main group

Lungs	main group
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Hyperplasia/hypertrophy, bronchioli; (multi)focal	0	0	0	5
·    Grade 1	0	0	0	5
Macrophages with particles; (multi)focal	0	3	0	0
·    Grade 1	0	3	0	0
Macrophages with particles; bronchial-associated lymphoid tissue; (multi)focal	0	1	4	5
·    Grade 1	0	1	4	5
Histiocytosis, alveolar, with particles; (multi)focal	0	2	5	5
·    Grade 1	0	2	5	0
·    Grade 2	0	0	0	5
Larynx (level I)	main group
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Epithelial alteration, (multi)focal	2	3	4	4
·    Grade 1	2	3	4	4
Tracheobronchial lymph nodes	main group
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Macrophages with particles; (multi)focal	0	1	3	4
·    Grade 1	0	1	3	3
·    Grade 2	0	0	0	1

 

 

 

 

Table 6: Histopathology findings after 3 weeks of recovery

Lungs	recovery group
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Hyperplasia/hypertrophy, bronchioli; (multi)focal	0	0	0	5
·    Grade 1	0	0	0	5
Macrophages with particles; (multi)focal	0	5	0	0
·    Grade 1	0	5	0	0
Macrophages with particles; bronchial-associated lymphoid tissue; (multi)focal	0	4	5	5
·    Grade 1	0	4	4	4
·    Grade 2	0	0	1	1
Histiocytosis, alveolar, with particles; (multi)focal	0	0	5	5
·    Grade 1	0	0	2	0
·    Grade 2	0	0	3	1
·    Grade 3	0	0	0	4
Hyperplasia, type ll pneumocytes; (multi)focal	0	0	0	5
·    Grade 1	0	0	0	5
Infiltrate; interstitial; lymphohistiocytic, (multi)focal	0	0	0	5
·    Grade 1	0	0	0	5
Tracheobronchial lymph nodes	recovery group
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Macrophages with particles; (multi)focal	0	3	4	4
·    Grade 1	0	3	4	1
·    Grade 2	0	0	0	3
Macrophage aggregates with particles; (multi)focal	0	0	1	4
·    Grade 1	0	0	1	3
·    Grade 2	0	0	0	1

Conclusions:

The no observed adverse effect concentration (NOAEC) for local effects was 5 mg/m³ for Pigment Yellow 139. The systemic NOAEC is above 60 mg/m³ (high concentration group). Target organ was the lung.

Executive summary:

The purpose of this study was to determine the pulmonary toxicity in rats using a short-term bioassay including bronchoalveolar lavage with clinico-chemical and cytological evaluation of lavage fluid and pathological examination of the lung. The No Observed Adverse Effect Concentration (NOAEC) after 5 days inhalation exposure to dust of the test substance was determined. In addition, recovery group animals were examined after an exposure-free period of 3 weeks to detect any reversibility or progression of potential toxic effects.

For this purpose, nine-week-old male Wistar rats (10 rats per concentration group) were nose only exposed to fresh air (control group) or dust of the test substance at concentrations of 5, 20, and 60 mg/m³ (low, mid, and high concentration) for 6 hours per day and 5 days. Body weight, mortality, and clinical observations were determined during the study. One half of the rats was examined at the end of the exposure period, whereas the other half was examined at the end of a 3-week post-exposure period by determining clinical pathology parameters including bronchoalveolar lavage with clinico-chemical and cytological evaluation of lavage fluid, organ weights and all histopathological changes.

The particle size resulted in MMADs between 0.82 and 1.28 µm with GSDs between 3.18 and 4.45. The calculated mass fractions of particles below 3 µm aerodynamic size is greater than 75 %. Thus, the aerosols were highly respirable for rats and a very high proportion of the aerosol particles reached the lungs. During the exposure period, the target concentrations were maintained as constant and stable as could be provided with dust generation techniques in the concentration range tested.

 

The exposure did not cause any clinical signs of toxicity, and there was no impairment of body weight development.

Regarding clinical pathology, no treatment related, adverse change was observed with blood parameters. After a 5-day administration period, in males of test group 3 (60 mg/m3) a slight local inflammation in the lungs can be assumed due to significantly increased total cell counts as well as neutrophil, lymphocyte and monocyte counts in the bronchoalveolar lavage fluid (BALF). Slightly increased gamma-glutamyl-transferase (GGT) and alkaline phosphatase (ALP) activities in BALF confirmed the very slight inflammation. CC-chemokine (cytokine-induced neutrophil chemoattractant-1 (CINC-1/IL8)) and CXC-chemokine levels (monocyte chemoattractant protein-1 (MCP-1)) as well as osteopontin levels in BAL were significantly increased in males of test group 3 indicating an activation of neutrophils as well as monocytes/macrophages and lymphocytes in the lungs. Neutrophil and lymphocyte counts in BALF were already marginally increased in males of test group 2 (20 mg/m³) but enzyme activities and cytokine as well as protein levels were not altered.

After a three-week recovery period, increased cell counts as well as increased MCP-1 and osteopontin levels in BAL of males in test group 3 (20 mg/m³) recovered partially, whereas enzyme activities in BAL recovered totally in this test group. Increased neutrophil and lymphocyte counts in BAL of males in test group 2 (20 mg/m³) recovered completely.

 

Regarding pathology, the main target organ was the lung. In the main group, animals of test groups 2 and 3 showed increased numbers of macrophages within alveoli and bronchioli (alveolar histiocytosis) with the cells containing yellowish to brownish particles within their cytoplasm. This finding correlated to the macroscopically observed discoloration. The particles were interpreted as the test material being phagocytized by the macrophages. In test group 3, a hyperplasia/hypertrophy of the bronchioli was observed in association with aggregates of macrophages. This reactive change is indicative of an underlying damage. Taken together, the findings were interpreted as treatment-related and adverse in test group 3. As no signs of lung damage were noted in test group 2, the findings were considered treatment-related, but not adverse. In test group 1, macrophage numbers were mostly within normal limits, but the cells also contained the yellowish to brownish particles. Particle-containing macrophages were also present in the bronchial-associated lymphoid tissue in all treated test groups. Overall, these findings were considered treatment-related, but not adverse as phagocytosis of foreign material entering the lung is part of the normal lung clearance. In the larynx (level I), minimal epithelial alteration was observed all test groups with a dose-dependent increase in severity. This well-known finding in inhalation studies was reversible after the recovery period and interpreted as treatment-related, but not adverse (Kaufmann et al., 2009).

 

Like in the lungs, particle-laden macrophages were also present in the mediastinal and tracheobronchial lymph nodes in test groups 2 and 3, causing the macroscopically observed discoloration. These findings were also considered part of the normal lung clearance with macrophages migrating to the draining lymph nodes following phagocytosis of the particles. Thus, the findings were considered treatment-related, but not adverse. After the recovery period, animals of test groups 2 and 3 revealed increased numbers of particle-containing macrophages, but they mostly formed aggregates within the bronchioli and alveoli. In test group 3, a hyperplasia/hypertrophy of the bronchioli and a type II pneumocyte hyperplasia of the alveoli was present in association with the macrophage aggregates. Further, an interstitial infiltration with lymphocytes and macrophages was present in test group 3. Overall, the findings were considered treatment-related and adverse in test group 3. As no signs of lung damage were noted in test group 2, the findings were considered treatment-related, but not adverse. In test group 1, macrophage numbers were comparable to control animals, but the cells contained yellowish to brownish particles. Particle-containing macrophages were also present in the bronchial-associated lymphoid tissue in all treated test groups. All these findings were considered to be part of the normal lung clearance and interpreted as treatment-related, but not adverse. Particle-laden macrophages were also present in the mediastinal and tracheobronchial lymph nodes in all test groups. In one animal of test group 2 and all animals of test group 3, formation of macrophage aggregates was noted. This is probably related to the increased amount of time the cells had to migrate to and accumulate within the lymph nodes. These findings were regarded treatment-related, but not adverse.

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.

 

In conclusion, inhalation exposure of rats to 60 mg/m³ of the test substance on 5 consecutive days caused increased total cell count, increased absolute and relative lymphocytes, neutrophils and monocyte counts in bronchoalveolar lavage. Moreover, several biochemical parameters (protein concentration, enzyme activities and cytokine concentrations) were significantly increased in lavage fluid. Histological examinations revealed alveolar histiocytosis with particles and hyptrophy/hyperplasia of bronchiole of the lung. At 20 mg/m³ only cytological parameters in lavage were significantly increased without histological correlate. No adverse effect was observed at 5 mg/m³. No systemic effect was observed up to the highest tested concentration of 60 mg/m³. Target organ was the lung. While parameters in lavage fluid were fully reversible within 3 weeks post-exposure period, the histological changes at 60 mg/m³ were not reversible.
Thus, under current study conditions, the no observed adverse effect concentration (NOAEC) for local effects was 5 mg/m³ and the systemic NOAEC is above 60 mg/m³ (high concentration group).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

5 mg/m³

Study duration:

subacute

Species:

rat

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

Repeated dose toxicity: oral

 

There is a reliable study available conducted with the test substance. The assessment of repeated oral dose toxicity may be extended to the overall data set for organic pigment which has been published by Stratmann et al in 2020. (“Indicators for lack of systemic availability of organic pigments” by Stratmann et al. published in Regulatory Toxicology and Pharmacology in 2020). The publication describes the overall search for experimental data on organic pigments in public databases and systematically analyses the public information on design and results. As in that time, no comprehensive information on organic pigment particle properties was required for health hazard related studies, the investigation could not include a nano/non-nano assessment of results. However, most of the pigments in the test set are expected to be nanomaterials according the EU recommendation as they are listed in EU nanomaterial inventories e.g. French nano inventory. Fifty-seven studies on repeated dose oral toxicity were available for 52 organic pigments. Of the 52 organic pigments, 44 showed no effects up to the limit dose of 1000 mg/kg bw and none of them are classified for repeated-dose toxicity for the oral route. Effects observed were related to susceptibility to extreme pH. There is no indication that for studies by the oral route, there is a difference between forms that are powders or "fine powders" (fulfilling the criteria of a nanomaterial).

 

Key study: Repeated dose toxicity, oral

The 28 day repeated dose study with rats was performed according to the OECD guideline 407 following GLP requirements (Mitsubishi, 2000). The study deviated from the guideline in that a functional observation battery was not included. The test substance (purity: 99.3 weight-%) was administered by gavage to male and female SD rats for 28 days at doses of 0, 8, 40, 200 and 1000 mg/kg bw to investigate its toxicity and, if necessary possible reversibility. A control group was dosed with a vehicle (0.5 % CMC-Na aqueous solution mixed with 0.1% Tween 80) only.

No changes attributable to the administration of the test substance were observed in any observations or measurements, namely, clinical sign, body weight, food consumption, hematology, blood chemistry, urinalysis, organ weight, necropsy or histology. Therefore, under the conditions of this test the NOEL of the test substance was determined to be 1000 mg/kg bw in both sexes.

 

 

Repeated dose toxicity: inhalation

 

Because there is no repeated dose inhalation toxicity study available with the test substance itself, a read-across is proposed. The read-across from analogue substance CAS 36888-99-0 to CAS 76199-85-4 is valid because the physico-chemical and toxicological properties of the particles are sufficiently similar.

The test item is a powder, and inhalation of particles is possible. Notably, the overall strategy for organic pigments includes studies for particle surface reactivity and cytotoxicity to lung macrophages (see IUCLID chapter 7.9 "Specific investigations") and a short-term inhalation study with BAL, investigating the repeated-dose inhalation toxicity after 5 days of exposure and after three weeks of recovery (see IUCLID chapter section 7.5.2). These studies were all conducted with the test substance itself, or in case of the short-term inhalation study with the structural analogue substance CAS 36888-99-0 because no repeated dose inhalation study is available for CAS 76199-85-4. The read-across from CAS 36888-99-0 to CAS 76199-85-4 is valid because the physico-chemical properties of these two particles are sufficiently similar.

 

Read-across from CAS 36888-99-0: Key study: Repeated dose toxicity, inhalation

The purpose of this study was to determine the pulmonary toxicity in rats using a short-term bioassay including bronchoalveolar lavage with clinico-chemical and cytological evaluation of lavage fluid and pathological examination of the lung (BASF, 2022). The No Observed Adverse Effect Concentration (NOAEC) after 5 days inhalation exposure to dust of the test substance was determined. In addition, recovery group animals were examined after an exposure-free period of 3 weeks to detect any reversibility or progression of potential toxic effects.

For this purpose, nine-week-old male Wistar rats (10 rats per concentration group) were nose only exposed to fresh air (control group) or dust of the test substance at concentrations of 5, 20, and 60 mg/m³ (low, mid, and high concentration) for 6 hours per day and 5 days. Body weight, mortality, and clinical observations were determined during the study. One half of the rats was examined at the end of the exposure period, whereas the other half was examined at the end of a 3-week post-exposure period by determining clinical pathology parameters including bronchoalveolar lavage with clinico-chemical and cytological evaluation of lavage fluid, organ weights and all histopathological changes.

The particle size resulted in MMADs between 0.82 and 1.28 µm with GSDs between 3.18 and 4.45. The calculated mass fractions of particles below 3 µm aerodynamic size is greater than 75 %. Thus, the aerosols were highly respirable for rats and a very high proportion of the aerosol particles reached the lungs. During the exposure period, the target concentrations were maintained as constant and stable as could be provided with dust generation techniques in the concentration range tested.

The exposure did not cause any clinical signs of toxicity, and there was no impairment of body weight development.

Regarding clinical pathology, no treatment related, adverse change was observed with blood parameters. After a 5-day administration period, in males of test group 3 (60 mg/m3) a slight local inflammation in the lungs can be assumed due to significantly increased total cell counts as well as neutrophil, lymphocyte and monocyte counts in the bronchoalveolar lavage fluid (BALF). Slightly increased gamma-glutamyl-transferase (GGT) and alkaline phosphatase (ALP) activities in BALF confirmed the very slight inflammation. CC-chemokine (cytokine-induced neutrophil chemoattractant-1 (CINC-1/IL8)) and CXC-chemokine levels (monocyte chemoattractant protein-1 (MCP-1)) as well as osteopontin levels in BAL were significantly increased in males of test group 3 indicating an activation of neutrophils as well as monocytes/macrophages and lymphocytes in the lungs. Neutrophil and lymphocyte counts in BALF were already marginally increased in males of test group 2 (20 mg/m³) but enzyme activities and cytokine as well as protein levels were not altered.

After a three-week recovery period, increased cell counts as well as increased MCP-1 and osteopontin levels in BAL of males in test group 3 (20 mg/m³) recovered partially, whereas enzyme activities in BAL recovered totally in this test group. Increased neutrophil and lymphocyte counts in BAL of males in test group 2 (20 mg/m³) recovered completely.

Regarding pathology, the main target organ was the lung. In the main group, animals of test groups 2 and 3 showed increased numbers of macrophages within alveoli and bronchioli (alveolar histiocytosis) with the cells containing yellowish to brownish particles within their cytoplasm. This finding correlated to the macroscopically observed discoloration. The particles were interpreted as the test material being phagocytized by the macrophages. In test group 3, a hyperplasia/hypertrophy of the bronchioli was observed in association with aggregates of macrophages. This reactive change is indicative of an underlying damage. Taken together, the findings were interpreted as treatment-related and adverse in test group 3. As no signs of lung damage were noted in test group 2, the findings were considered treatment-related, but not adverse. In test group 1, macrophage numbers were mostly within normal limits, but the cells also contained the yellowish to brownish particles. Particle-containing macrophages were also present in the bronchial-associated lymphoid tissue in all treated test groups. Overall, these findings were considered treatment-related, but not adverse as phagocytosis of foreign material entering the lung is part of the normal lung clearance. In the larynx (level I), minimal epithelial alteration was observed all test groups with a dose-dependent increase in severity. This well-known finding in inhalation studies was reversible after the recovery period and interpreted as treatment-related, but not adverse (Kaufmann et al., 2009).

Like in the lungs, particle-laden macrophages were also present in the mediastinal and tracheobronchial lymph nodes in test groups 2 and 3, causing the macroscopically observed discoloration. These findings were also considered part of the normal lung clearance with macrophages migrating to the draining lymph nodes following phagocytosis of the particles. Thus, the findings were considered treatment-related, but not adverse. After the recovery period, animals of test groups 2 and 3 revealed increased numbers of particle-containing macrophages, but they mostly formed aggregates within the bronchioli and alveoli. In test group 3, a hyperplasia/hypertrophy of the bronchioli and a type II pneumocyte hyperplasia of the alveoli was present in association with the macrophage aggregates. Further, an interstitial infiltration with lymphocytes and macrophages was present in test group 3. Overall, the findings were considered treatment-related and adverse in test group 3. As no signs of lung damage were noted in test group 2, the findings were considered treatment-related, but not adverse. In test group 1, macrophage numbers were comparable to control animals, but the cells contained yellowish to brownish particles. Particle-containing macrophages were also present in the bronchial-associated lymphoid tissue in all treated test groups. All these findings were considered to be part of the normal lung clearance and interpreted as treatment-related, but not adverse. Particle-laden macrophages were also present in the mediastinal and tracheobronchial lymph nodes in all test groups. In one animal of test group 2 and all animals of test group 3, formation of macrophage aggregates was noted. This is probably related to the increased amount of time the cells had to migrate to and accumulate within the lymph nodes. These findings were regarded treatment-related, but not adverse.

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.

In conclusion, inhalation exposure of rats to 60 mg/m³ of the test substance on 5 consecutive days caused increased total cell count, increased absolute and relative lymphocytes, neutrophils and monocyte counts in bronchoalveolar lavage. Moreover, several biochemical parameters (protein concentration, enzyme activities and cytokine concentrations) were significantly increased in lavage fluid. Histological examinations revealed alveolar histiocytosis with particles and hyptrophy/hyperplasia of bronchiole of the lung. At 20 mg/m³ only cytological parameters in lavage were significantly increased without histological correlate. No adverse effect was observed at 5 mg/m³. No systemic effect was observed up to the highest tested concentration of 60 mg/m³. Target organ was the lung. While parameters in lavage fluid were fully reversible within 3 weeks post-exposure period, the histological changes at 60 mg/m³ were not reversible.
Thus, under current study conditions, the no observed adverse effect concentration (NOAEC) for local effects was 5 mg/m³ and the systemic NOAEC is above 60 mg/m³ (high concentration group).

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. There were no toxic effects observed up to the highest tested dose of 1000 mg/kg bw/day after subacute oral exposure in rats. Moreover, no systemic effect was observed up to the highest tested concentration of 60 mg/m³ in a repeated dose inhalative toxicity study. As a result, the substance is not classified for repeated dose toxicity under Regulation (EC) No. 1272/2008, as amended for the seventeenth time in Regulation (EC) No. 2021/849.