7,14,25,32-tetraazaundecacyclo[21.13.2.2²,⁵.0³,¹⁹.0⁴,¹⁶.0⁶,¹⁴.0⁸,¹³.0²⁰,³⁷.0²⁴,³².0²⁶,³¹.0³⁴,³⁸]tetraconta-1(36),2(40),3,5(39),6,8(13),9,11,16,18,20,22,24,26(31),27,29,34,37-octadecaene-15,33-dione; 7,14,25,32-tetraazaundecacyclo[21.13.2.2²,⁵.0³,¹⁹.0⁴,¹⁶.0⁶,¹⁴.0⁸,¹³.0²⁰,³⁷.0²⁵,³³.0²⁶,³¹.0³⁴,³⁸]tetraconta-1(36),2(40),3,5(39),6,8(13),9,11,16,18,20,22,26(31),27,29,32,34,37-octadecaene-15,24-dione

Endpoint summary

Description of key information

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

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

Study period:

04 November 2005 - 04 March 2006 (experimental)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(1995)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

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

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

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

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

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

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

Details on oral exposure:

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

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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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

Duration of treatment / exposure:

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

Frequency of treatment:

daily for at least 28 days, 7 days per week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

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

Control animals:

yes, concurrent vehicle

Details on study design:

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

Positive control:

none

Observations and examinations performed and frequency:

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

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

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

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

WATER CONSUMPTION: Yes (but no quantification)

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

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

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

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

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

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

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

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

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

Statistics:

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

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

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

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

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

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

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

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

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

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

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

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

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

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

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Conclusions:

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

Executive summary:

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral toxicity

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

 

Further toxicological data of category members:

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

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

 

 

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

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

 

Particle specific investigations:

Solubility:

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

Surface Reactivity:

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

 

Toxicological in vivo data:

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

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

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

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

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

 

Conclusion:

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

 

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

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

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

 

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

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

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

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

 

References

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

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

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

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008.

No data on repeated inhalation toxicity is available for the test substance, but data are available for the category member CAS 3049-71-6 (Pigment Red 178). In this study, repeated inhalation exposure to Pigment Red 178 was shown to induce local inflammation processes in the lung at concentrations of 5 mg/m³ and above, but no systemic toxicity was observed in this subchronic inhalation study or any other of the toxicological in vivo studies available for all category members.

The induction of local inflammation in the 90-day inhalation study, however, is most likely induced by the surface activity of the respective particles, as indicated in the results of the EPR (Please also refer to the Assessment Report in Chapter 13.2. or detailed explanations). However, the values of the red and violet Perylenes were significantly higher than those of the black Perylenes in this assay. This means that although all category members are enormously similar in structural and physicochemical and toxicological parameters, there are differences between the substances in this property. The black pigments appear to be significantly less surface active than the red and violet ones and therefore are expected not to be less toxic to the lung.

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

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

As a result, the test substance is not considered to be classified for repeated dose toxicity under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2020/217.